Reproducibility of the cobas 4800 HPV test in the "grey zone".

In cervical cancer screening, standardized methods are used for the detection of human papillomavirus (HPV). The most clinically validated method is currently Hybrid Capture 2 (HC2, Qiagen, Hilden, Germany). However, this technique produces equivocal results in the so-called grey zone (results between 2.5 and 0.8 relative light units),1-4 especially when liquid-based cytology is used. The cobas 4800 HPV test (Roche Molecular Systems, Pleasanton, California, USA) is an emerging technology that can be used as an alternative to the HC2 test for screening purposes.5 It is a real-time, polymerase chain reaction (PCR)–based technology that independently analyzes the presence of HPV 16, HPV 18, and other high-risk HPV types. The present study addresses the reproducibility of the cobas 4800 HPV test results in the grey zone. We considered the grey zone to comprise those cases that become positive after 35 cycles of PCR amplification. The cycle threshold obtained in real-time PCR is inversely correlated with the amount of target DNA present in the samples. Results obtained after cycle 35 are therefore obtained when a low amount of target DNA (e.g., HPV) is present in the sample. In addition, cases in which amplification is observed between cycles 35 and 40 show a higher variation among the cycle threshold replicate values, as PCR becomes more stochastic in the presence of a low amount of HPV DNA. For this reason we aimed at determining the reproducibility of cases with a low amount of HPV present in the sample, that is, cases amplifying any of the HPV tested between cycles 35 and 40.6,7 A total of 79 liquid-based cytology cases were included in the study: 64 cases were positive for HPV after > 35 cycles, and 15 cases were negative for all the 3 determinations (16, 18, and other high-risk HPV). All of the cases were retested by cobas 4800. Regarding global results, a case was considered positive when at least 1 of the 3 determinations was positive. In the second test, 78 out of 79 cases showed the same results as the first determination (Table I) (kappa = 0.960, 95% CI 0.920–1), but it has to be taken into account that in some cases more than one HPV determination was positive and that one of the positive values could be at cycles below 35. When analyzed by genotypes, regarding HPV 16 the results of 4 out of 79 cases changed (from positive to negative) in the second test (kappa = 0.877, 95% CI 0.818–0.936). Among HPV 18–positive cases only 2 of them changed to negative in the second determination (kappa = 0.928, 95% CI 0.878–0.978). Finally, among the other high-risk HPV-positive cases, 4 out of 79 patients changed (positive to negative) in the second test (kappa = 0.874, 95% CI Analytical and Quantitative Cytopathology and Histopathology ®