• Self-administered survey

• Cervical mucus collection with Weck-cel sponge

• Pap smear in ThinPrep fluid

• Additional Pap smear in RNAlater

• One time blood draw (or saliva collection) on the initial visit

We collect cervical smear samples from all study participants: these consist of a preliminary swab (painless and non-invasive) to collect cervical mucus, a Pap test performed with the classic ThinPrep method, from which we obtain the cells that remain after all diagnostic procedures are conducted, and a second Pap test collected into “RNA-later” (Ambion) sample buffer, which allows for the recovery of intact RNA suitable for RT-PCR and microarray analysis from the exfoliated cells. In addition, a blood sample is collected from each participant at the first visit only, and serum and blood cells separated and stored.

Samples will be collected every six months from study participants. In addition, at the first visit we administer a complete questionnaire to assess life style and environmental factors that may influence human papillomavirus (HPV) persistence and the progression of the disease (including, but not limited to: age, number of sexual partners, age at first intercourse, age at menarche, smoking, history of contraceptive use, STD history, physical activity, dietary factors, and stress assessment questionnaires). A less complex, follow-up questionnaire is administered at each follow-up visit, to monitor changes in life style factors that may have taken place.

   Data management is an important concern for the Carolina Women’s Care Study (CWCS).  The study generates large amounts of data from the numerous samples collected at each clinic visit, multiple tests being done with each of these samples and the large amount of survey data collected at each visit.  As a means of securely housing and managing the data, CWCS personnel have designed a database.  The database allows data to be quickly queried from multiple sources and allows for the sharing of data between study personnel.

Clinical data such as pap date, pap results, and other non-identifying data collected during the visit to the study site are added to the database using a file export in which all participant identifying information is excluded.  Clinical personnel have access to participant identifying data so patients can receive proper care but non-clinical study personnel have no access to participant identifying information.

• Cytology, and biopsy-confirmed disease status.

• HPV status by PCR with the MY09/MY11 L1-directed consensus primer set and sequencing of the amplimers, for the identification of specific HPV types. Samples with equivocal results due to infection by multiple HPV types are analyzed by sequencing and the predominant types listed.

• Viral load, assessed by real-time PCR with type specific L1 primers, in relation to beta-globin control primers, on the DNA sample from the Thin-Prep, and also on DNA extracted from the second specimen, in each HPV-positive case. This is done to obviate a concern with taking two consecutive samples from a diseased cervix: the two samples may or may not have equivalent amounts of abnormal/HPV infected cells. In this way, we also gain an internal assessment of the reproducibility of results between two consecutive samples, which is valuable information on its own.

• E7 expression by Real Time RT/PCR with type specific primers in RNA extracted from the second sample in RNA-later (Ambion), in relation with β-actin and RPLPO mRNA levels, used here as housekeeping controls.

• Cytokine profile in the cervical mucus, as an expression of the specific type of immune competence on the cervix. The custom cytokine assay kit we use is produced by Pierce, and designed to measure the levels of IL-1a, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α, GM-CSF, Eotaxin, IFN-α, IFN-γ, IP-10, MCP-1, and RANTES in biological specimens.