Of the 226 specimens tested by the Invader and Cobas for HR HPV, 92 were previously positive and 134 negative by the Invader assay. No specimens were excluded for inadequate cellularity/DNA content. Of the 92 Invader positive samples, 58 (63%) were positive on the Cobas 4800, while 34 (37%) were negative. Of the 134 Invader negative samples, 6 (4%) were positive by the Cobas 4800 while 128 (96%) were negative (see Table 1). In total there were 40 (18%) discordant results. This represented a significant discordance between the assays (p < 0.001; exact McNemar X² test), with overall agreement of only 82%. Of the 20 randomly-selected Invader positive discordant samples analyzed by Linear Array HPV genotype testing, 4 were low-risk HPV and 12 were negative for HPV DNA. One sample lacked sufficient cellularity to obtain a result. The remaining 3 samples genotyped as HPV types of uncertain oncogenic risk (HPV types 55, 67 and IS39).

There was 100% concordance between the 31 positive samples run in duplicate on the Cobas 4800. The mean difference between the cycle threshold (Ct) of the two runs was 0.4 with a standard deviation of 1.4. The run-to-run coefficient of variation was 3.5%. There was not a significant difference between the runs (p = 0.9). All 10 HR HPV control samples tested positive for HR HPV with a mean Ct of 33.33 and standard deviation of 0.50 and a coefficient of variation of 1.5%. All interspersed negative samples tested negative.

Of 10 the samples re-processed on the Cytyc T-3000, the 6 spiked positive HR control samples all tested positive for HR HPV on the Cobas 4800 while the 4 interspersed negative samples all tested negative.

Several studies have been published showing good agreement between the Cobas 4800 test and the Hybrid Capture 2 test (Qiagen, Gaithersburg, MD) [7,8,9]. In the present study, the comparison of the Cobas 4800 HR and Invader HPV ASR assays showed poor agreement. There was a striking difference between the agreement in the Invader positive and Invader negative groups. A high discrepancy was found in the Invader positive group with only 63% of the 92 specimens testing positive by Cobas 4800 HR analysis. This discordance may be attributed to the previously described high false positive rate of the Invader ASR assay [10]. Indeed, the majority of the 20 discordant samples selected for Linear Array analysis were, in fact, Invader false positives with 4 genotyping as low-risk HPV and 12 as HPV negative. Of the remaining 4 samples, 1 lacked sufficient cellularity to obtain a result and 3 samples genotyped as HPV types of uncertain oncogenic risk (HPV types 55, 67 and IS39). The Invader negative samples had a much higher agreement of 96% with the Cobas 4800 assay, suggesting a similar sensitivity between the two assays. In a comparison study between the FDA-approved Cervista HPV HR assay and the Digene hc2 assay, Youens et al., show a lower potential false positive rate with the Cervista assay [11]. This was attributed to a possible higher sensitivity of hc2 than Cervista or due to cross reactivity of the hc2 assay. A major difference between the Youens study and our current study was that the former conclusions were based on positivity rates versus our study which were based on testing of identical samples. These differences may reflect preanalytical differences in positivity rates of the different populations or analytical variables as described. In addition, our study compared the Cobas 4800 HR assay to the Invader HPV ASR which was validated in the laboratory as a LDT and has been in use in the laboratory for the past several years. The performance of the Invader ASR may not reflect the same performance of the FDA-approved Cervista HR HPV assay.

As noted by Kinney et al., the false-positive rate of any HPV screening assay is problematic as it can lead to over-referral to colposcopy and over-treatment causing both psychological stress for the women involved and inefficient use of health care resources [10]. The Cobas 4800 assay may offer improved specificity without sacrificing sensitivity in detecting HR HPV.

We recognize the small sample size as a limitation of this study and the fact that the Cobas testing was performed on post-aliquot residual liquid cytology samples. Additionally, only 20 of the 40 discordant results were genotyped by the Linear Array test. However, the fact that the vast majority of these 20 discordant samples randomly chosen for Linear Array testing indicated false positive Invader results supports the above hypothesis. The possibility exists that discordance is not solely due to false positive Invader results and could be due to sampling error. It is also possible that the Cobas 4800 test produced false negative results. However, the detection of human beta-globin DNA helps to reduces false negatives by serving as an internal control of adequate sample cellularity and extraction.

The FDA approved the Cobas 4800 HPV assay in the setting of pre-aliquot samples, those that have not been processed for cytology by an instrument such as the Cytyc T-3000. However, obtaining “pre-aliquot” samples for HR HPV testing presents logistical barriers for many laboratories, due in part to the “reflex” nature of a large part of HPV testing. Because the PreservCyt specimens are first and foremost cytology specimens and subsequent HPV testing is often a “reflex” test performed only after a cytologic diagnosis of ASC-US has been rendered, obtaining pre-aliquot specimens for HPV testing is impractical and could compromise the cytology exam. We observed no detectable carryover contamination from cytology processing with the Cytyc T-3000 when samples were subsequently analyzed on the Cobas 4800 and HPV detection rates were as expected.

The Cobas 4800 achieved excellent precision with a mean run-to-run difference in cycle threshold of 0.4 and a run-to-run coefficient of variation of 3.5%. In general clinical testing, a coefficient of variation of less than 5% is considered desirable. Precision in testing is an important quality for any laboratory test as it indicates that the chance of random variation in test results is small.

Our laboratory validated the Invader HPV ASR for clinical use in screening Surepath and PreservCyt liquid cytology samples before the assay was FDA approved as the Cervista HR HPV assay. The test uses the Invader technology to qualitatively detect HR HPV DNA from a pool of 14 HR HPV strains (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68). The assay requires a minimum volume of 2 mL of PreservCyt liquid cytology specimen and incorporates a Tecan EVO 150 robotic workstation using sample DNA extracted with the Agencourt Genfind magnetic bead separation method. After extraction, the HPV-specific reagents are then presented in three probe pools that correspond to four HPV virus groups based on phylogenetic relatedness. Additionally, a human DNA reagent specific for the human histone 2 gene (HIST2H2BE) serves as an internal control assuring the sample contains sufficient cellularity/DNA and that no inhibitory substances are present. If the internal control is negative, results are reported as insufficient specimen quality or quantity. In this study, all samples were analyzed according to the laboratory developed and validated protocol using the ASR.

The Cobas 4800 HPV assay is a qualitative test for the presence of HPV 16, 18 and a pool of 12 other HR HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. This assay requires 1 mL of liquid cytology sample. DNA is extracted, purified and prepared for PCR from PreservCyt cytology specimens by the Cobas x480 instrument which is integrated into the Cobas 4800 platform. Target DNA is then amplified by PCR with subsequent nucleic acid hybridization on the Cobas z480 analyzer. The Cobas 4800 assay simultaneously tests for human beta-globin DNA as an internal control of sufficient specimen cellularity. In addition, a positive and negative control specimen is included in each run. In this study, the Cobas 4800 HPV test was performed according to the recommendations of the manufacturer with the exception of using post-aliquot samples.

The Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Inc) was used to amplify target DNA by PCR and hybridize nucleic acids with probes designed to detect thirty-six HPV genotypes and one HPV 82 variant [6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), IS39, and CP6108] [12]. The assay also contains an internal control in the form of the human β-globin gene. The HPV Linear Array test was performed according to the manufacturer’s recommendation with the following exception: DNA extracted on the Tecan EVO150 with the Agencourt Genfind reagents.

At the time of this study, our laboratory performed reflex testing for HR HPV using the Invader HPV ASR assay on specimens with a cytologic diagnosis of ASC-US as well as co-testing for women over the age of 30 years upon clinician request. To perform this HR HPV testing, 4 mL of residual (“post-aliquot”) cytology specimen in PreservCyt medium were aliquotted from the original collection vial and 1 mL of this sample was then used in the assay. Before HR HPV testing, these samples had been previously processed with the Cytyc T-3000 (Hologic, Bedford, MA), an automated slide preparation technology for liquid-based cytology. In this study, we identified consecutive cervical cytopathology specimens (n = 226) between October 2011 and December 2011 that had already undergone Invader HPV testing and that had adequate residual volume in the PreservCyt medium for additional testing.

Each of the residual PreservCyt cytology specimens was analyzed with the Cobas 4800 assay within three months of initial collection. PreservCyt specimens were stored at 4 °C. Twenty Invader-positive discordant samples were randomly selected and analyzed using the Roche HPV Linear Array to detect the presence or absence of HPV DNA and to genotype positive samples.

To assess precision, 31 Invader positive samples were run in duplicate on the Cobas 4800 by different operators over multiple days. Additionally, HPV High Risk control material was purchased from Acrometrix, Inc and was analyzed on the Cobas 4800 in 10 replicates within the same run with previously tested negative samples between each positive control.

To control for possible sample-to-sample cross-contamination during cytology processing by the Cytyc T-3000, 6 Invader negative samples were spiked with different volumes of Acrometrix HPV High Risk Positive Control: 2 samples with 4 mL, 2 with 2 mL and 2 with 1 mL. These 6 spiked samples were re-processed on the Cytyc T-3000 with 4 previously negative samples interspersed between the controls in the following order: 4 mL, blank, 2 mL, 1 mL, blank, 4 mL, blank, 2 mL, 1 mL, blank. Each sample was analyzed for HR HPV on the Cobas 4800.