128 Chapter 5 SUPPLEMENTARY MATERIALS Supplemental Methods Sample collection and processing Urine and cervicovaginal self-samples were collected at home for which all participants received a package including materials needed for collection and transport. Participants were instructed to collect urine before the cervicovaginal self-sample. Cervicovaginal self-samples were collected according to the provided user manual using the Evalyn® brush (Rovers Medical Devices, Oss, The Netherlands), which is a clinically validated self-sampling method (1). Urine was collected in 3x30 mL tubes containing the storage buffer Ethylenediaminetetraacetic acid (EDTA; final concentration 40 mM) to preserve nucleic acids during transport. Clinician-taken cervical scrapes were collected prior to surgery using a Cervex-Brush (Rovers Medical Devices) and directly placed in 10 mL Thinprep PreservCyt medium (Hologic, Marlborough, MA, US). Samples were sent to the Pathology department of Amsterdam UMC, location VUmc, within 72 hours by regular mail and processed directly after arrival. Urine was processed as described in our previously validated processing and storage protocol (2). Briefly, a total of 15 mL of urine was centrifuged at 3000g for 10 min to separate the urine into two fractions: the urine supernatant and urine sediment. Both fractions and the remaining full void (i.e. unfractionated) urine were stored at -20°C. Cytological samples were processed as described previously for cervical (3) and endometrial cancer (4). Cervicovaginal self-samples were stored in 1.5 mL ThinPrep PreservCyt medium upon arrival. Cervicovaginal self-samples and cervical scrapes were stored at 4°C. Formalin-fixed paraffin-embedded (FFPE) and fresh frozen tissue specimens were consecutively sectioned of which the first and last sections were Hematoxylin and Eosin (H&E) stained for histopathological review by a pathologist to confirm the presence of ovarian cancer or normal fallopian tube tissue. DNA extraction and bisulfite modification DNA from full void urine (30 mL patients diagnosed with ovarian mass; 40 mL controls), urine sediment (15 mL original volume), and urine supernatant (15 mL) was extracted as described previously (5, 6). In short, both full void urine and urine supernatant were isolated with the Quick DNA urine kit (Zymo Research, Irvine, CA, US) and urine sediment using the DNA mini and blood mini kit (Qiagen, Hilden, Germany). DNA from cervicovaginal self-samples and clinician-taken cervical scrapes was isolated as
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