Tsiodras et al. BMC Cancer 2010, 10:53 http://www.biomedcentral.com/1471-2407/10/53 Capture 2 and PCR. Furthermore, we correlate these findings with histological diagnosis. Methods This is a cross-sectional study performed at all speci- mens sent for diagnostic cytology at the Departments of Histology and Embryology, and the Department of Cytopathology of the National and Capodistrian Uni- versity of Athens Medical School. Samples are addressed to these departments every day from many community and hospital-based Gynecology clinics cov- ering the metropolitan area of Athens (roughly a popu- lation of 3.8 million people). Informed consent was obtained from all women included in this study that was performed between January 2000 and December 2003. Cytological diagnosis Samples for the ThinPrep Pap test were collected by means of a Brun’s-like brush. The PreservCyt® vials (Cytyc Inc, Boxborough, MA, USA) containing the cell samples were addressed to the aforementioned Depart- ments for preparation of thin-layer slides using the ThinPrep 2000 Automated Slide Processor® (Cytyc, Boxborough, MA, USA) according to the manufac- turer’ instructions. Cytological findings were inter- preted according to the Bethesda classification system [10] and were classified as follows a) within normal limits; b) atypical squamous cells of undetermined sig- nificance (ASC-US); c) low-grade squamous intrae- pithelial lesion (LSIL); d) high-grade squamous intraepithelial lesion (HSIL); e) squamous cell carci- noma (SCC) or adeno-carcinoma. An experienced cytopathologist whose diagnostic experience exceeds 10 years examined all samples. Histological diagnosis A cervical biopsy was performed if ThinPrep testing revealed ASCUS and above cytological categories or there was positive HPV testing or there was a visible lesion upon colposcopy. Biopsy was performed by an experienced colposcopist (in practice for more than 10 years) as part of the study protocol. All women with indications consented to this procedure. The research was performed with the approval of the Institutional Review Board and in compliance with the Helsinki declaration. The three-tiered cervical intraepithelial neo- plasia (CIN) grading system was used for histological diagnosis [11]. All cases were diagnosed by an experi- enced pathologist (more than 10 years experience). In case histology showed a CIN 2 or CIN 3 or invasive car- cinoma the patient was referred for appropriate treatment. Page 2 of 8 HPV DNA detection Two techniques were used simultaneously: a) The commercially available Hybrid Capture 2 (HC2) kit that was used according to the manufacturer’s instructions (Digene Corporation, Gaithersburg, MD). Briefly, HC2 is a signal amplification assay that uses a combination of antibody capture and chemiluminescent signal detection. An RNA probe cocktail (full genome probes) that detects 13 high-risk HPV types (16/18/31/ 33/35/39/45/51/52/56/58/59/68) and 5 low-risk (6/11/ 42/43/44) HPV types is used in the reaction with the target DNA. The RNA:DNA hybrids are captured onto a solid phase coated with universal capture antibodies specific for the RNA:DNA hybrids. Capture RNA:DNA hybrids are detected with multiple antibodies conjugated to alkaline phoshatase. The signal resulting from the chemiluminescent reaction is read and the results are interpreted. For each specimen relative light unit/cutoff values were calculated as the ratio of the specimen lumi- nescence relative to the luminescence of the 1.0 pg/ml HPV-16 cut-off standard and reflect a semi-quantitative value of the cumulative viral burden from one or more of the examined genotypes. A relative light unit/cutoff value of ≥ 1 was considered as a positive result. Speci- mens testing positive for high-risk HPV types or low risk HPV types only were classified as HPV-high risk or HPV-low risk respectively, whereas specimens testing positive for both low and high risk HPV types were clas- sified as HPV low-high risk. b) An in house Polymerase Chain Reaction (PCR) to detect HPV DNA presence. DNA was extracted and purified from 3 ml of residual ThinPrep samples using a commercially available kit according to the manufac- turer’s instructions (QIAamp DNA mini kit, Tissue pro- tocol, QIAGEN Inc, Valencia, Ca). The quantity and integrity of the DNA extracts was monitored through spectrophotometer readings and amplification of b-glo- bin gene (endogenous control). The extracted DNA was tested for the presence of HPV DNA by PCR using the consensus (general) primers GP5+/GP6+ following a previously published protocol[12]. These primers amplify a fragment of approximately 150 base pairs (bp) from the L1 region of 22 anogenital HPV genotypes (types 6, 11, 13, 16, 18, 30-33, 35, 39, 40, 43, 45, 51, 52, 54, 55, 56, 58, 59 and 66). Positive PCR products (approximately 150 bp) visualized under UV light after electrophoresis in a 1% agarose gel in TBE buffer (0.1 mol/L Tris, 0.09 mol/L Borate, 1 mol/L EDTA) stained with ethidium bromide (0,5 μl/ml) were further analyzed using type-specific primers targeting the E6 region of the HPV genome of HPV types 6,11,16,18,31 and 33 according to a previously published protocol[13]. DNA extracted from HeLa cells infected with HPV18