Comparison of the Cobas HPV Test and Hybrid Capture 2 High-Risk HPV DNA Test

Testing for high risk (HR) HPV and cytology are recommended for cervical cancer screening in women over 30 years. The Hybrid Capture 2 assay (hc2, Qiagen) targets 13 HR-HPV types, the cobas HPV test (cobas, Roche) detects 14 HR-HPV types with optional genotyping for HPV 16 and 18. We compared both assays using 4 sets of samples: 1) consecutive (unselected) liquid-based cervical cytology samples, 2) cervical cytology samples with ~50% hc2-positivity rate, 3) vaginal, and 4) anal cytology samples. Revised Abstract


NAME AND INTENDED USE
The hc2 High-Risk HPV DNA Test ® using Hybrid Capture ® 2 (hc2) technology is an in vitro nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of thirteen high-risk types of human papillomavirus (HPV) DNA in cervical specimens. The HPV types detected by the assay are the high-risk HPV types 16/18/31/33/35/39/45/51/52/56/58/59/68. The hc2 High-Risk HPV DNA Test cannot determine the specific HPV type present.

Caution: Federal law restricts this device to sale by or on the order of a physician.
Cervical specimens that may be tested with the hc2 High-Risk HPV DNA Test include the following: • Specimens collected with the hc2 DNA Collection Device • Biopsies collected in Specimen Transport Medium ™ (STM) • Specimens collected using a broom type collection device and placed in Cytyc ThinPrep ® Pap Test PreservCyt ® Solution (refer to the hc2 Sample Conversion Kit package insert for complete details).
The use of this test is indicated: 1. To screen patients with ASC-US (atypical squamous cells of undetermined significance) Pap smear results to determine the need for referral to colposcopy. The results of this test are not intended to prevent women from proceeding to colposcopy.
2. In women 30 years and older the hc2 High-Risk HPV DNA Test can be used with Pap to adjunctively screen to assess the presence or absence of high-risk HPV types. This information, together with the physician's assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management.

WARNING
• The hc2 High-Risk HPV DNA Test is not intended for use as a screening device for Pap normal women under age 30 and is not intended to substitute for regular Pap screening. • There is insufficient evidence to indicate whether a single WNL Pap result with concurrent negative high-risk HPV result confers low risk similar to consecutive annual, technically adequate WNL Pap results. • Detection of HPV using the hc2 High-Risk HPV DNA Test does not differentiate HPV types or infection with more than one type, and cannot evaluate persistence of any one type. • The use of this test has not been evaluated for the management of women with prior cytologic or histologic abnormalities, hysterectomy, who are postmenopausal, or who have other risk factors (e.g., HIV+, immunocompromised, DES exposure, history of STI).
The hc2 High-Risk HPV DNA Test is designed to augment existing methods for the detection of cervical disease and should be used in conjunction with clinical information derived from other diagnostic and screening tests, physical examinations and full medical history in accordance with appropriate patient management procedures.
hc2 High-Risk HPV DNA Test results should not be used as the sole basis for clinical assessment and treatment of patients.
Another Digene HPV kit, the hc2 HPV DNA Test ( REF 5198-1220), which detects both high-risk and some low-risk HPV types should not be used as an adjunct for screening because low-risk types are not associated with risk of cervical cancer. Only the hc2 High-Risk HPV DNA Test should be used as an adjunct for screening.

SUMMARY AND EXPLANATION
In women, human papillomaviruses (HPVs) can infect the cervix, vagina, vulva, urethra, or the area around the anus. More than 70 types of HPV have been identified, and are generally classified as high-risk or low-risk depending on their known association or lack of association with cancer and its precursor lesion, high-grade cervical intraepithelial neoplasia (CIN 2-3). The presence of certain HPV types in the female genital tract is associated with a number of diseases, including condyloma, Bowenoid papulosis, cervical, vaginal, and vulvar intraepithelial neoplasia and cancer. 1,2 It is generally accepted that these viruses are predominantly sexually transmitted and that high-risk HPV types are a major recognized risk factor for development of cervical cancer. [2][3][4][5][6] Infection of the cervix with high-risk HPV types can be associated with cytological and histological changes that are detected by Pap screening, colposcopy, or biopsy. The natural history of how HPV infection progresses to cancer, however, is not completely understood. Low-risk HPV types 6 and 11 have been associated with the presence of genital warts, or condylomas, but have been linked infrequently with precancerous or cancerous cervical changes. There are many other low-risk HPV types that are not associated with genital warts or cervical cancer. 7,8 Human papillomaviruses are composed of an icosahedral viral particle (virion) containing an 8000 base pair double-stranded circular DNA molecule surrounded by a protein capsid. Following infection of epithelial cells, the viral DNA becomes established throughout the entire thickness of the epithelium, but intact virions are found only in the upper layers of the tissue. Thus, viral DNA can be found either in virions or as episomal or integrated HPV sequences, depending upon the type and grade of lesion.
To date, HPV cannot be cultured in vitro, and immunological tests are inadequate to determine the presence of HPV cervical infection. Indirect evidence of anogenital HPV infection can be obtained through physical examination and by the presence of characteristic cellular changes associated with viral replication in Pap smear or biopsy specimens. Alternatively, biopsies can be analyzed by nucleic acid hybridization to directly detect the presence of HPV DNA.
Historically, HPV 16 and HPV 18 have been regarded as high-risk cancer associated HPV types. 2,9,10 HPV types 31, 33, and 35 have been demonstrated to have an intermediate association with cancer. 2,11 This intermediate association is due to the fact that these types are more frequently detected in CIN 2-3 rather than in cancers. Therefore, cancers associated with the presence of these types are less common than cancers that are associated with high-risk HPV DNA types 16 and 18. 2,12 These five HPV types together account for about 80% of cervical cancers. 2,13,14 Additional high-and intermediate-risk HPV DNA types, including types 39, 45, 51, 52, 56, 58, 59 and 68, have been identified as the principal HPVs detectable in the remaining cancers. 2,[14][15][16][17][18][19][20] HPV infection is common in adults who have had more than one sexual partner (or a single partner who has had multiple partners) and can persist for years with no symptoms. Infection with some HPV types is an important risk factor for cervical cancer; however, most women with HPV infection do not develop cervical cancer or CIN 2-3, and infections regress. Most infections cause mild cytologic changes that resolve. HPV DNA has been shown to be present in approximately 10% of women with normal cervical epithelium but the actual prevalence in specific groups of women is strongly influenced by age and other demographic variables. 2,13,21 Prospective studies (age 16-60 years) have shown that 15-28% of HPV DNA positive women developed squamous intraepithelial lesions (SIL) suggestive of CIN 1-3 or cancer within 2 years compared to only 1-3% of HPV DNA negative women. 4,22,23 In particular, the risk of progression for HPV types 16 and 18 was greater (approximately 40%) than for other HPV types. 4,6,10,23,24 Most SIL was low-grade.
Very few HPV DNA positive women develop cytologic high-grade SIL (HSIL) indicating underlying CIN 2-3 or cancer. 25 The absolute risk of developing an incident cytologic abnormality following an HPV infection with types detected by hc2 has not been adequately described, and is known to vary in different populations. 6 Although current scientific literature suggests that persistent infection with high-risk HPV is the main risk factor for development of highgrade cervical neoplasia and cancer 2,4,5,10,24,26-31 , apparent persistence may represent continuous infection with a single HPV type, with multiple HPV types, or reinfection. Nonetheless, women who are repeatedly Pap negative and High-Risk HPV negative appear to be at low risk for having or developing cervical precancerous lesions. 5,24,32,33 A negative hc2 High-Risk HPV DNA Test result with a concurrent normal Pap result implies low risk at a single point in time for the development of cervical neoplasia and is therefore clinically meaningful for assessing risk; however there are insufficient data to establish a definitive time period over which this lower risk is clinically relevant.

PRINCIPLE OF THE PROCEDURE
The hc2 High-Risk HPV DNA Test using Hybrid Capture 2 technology is a nucleic acid hybridization assay with signal amplification that utilizes microplate chemiluminescent detection. Specimens containing the target DNA hybridize with a specific HPV RNA probe cocktail. The resultant RNA:DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA:DNA hybrids, and detected with a chemiluminescent substrate. Several alkaline phosphatase molecules are conjugated to each antibody. Multiple conjugated antibodies bind to each captured hybrid resulting in substantial signal amplification. As the substrate is cleaved by the bound alkaline phosphatase, light is emitted that is measured as relative light units (RLUs) on a luminometer. The intensity of the light emitted denotes the presence or absence of target DNA in the specimen.
An RLU measurement equal to or greater than the Cutoff Value (CO) indicates the presence of high-risk HPV DNA sequences in the specimen. An RLU measurement less than the Cutoff Value indicates the absence of the specific high-risk HPV DNA sequences tested or HPV DNA levels below the detection limit of the assay. • Pipette High-Risk HPV Probe into Probe Diluent by placing the pipette tip against the inner wall of the tube just above the meniscus and expelling the contents. Do not immerse the tip into Probe Diluent.

REAGENTS AND MATERIALS PROVIDED
• Vortex for at least 5 seconds at maximum speed to mix thoroughly. A visible vortex must be produced. Label as "High-Risk HPV Probe Cocktail." Unused Probe Mix should be discarded. It is recommended that the Wash Apparatus and tubing be cleaned with bleach and rinsed thoroughly with distilled or deionized water once every three months to prevent possible contamination from alkaline phosphatase present in bacteria and molds.

SPECIMEN COLLECTION AND HANDLING
The types of cervical specimens recommended for use in the hc2 High-Risk HPV DNA Test are listed below. Specimens taken with other sampling devices or transported in other transport media have not been qualified for use with this assay. The hc2 High-Risk HPV DNA Test's performance characteristics with other specimen types and collection devices are unknown.
Cervical specimens must be collected prior to the application of acetic acid or iodine if colposcopic examination is being performed. See the hc2 DNA Collection Device package insert for additional specimen collection and handling procedures.

CERVICAL BRUSHES*
The hc2 High-Risk HPV DNA Test is designed for use with specimens collected and transported using the hc2 DNA Collection Device (cervical brush and STM). Specimens may be held for up to two weeks at room temperature and shipped to the testing laboratory, after which specimens can be stored an additional week at 2-8°C. If the assay will be performed more than 3 weeks from collection, specimens can be placed at -20°C for up to three months prior to testing. A preservative has been added to the STM to retard bacterial growth and to retain the integrity of DNA. It is not intended to preserve viability of organisms or cells. The hc2 DNA Collection Device should not be used for collection of specimens from pregnant women.

Time Prior to Testing Storage Duration
Storage Temperature Up to 2 weeks Room Temperature 3 weeks Up to an additional week 2-8°C Greater than 3 weeks Up to three months -20°C Specimens may be shipped without refrigeration to a testing laboratory; however, specimens should be shipped in an insulated container using either an overnight or 2-day delivery vendor.

CERVICAL BIOPSIES*
Freshly collected cervical biopsies, 2-5 mm in cross-section, may also be analyzed with the hc2 High-Risk HPV DNA Test. The biopsy specimen must be placed immediately into 1.0 ml of STM and stored frozen at -20°C. Biopsy specimens may be shipped at 2-30°C for overnight delivery to the testing laboratory and stored at -20°C until processed. Biopsies less than 2 mm in diameter should not be used.

SPECIMENS IN CYTYC PRESERVCYT SOLUTION
Specimens collected with a broom-type collection device and placed in Cytyc PreservCyt Solution for use in making Cytyc ThinPrep ® Pap Test slides can be used in the hc2 High-Risk HPV DNA Test. Specimens should be collected in the routine manner, and the ThinPrep Pap Test slides should be prepared according to Cytyc instructions.
There must be at least 4 ml of PreservCyt Solution remaining for the hc2 High-Risk HPV DNA Test. Specimens with less than 4 ml after the ThinPrep Pap Test has been prepared may contain insufficient material and could be falsely negative with the hc2 High-Risk HPV DNA Test.
PreservCyt Solution specimens may be held for up to three months at temperatures between 2°C and 30°C, following collection and prior to processing for the hc2 High-Risk HPV DNA Test. PreservCyt Solution specimens cannot be frozen. To process these specimens, refer to the hc2 Sample Conversion Kit package insert. For convenience, the specimen processing steps have also been included in the Test Procedure section below.

TEST PROCEDURE
Specimens may contain infectious agents and should be handled accordingly.
The hc2 High-Risk HPV DNA Test can be performed manually as instructed in this package insert or using the Rapid Capture System instrument for high-volume sample-throughput testing.

High-Volume Sample-Throughput Testing Using the RCS Instrument Application
The Rapid Capture System is a general-use automated pipetting and dilution system that can be used with the hc2 High-Risk HPV DNA Test for high volume sample-throughput testing. This system handles up to 352 specimens in eight hours, including a 3.5-hour period during which user intervention is not required; up to 704 specimen results can be generated in 13 hours. Denaturation of the specimens in preparation for testing is performed independently of the RCS, in the primary collection tube, as performed for the manual method of the hc2 High-Risk HPV DNA Test described below, prior to placing on the RCS platform. In addition, chemiluminescent signal detection and result reporting are performed using the offline luminometer system common to both the manual and RCS methods. Each of the hc2 High-Risk HPV DNA Test's procedural steps is performed in the exact sequence as the manual test procedure. The RCS Application allows for the staggered processing of up to 4 microplates, each plate containing specimens and the required assay calibrators and quality controls.
When using the Rapid Capture System, refer to the Rapid Capture System User Guide provided with the instrument, in addition to this package insert, for necessary procedural and descriptive information. SETUP 1. Allow 60 minutes for the HCS Microplate Heater I to equilibrate to 65 ± 2°C from a cold start. Consult the Microplate Heater I Operator's Manual for details. Confirm a water bath is at 65°C and the water level is high enough to immerse the entire specimen volume in the specimen tubes.
2. Remove the specimens and all required reagents from the refrigerator prior to beginning the assay. Allow them to reach 20-25°C for at least 15 to 30 minutes.
Note: Prepare PreservCyt Solution specimens prior to equilibrating any previously denatured specimens and kit reagents to room temperature.
3. Create a plate layout using the Digene assay analysis software with Digene assay protocols for HPV. See the applicable software user guide for details.

Place Calibrators, Quality Controls
, and specimens to be tested in a test tube rack, in the same order in which they will be tested.  5. NC and HRC are tested in triplicate with High-Risk HPV Probe Cocktail. Digene assay analysis software determines the calibrator and quality control positions in the microplate. See the applicable Digene assay analysis software user guide for proper Calibrator/Quality Control/specimen setup in the software.

Notes:
• Caution: Denaturation Reagent is corrosive. Use care and wear powder-free gloves when handling.
• Important: Some cervical specimens may contain blood or other biological material, which may mask the color changes upon addition of Denaturation Reagent. Specimens that exhibit a dark color prior to the addition of Denaturation Reagent may not give the proper color changes at this step. In this case, failure to exhibit the proper color change will not affect the results of the assay. • Do not remove hc2 specimen collection device prior to denaturation.
• During the denaturation step, be sure that the water level in the water bath is adequate to immerse the entire volume of specimen in the tube. • Calibrators, Quality Controls, and specimens may be prepared up through the denaturation step and stored at 2-8°C overnight, or at -20°C for up to 3 months. A maximum of 3 freeze/thaw cycles may be performed with a maximum of 2 hours at room temperature during each thaw cycle. Mix well before using. • To avoid false-positive results, it is critical that all Calibrator, Quality Control, and specimen material come into contact with Denaturation Reagent. Mixing after Denaturation Reagent addition is a critical step; if using the Hybrid Capture System Multi-Specimen Tube (MST) Vortexer 2, make sure it is set to 100 (maximum speed) and a visible vortex of liquid is observed during mixing such that the liquid washes the entire inner surface of the tube. If performing manual vortexing, make sure that each Calibrator, Quality Control, and specimen is mixed individually by vortexing each for at least 5 seconds at full speed such that the liquid vortex washes the entire inner surface of the tube, followed by inverting the tube once. • Following denaturation and incubation, the specimens are no longer considered infectious. 36 However, laboratory personnel should still adhere to practical universal precautions.

Calibrators, Quality Controls, and STM specimens (including biopsy hc2 DNA Collection Device specimens)
Note: This procedure is not for preparation and denaturation of PreservCyt Solution specimens.
1. Remove and discard caps from Calibrator, Quality Controls, and specimen tubes.
2. Pipette Denaturation Reagent with Indicator Dye into each Calibrator, Quality Control, or specimen using a repeating or adjustable pipettor. Take care not to touch the sides of the tube or cross-contamination of specimens could occur. The volume of Denaturation Reagent needed is equivalent to half the specimen volume. The exact volume for each type of Calibrator, Quality Control, and specimen is listed in the table below. • Dilute remaining Denaturation Reagent in bottle prior to disposing. Dispose of in accordance with local, state and federal regulations.

Refer to the
3. Mix the specimens using one of the two methods below. a. Cover the Calibrator/Quality Control/Specimen tubes with DuraSeal™ Tube Sealer film by pulling the film over the tubes in the rack. b. Place the rack lid over the film-covered tubes and lock the lid into place with the two side clamps. Cut the film with the cutter device after the lid is securely fastened. c. Move the red-handled lever up so that it is in a horizontal position. d. Place the MST or Digene Specimen Rack and Lid on the MST Vortexer 2 so that the largest notched corner of the rack is located in the right front corner. Position the rack and lid on the MST Vortexer 2 platform so that it fits securely within the guides. Secure the rack in place by moving the red-handled lever down to the vertical position. This will lock the rack in place. e. Verify that the speed setting is at 100 (maximum speed) and the Pulser button is in the OFF position. f. Turn the Vortexer power switch to the ON position. Vortex the tubes for 10 seconds. g. Turn the Vortexer power switch to the OFF position. h. Remove the Rack from the Vortexer by lifting up on the red-handled lever. i. Incubate in a 65 ± 2°C water bath for 45 ± 5 minutes (denatured Calibrators, Quality Controls, and specimens may be tested immediately, or stored as described in Notes above). Prepare the High-Risk HPV Probe cocktail during this incubation. See Reagent Preparation and Storage section. j. Remove rack from water bath, dry the rack, and secure on the vortexer. k. Turn the Vortexer power switch to the ON position. Vortex the tubes for 5 seconds. l. Turn the Vortexer power switch to the OFF position. Remove the rack. m. Immediately remove the rack lid and DuraSeal tube sealer film from the specimens. n. Proceed to the Hybridization step below or see Optional Stop Point for storage and treatment of denatured specimens.
Independent of the vortexing method utilized, there must be a visible vortex of liquid inside each tube during mixing such that the liquid washes the entire inner surface of the tube. The Calibrators, Quality Controls, and specimens should turn purple.

PreservCyt Solution Specimen Preparation and Denaturation Procedure
Notes: • Consult the hc2 Sample Conversion Kit package insert for complete details.
• Processing a 4-ml aliquot of PreservCyt Solution produces enough for 2 tests, when tested manually. The minimum volume that can be processed is 4 ml.
• Prepare PreservCyt Solution specimens in batches of 36 or fewer; otherwise, pellets may become dislodged when decanting the supernatant. This is important for maintaining the integrity of the cell pellet during the decanting step. If preparing additional PreservCyt Solution vials, do not start to prepare them until after completing the preparation of the first batch (see the table below).

Reagent Preparation
If not performed previously, prepare the Denaturation Reagent (DNR) from the hc2 Sample Conversion Kit by adding 3 drops of Indicator Dye to the bottle of DNR and mix well. The solution should be a uniform, dark purple color.
Volume requirements are determined based on the number of replicates to be tested per specimen. Use the table below.

Volume Requirements: Reagent Preparation
1. Label the 10-ml conical Sarstedt tube or a 15-ml VWR or Corning brand conical tube (required for MST Vortexer 2 method) with the appropriate specimen identification number.
2. Handling one specimen at a time: a. For 5-10 seconds, vigorously shake the PreservCyt vial by hand or vortex each vial individually using a vortex mixer at maximum speed setting. Do this to resuspend cells and ensure homogeneity.
b. Immediately, as cells settle very quickly, pipette the appropriate volume of the PreservCyt specimen into the labeled tube. Deliver the PreservCyt solution to the bottom of the conical tube to minimize cellular material adhering to the inside of the tube.
3. Add the appropriate volume of Sample Conversion Buffer to each tube (see the 4. Recap and mix the contents of each tube thoroughly by using a vortex mixer with cup attachment.
5. Centrifuge the tubes in a swinging bucket rotor at 2,900 ± 150 x g for 15 ± 2 minutes.
Note: Solution must be prepared fresh each day the test is being performed.
a. To determine the total volume of STM/DNR mixture required, use the starting volume of the PreservCyt Solution specimen as a guide and then multiply the STM and DNR "per tube" volumes by the number of specimens to be processed (see the table, below).

Notes:
• Do not blot in the same area of the absorbent low-lint paper towel.
• It is important to remove the maximum amount of PreservCyt Solution by blotting. However, it is normal to see residual PreservCyt Solution after blotting.
Manual/Individual Tube Vortexing Method 1. Add the appropriate volume of Specimen Transport Medium + Denaturation Reagent to each pellet. Resuspend each pellet by vortexing each tube individually for at least 30 seconds at the highest speed setting. If a pellet is difficult to resuspend, vortex for an additional 10-30 seconds or until the pellet floats loose from the bottom of the tube. If a pellet remains undissolved after additional vortexing (a total of 2 minutes maximum), note the specimen identification and proceed to the next step.
2. Place tubes in 65 ± 2°C water bath for 15 ± 2 minutes. Ensure that the water level is sufficient to cover all liquid in the tubes.
3. Remove the rack with specimens from the water bath and vortex specimens individually for about 15-30 seconds.
Note: Make sure that all pellets are completely resuspended at this point. Specimens that still have visible pellets are not acceptable for testing.
4. Return the rack to the 65 ± 2°C water bath and continue denaturation for another 30 ± 3 minutes.
5. Proceed to the Hybridization step below or see Optional Stop Point for storage and treatment of denatured specimens.

Multi-Specimen Tube (MST) Vortexer 2 Method
Notes: • The Hybrid Capture System Multi-Specimen Tube (MST) Vortexer 2 Method is validated for the processing of PreservCyt Solution specimens following centrifugation and decanting of the supernatant.
• The MST Vortexer 2 procedure has not been validated for vortexing PreservCyt Solution specimens with Sample Conversion Buffer prior to centrifugation.
• Only the MST Vortexer 2 is designed for PreservCyt Solution specimen processing.
• The Conversion Rack and Lid are specifically designed to accommodate VWR or Corning brand 15-ml conical tubes. The user should use only one tube type on the Conversion Rack at a time. Other brands are not validated for use.
• Strict adherence to the specified vortexing times of the Conversion Rack and Lid is required.
• The Conversion Rack and Lid cannot be used to vortex the hc2 DNA Test kit Calibrators or Quality Controls. The height of the STM tubes prevents adequate Vortexing using the Conversion Rack.
1. After blotting each labeled 15-ml conical tube, place each in its proper position in the Conversion Rack. 2. Add the appropriate volume of Specimen Transport Medium + Denaturation Reagent mixture to each pellet. 3. Cover the 15-ml conical tubes with DuraSeal tube sealer film by pulling the film over the tubes in the rack. 4. Place the rack lid over the film-covered tubes and lock the lid into place with the two side clamps. Cut the film with the cutter device after the lid is securely fastened. 5. Move the red-handled lever up so that it is in a horizontal position. 6. Place the Conversion Rack and Lid on the MST Vortexer 2 so that the largest notched corner of the Conversion Rack is located in the right front corner. Position the rack and lid on the MST Vortexer 2 platform so that it fits securely within the guides. Secure the rack in place by moving the red-handled lever down to the vertical position. This will lock the rack in place. 7. Verify that the speed setting is at 100 (maximum speed) and the Pulser button is in the OFF position. 8. Turn the Vortexer power switch to the ON position. Vortex the tubes for 30 seconds. 9. Turn the Vortexer power switch to the OFF position. 10. Remove the Conversion Rack from the Vortexer by lifting up on the red-handled lever. 11. Place the rack in the 65 ± 2°C water bath for 15 ± 2 minutes. Be sure the water level completely covers all liquid in all of the tubes. 12. After the 15-minute incubation, remove the rack with specimens from the water bath. 13. To prevent splashing, dry the rack of excess water prior to placing it on the MST Vortexer 2. 14. Secure the Conversion Rack on the MST Vortexer 2 as described in Step 6. 15. Verify speed setting is at 100, and turn the vortexer power switch to the ON position. Vortex the tubes for 1 minute. 16. Turn the power switch to the OFF position. 17. Return the rack to the 65 ± 2°C water bath, and continue denaturation for 30 ± 3 minutes. 18. Remove rack from water bath, dry the rack, and secure on the vortexer. 19. Turn the Vortexer power switch to the ON position. Vortex for 10 seconds at the maximum setting. 20. Turn the Vortexer power switch to the OFF position. Remove the rack. 21. Immediately remove the rack lid and DuraSeal tube sealer film from the specimens. 22. Proceed to the Hybridization step below or see Optional Stop Point for storage and treatment of denatured specimens.

Optional Stop Point
After denaturation, all specimens may be stored at 2 -8°C overnight or at -20°C for up to 3 months. For overnight refrigeration, specimens may be left in the MST, Digene Specimen, or Conversion Rack with new DuraSeal film and Rack Lid replaced. Prior to storage at -20°C, the Rack Lid and DuraSeal film must be removed, and caps placed on the tubes. If the manual vortex procedure was used, place the rack of capped tubes in the desired storage temperature. In either case, the specimens must be equilibrated to room temperature (20 -25°C) and thoroughly vortexed before proceeding to the Hybridization step.
Note: Do not store or ship denatured specimens on dry ice.
A maximum of 3 freeze/thaw cycles may be performed with a maximum of 2 hours at room temperature during each thaw cycle. For specimens processed using the MST Vortexer 2, remove the Conversion Rack Lid and DuraSeal tube sealer film from the 15-ml conical tubes and cap each tube before storing specimens at -20°C.

Notes:
High-Risk HPV Probe mix is viscous. Care should be taken to ensure thorough mixing and that the required amount is completely dispensed into each microplate well. See Reagent Preparation and Storage section.
Important: Some cervical specimens may contain blood or other biological material that may mask the color changes upon addition of Probe Mix. Specimens that exhibit a dark color prior to the addition of Denaturation Reagent may not give the proper color change at this step. In these cases, failure to exhibit the proper color change will not affect the results of the assay. Proper mixing can be verified by observing the color change of the Calibrators and Quality Controls.

Notes:
• Specimens collected with the hc2 DNA Collection Device in STM and processed using the MST Vortexer method can be hybridized utilizing the Microplate Heater I method only. • If the denatured specimen has been frozen or refrigerated, equilibrate to 20-25°C and vortex on the maximum speed setting for 5 seconds if Vortexing manually, or 10 seconds if using the MST Vortexer 2 with PreservCyt specimens. • Preheat the Microplate Heater I to 65 ± 2°C for 60 minutes prior to use. See the Microplate Heater I Operator's Manual for further instructions, as needed.
1. Obtain and label a Hybridization Microplate.
2. Pipette 75 µl of each Calibrator, Quality Control, or specimen into the bottom of an empty hybridization microplate well following the plate layout created under Setup. Avoid touching the sides of the wells and limit formation of air bubbles. Use a clean extra-long pipette tip for each transfer to avoid cross-contamination of Calibrators, Quality Controls, or specimens. It is not necessary to remove the specimen collection device from the specimen transport tube. Denatured specimens may be capped with specimen collection tube screw caps and stored with specimen collection devices remaining in the tubes. 5. Cover the Hybridization Microplate with a plate lid and shake on Hybrid Capture System Rotary Shaker I set at 1100 ± 100 rpm for 3 ± 2 minutes. The Calibrators, Quality Controls, and specimens should turn yellow after shaking. Wells that remain purple may not have received the proper amount of Probe Mix. Add an additional 25 µl of Probe Mix to specimens that remain purple and shake again. If wells remain purple after following this procedure, specimens should be retested.

Notes:
• After shaking, PreservCyt Solution specimens should turn pink instead of yellow.
• When placing the Hybridization Microplate in the Microplate Heater I, care should be taken not to cause splashing. 6. Incubate in a preheated and equilibrated to 65 ± 2°C Microplate Heater I for 60 ± 5 minutes.
Note: Create a plate layout file using the Digene assay analysis software with Digene assay protocols for HPV if this has not been completed earlier.

Hybridization Method using Microtubes and Water Bath
Notes: • The processing of specimens collected with the hc2 DNA Collection Device in Specimen Transport Medium (STM) using the MST Vortexer 2 method for mixing and the water bath method for hybridization has not been validated.
Specimens collected with the hc2 DNA Collection Device in STM and processed using the MST Vortexer 2 method can be hybridized using the Microplate Heater I method only. • If the denatured specimen has been stored at -20°C, allow the specimen to thaw to 20-25°C, and thoroughly vortex the specimen before proceeding with hybridization.
1. Label and place the required number of clean hybridization microtubes into the microtube rack.
2. Remove Calibrators, Quality Controls, and specimens from the water bath after incubation. Vortex each tube individually for at least 5 seconds just prior to removing aliquots.
3. Pipette 75 µl of each Calibrator, Quality Control, or specimen into the bottom of empty hybridization microtubes following the plate layout created under Setup. Use a clean extra-long pipette tip for each transfer to avoid cross-contamination of Calibrators, Quality Controls, or specimens. It is not necessary to remove the specimen collection device from the specimen transport tube. Denatured specimens may be stored with the specimen collection devices remaining in the tubes. 6. Cover the microtubes with a plate sealer. Place rack cover on top of rack. Shake the microtube rack on Rotary Shaker I set at 1100 ± 100 rpm for 3 ± 2 minutes. The Calibrators, Quality Controls, and specimens should turn yellow after shaking. Tubes that remain purple may not have received the proper amount of Probe Mix. Add an additional 25 µl of Probe Mix to specimens that remain purple and shake again. If tubes remain purple after following this procedure, specimens should be retested.

Note:
After shaking PreservCyt Solution specimens should turn pink instead of yellow.
7. Incubate in a 65 ± 2°C water bath for 60 ± 5 minutes. Ensure that the water level in the water bath is sufficient to cover the entire volume of hybridization mixture. The microtube rack may be allowed to float in the water bath.
Note: Create a plate layout file using the Digene assay analysis software with Digene assay protocols for HPV if this has not been completed earlier.

HYBRID CAPTURE
1. Remove all but the required number of Capture Microplate wells from the plate frame. Return the unused microwells to the original bag and reseal. With a marker, number each column 1, 2, 3. . . . and label the microplate with an appropriate identifier. The specimens will be added to the wells according to the example layout prepared under Setup.
2. Carefully remove Hybridization Microplate containing Calibrators, Quality Controls, and specimens from the Microplate Heater I. Immediately remove Plate Lid and place it on clean surface. Alternatively, remove microtube rack from the water bath. Immediately remove the rack lid and slowly pull the plate sealer up and across the rack.

Notes:
• Make additions across the plate in a left-to-right direction using an 8-channel pipettor.
• It is recommended that the reverse pipetting technique be utilized to improve consistency of reagent delivery. With this technique, the pipette tips are initially over-filled by using the second stop on the pipettor's aspirate/dispense control (plunger). See procedure below. Wipe tips on disposable Reagent reservoir or on a clean low-lint pad to remove excess reagent before delivery to plate. • If desired, the pipettor may be steadied by resting the middle of the pipette tips on the top edge of the microwells. Take care not to touch the sides of the microwells or cross-contamination of specimens could occur. Refer to Diagram 1 above. • Verify that the Waste Reservoir is empty and the cap is securely fastened.
• The Automated Plate Washer will automatically prime itself before each wash, and rinse after each wash.
1. Remove plate lid and place plate on Automated Plate Washer platform.
2. Verify that the power is on, and that the display reads "Digene Wash Ready" or "P1".
Note: If only a partial strip of capture wells is being used, empty microplate wells will need to be placed in capture plate to complete the column prior to washing. See Accessories section for ordering information.
3. Select the number of strips to be washed by pressing the ROWS key and then + or -to adjust. Press ROWS key to return to "Digene Wash Ready" or "P1".
4. Press START/STOP to begin.
5. The Automated Plate Washer will perform six fill and aspirate cycles taking approximately 10 minutes. There will be a brief pause during the program so be sure not to remove the plate prematurely. When the Automated Plate Washer is finished washing, it will read "Digene Wash Ready" or "P1".
6. Remove the microplate from the Automated Plate Washer when the program is finished. Plate should appear white, and no residual pink liquid should remain in the microwells.
Manual Washing Method 1. Remove Detection Reagent 1 from the wells by placing clean Kimtowels Wipers or equivalent low-lint paper towels on top of the plate and carefully inverting. Before inverting, ensure that the paper is in contact with the entire surface area of the plate. Allow the plate to drain for 1-2 minutes. Blot well on clean Kimtowels Wipers or equivalent low-lint paper towels. Carefully discard the used Kimtowels Wipers or equivalent low-lint paper towels to avoid alkaline phosphatase contamination of later steps.
2. Using the Wash Apparatus, hand wash the plate 6 times. Each well must be washed to overflowing to remove Detection Reagent 1 from the top of the wells. Washing begins at well A1 and continues in a serpentine fashion to the right and downward. After all wells have been filled, decant liquid into sink with a strong downward motion. The second wash is started at well H12 moving in a serpentine motion to the left and upward. This sequence of 2 washes is repeated 2 more times for a total of 6 washes per well.
3. After washing, blot the plate by inverting on clean Kimtowels Wipers or equivalent low-lint paper towels and tapping firmly 3-4 times. Replace the toweling and blot again. Leave plate inverted and allow to drain for 5 minutes. Blot the plate one more time. 3. Read the microplate on the luminometer after 15 minutes of incubation (and no later than 30 minutes of incubation).
4. If a full microplate was not used, remove used microwells from the microplate holder, rinse the holder thoroughly with deionized water, dry and reserve for next assay.

ASSAY CALIBRATION VERIFICATION CRITERIA
Assay Calibration Verification is performed to ensure that the reagents and furnished Calibrator material are functioning properly, permitting accurate determination of the assay cut-off value. The hc2 High-Risk HPV DNA Test requires calibration with each assay, therefore it is necessary to verify each assay using the following criteria. This verification procedure is not intended as a substitute for internal quality control testing. The Digene assay analysis software with version 4.01 or higher Digene assay protocols for HPV automatically verify the criteria below.

Negative Calibrator
The Negative Calibrator must be tested in triplicate with each assay. The Negative Calibrator mean must be ≥ 10 and ≤ 250 RLUs in order to proceed. The Negative Calibrator results should show a coefficient of variation (%CV) of ≤ 25%. If the %CV is > 25%, discard the Calibrator value with a RLU value farthest from the mean as an outlier, and recalculate the mean using the remaining two values. If the difference between the mean and each of the two values is ≤ 25%, proceed to step 2; otherwise, the assay calibration verification is invalid and the test must be repeated for all patient specimens. Accordingly, patient specimen results should not be reported.

High-Risk Calibrator
The High-Risk HPV Calibrator (HRC) must be tested in triplicate with each assay. The Calibrator results should show a coefficient of variation (%CV) of ≤ 15%. If the %CV is > 15%, discard the Calibrator value with a RLU value farthest from the mean as an outlier, and recalculate the mean using the remaining two Calibrator values. If the difference between the mean and each of the two values is ≤ 15%, proceed to step 3; otherwise, the assay calibration verification is invalid and the test must be repeated for all patient specimens. Accordingly, patient specimen results should not be reported.
The assay calibration verification described above for the Calibrators is performed automatically by the Digene assay analysis software and printed in test result reports. The Digene assay analysis software with version 4.01 or higher Digene assay protocols for HPV automatically verify that the High-Risk Calibrator %CV is ≤ 15%. However, previous versions (1.0.2 and 1.0.3) of the Digene Qualitative Software will NOT invalidate the assay unless the %CV is >25% for the High-Risk HPV Calibrator. Therefore, the user of version 1.0.2 or 1.

Situation
1 ≤ 15% Assay reported as "Valid" Results may be reported; no further action required.
2 Between 15% and 25% No outliers removed and assay reported as "Valid" Remove the Calibrator RLU value farthest from the mean. Recalculate the %CV of the Calibrator with the two remaining values. If the %CV of the two remaining RLU values is >15%, the assay is invalid. The results must not be reported. If the %CV of the two remaining RLU values is ≤15%, recalculate the assay cutoff, then recalculate the RLU/cutoff ratio for each specimen using this cutoff. These recalculated values may be reported. 3 Between 15% and 25% One outlier removed and assay reported as "Valid" Assay is invalid, results must not be reported. Assay must be repeated.

> 25%
One outlier removed and assay reported as "Invalid" Assay is invalid, results must not be reported. Assay must be repeated.
In order to manually calculate the %CV as required in Situation 2 above, the user should divide the standard deviation (n-1) of the two remaining replicate RLU values by the mean of the two remaining replicate RLU values (HRC) and multiply that result by 100.
To calculate the %CV using Microsoft ® Excel ® (supplied with the Digene Qualitative Software), the user can calculate the standard deviation of the Calibrator replicates using the formula "STDEV" and determine the mean RLU of the Calibrator using the formula "AVERAGE". Once these two values are obtained, divide the STDEV by the AVERAGE and multiply the result by 100 to obtain the %CV.

If there are any questions related to calculating %CV's, recalculating the assay cutoff, or recalculating the RLU/cutoff of the specimens, please call Digene Technical Services.
To determine High-Risk Calibrator reproducibility and estimate the frequency in which manual recalculations may be necessary, the results from three clinical evaluations involving 152 assays performed with the hc2 High-Risk HPV DNA Test were compiled. The results showed that the average %CV for these 152 assays was 8.1%. Considering all 3 replicates of the Calibrator per assay, Calibrator reproducibility of greater than 15%CV was observed for only 17 out of 152 (11.2%), with 10 out of these 17 resulting in %CV between 15-25% (Situation 2). For the 17 assays that yielded a %CV greater than 15, a single outlier was removed and the %CV recalculated. Following the User Action for Situation 2, only one of the %CV's remained greater than 15%, invalidating the assay. The %CVs of the remaining 151 assays were calculated for an average %CV of 6.0.

3.
The Calibrator mean (HRC ) and Negative Calibrator mean (NC ) results are used to calculate the HRC /NC ratio. Version 4.01 or higher Digene assay protocols for HPV automatically verify the HRC /NC ratio acceptable range within 2-15. However, previous versions (v1.0.2 and v1.0.3) of the Digene Qualitative Software assay protocols do not verify the upper limit of this range. This ratio must meet the following criteria to verify the assay calibration before the specimen results can be interpreted: Assay Calibration Verification Acceptable Ranges 2.0 ≤ HRC / NC ≤ 15.0

4.
Calculate the HRC /NC ratio. If the ratio is ≥2.0 and ≤15.0, proceed to the next step. If the ratio is <2.0 or >15.0, the assay calibration is invalid and must be repeated. All patient specimens should be repeated within the assay.
Note: Acceptable ranges for the Calibrators have been established only for Digene-approved luminometers.

CUTOFF CALCULATION
Once an assay has been validated according to the criteria stated above, the Cutoff Value for determining positive specimens is the HRC .

QUALITY CONTROL
Quality control specimens are supplied with the hc2 High-Risk HPV DNA Test. Consult the applicable Digene assay analysis software user guide for instructions on how to input the Lot Numbers and Expiration Dates of the Quality Controls. These controls must be included in each assay, and the RLU/CO of each control must fall within the following acceptable ranges for it to be considered valid. Version 4.01 and higher Digene assay protocols for HPV will automatically invalidate an assay if the controls are not within the specified limits. Earlier protocol versions 1.0.2 and 1.0.3 will not automatically invalidate an assay if the quality controls are not within the specified limits. If the quality controls do not fall within these ranges, the assay is invalid and must be repeated. Accordingly, no patient results should be reported for any invalid assay. 1. The High-Risk HPV Quality Control provided in the kit is cloned HPV DNA target and is not derived from wild-type HPV. The QC2-HR is 5 pg/ml HPV 16 DNA while the Calibrator contains 1 pg/ml of this same material.

Control
2. This control material will not act as an appropriate processing control for the PreserveCyt specimen.
3. The controls provided with this test kit must be used for internal quality control. Alternatively, external controls may be tested according to guidelines or requirements of local, state, and/or country regulations or accrediting organizations.
Users may develop these external quality control material, as defined by NCCLS C24-A (currently known as CLSI). 37 Please refer to NCCLS C24-A for additional guidance on appropriate internal quality control testing practices.

INTERPRETATION OF SPECIMEN RESULTS
1. Specimens with RLU/CO ratios ≥ 1.0 are considered positive.
2. Specimens with RLU/CO ratios < 1.0 are considered negative or non-detected for the 13 HPV types tested. High-risk HPV DNA sequences are either absent or the HPV Solution DNA levels are below the detection limit of the assay.
3. When testing PreservCyt Solution specimens, if the RLU/CO ratio of a specimen is ≥ 1.0 and < 2.5, the specimen must be retested.
If the initial retest result is positive (≥ 1.0 RLU/CO), the specimen can be reported as positive and no further retesting needs to be completed. However, if the first retest result is negative (< 1.0), then a second retest (third result) needs to be completed to generate a final result. The result of the second retest is considered the final result and is to be reported (see Table 1 The effects of age and HPV positivity are not fully known. It has been demonstrated in studies that HPV prevalence will decrease with age. 38 For information on the age-specific performance of the hc2 High-Risk HPV DNA Test versus a histological diagnosis of high-grade neoplasia, please refer to Table 8 of this package insert.
Additional testing is recommended in any circumstance when false-positive or false-negative results could lead to adverse medical, social or psychological consequences.
Results of this test should be interpreted only in conjunction with information available from clinical evaluation of the patient and from other procedures.
Results of this test are not intended to prevent women from proceeding to colposcopy or from continuing regular cervical cancer screening. This test is not intended for use in women with normal cytology who are under age 30.

DIAGNOSTIC ALGORITHM
This algorithm is used to interpret the results of the hc2 High-Risk HPV DNA Test in conjunction with Pap test results as an aid in determining appropriate patient management. Results should be interpreted only in conjunction with information available from clinical evaluation of the patient including other procedures, patient history and demographics. Device with Specimen Transport Medium (STM) or cervical cytologic specimens collected using a broom-type collection device and placed in PreservCyt Solution. Biopsy specimens may be assayed only if they are placed immediately in STM and stored at -20°C until assayed. • The hc2 DNA Collection Device should not be used for collection of specimens from pregnant women.
• Infection with HPV is not an indicator of cytologic HSIL or underlying high-grade CIN, nor does it imply that CIN 2-3 or cancer will develop. Most women infected with one or more high-risk HPV types do not develop CIN 2-3 or cancer. • A negative High-Risk HPV result does not exclude the possibility of future cytologic HSIL or underlying CIN 2-3 or cancer. A small proportion of high-grade lesions occur in women who are High-Risk HPV negative by existing technologies. 6 • A small amount of cross-hybridization between HPV types 6 and 42 (low-risk HPV types) and the High-Risk HPV Probe exists.
Specimens with high levels (4 ng/ml or higher) of HPV 6 or HPV 42 DNA may be positive. It has also been reported in the literature that complex probe cocktails similar to that used in this test may cause false-positive results due to cross-hybridization with HPV types 11, 40, 53, 54, 55, 66, MM4, MM7, MM8, or MM9. 39 Although several of these HPV types are rare or novel types not often encountered with high-grade disease, patients whose specimens contain high levels of these HPV DNA types may incorrectly be reported as positive in the hc2 High-Risk HPV DNA Test. 12 • Cross-reactivity between the hc2 high-risk HPV DNA Test probe and the plasmid pBR322 is possible. The presence of pBR322 homologous sequences has been reported in human genital specimens and false-positive results could occur in the presence of high levels of bacterial plasmid. • There is no known utility for HPV testing in Pap AGUS results.
• When processing PreservCyt Solution specimens, false-negative results could occur if the cell pellet is not visible after centrifugation. This observation is indicative of insufficient cellular material available to obtain a reliable test result. • PreservCyt Solution specimens containing volumes less than 4 ml after the ThinPrep Pap Test slides are prepared are considered inadequate for the hc2 DNA Tests. • Prepare PreservCyt Solution specimens in batches of 36 or fewer. If processing more than 36 specimens at the same time, the additional pellets formed after centrifugation may loosen and be inadvertently discarded during the decanting step. • The denaturation step of the specimen processing procedure must be performed as directed in this package insert. Improper execution of the denaturation step of the hc2 High-Risk HPV DNA Test Procedure may lead to false-positive results. Improper specimen vortexing, tube inversion and agitation could result in incomplete denaturation of non-specific RNA/DNA hybrids endogenous to cervical specimens. False-positive results could occur due to contamination of the hc2 High-Risk HPV DNA Test specimen with these non-specific RNA/DNA hybrids. In order to prevent possible carryover of this non-denatured cellular material, it is important that the micro-pipette tip not touch the sides of the specimen denaturation tube during transfer of the denatured specimen to the microtube or microplate well used for HPV probe hybridization.

HIGH-RISK HPV PREVALENCE
The prevalence of infection by HPV type, as measured by the detection of an HPV DNA risk group, varies with the patient population. Important variables include age at first intercourse, number of sexual partners, concurrent sexually transmitted diseases and history of abnormal Pap smears. 2,24,31,41 Also, it has been reported that the prevalence of HPV infection decreases dramatically with age. 2,38 Hence, it is not possible to define a single typical pattern of prevalence for HPV infection. Table 3 shows the prevalence in the United States of each high-risk HPV type detected by the hc2 High-Risk HPV DNA Test as reported by two independent researchers. These prevalence values are representative only of the populations tested and may vary in specific areas of the country.  Table 4 shows high-risk HPV prevalence results compiled from several groups of women referred to three gynecology clinics within metropolitan medical centers (high prevalence for HPV infection) for cervical abnormality and tested using the hc2 High-Risk HPV DNA Test. These results demonstrate a fairly consistent pattern of HPV positivity across sites.  Table 5 shows the prevalence of single or combined high-risk HPV types as detected by the hc2 High-Risk HPV DNA Test as reported by six independent researchers. These prevalence values are representative only of the populations tested and may vary from prevalence found in specific areas of the United States.

PERFORMANCE CHARACTERISTICS CLINICAL SENSITIVITY AND SPECIFICITY FOR SCREENING PATIENTS WITH ASC-US PAP SMEAR RESULTS TO DETERMINE THE NEED FOR REFERRAL TO COLPOSCOPY
A study entitled "Utility of HPV DNA Testing for Triage of Women with Borderline Pap Smears" was conducted in 1996 under the direction of the Kaiser Foundation Research Institute and the Kaiser Permanente Medical Group. Cervical specimens for routine Pap smear and for the hc2 High-Risk HPV DNA Test were obtained from women attending several Kaiser clinic facilities. Initial Pap smears were evaluated according to the Bethesda Classification. Women (15 years or older) with Pap smear results of ASC-US returned for colposcopy and biopsy. Colposcopically directed histological specimens were examined by pathologists and an initial diagnosis was made. Each histologic specimen was also reviewed by an independent pathologist and discrepancies between the initial review and the independent review were adjudicated by a third pathologist.
The hc2 High-Risk HPV DNA Test was performed on the initial specimen. HPV DNA testing was performed with a prototype of the hc2 High-Risk HPV DNA Test that contained probes to 11 of the 13 HPV types included in the hc2 High-Risk HPV DNA Test, but did not contain probes to HPV types 59 and 68. This difference would not be expected to result in significantly different performance profiles for the two assays.
hc2 High-Risk HPV DNA Test results and histological diagnoses were available from 885 women with ASC-US Pap smears. Testing on the majority of patients was performed with specimens collected in both STM and PreservCyt Solution. Due to the similarities between the hc2 High-Risk HPV DNA Test's performance characteristics for STM and PreservCyt Solution, assay performance is presented for only the PreservCyt Solution. Table 6 shows that among those presenting with an ASC-US referral Pap smear, the negative predictive value of the hc2 High-Risk HPV DNA Test for having HSIL or greater disease at colposcopy is 99.0%.  Table 7 shows theoretical positive and negative predictive values based on various prevalence results for an initial ASC-US being found to be CIN 2-3 or cancer based on hc2 High-Risk HPV DNA Test results.  Table 8 illustrates the variation between the various age groups contained in this study:

Test Performance in Clinical Specimens
Although no clinical trial was performed specifically to support the use of hc2 High-Risk HPV DNA Test as an adjunct to the Pap test, compared with Pap test alone, consistent data obtained from multiple cross-sectional and prospective cohort studies conducted with a variety of cell sampling methods and utilizing the hc2 HPV DNA Tests and several research-use testing methods provide strong evidence that a negative HPV DNA test implies very low risk of prevalent or incipient CIN 2-3 or cancer when Pap results are normal (WNL). 6,25,[42][43][44][45][46][47][48][49]

Internal Study using Plasmid DNA
A non-clinical panel of cloned HPV plasmid DNA was tested to determine if each of the 13 HPV types are detectable by the hc2 High-Risk HPV DNA Test and to determine the analytical sensitivity of the assay for each of the HPV types. Each HPV target concentration (100 pg/ml, 10 pg/ml, 2.5 pg/ml, 1.0 pg/ml, 0.5 pg/ml, and 0.2 pg/ml targets of each of the 13 HPV DNA types (16,18,31,33,35,39,45,51, 52, 56, 58, 59 and 68) was run in triplicate. The mean signal (in Relative Light Units, RLU) for each concentration of each HPV type was calculated and compared to the HRC x .
The detectable limit of each HPV type is shown in Table 9. The detectable limits varied from 0.62 pg/ml to 1.39 pg/ml depending on the HPV type tested. All HPV types were detectable at an estimated level of 1.08 pg of HPV DNA target per 1 ml of specimen. The mean detectable limit of all 13 HPV DNA types was 1.08 pg/ml with a standard deviation of 0.05 pg/ml. Note: These analytical detection levels have also been clinically validated for the hc2 HPV DNA Test; however, analytical performance can be poorly correlated to clinical performance if there is inadequate attention devoted to setting a correct threshold for positive results, particularly for molecular test methods with high analytical sensitivity. It has recently been stated in the published literature that a low threshold appears to exist under which levels of HPV infection are not associated with cervical disease, rendering detection at such levels clinically irrelevant. This study concluded further that it is important to make a clear distinction between clinically relevant and irrelevant high-risk HPV infections when considering HPV tests for cervical cancer screening programs. 50

External Study Using Clinical Specimens
Note: The following information is provided for analytical purposes only to demonstrate that the hc2 assay detects the thirteen HPV types for which it was designed and does not infer any correlation to clinical performance.
In addition to the constructed HPV plasmid data shown above, the ability of the hc2 HPV DNA test to detect High-Risk HPV DNA from archived clinical specimens characterized by type-specific PCR was evaluated. In a study conducted by Digene and the National Cancer Institute (NCI) involving 209 PreservCyt specimens, a research use type-specific HPV polymerase chain reaction (PCR) test method was utilized by NCI to determine concordance with the hc2 High-Risk HPV DNA Test. Specimens were selected specifically to demonstrate detection of the thirteen high-risk types of HPV recognized by the hc2 assay. The NCI PCR result was used as the sole determinant for the presence of HPV DNA. Of the 209 specimens, the proportion of PCR negative results that were positive by hc2 High-Risk HPV DNA Test was 31/56. Conversely, the proportion of PCR positive results that were negative by hc2 was 5/153 (see Table 10 below). When analyzed in this manner, an overall 82.8% agreement (173/209; 95% CI = 77.0-87.6) was observed between the methods and a positive and negative agreement of 96.7% and 44.6%, respectively (95% CI =92.5-98.9 and 31.3-58.5). When comparing the test methods described above, PCR was used as an indicator of HPV DNA detection. However, analytical PCR test performance can vary greatly due to a lack of test method standardization and inherent issues known to affect PCR method performance. In addition, this study was conducted prior to the introduction of a procedural modification to the hc2 test that demonstrated improved assay reproducibility around the test cut-off and a reduction in apparent false positive HPV results due to possible technique-related variability when performing the test with specimens collected in PreservCyt.

EQUIVALENCE BETWEEN STM AND PRESERVCYT SOLUTION SPECIMENS
Equivalence between STM and PreservCyt Solution specimens was examined for equal recovery of HPV 18 DNA from approximately 10 6 positive HeLa cells containing integrated HPV 18 genomes spiked into STM and into a negative cell pool in PreservCyt Solution. Each specimen type was processed according to its respective processing/denaturation procedures described in this package insert and tested with the hc2 High-Risk HPV DNA Test. The results demonstrated that recovery of HPV 18 DNA from human carcinoma cells is equivalent for the two media and that the PreservCyt Solution preparation procedure does not affect the analytical sensitivity of the hc2 High-Risk HPV DNA Test.

REPRODUCIBILITY
A multicenter reproducibility study was performed to determine the between days, between sites, and overall reproducibility of the hc2 High-Risk HPV DNA Test using a panel of HPV DNA targets and HPV-positive and HPV-negative clinical specimens.
Three external laboratories performed the testing with the same lot of hc2 High-Risk HPV DNA Test kits on three different days with an identical reproducibility panel. The reproducibility panel included the following specimens: 12 denatured clinical STM specimen pools; three undenatured clinical PreservCyt Solution specimen pools; Negative Calibrator; and Positive High-Risk HPV Calibrator at concentrations of 0.5 pg/ml, 1 pg/ml, 2.5 pg/ml, 5 pg/ml, and 10 pg/ml. All panel members were tested each day in triplicate. The results are shown in Table 11. This indicates that the reproducibility of the hc2 High-Risk HPV DNA Test with clinical specimens is very good.
A second study was performed using simulated PreservCyt Solution specimens and conducted at two external laboratories and Digene. Each testing laboratory performed two hc2 HPV DNA Test assays per day on five different days. For each assay, a reproducibility panel of six simulated PreservCyt Solution specimens was individually processed and tested in quadruplicate. Each panel member was formulated by spiking cultured cells into PreservCyt solution to yield an approximate RLU/CO value simulating two negatives (1N, 2N), two low positives (3P, 4P), one mid positive (5P) and one high positive (6P). Results are shown in Table 12. An additional in-house reproducibility study was performed using clinical PreservCyt specimens obtained predominately from 252 women with cytology of ASC-US or greater (HPV prevalence 57%). Specimens were divided into two aliquots; each aliquot was then processed individually using the hc2 Sample Conversion Kit and then tested in duplicate with the hc2 High-Risk HPV DNA Test. As with other qualitative IVDs, variability of hc2 High-Risk HPV DNA Test results obtained from clinical specimens is associated primarily with one or a combination of the following: 1) Specimen collection; 2) specimen processing prior to testing; and 3) the testing procedure. Since the test results under comparison were obtained from the same clinical specimen, the experimental design controlled for variability due to specimen collection. The reproducibility of results obtained from two individually processed specimen aliquots from the same clinical specimen (referred to as "Between Processed Aliquots") reflects variation due to the combination of PC specimen conversion processing and the hc2 test procedure. In contrast, the reproducibility of replicate results obtained from the same processed specimen aliquot (referred to as "Within Processed Aliquot") reflects variation from the hc2 test procedure alone. The results are shown in Table 13.  14 -98.32 Since each specimen in the study generated four test results, there was insufficient volume remaining to allow for retesting of specimens in the defined Cutoff Region Retest Area; therefore, Table 12 presents initial data only. In Table 13, the results from this study are tabulated where only results outside the 1.0 to 2.5 RLU/CO Cutoff Region are considered in the analysis. An assay user employing the Cutoff Region Retest Algorithm given in the Interpretation of Specimen Results section of this Package Insert would be expected to obtain results between those seen in Tables 13 and 14. A multi-center clinical study was also conducted to estimate the additional contribution of cervical specimen sampling to hc2 High-Risk HPV DNA Test result variability. These results are summarized in Table 15. Paired PreservCyt specimens were taken from each patient, processed separately using the hc2 Sample Conversion Kit, then tested separately. Paired STM specimens were also collected and tested separately. Specimens were collected from female patients attending an OB/GYN clinic, colposcopy clinic, STD clinic, hospital, or family planning center. Four geographically diverse sites within the United States collected the PreservCyt specimens, and the STM specimens were all collected from a separate population from multiple clinics in metropolitan San Diego. Testing was performed at four accredited U.S. laboratories. Results for each specimen type were interpreted as recommended, i.e., PC specimen testing employed a Cutoff Region Re-test Algorithm in the 1.0 to 2.50 RLU/CO range, while the initial test results were compared for STM specimens.
Note: These data do not equate to clinical false-positive or false-negative results due to the nature of the paired study design, which assesses duplicate specimen testing agreement. These results reflect variability associated with specimen collection, in addition to the variability due to specimen processing and the assay procedure. Further inspection of these results revealed that result variability was concentrated in the assay cutoff region. When only specimens yielding results outside the cutoff region are included in the analysis, positive agreement for PC specimens increases to 94.0% while negative agreement increases to 99.4%; while similarly for STM specimens, the positive and negative agreement values increase to 97.5% and 99.6% respectively.

EFFECT OF BLOOD AND OTHER SUBSTANCES ON STM SPECIMENS
The effect of blood and other potentially interfering defined or undefined substances was evaluated in the hc2 High-Risk HPV DNA Test . Whole blood, douche, anti-fungal cream and contraceptive jelly (agents that may commonly be found in cervical specimens) were added to STM negative and positive specimens (clinical specimen pools and non-clinical specimens) at concentrations that may be found in cervical specimens. No false-positive results were observed with any of the four agents at any concentration. However, a false-negative result may be reported in clinical specimens with HPV DNA levels close to that of the positive cutoff for the assay (1 pg/ml) if high levels of anti-fungal cream or contraceptive jelly were present. However, it is very unlikely that a clinical specimen will consist almost entirely of one of these substances because the cervix is routinely cleared prior to obtaining specimens for Pap smear and for HPV testing.

EFFECT OF BLOOD AND OTHER SUBSTANCES ON PRESERVCYT SOLUTION SPECIMENS
The effect of blood and other potentially interfering defined or undefined substances potentially present in PreservCyt Solution clinical specimens was evaluated in the hc2 High-Risk HPV DNA Test. Whole blood, douche, anti-fungal cream and contraceptive jelly (agents that may commonly be found in cervical specimens) were added to PreservCyt Solution negative and positive clinical specimen pools at concentrations that may be found in cervical specimens. No false-positive or false-negative results were observed with any of the four agents at any concentration. Furthermore, substances inherent in some clinical specimens do not inhibit the detection of the HPV DNA by the hc2 High-Risk HPV DNA Test.

REPRODUCIBILITY OF hc2 HIGH-RISK HPV DNA TEST WITH CLINICAL SPECIMENS COLLECTED IN STM
The reproducibility of the hc2 High-Risk HPV DNA Test with clinical specimens collected in STM was determined in a study using 20 clinical pools (ten positive and ten negative) prepared by combining previously denatured and tested cervical brush specimens collected in STM. Specimens were tested in replicates of four on each of five days for a total of 20 replicates per specimen. Testing was performed using a combined probe cocktail consisting of the hc2 High-Risk HPV DNA Test probe and low-risk HPV type probes. Mean, standard deviation and 95% confidence interval about the mean (CI) were calculated for each specimen within day and over five days and results are shown in Table 16 below. The reproducibility of the assay would not be expected to differ when using only the high-risk HPV type probe in this kit. For the five specimens with a mean RLU/CO at 20% or more above the cutoff (Nos. [1][2][3][4][5], 100 of 100 replicates (100.0%) were positive. For the five specimens with a mean RLU/CO within 20% above or below the assay cutoff (Nos. 6-10), 60 of 100 (60%; 95% CI = 49.7-69.6) of the replicates were positive and 40 of 100 (40%) were negative. For the 10 specimens with the mean RLU/CO at more than 20% below the assay cutoff, 200 of 200 replicates (100%) were negative.
Thus, specimens with a mean RLU/CO of 20% or more above the cutoff were positive 100% of the time, while specimens with a mean RLU/CO of 20% or more below the cutoff were negative 100% of the time, indicating that specimens at 20% or more away from the cutoff can be expected to yield consistent results. Specimens close to the cutoff yielded approximately equal numbers of positive and negative results. These data demonstrate that STM specimens yield reproducible results in the hc2 High-Risk HPV DNA Test.

REPRODUCIBILITY OF PRESERVCYT SOLUTION SPECIMENS IN THE hc2 HIGH-RISK HPV DNA TEST
The reproducibility of clinical specimens in PreservCyt Solution in the hc2 High-Risk HPV DNA Test was determined in a study using 24 mock specimens at a concentration spanning a range of HPV DNA concentrations. Specimens consisted of PreservCyt Solution and white blood cells, with and without HPV 16 plasmid-containing bacteria.
Specimens were tested in replicates of four on each of five days, for a total of 20 replicates per specimen. On each of the five days of the study, an 8-ml aliquot from each specimen was processed and tested according to the hc2 Sample Conversion Kit package insert instructions. Mean, standard deviation, and 95% confidence interval (CI) were calculated for each specimen within day and over all five days and replicates. The mean RLU/CO, confidence interval about the mean, and the percent of positive replicates are shown below in Table 17 for each specimen, in descending order based on the mean RLU/CO. For the six specimens with a mean RLU/CO at 20% or more above the cutoff (Nos. 1-6), 114 of 120 replicates (95.0%) were positive. For the seven specimens with a mean RLU/CO within 20% above or below the assay cutoff (Nos. 7-13), 88 of 139 (63.3%; 95% CI = 54.3 -70.9) of the replicates were positive and 51 of 139 (36.6%) were negative. For the 11 specimens with the mean RLU/CO at more than 20% below the assay cutoff, 220 of 220 replicates (100%) were negative.
Thus, specimens with a mean RLU/CO of 20% or more above the cutoff were positive greater than 95% of the time, while specimens with a mean RLU/CO of 20% or more below the cutoff were negative 100% of the time, indicating that specimens at 20% or more away from the cutoff can be expected to yield consistent results. Specimens close to the cutoff yielded approximately equal numbers of positive and negative results. These data demonstrate that PreservCyt Solution specimens yield reproducible results in the hc2 High-Risk HPV DNA Test.

hc2 HIGH-RISK HPV DNA TEST TROUBLESHOOTING GUIDE Observation Probable Causes Solutions
Denaturation Reagent not added, or Denaturation Reagent not prepared properly.
Verify that the Denaturation Reagent contains the Indicator Dye and is a dark purple color.
Verify that Denaturation Reagent was added to the specimen by measuring the specimen volume (1.5 ml is expected). If the volume indicates that Denaturation Reagent was not added, make the appropriate addition, mix and proceed with the assay if the proper color change is then observed.
Specimen contains blood or other materials that mask the color change.
The exact color change described is not expected with these types of specimens; assay test results should not be adversely affected.

Improper or no color change observed during denaturation.
Specimen pH may be unusually acidic.
If neither of the other causes apply, the specimen may be unusually acidic, and the expected color change will not occur. Collect a new specimen prior to the application of acetic acid to the cervix because improper specimen pH will adversely affect the test results.
Incorrect software protocol chosen for test (e.g., used LR protocol for HR method) If the software protocol is incorrect for the test being performed, the plate should be read again within 30 minutes after Detection Reagent 2 addition and with the correct protocol.

Quality Controls give incorrect results
Reverse placement of QC1-LR and QC2-HR Retest Specimens.
Inadequate mixing of Probe Cocktail with denatured Quality Controls and/or specimens; or, Probe Cocktail not added; or, incorrect volume of reagent added.
Shake hybridization microplate or microtube rack for an additional 2 minutes. If there are wells that still remain purple, add an additional 25 µl of the appropriate Probe Cocktail and mix well. If upon probe addition and remixing, the proper color change does not occur, and the specimen did not contain blood or other materials, retest the specimen.
Specimen contains blood or other materials that mask the color change.
The exact color change described is not expected with these types of specimens; assay test results should not be adversely affected. Luminometer malfunction or incorrect programming.

Improper
Refer to the maintenance/service and troubleshooting sections in the applicable Digene assay analysis software user guide for further instructions, or call Digene Technical Services. Wide %CVs between replicates.

Observation Probable Causes Solutions
Detection Reagent 1 contamination of microwells.
Ensure all work surfaces are clean and dry. Use care when using Detection Reagent 1. Avoid aerosols.
Detection Reagent 2 contaminated. Be careful not to cross-contaminate specimens as you aliquot Detection Reagent 2 between specimens. If only using part of a kit, aliquot the volume needed for that assay into a clean disposable reagent reservoir prior to filling the pipettor.
Wash microwells thoroughly with Wash Buffer 6 times, filling to overflowing each time or using Automated Plate Washer. There should be no residual pink liquid visible in the microwells after washing.
Contamination of pipette tip with undenatured material during transfer of denatured specimen to the microtube or microplate well used for HPV probe hybridization.
The denaturation step of the specimen processing procedure must be performed as directed in this package insert. Improper specimen vortexing, tube inversion and agitation can result in incomplete denaturation of non-specific RNA/DNA hybrids endogenous to cervical specimens. When using PreservCyt Solution specimens in particular, these hybrids are likely to be present on the inside walls of the specimen denaturation tube. In order to prevent possible carryover of this non-denatured cellular material, the micro-pipette tip must not touch the sides of the specimen denaturation tube during transfer of the denatured specimen to the microtube or microplate well used for HPV probe hybridization.
During decanting and blotting of the capture plate, the plate was blotted on the same area of the Kimtowels Wipers or equivalent low-lint paper towels.
Do not blot on area that has been previously used as cross-contamination could occur.
Inadequate specimen preparation. Add the appropriate volume of Denaturation Reagent and mix thoroughly by vortexing.
To avoid false-positive results, make sure liquid washes entire inner surface of tube with both the manual and MST Vortexer methods (for the manual vortex method, invert tube one time). For PreservCyt Solution specimens, ensure proper mixing and resuspension of the cell pellet is completed prior to denaturation incubation. Consult hc2 Sample Conversion Kit package insert for protocol details. For all specimens, a distinct color change to dark purple should be seen. Incubate for 45 ± 5 minutes at 65 ± 2°C.
False-positive results obtained from known negative specimens.
Improper washing conditions. Wash microwells thoroughly with Wash Buffer 6 times, filling the wells to overflowing each time or using the Automated Plate Washer and proper Automated Plate Washer protocols.

Observation Probable Causes Solutions
Detection Reagent 2 was incubated at a temperature greater than 20-25°C.
Repeat the test, and ensure that Capture and Detection steps are incubated at 20-25°C.
Detection Reagent 2 was incubated longer than 30 minutes.
Read plate after 15 minutes of incubation (and no later than 30 minutes of incubation) at 20-25°C.