CHAPTER 8 N. Esther Babady Memorial Sloan Kettering Cancer Center, New York, NY, USA Human papillomaviruses (HPVs) are small (7900 bp), circular, double stranded DNA viruses belonging to the Papillomaviridae family, which is further divided into genera, species, types, and subtypes [34]. The HPV genome consists of eight open reading frames (ORF) encoding three oncogenes (E5, E6, and E7), two regulatory proteins (E1 and E2), and two capsid proteins (L1 and L2). These ORFs belong to either the early region (E: 4500 bp), the late region (L: 1500 bp), or the long control region (LCR: 1000 bp), a noncoding region located between the early and late regions [10]. To date, there are more than 150 HPV types based on a difference of at least 10% within the L1 ORF [34]. HPVs primarily infect the epithelial cells of the skin and mucosal surfaces and cause a range of infections from asymptomatic to benign skin warts to invasive cervical cancer [10]. Cutaneous HPVs (types 1, 2, 3, 4, 5, 7, 8, 10, 26–29, 41) are associated with various conditions including common warts, “butcher’s warts”, plantar warts, flat warts, and epidermodysplasia verruciformis, a condition most commonly found in patients with autosomal recessive mutations on chromosome 17 [13, 57]. Mucosal HPVs are classified as either low-risk (LR) or high-risk (HR) HPV types based on their oncogenic potential. Approximately 40 HPV types, both low- and high-risk HPVs, can infect mucosal membranes, resulting in warts (LR HPVs) or intraepithelial neoplasia and cancers (HR HPVs) [58, 81]. Although multiple types exist, the two most common LR HPV types are HPV-6 and HPV-11. Both of these types are found in 90% of condyloma acuminata, a common type of genital warts that affect both men (coronal sulcus, the glans penis, and penil shaft) and women (cervix and vulva) [49]. Other low-risk HPVs that have been associated with condylomata acuminata include HPVs 2, 40–45, 51–56, 61, 70, 72, and 81 and coinfections with intermediate- and high-risk HPV types are not uncommon [13, 49, 57]. HPV-6 and HPV-11 are also associated with infections of the oral mucosa, including recurrent respiratory papillomatosis (laryngeal papillomatosis), a benign infection of the upper respiratory tract thought to be transmitted from mother to child during delivery [73, 119]. Infections of the cervix with high-risk HPV types tends to be persistent and often result in precancerous cervical lesions and invasive cervical cancer [12]. The association between HPV and cervical cancer is well established. High-risk HPVs account for approximately 10% of cancer worldwide [90]. HPV-16 is the most common HPV genotype found in cervical cancer, encompassing 55–60% of all cases, followed by HPV-18, which is responsible for 10–15% of all cases, with other high-risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 66) accounting for the other 30% of cases [33, 81, 122]. Following acquisition and persistence of HPV infection, cervical cancer develops through progression of the infected cells into precancerous cells and eventually invasion of the cervix [103]. Cervical intraepithelial neoplasia (CIN) is a histological abnormality of the cervical squamous epithelium and is considered a precursor to cancer, although CIN can regress spontaneously within 1–2 years without treatment. CIN is graded from CIN1 to CIN3 based on the degree of invasion of the epithelium, with CIN1 representing mild dysplasia with invasion of one-third of the epithelium, CIN2 representing moderate dysplasia with invasion of two-thirds of the epithelium, and CIN3 representing severe dysplasia with complete invasion of the epithelium [64, 124]. Similar to cervical cancer, high-risk HPV types are also associated with anal and oropharyngeal cancers. In a study of women with high-grade genital neoplasia, anal HPV DNA was detected in 51% of women, with 70% of infected women positive for a HR HPV subtype; 34% HPV-16 and HPV-18 and 36 % other HR HPV types including HPV-31, HPV-53, and HPV-59 [89]. Anal HPV infections are common in HIV positive men who have sex with men (MSM) [28, 85, 86]; in non-HIV infected MSM, high-risk HPV types can be detected in up to 75% of men screened, with multiple types being detected and HPV-16 being the most commonly detected genotype [21, 39]. About 25% of squamous cell carcinoma of the head and neck are associated with the presence of HPV. Approximately 60–70% of those are caused by high-risk HPV types, with HPV-16 being responsible for greater than 90% of the cases and other types, including HPV-18, 31, 33, and 35, accounting for the remaining cases [121]. In a recent study, Paolini et al. showed that both cutaneous and mucosal HPV infect the oral epithelium, but only mucosal HPV, particularly the high-risk HPV-16 type, is associated with cancer [87]. The goal of screening is to identify women with precancerous lesions, mainly CIN3, who are at high risk of developing cervical cancer. Screening guidelines for cervical cancer are established by the American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology (ASCP). The most recent guidelines recommend that women be screened starting at age 21 years, with frequency based on age; every 3 years by cytology for women 21–29 years of age; every 5 years by cytology and HPV testing or every 3 years by cytology alone for women 30–65 years of age; and no testing for women over 65 years of age and no history of positive HPV tests and abnormal cytology [101]. Women testing HPV positive by co-testing (i.e., HPV testing positive, cytology negative) should further be evaluated by either repeat co-testing within 12 months or immediate HPV genotyping for either HPV-16 alone or both HPV-16 and 18 followed by colposcopy for any positive results [101]. On the other hand, women with a negative HPV test but positive cytology should continue with routine surveillance screening as there is no evidence of decreased risk of cancer by increasing testing frequency in this subgroup of women [101, 128]. Although HPV testing has increased sensitivity over cytology for the detection of CIN2 and CIN3 lesions [3, 66, 68, 96], the lower specificity and inability to always ensure specimen adequacy had resulted in a recommendation that HPV testing alone not be used for cervical cancer screening [101]. In the latest screening guidelines (2012), HPV co-testing with cytology or cytology alone remains the recommended methods for cervical cancer screening. However, in April 2014, the Cobas 4800 HPV (Roche Diagnostics, Indianapolis, IN) became the first molecular HPV test to be approved by the US Food and Drug Administration (FDA) as a first-line primary test for screening in women 25 years and older. The addition of this claim to a molecular HPV test will certainly impact the next set of recommendations for cervical cancer screening. Both low- and high-risk HPV types can cause abnormal cervical cytology. Abnormal cervical cytology diagnoses are reported as atypical squamous cells (ASC), low-grade squamous intraepithelial lesions (LSIL) or high-grade squamous intraepithelial lesions (HSIL), and squamous cell carcinoma. The two most common subcategories of ASC are ASC of undetermined significance (ASC-US) and ASC cannot exclude HSIL (ASC-H) [124]. In addition to abnormality in squamous cells, cervical screening can also reveal abnormalities in glandular cells, which are divided in increasing order of severity from atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS) and adenocarcinoma [124]. Conventional cytology, commonly known as the Papanicolaou (Pap) smear, has been in use since the 1950s [88]. Pap smears are performed by manually collecting a cervical sample using a cervix brush and directly transferring cells onto a glass slide for subsequent staining and analysis [61]. Pap smears remain the gold standard for cervical cancer. However, the test is laborious and prone to false-negative results due either to sampling errors or detection errors, with reported sensitivity varying from 30 to 87% [51]. Liquid-based cytology (LBC) was developed to improve the quality of specimen sampled for cervical screening and concomitantly allows co-testing by HPV DNA testing and cytology. Currently, two LBC devices have been approved by the US Food and Drug Administration (FDA) as of August, 2014, and are commercially available (Table 8.1). Other methods and products may also be available. The ThinPrep Pap Test (Hologic Inc., Marlborough, MA) was FDA approved in 1996 and works by using a brush, spatula, or broom-like device to collect cells from the cervix. The collected cells are then transferred from the collection device into a vial containing PreservCyt solution. In the laboratory, the PreservCyt sample is set-up on a ThinPrep 2000 Processor (Hologic Inc., Marlborough, MA), which gently disperses the specimen, collects diagnostic cells on a filter and transfers them onto a glass slide for downstream analysis. The ThinPrep Pap test is also FDA approved for use in additional testing, including glandular lesions, HPV testing, and Chlamydia trachomatis and Neisseria gonorrhoeae testing. Table 8.1 Liquid-based cytology devices * As of August, 2014. The second LBC device to be FDA approved was the SurePath liquid-based Pap test in 1999 (Becton Dickinson, Franklin Lakes, NJ). Similar to the ThinPrep Pap test, the SurePath LBC uses a brush, a spatula or broom-like device to collect cervical cells, followed by transfer to a vial containing BD CytoRich Red preservative fluid. Once in the laboratory, the specimen is placed on the PrepStain slide processor where it is vortexed and transferred to a PrepStain Density reagent for enrichment by centrifugal sedimentation. The sediment obtained is resuspended and moved to a PrepStain settling chamber for transfer onto a glass slide for downstream processing, including a modified Papanicolaou stain procedure [9]. The BD SurePath Pap test is also FDA approved for out-of-vial testing of Chlamydia trachomatis and Neisseria gonorrhoeae. Although there is clear improvement in the quality of the smear obtained using LBC compared to conventional Pap smear [112], studies on the superiority of LBC in providing increased diagnostic sensitivity have yielded conflicting results. In one meta-analysis study, the ThinPrep Pap test had double the rate of diagnosis for HSIL lesions compared to conventional Pap smear (0.71% incidence vs. 0.30% incidence) but no significant difference in the detection of atypical squamous cells of undetermined significance (ASCUS) [8]. Similar findings were obtained in a more recent study showing a significantly higher detection rate by SurePath for ASCUS (2.07% vs. 0.87%) and a slightly higher rate of detection for LSIL (0.27% vs. 0.22%) and HSIL (0.64 vs. 0.56) [6]. A meta-analysis and a randomized controlled trial from the Netherlands comparing the positivity rate of the ThinPrep Pap test to that of conventional cytology did not show a statistically significant difference between the two methods for the diagnosis of CIN, ASCUS, LSIL, or HSIL although the number of adequate slides was greater by LBC [1, 105, 106]. Only a few reports of head-to-head comparison of the ThinPrep versus SurePath have been published [45, 127, 131]. Zhao and colleagues compared the two methods for rates of unsatisfactory smears, residual specimens available for additional testing, and accuracy in detection of significant cervical lesions [131]. The two methods performed similarly, except SurePath resulted in less unsatisfactory smears than ThinPrep (0.2% vs. 1.5%). A study by Wright et al. also showed equivalence for both ThinPrep and SurePath in terms of predictive value for CIN2, high-grade, and low-grade dyskaryosis [127]. A recent meta-analysis demonstrated that SurePath LBC yields fewer unsatisfactory smears than ThinPrep, although multiple factors including year of publications, country of origin, and criteria to determine adequacy might have affected the outcome of the analysis [45]. Reprocessing of unsatisfactory slides obtained using ThinPrep Pap with 10% glacial acetic acid was approved by the FDA and was shown in one study to decrease the unsatisfactory rate by 34% [62]. The most common target for HPV immunohistochemistry (IHC) assays is the cyclin-dependent kinase inhibitor p16 (INK4a), which participates in the control of the cell cycle by the retinoblastoma (Rb) protein. In cervical lesions due to high-risk HPV, the p16 protein accumulates due to lack of transcription inhibition from an inactivated Rb by the HPV E7 oncoprotein [83]. Detection of the overexpressed p16 is used as a surrogate marker of HPV infection and is accomplished by IHC. Specific monoclonal antibodies are commercially available as part of a kit from several manufacturers. Following a series of washes and incubations with secondary antibodies, horseradish peroxidase, and chromogenic substrates, positive p16 IHC are visualized as brown spots that can have either a diffuse or strong focal staining pattern [70]. The p16 IHC has been evaluated against other HPV detection methods including cytology, nucleic acid testing, and in situ hybridization. Reported sensitivity and specificity of p16 IHC varied from 75 to 80% and 62 to 71% respectively, for detection of p16 in ASCUS cytology with underlying CIN2+ and CIN3+ [35, 78]. In other studies, the sensitivity and specificity of p16 IHC in LSIL ranged from 82.1% to 100% and 93.3% to 100% respectively [70, 123]. Szarewski et al. compared the performance of p16 IHC against several assays using CIN2 as a cutoff and showed the sensitivity and specificity of p16 IHC to increase from 59.1% for CIN2, to 83.0% for CIN2+ and 92.7% for CIN3+, with specificity of 68.7% and 65.8% for CIN2+ and CIN3+ to 92.2% [113]. In a subsequent study by the same group the sensitivity of the p16 IHC increased slightly for CIN2 (78.2% vs. 59.1%) with a decrease in the overall specificity of the assay (54.7%) [113, 114, 123]. In situ hybridization (ISH) allows for the direct visualization of target nucleic acids in tissues or cells through binding of labeled probes. Commercial reagents available for ISH HPV include the INFORM HPV II Family 6 Probe and the INFORM HPV III Family 16 Probe (Ventana Medical/Roche Diagnostics, Tucson, AZ) for cytological specimens (C) and tissues samples (B) and the GenPoint Catalyzed Signal Amplification System (DAKO, Carpinteria, CA). The INFORM HPV II Family 6 Probe consists of a cocktail of HPV genomic probes directed against the most common low-risk HPV types (6 and 11) while the INFORM HPV III Family 16 consists of a cocktail of HPV genomic probes directed against the most common high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66). Additional reagents include the ISH Protease for digestion of proteins surrounding the HPV DNA and the iVIEW Blue Plus detection kit (Ventana Medical/Roche Diagnostics, Tucson, AZ), which uses biotin labeled antifluorescein antibody to detect the fluorescein labeled probes followed by addition of streptavidin, nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate, and alkaline phosphatase, and yields an intense blue color when probes hybridize to target DNA and finally the Ventana Red counterstain [70]. Positive HPV ISH pattern can be either episomal or integrative. The GenPoint Catalyzed Signal Amplification System targets high-risk HPV genotypes, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and code Y1443 as well as the low-risk HPV types including types 6 and 11. Detection is performed using GenPoint Tyramide signal amplification system for biotin labeled probes, peroxidase-conjugated streptavidin, diaaminbenzide as the chromogenic substrate, and hematoxylin as the counterstain [32, 67]. Although labor-intensive and prone to subjective interpretation, HPV ISH assays are highly specific and have the advantage of detecting HPV DNA in the right histological context of HPV genome integration within the nucleus [11, 32]. In one study, the results obtained using ISH (DAKO), cytology, and polymerase chain reaction (PCR) were comparable, with concordance between ISH and PCR of greater than 90% for CIN3+ and HSIL lesions [67]. The sensitivity and specificity of ISH for detection of CIN2+ was 89.5% and 39.9% respectively, emphasizing the need for additional testing. Kong et al. compared the performance of INFORM HPV assays, a research-use only (RUO) version of the DAKO ISH assay, and p16 IHC and PCR in a set of specimens that included LSIL, HSIL, and negative cervical specimens [70]. The specificity of all ISH assays was 100% with the sensitivity of DAKO, INFORM HPV II, and INFORM HPV III being 55.6%, 53.6%, and 69.2% respectively for SIL. In this study, compared to p16 IHC, all ISH assays were less sensitive (IHC sensitivity 82.1%). Currently, there are four US FDA-cleared assays for high-risk HPV testing including the Digene Hybrid capture assay 2 (HC2) HR (Qiagen Inc., Germantown, MD), the Cervista HR HPV (Hologic Inc., Bedford, MA), the APTIMA HPV assay (Hologic Gen-Probe Inc., San Diego, CA) and the Cobas 4800 HPV (Roche Diagnostics, Indianapolis, IN) (Table 8.2). These assays are approved for co-testing along with cytology and for patients with ASCUS on cytology [101]. The Roche Cobas 4800 HPV test is additionally approved as a first-line cervical cancer screening assay in women 25 years of age or older. Table 8.2 Commercial assays for HPV nucleic acids detection * As of August, 2014. STM: specimen transport medium; TC: target capture; TMA: transcription mediated amplification; HPA: hybrid protection assay; HC: hybrid capture; CE: Conformité Européenne; FDA: Food and Drug Administration. The first molecular assay approved by the US FDA for the detection of HPV infection was the Hybrid Capture assay (Digene Corporation, Gaithersburg, MD). The HC2 assay is a hybridization assay with signal amplification detection. Two sets of single-stranded RNA probes, one for low-risk HPV subtypes and one for high-/intermediate-risk HPV subtypes, are incubated with denatured DNA from specimens suspected of harboring HPV DNA. The resulting RNA/DNA hybrids are captured by an antibody conjugated to alkaline phosphatase enzyme and detected by addition of a chemiluminescent substrate that emits light when cleaved by the alkaline phosphatase enzyme. The reaction is measured using a luminometer and the results expressed as relative light unit (RLU). The first generation of the HC assay, the HC1 assay, was designed to detect 14 HPV subtypes including nine high-risk types (16, 18, 31, 33, 35, 45, 51, 52, and 56) and five low-risk types (6, 11, 42, 43, and 44) and had a sensitivity for the detection of high-grade SIL varying from 71.2 to 93% with a positive predictive value of approximately 17.8% [23, 27, 43]. The HC1 assay was subsequently replaced by the second-generation assay, the HC2 assay, which detects additional HPV types including 39, 58, 59 and 68 and has a reported sensitivity up to 98.1% for detection of high-grade cervical lesions [19, 24, 25]. The HC2 assay differentiates between low- and high-risk HPV infection but does not provide specific HPV genotypes. Another formulation of the HC2, the digene HC2 High-Risk HPV DNA, is designed to detect 13 high-risk HPV types including the nine types detected by the HC2 LR and HR assay plus HPV types 39, 48, 59, and 68. Limitations of the HC2 assay include cross-reaction with untargeted low-risk HPV types (6, 11, 26, 30, 40, 42, 53, 54, 61, 67, 70–73, 81, 83, 84, 87, and 91), resulting in reduced test specificity [18, 52, 93]. Additional limitations of the HC2 assay include the requirement for large sample volume (at least 4 mL) and the need to retest any results falling within the gray zone [80]. The APTIMA HPV assay is a target capture assay designed to amplify the messenger RNA (mRNA) transcript of the E6/E7 gene by transcription-mediated amplification and detect the amplified product by hybridization protection assay [36]. The assay targets 14 high-risk HPV including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The assay does not discriminate among the 14 genotypes. The lower limit of detection of the APTIMA HPV test is between 17 and 488 copies/mL depending on the genotype and the assay can be fully automated on the TIGRIS DTS platform or the PANTHER System (Hologic Gen-Probe Inc., San Diego, CA) [36, 37, 50]. In a recent meta-analysis study, the pooled sensitivity of the APTIMA HPV assay was similar to that of HC2 assay for detection of underlying CIN2 or CIN3 in cytology specimens showing ASCUS (95.7–96.2% vs. 93.8–95.5%) and LSIL (91.0–96.7 % vs. 95.5–98.8%) but the APTIMA assay had a significantly higher specificity than HC2 assay for both ASCUS (54.9–56.4% vs. 44.9–46.8%) and LSIL (38.7–42.5% vs. 27.8–28.6%) [2]. The Cervista HPV HR gained FDA approval in 2009. The assay used hybridization probes and signal amplification to detect 14 high-risk HPV types including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. In a multicenter evaluation, the Cervista HPV HR test showed a lower limit of detection varying from 1250 to 7500 copies/mL depending on the genotype [30]. The clinical sensitivity and specificity of the Cervista HPV HR assay for the detection of CIN2 and CIN3 from ASCUS specimens was 92.8 and 44.2% for CIN2 and 100% and 43% for CIN3, respectively [42]. In a recent head-to-head comparison of the Cervista HPV HR assay to the HC 2 assay, the two assays showed comparable sensitivity in detecting HR HPV in specimens with negative cervical cytology, although the positive agreement between the two assays was only 70% [71]. In other studies, the Cervista HPV HR assay showed comparable sensitivity but increased specificity for the detection of CIN3 or cancer, which was hypothesized to be due to the higher viral load cutoff for Cervista HPV HR (3.2 pg vs. 1.0 pg) and the lack of cross-reactivity of the assay with LR HPV types [7]. However, cross-reactivity of the Cervista HR is seen with low-risk HPV types 67 and 70, in addition to HPV types 6, 73, 84, and 91 [52]. In 2011, the FDA approved the Cobas 4800 HPV test (Roche Diagnostics, Indianapolis, IN) for the detection of 14 high-risk HPV including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. In 2014, the Cobas 4800 HPV received FDA approval for use as a first-line primary test for screening in women 25 years and older. The assay is fully automated and combines specimen extraction (PreservCyt) followed by multiplexed real-time PCR amplification and detection. The clinical sensitivity and specificity of the Cobas 4800 HPV test for detection of CIN2+ was comparable to that of the HC2 assay when tested on archived cervical scrapes [59] or ThinPrep specimens [72, 111]. In two recent studies, the agreement between the Cobas 4800 HPV test and HC2 was shown to be moderate (85.9%) [47] or high (93.8%) [125] but the results of the detection rate were conflicting, with one study showing higher detection rate for Cobas 4800 HPV and the other study showing higher rate for the HC2 assay [47, 125]. The following section covers assays that have not yet been approved for use by the US FDA, as of this writing, but are undergoing evaluation, or have already been Conformité Européenne (CE) marked or cleared for use in Europe, Canada, and other countries outside of the United States (Table 8.2). The Roche Amplicor HPV test includes a conventional PCR step followed by hybridization with a cocktail of probes and detection of HPV DNA on a plate reader at 450 nm. The assay targets the following 13 high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 without differentiating among them [116]. In studies comparing the Amplicor HPV test to the HC2 assay, the two tests showed comparable sensitivity and specificity for detection of HPV DNA with agreement ranging from 82.6–89.2% [14, 40, 100]. Similar to the HC2 assay, the Amplicor HPV test can cross-react with low-risk HPV types including HPV-6, 11, 42, 53, 54, 62, 71, 81 and 83, resulting in lower specificity [100]. The Abbott Real
Human Papillomaviruses
8.1 Background
8.1.1 Low-risk HPV and related infections
8.1.2 High-risk HPV and related infections
8.1.3 Testing utility and recommendations
8.2 Commercial assays
8.2.1 Non-nucleic acid amplification tests
8.2.1.1 Cytology-based assays
Name
Manufacturer
Country
Method
Automation
FDA status*
ThinPrep Pap
Hologic Inc.
USA
Filtration
ThinPrep 2000 Processor
Approved
SurePath Pap
Becton, Dickinson and Co.
USA
Centrifugation
BD PrepStain slide processor
Approved
CYTO-screen
Seroa
Monaco
Centrifugation
None
Not approved
Turbitec
Labonord
France
Centrifugation
None
Not approved
(CE marked)
CellSolution
Menarini Diagnostics
Italy
Centrifugation
CellSolutions 30
Not approved
Shandon Papspin
ThermoFisher Scientific
USA
Centrifugation
None
Not approved
DNA-Citoliq
Digene
Brazil
Centrifugation
None
Not approved
8.2.1.2 Immunohistochemistry assays
8.2.1.3 In situ hybridization assays
8.2.2 Nucleic acid amplification tests
8.2.2.1 FDA-cleared assays
Test name
Technology
Target
Internal control
HPV Genotypes
Specimen
Regulatory status*
Extraction
Detection
Aptima HPV
(Hologic Gen-Probe, Inc.)
TC
TMA
HPA
mRNA
Exogenous noninfectious RNA target
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
PreservCyt solution
FDA
CE
Automated target capture on TIGRIS DTS system
TIGRIS DTS System or Panther System
Cervista HPV HR
(Hologic, Inc.)
Invader chemistry (isothermal signal amplification)
DNA
Endogenous (Human histone 2
gene)
16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
PreservCyt solution
FDA
CE
Manual
(Gen-find DNA extraction kit)
Fluorescent plate reader
Digene HC2 HR and LR
(Qiagen, Inc.)
Hybridization HC
Signal amplification
DNA
None
6, 11, 42, 43, 44, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
PreservCyt solution
hc2 DNA Collection Device
Biopsies collected in STM
FDA
CE
Manual
Luminometer
COBAS HPV Test (Roche Molecular Diagnostics)
Multiplex real-time PCR
DNA
β-globin
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
PreservCyt solution
FDA
CE
Cobas x 480
Cobas z 480
Abbott RealTime High Risk HPV
(Abbott Molecular )
Multiplex Real-time PCR
DNA
β-globin
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
PreservCyt Solution
SurePath Preservative Fluid
Abbott Cervi-Collect Specimen Collection Kit
CE
m2000sp
m2000rt
Roche Amplicor HPV test (Roche Molecular)
PCR
NA hybridization
DNA
β-globin
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
PreservCyt Solution
SurePath Preservative Fluid
CE
Manual
Plate reader
The PreTect HPV-Proofer (Norchip AS)
Real-time multiplex NASBA
mRNA
Endogenous (U1A)
16, 18, 31, 33, 45
PreservCyt Solution
SurePath Preservative Fluid
PreTect TM
CE
Manual or automated
Fluorescence reader
NucliSENS EasyQ HPV (bioMérieux)
Multiplex NASBA
mRNA
Endogenous (U1A)
16, 18, 31, 33, 45
PreservCyt Solution
CE
NucliSENS MiniMag
Easy Mag
NucliSENS
EasyQ
HPV OncoTect E6/E7 mRNA (IncellDx)
Flow cytometry
In situ hybridization
mRNA
PreservCyt Solution
SurePath Preservative Fluid
CE
None
FACScan cytometer
8.2.2.2 Non-FDA-cleared assays
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