Since several countries adopted HPV testing as a primary screening method for cervical cancer, different triage strategies for high-risk HPV positive women are being evaluated in order to avoid misinterpretation and mismanagement of patients. Reflex cytology, partial HPV genotyping and host and viral methylation are the most commonly evaluated strategies to date. The aim of our study was to assess whether there is a correlation between the beta-globin cycle threshold (Ct) value and the concentration of extracted DNA in order to predict sufficient yield of DNA for methylation.
For the purpose of this study, we have evaluated 195 samples that were tested with clinically validated HPV test Abbott RealTime High Risk HPV test (RealTime; Abbott, Wiesbaden, Germany) at the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia between January 2016 and March 2016. The internal control of the RealTime is based on amplification of the 136-bp region of the beta-globin gene. Furthermore, DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and DNA concentration was measured with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, USA). Samples with DNA concentration above 2.5 ng/ul were considered eligible for further HPV methylation analysis.
Ct values for beta-globin ranged between 20.26 to 31.25, with an average Ct value of 24.57. Of the 195 samples tested, 92 had DNA concentration above 2.5 ng/uL, 98 samples below 2.5 ng/uL and 5 below measurable concentration (range: 0.144-40,0 ng/ul). Our analysis showed that 93.8% of the samples with Ct values below 22.00 (15/16), 87.7% of the samples with Ct values between 22.00 and 24.00 (57/65) and 29.0% of the samples with Ct values between 24.00 and 26.00 (18/62) had sufficient DNA yield for methylation, respectively. Additionally, only two out of 52 samples with the Ct value above 26.00 had a concentration above the cut-off value (one sample with Ct value 26.15 had DNA concentration 3.42 ng/ul and one sample with Ct value 26.28 had DNA concentration 4.28 ng/ul).
In HPV primary screening settings a well-balanced triage strategy will be needed to identify women with higher risk of underlying cervical intraepithelial neoplasia grade 2 or worse. Our results suggest that Ct values of beta-globin of clinically validated HPV test RealTime can be used as a preliminary indicator for sufficient DNA concentration needed for methylation analysis.