114 Chapter 5 diagnostic and screening settings. METHODS CERVICAL SAMPLES Clinician-collected cervical samples in PreservCyt solution (Hologic Inc., San Diego, CA, USA) were obtained from the Scottish HPV Archive of the University of Edinburgh, Scotland, according to the Research Tissue Bank status as approved by the Lothian NRS BioResource (15/ES/0094). Aliquots of 2-4 mL were available for this study. In light of the HPV triage application, the series consisted of cervical samples of 120 HPV-positive women, comprising of 14 women with histologically confirmed CIN3 (median age 38; range 32-45; IQR 32-45), 10 women with histologically confirmed CIN2 (median age 33.5; range 25-54; IQR 27.8-42.3) and 96 women defined as controls (median age 35; range 22-63; IQR 28-44), who either had histologically confirmed CIN0 (n = 28) or CIN1 (n = 10), or were considered to have no evidence of CIN2 or worse (CIN2+) as they displayed HPV clearance combined with normal cytology in follow-up (n = 58). DNA ISOLATION Extraction of DNA from 1 mL PreservCyt sample was performed using an automated extraction system (NucleoMag 96 tissue kit, Macherey-Nagel GmbH&Co. KG, Düren, Germany) and a Microlab Star robotic system (Hamilton, Gräfelfing, Germany) according to manufacturer’s protocol. The concentration of extracted DNA was measured using a Qubit fluorometer (Qubit, ThermoFisher Scientific, MA, USA). BISULPHITE TREATMENT For bisulphite conversion of DNA, the reference protocol (EZ DNA Methylation Zymo kit; Zymo Research, CA, USA) was performed according manufacturer’s recommendation using 250 ng of isolated genomic DNA as input material 13. This bisulphite-conversion kit is verified for use with the QIAsure Methylation Test 14, but not compatible with direct cell input. A direct conversion protocol on the cervical cells used the Epitect Fast 96 Bisulphite conversion kit (QIAgen, Hilden, Germany) 15. Cervical cells from 500 µl (1/40th of the original sample) PreservCyt sample was used as input. For invalid samples an input of 2 mL (1/10th of the original sample) PreservCyt sample was used. Samples were centrifuged and the cell pellet was resuspended in 20 µl PreservCyt prior to bisulphite conversion, performed according to manufacturer’s instructions, except for elution in 50 μl Elution Buffer and omission of carrier RNA. Details on both protocols are outlined in Figure 5.1.
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