Att G_Laboratory protocol_0209

Laboratory Protocol

On-site testing. Once clinic staff have determined that a patient is eligible for the study and she has signed the consent form, clinic staff will assign a patient ID number. Clinic staff will affix a bar-coded label containing the patient ID number to the patient consent form, patient enrollment form, patient chart, and the HPV test tube. The completed enrollment forms will be faxed to Battelle for data entry. The patient enrollment forms will provide Battelle with names and contact information necessary for reporting HPV test results to the clinics and for conducting the patient follow-up surveys.

At the time of the pelvic examination, the health care provider will collect cervical specimens for routine Pap test and HPV testing. After visualization of the cervix, two samples of exfoliated cervical cells will be collected. The first sample will be for cytology diagnosis and will use the standard collection device and preparation method employed by the clinic for routine Pap testing.

The second specimen will be collected using the Digene test kit provided as part of this study. Briefly, the conical brush is inserted 1-1.5 cm into the cervical os until the largest outer bristles of the brush touch the ectocervix. The brush is rotated three full turns in a counter-clockwise direction. The brush is then removed from the canal, avoiding contact with any object. The brush is then inserted to the bottom of the transport tube. The handle of the brush is snapped off at the score line and discarded. The vial is sealed with the lid and the specimen is labeled with study ID (bar code) and stored at room temperature until transported to the CDC.

Local laboratory shipment procedures. The Pap test specimen will be labeled with the patient’s name and clinic number and be handled as a routine diagnostic sample in accordance with the clinic’s standard practice. When the Pap test results are received from the local lab, clinic staff will access LUNA, enter the patient ID number, and enter the results of the Pap test.

CDC laboratory shipment procedures. Clinic staff will also be responsible for the labeling and packaging of specimens for HPV testing. All specimens will be shipped in accordance with International Air Transport Association (IATA) and CDC specimen shipping regulations to the CDC HPV lab. The scanning tunneling microscopy (STM) samples will be shipped at ambient temperature for overnight delivery. HPV tests will not be individually sent—they will be shipped in batches. Shipments will be sent by Federal Express Monday through Thursday. The frequency of shipments will vary depending on the volume of tests done at each clinic. To ensure that test results are provided in a timely manner, clinics will be asked to ship tests at least twice per week.

Tests sent to the CDC HPV lab will not include patient names and will be identified only with the bar-coded patient identification number. Upon arrival at the CDC laboratory, the CDC HPV Lab will access LUNA to confirm receipt of the HPV tests. In addition to scanning the ID numbers into LUNA, the numbers will be entered into an MS access table as an internal laboratory record.

The specimens will be stored at 4ºC until processed. At the time of processing, the STM sample will be aliquotted in a biologic safety cabinet with precautions to avoid sample contamination; 250 µL for Digene test; 150 µL for extraction. The original sample vial with residual material and brush will be stored at -20ºC until the end of the study.

The Digene Hybrid Capture 2 High Risk HPV DNA test (hc2 hr HPV; Qiagen Inc., Valencia CA) will be used to provide clinical HPV results. This is the only test currently approved by the US FDA for clinical applications. The test will follow the manufacturer’s protocol modified to test a 250 µL aliquot. Briefly, the volume of denaturating fluid is reduced to match the volume of the sample. The rest of the assay is unchanged. DNA from the 150 µL aliquot will be extracted using an automated method on a MagNA Pure LC instrument (Roche Applied Science) resulting in a 100 µL eluate. A portion of the eluate will be used in one or more PCR-based HPV typing assays.

The CDC HPV Lab will analyze the HPV tests. The anticipated turn-around time for the clinical test is within one week of sample collection. The hc2 hr HPV test will be reported as positive or negative following the manufacturer’s specified cut-off values. These results will be input into LUNA. Battelle will merge the test result data from LUNA with patient names reported on the patient enrollment form and prepare a laboratory report for each patient. The reports will then be sent to the clinic/provider. The clinic/provider will inform the patient of the results of the HPV test. HPV typing results will not be provided to clinics or providers. Lab staff will send the typing results to Battelle via a formatted spread sheet that will be submitted monthly.

Specimen storage and possible future research testing. CDC staff will store all cervical specimens collected and submitted to the CDC HPV lab as part of this study at least through the end of the study. In addition, study participants will be asked to provide written informed consent for their specimens to be stored after the end of the study for possible future testing, but no longer than 10 years. For participants who agree, residual DNA extracts will be aliquoted into cryo-vials and kept at -70ºC for 10 years. The specimens of participants who do not consent to long-term storage and additional testing will be destroyed at the end of the study.

Attachment G. Laboratory protocol 2