The presence of high risk (HR) and low risk (LR) HPVs in cervical specimens was evaluated by Hybrid Capture II® (HCII) (Qiagen, Gaithersburg, USA) [12–17] using probemix B, specific for 13 HR HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and probemix A, specific for 5 LR HPV types: 6, 11, 42, 43 and 44. To lyse cells and release DNA, specimens collected in PreservCyt® (no more than 20 samples at a time) were processed with Digene Sample Conversion kit (Qiagen, Gaithersburg, USA), according to the manufacturer's instructions. HCII assay followed. HPV DNA testing was done locally in 3 sites (one in Abruzzo and two in Rome) while samples collected in Cagliari and Catania were analysed in Florence (ISPO). Results of HCII tests for HPV DNA were expressed as relative light units (RLU): specifically, the ratio of the specimen light emission to the average emission of three concurrently tested positive controls (1 pg/ml HPV DNA). All samples with a ratio value over 1 were considered HPV positive. HCII quality assurance procedures were performed as previously described .
An additional internal control was then used in each HCII test plate. At the beginning of the study, each laboratory denatured a quality control LR HPV sample (HPV type 6 DNA) and a quality control HR HPV sample (HPV type 16 DNA) provided with the kit. 100 μl of denaturated control sample were aliquoted in 15 tubes and stored at -20°C. Single aliquots were then used in every HCII plate. Quality controls for both LR and HR HPV had a specific concentration of virus DNA (5 pg/ml) and therefore an expected ratio value ranging from 2 to 8.
For external quality assurance, 22 STM samples (100 μl) were sent to one of the laboratories involved in the study (Florence, ISPO) and samples had to be tested twice. Laboratories that used PreservCyt® medium, sent 15 additional ThinPrep samples (4 ml) to the same facility, where the whole HCII procedure was repeated.
Results were expressed as specimens in RLU/CO; a modal value (i.e. the most frequently positive or negative value reported) was defined for each case in the external control set and considered as the reference result. The result provided from each single laboratory was compared with the modal value.
Typing procedures were centralized in Florence at the ISPO laboratory and all the centres involved in the study sent HR or LR HPV positive samples there.
For DNA extraction, 1.5 ml of samples in PreservCyt® solution and 200 μl of STM samples were used to collect DNA with a QIAamp DNAMini Kit. Samples were eluted in 100 μl AE buffer preheated at 70°C. An HPV negative sample was included in each batch of extraction to exclude any contamination during this phase.
HCII HR HPV positive specimens were amplified and typed with "consensus High Risk HPV genotyping kit" (Qiagen); this system uses PCR with biotinilated GP5+/GP6+ primers, followed by reverse Line Blot for 18 high risk HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.
PCR was performed in 50 μl final volume with 10 μl DNA, 5 μl 10× buffer II, 7 μl 25 mM MgCl2, 10 μl 1 mM dNTPs, 0,3 μl AmpliTaqGold (Applied Biosystem) DNA polymerase (5 U/μl) and 2 μl of Qiagen Primer set, as recommended by the manufacturer. The thermal cycler program contained ramping times between the temperature used for denaturation, annealing and elongation that appeared essential for optimal performance . Activation of the Taq polymerase (9 min at 94°C) was followed by 40 PCR cycles: denaturation at 94°C for 20 seconds, with 2,8°C/sec ramp speed, annealing at 38°C for 30 seconds, with 1,8°C/sec ramp speed, and elongation at 71°C for 80 seconds with 1,8°C/sec ramp speed, and a final extension at 71°C for 4 minutes . Amplified products were run on 2% agarose gels containing ethidium bromide and visualized by ultraviolet light.
All samples, independently from gel results, underwent reverse line blot analysis. Biotinylated PCR products were hybridized with specific oligonucleotide probes bound to membrane strips. After stringent washing, streptavidin-conjugated alkaline phosphatase was added and a non-radioactive reaction performed using a chromogenic substrate. Strips containing the resulting purple precipitates were analysed visually from an interpretation grid supplied with the kit. A biotinylated poly(dT) control for conjugate reaction is present in each strip to ensure good test performance and a proper alignment of the strips on the interpretation sheet. Samples were considered positive for a specific HPV type if the precipitate was viewed in the specific position of the strip. In each run we included two negative controls (a purified HPV negative DNA sample extracted in the same batch as the test ones, and a DNA-free sample) and two positive controls (HPV16 plasmid purchased with the kit and a known non-16 HR HPV DNA).
GP5+/6+PCR-negative and reverse Line Blot-negative samples were amplified for the β-globin gene using primers GH20-PC04 (268 bp amplicon lenght) to assess the integrity of DNA .
β-globin positive samples were re-typed with "INNO-LiPA HPV genotyping Extra Amp" (Innogenetics, Ghant, Belgium) following the manufacturer's instructions, because this system can detect 28 HPV HR or LR types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82. The β-globin negative samples were extracted and typed again.
The remaining un-typed samples (GP5+/6+PCR-positive, reverse Line Blot-negative and INNO-LiPA negative) were considered HPV X type.
HCII LR HPV positive specimens (probe A specific for 5 LR types: 6,11,42,43,44) were typed using HPV 6 and HPV 11 specific primers and resulting negative samples were considered positive for the other LR types included in probemix A (HPV 42,43,44). No LR type, other than 6 and 11, was searched for.
Primer sequences for HPV 6 were 5'-TAGTGGGCCTATGGCTCGTC-3' (sense) and 5'-TCCATTAGCCTCCACGGGTG-3' (antisense) which amplified a 289 bp product.
Primer sequences for HPV 11 were 5'-GGAATACATGCGCCATGTGG-3' (sense) and 5'-CGAGCAGACGTCCGTCCTCG-3' (antisense) which amplified a 360 bp fragment .
PCR reactions were performed in 25 μl reaction volume using 5 μl DNA, 2,5 μl 10× buffer II, 1,5 μl 25 mM MgCl2, 2 μl 2,5 mM dNTPs, 1,25 μl 10 μM primers, 0,2 μl Taq Gold (Applied Biosystems). Hot-starting at 94°C for 6 min was followed by 40 cycles as follows: 95°C for 30 sec, 58°C for 30 sec, 72°c for 30 sec and an hold at 72°C for 5 min. Amplified products were run on 2% agarose gels containing ethidium bromide and visualized by ultraviolet light.