Cervista ® HPV HR 92-011, PRD-01560
Contents
1. Copies Reaction or Cells mL HPV Positive HPV Negative Target Extracted N n n 5 000 84 60 71 24 29 10 000 84 84 100 0 0 2 500 84 52 62 32 38 HPV 39 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 84 100 0 0 HPV 45 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 77 92 7 8 HPV 51 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 21 25 63 75 HPV 52 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 64 76 20 24 HPV 56 5 000 84 83 99 1 1 10 000 84 84 100 0 0 2 500 84 60 71 24 29 HPV 58 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 16 19 68 81 HPV 59 5 000 84 79 94 5 6 10 000 84 84 100 0 0 2 500 84 40 48 44 52 HPV 66 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 43 51 41 49 HPV 68 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2500 SiHa 97 500 Jurkat 84 0 0 84 100 SiHa Jurkat 5000 SiHa 95 000 Jurkat 84 15 18 69 82 20 000 SiHa 80 000 Jurkat 84 84 100 0 0 1 250 HeLa 98 750 Jurkat 84 65 77 19 23 HeLa Jurkat 2 500 HeLa 97 500 Jurkat 84 84 100 0 0 10 000 HeLa 90 000 Jurkat 84 84 100 0 0 10 000 84 2 2 82 98 Jurkat 20 000 84 0 0 842. Cervista Cleavase Invader Invader Call Reporter PreservCyt and ThinPrep are registered trademarks of Hologic Inc All other Trademarks Registered Trademarks referenced within this product insert are the property of each of their respective companies Some components of nucleic acid analysis such as specific methods and compositions for manipulating or visualizing nucleic acids for analysis may be covered by one or more patents owned by other parties Similarly nucleic acids containing specific nucleotide sequences may be patented Making using or selling such components or nucleic acids may require one or more licenses Nothing in this document should be construed as an authorization or implicit license to make use or sell any so covered component or nucleic acid under any such patents 2011 Hologic Inc Part Number 15 3053 Revision 103 HOLOGIC 25
3. CUSTOMER AND IS NOT GIVEN TO NOR MAY IT BE RELIED UPON BY ANY THIRD PARTY INCLUDING WITHOUT LIMITATION CUSTOMERS OF CUSTOMER THIS WARRANTY IS VOID UPON TRANSFER OF PRODUCT BY CUSTOMER TO ANY ENTITY WHO HAS LESS THAN FIFTY 50 PERCENT OWNERSHIP IN THE PRODUCT SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSIONS MAY NOT APPLY TO YOU YOU MAY ALSO HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE These warranties do not apply to any item that is a repaired moved or altered other than by Hologic authorized service personnel b subjected to physical including thermal or electrical abuse stress or misuse c stored maintained or operated in any manner inconsistent with applicable Hologic specifications or instructions or d designated as supplied subject to a non Hologic warranty or on a pre release or as is basis WARRANTY CLAIMS AND REMEDIES In the event of any warranty claim Hologic will replace with new or repaired items any Equipment part component or consumable supply that is in breach of warranty and will use reasonable efforts to promptly fix or provide a workaround for any Software defect or bug which prevents operation in substantial conformity with functional specifications Alternatively Hologic may elect to repay or credit to Customer an amount equal to the purchase price of the defective Equipment component Software consumable supply or Service Items replaced shall become Hologic pr
4. HPV Oligo HPV types 18 39 45 59 and 68 Mix 2 Yellow cap and 1 100 HL OR TRUR HI suspended in water and MOPS yellow stripe buffer pH 7 5 03 Oligonucleotides with affinity to HPV Oligo Orange ca HPV types 16 31 33 35 52 and Mix 3 l g 3 1S AUO HE SEON 58 suspended in water and MOPS and orange stripe buffer pH 7 5 Cleavase Enzyme suspended in Cleavase E 140 mM MgCl 10 mM Tris pH Enzyme Purple cap and 1X 1100 uL 8 x 970 uL 8 0 25 mM KCI 0 25 Tween 20 Solution purple stripe 0 25 Nonidet P40 25 Glycerol and 0 05 mg mL BSA 1000 copies uL cloned HPV type 51 HPV C1 DNA and 3000 copies uL cloned Control 4 Clear cap and 1 x 350 uL 8 x 350 uL HIST2H2BE DNA in yeast tRNA black stripe and 10 mM Tris 0 1 mM EDTA Buffer 1000 copies uL cloned HPV type 18 apy C2 DNA and 3000 copies pL cloned Control 2 Clear cap and 1 x 350 uL 8 x 350 uL HIST2H2BE DNA in yeast tRNA black stripe and 10 mM Tris 0 1 mM EDTA Buffer 1000 copies uL cloned HPV type 16 doy C3 DNA and 3000 copies uL cloned Control 3 Clear cap and 1 x 350 uL 8 x 350 uL HIST2H2BE DNA in yeast tRNA black stripe and 10 mM Tris 0 1 mM EDTA Buffer No T t ae Yeast tRNA and 10 mM Tris 0 1 r a an mM Tris 0 Control Clearcapand 1x350 ud Bx cote met MM EDTA Buffer black stripe WARNINGS AND PRECAUTIONS 1 For in vitro diagnostic use 2 Universal safety precautions should be used when handling any human tissues o
5. 02378 002 for the Cervista MTA system 2 All quality control requirements should be performed in conformance with local state and federal regulations as well as accreditations requirements LIMITATIONS 1 The Cervista HPV HR test detects DNA of high risk HPV types 16 18 31 33 35 39 45 51 52 56 58 59 66 and 68 This test does not detect DNA of HPV low risk types e g 6 11 42 43 44 2 The Cervista HPV HR test exhibits cross reactivity to two HPV types of unknown risk An HPV positive result was observed with 5000 copies reaction of HPV type 67 and 50 000 copies reaction of HPV type 70 3 A negative result does not exclude the possibility of HPV infection because very low levels of infection or sampling error may cause a false negative result 4 The test has been validated for use only with cervical cytology specimens collected in PreservCyt Solution or SurePath Preservative Fluid 5 The performance of the Cervista HPV HR test was established using DNA extracted with the Genfind DNA Extraction Kit 6 Interference was observed in cervical specimens collected into PreservCyt Solution contaminated with high levels 2 of contraceptive jelly and or anti fungal creams when DNA was isolated with the Genfind DNA Extraction Kit Under these conditions false negative results may be obtained 7 Interference was observed in cervical specimens collected into SurePath Preservative Fluid contaminated wi
6. 100 100 000 84 0 0 84 100 Performance of the Cervista HPV HR test on specimens collected into SurePath Preservative Fluid compared to specimens collected into PreservCyt Solution A total of 418 subjects were enrolled in a co collection study to obtain paired cervical specimens collected into SurePath Preservative Fluid and PreservCyt Solution from each subject Each pair of specimens was tested with the Cervista HPV HR test A total percent agreement of 92 was observed for the results obtained for specimens collected into the SurePath Preservative Fluid compared to the results obtained for specimens collected into the PreservCyt Solution 17 Table 16 Summary of Cervista HPV HR results from cervical specimens co collected into SurePath Preservative Fluid and PreservCyt Solution SurePath Specimen PreservCyt Specimen Results Results Total 418 418 Positive 29 4 29 2 Negative 69 9 70 6 Indeterminate 0 7 0 2 References 1 2 National Cancer Institute website www cancer gov 2008 Meijer CJ Snijders PJ and Castle PE 2006 Clinical utility of HPV genotyping Gynecol Oncol 103 12 17 Wright TC Jr Massad LS Dunton CJ Spitzer M Wilkinson EJ and Solomon D 2007 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests Am J Obstet Gynecol 197 4 346 55 Sherman ME Schiffman M and Cox
7. OF THE PROCEDURE Cervista HPV HR is a qualitative in vitro diagnostic test for the detection of DNA from 14 high risk HPV types namely types 16 18 31 33 35 39 45 51 52 56 58 59 66 and 68 The Cervista HPV HR test uses the Invader chemistry a signal amplification method for detection of specific nucleic acid sequences This method uses two types of isothermal reactions a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal See Figure 1 In the primary reaction two types of sequence specific oligonucleotides i e a probe oligonucleotide and an Invader oligonucleotide bind to the DNA target sequence When these oligonucleotides overlap by at least one base pair on the target sequence an invasive structure forms that acts as a substrate for the Cleavase enzyme The enzyme cleaves the 5 portion flap of the probe at the position of the overlap The probes are present in large molar excess and cycle rapidly on and off the target sequence so that many cleaved 5 flaps are generated per target sequence The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer FRET oligonucleotide creating another invasive structure that the Cleavase enzyme recognizes as a substrate The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces fluorescence signal as the cleaved flaps cycle on and o
8. mineral oil and slowly pipette up and down 3 4 times e Make sure that all liquid is expelled from the pipette tip during additions e Verify that the correct control was added to each well e Verify that the correct control volume was added to each well e Verify the calibration information on equipment e Visually inspect plate for consistent volumes between wells Correct control s was not added to the plate or was not added to the correct plate position Verify the correct controls were added to the correct plate positions 20 Incubation period was shorter or longer than the specified length of time recommended Confirm that the incubation was performed for the specified length of time and at the specified temperature Suspected contamination during sample addition Use nuclease free aerosol barrier tips and sterile tubes during set up Wear gloves when setting up the test Make sure that pipette tips touch only the solution being dispensed Do not touch pipette tips with hands Clean lab surfaces using appropriate materials Sample evaporation Verify mineral oil addition to each well Improper plate orientation When scanning the plate orient the plate so well A 1 is in the upper left hand corner Bubbles in the reaction plate wells If possible spin down plates prior to fluorescence scanning Prepared reaction mixes were not used with
9. types of vaginal douches contraceptive jelly two types of anti fungal creams and negative clinical specimens that visually contained blood and mucous The PreservCyt Solution douche contraceptive jelly and anti fungal creams were added at two levels 0 5 and 2 These levels were chosen in order to represent extreme situations that could potentially occur during specimen collection if the cervix was not cleared prior to obtaining the specimen DNA was isolated from pure and impure samples using the Genfind DNA Extraction Kit and was tested with the Cervista HPV HR test to assess interference caused by the introduced substances Contraceptive jelly and the anti fungal creams containing either clotrimazole or miconazole at a 2 sample concentration resulted in indeterminate and false negative results During DNA extraction the contraceptive jelly interfered with the magnetic bead separation in the 10 mM Tris buffer causing low DNA recovery and insufficient DNA sample for testing This interference was visually detectable The levels of the above substances that are required to cause testing failure are unusually high and should not be encountered in actual clinical specimens if the clinician follows the proper Pap sampling procedure of clearing the cervix before obtaining the cell sample for Pap analysis The Cervista HPV HR test was also tested with components that could potentially be transferred inadvertently during sample extraction us
10. 00 1 34 0 09 39 2500 5000 1 30 0 06 45 1250 2500 1 31 0 06 51 2500 5000 1 35 0 09 52 1250 2500 1 28 0 04 56 1250 2500 1 37 0 10 58 2500 5000 1 35 0 09 59 2500 5000 1 35 0 09 66 2500 5000 1 30 0 06 68 2500 5000 1 30 0 06 Mean 1 324 0 074 Accuracy and Specificity Compared to a PCR DNA Sequencing Method A study designed to evaluate the ability of the Cervista HPV HR test to detect High Risk HPV DNA from clinical specimens was performed The specimens were characterized using a research use HPV genotyping method that utilized degenerate PCR amplification followed by HPV type specific sequencing The PCR sequencing method was used as the sole determinant for the presence of HPV DNA The study involved 192 specimens stored in PreservCyt Solution of which 189 had clear sequencing results Of these 189 samples two samples were indeterminate by the Cervista HPV HR test Indeterminate results were not included in the comparative analysis of the Cervista HPV HR test and PCR sequencing methods The proportion of PCR sequencing negative results that were positive by the Cervista HPV HR test was 5 187 Conversely the proportion of PCR sequencing positive results that were negative by the Cervista HPV HR test was 11 187 see Table 9 When analyzed in this manner an overall 91 4 agreement 171 187 95 Cl 86 5 95 0 was observed between the methods with a positive and negative agreement of 89 8 and 93 7 respectiv
11. 4 S4 S12 S12 S12 S20 S20 S20 S28 S28 S28 Figure 2 Cervista HPV HR test plate layout Overlay each well with 20 uL of mineral oil and plate sealing tape to minimize evaporation Incubate the samples at 95 C for 5 minutes in a thermal cycler Mix the reagents and reaction mixes thoroughly and consistently prior to use Prepare the reaction mixes as indicated in the Mix Preparation sheet printed from the Invader Call Reporter software or according to the calculations in Table 2 Prepare one reaction mix for each of the three HPV Oligo Mixes Table 2 Reaction Mix Preparation Instructions Component HL Well Number of Reactions 25 Samples amp Controls k Overage tal Volume HPV Oligo Mix 1 2 or 3 8k 1 25 Cleavase Enzyme Solution 2k 1 25 Total Mix Volume 10k 1 25 uL Decrease thermal cycler temperature setting to 63 C 7 Add 10 uL of the appropriate reaction mix to each well containing a control or sample see Figure 2 8 taking care to pipette below the mineral oil Incubate the plate at 63 C setting for 4 hours Data Collection 1 Always bring the plate to room temperature before reading If the plate cannot be read immediately store it at 2 8 C it is recommended to read the plate within 24 hours of test completion Place the 96 well plate well A1 must be in the upper left corner in the plate holder of the fluorescenc
12. Cerist HPV HR Cervista HPV HR 92 011 PRD 01560 INTENDED USE The Cervista HPV HR test is intended for two uses 1 In combination with cervical cytology screening for women age 30 and above to guide patient management 2 To triage patients with atypical squamous cells of undetermined significance ASC US Pap test results to determine the need for referral to colposcopy YZ 15 C 92 011 V96 30 C PRD 01560 Vos Hologic UK Ltd Link 10 Napier Way EC REP Crawley West Sussex RH10 9RA UK 44 0 1293 522080 Do not store in a frost free freezer Protect from light FOR EXPORT ONLY NOT FOR SALE IN THE UNITED STATES OF AMERICA OR CANADA TABLE OF CONTENTS ABBREVIATIONS USED HARMONIZED SYMBOLS USED SUMMARY AND EXPLANATION OF THE TEST PRINCIPLES OF THE PROCEDURE REAGENTS PROVIDED WARNINGS AND PRECAUTIONS STORAGE AND HANDLING REQUIREMENTS ADDITIONAL REAGENTS AND MATERIALS MATERIALS REQUIRED BUT NOT PROVIDED SPECIMEN COLLECTION DNA EXTRACTION AND STORAGE FOR ANALYSIS TEST PROCEDURE PROCEDURAL NOTES INTERPRETATION OF RESULTS QUALITY CONTROL LIMITATIONS PERFORMANCE CHARACTERISTICS REFERENCES TROUBLESHOOTING GUIDE ABBREVIATIONS USED ASC US Atypical squamous cells of undetermined significance CIN Cervical intra epithelial neoplasia DNA Deoxyribonucleic acid FAM Carboxyfluorescein dye FRET Fluorescence resonance energy transfer FOZ Fold over zero sample or control signal divid
13. Contact your local representative to order the Genfind DNA Extraction Kit REF 95 449 MATERIALS REQUIRED BUT NOT PROVIDED Consumable Supplies Pipette tips filter barrier and nuclease free 96 well polypropylene plates Clear Plate Sealers Mineral oil molecular biology grade 2 0 mL sterile polypropylene tubes and screw caps Equipment Cervista MTA System for automation users Pipettes Vortex Tecan Infinite F200 Tecan GENios or BioTek FLx800 fluorescence plate reader Desktop PC with Microsoft Windows XP operating system and Microsoft Excel and Adobe Reader software e Thermal cycler or oven capable of maintaining appropriate reaction temperatures SPECIMEN COLLECTION DNA EXTRACTION AND STORAGE FOR ANALYSIS Cervical specimens that may be tested with the Cervista HPV HR test include the following e Specimens collected in PreservCyt Solution the ThinPrep Pap test preservation system using an approved collection device h e Specimens collected in SurePath Preservative Fluid using an approved collection device Cervical specimens in PreservCyt Solution can be stored at room temperature 20 30 C for up to 24 weeks prior to performing the test Cervical specimens in SurePath Preservative Fluid can be stored at room temperature 20 30 C for up to 6 weeks prior to performing the test The Genfind DNA Extraction Kit REF 95 449 has been validated for use with th
14. If the HPV FOZ Ratio is greater than or equal to 1 525 then the sample is positive for HPV However in a subset of mixed infections all three reaction wells may generate a signal much higher than background In some cases these mixed infections may generate positive signals of similar intensity in all three reaction wells and therefore a HPV FOZ Ratio of less than 1 525 In order to avoid the chance of a false negative due to the triple positive scenario described above a second calculation is applied as follows when the FOZ ratio is less than 1 525 but all three individual reaction FOZ values are greater than or equal to a second cutoff value of 1 93 the sample is positive for HPV 8 An indeterminate call is generated in three different scenarios 1 when the CV between the gDNA FOZ values is 225 0 High CV 2 when all three HPV FOZ values are lt 0 7 Low HPV FOZ and 3 when average gDNA FOZ of a negative sample is lt 1 5 low gDNA A summary of the sample call criteria described above is shown in Figure 3 Terminology HPV FOZ For each HPV Oligo Mix the FAM signal of the sample divided by the FAM signal of the No Target Control HPV FOZ Ratio The highest HPV FOZ of the three HPV Oligo Mixes divided by the lowest HPV FOZ of the three HPV Oligo Mixes normalized to 1 0 if FOZ is lower than 1 0 Average gDNA FOZ The average value determined from the three genomic DNA FOZ values obtained from each of the three reaction mi
15. R test was demonstrated in a 21 day study with three alternating operators each performing two runs per day on individually assigned sets of equipment Each run consisted of four plates Different plate layouts were used for the runs within a day Each run consisted of genomic DNA samples isolated from two HPV positive cell lines SiHa Type 16 and HeLa type 18 an HPV negative cell line Jurkat and contrived samples containing HPV 16 HPV18 HPV31 HPV33 HPV35 HPV39 HPV45 HPV51 HPV52 HPV56 HPV58 HPV59 HPV66 or HPV68 plasmid DNA and Jurkat DNA Each sample was tested in duplicate at three concentrations At 2500 copies reaction the plasmid DNA samples yielded 57 4 675 1176 positive results At 5000 copies reaction the plasmid DNA samples yielded 97 2 1143 1176 positive results At 10000 copies reaction the plasmid DNA samples yielded 100 0 1176 1176 positive results see Table 15 Table 15 Summary of positive and negative values for each sample condition tested Target Extracted N n n 2 500 84 82 98 2 2 HPV 16 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 64 76 20 24 HPV 18 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 58 69 26 31 HPV 31 5 000 84 84 100 0 0 10 000 84 84 100 0 0 2 500 84 13 15 71 84 HPV 33 5 000 84 81 96 3 4 10 000 84 84 100 0 0 HPV 35 2 500 84 1 1 83 99
16. Referral rate for women 30 years of age and older was 43 12 Analytical Sensitivity Cloned HPV plasmid DNA representing the 14 HPV types detected by the Cervista HPV HR test was tested to determine the individual analytical sensitivity for each specific type Individual Limit of Detection LoD values were calculated for the 14 HPV types 16 18 31 33 35 39 45 51 52 56 58 59 66 68 as a function of a Limit of Blank LoB measurement and a population variance measurement SD from multiple concentrations of the specific HPV target CLSI NCCLS guideline EP17 A Vol 24 No 34 Nine HPV negative characterized DNA samples isolated from cervical specimens were used to determine the LoB value FAM FOZ Ratio 1 20 Each HPV plasmid DNA was tested at concentrations of 7500 5000 2500 and 1250 copies per reaction each in a background of three genomic DNA concentrations isolated from an HPV negative cell line 10 ng 100 ng and 1 ug per reaction All positive and negative samples were tested in replicates of eight The Limit of Detection for each HPV type is referenced in Table 8 Limits are described in terms of the FAM FOZ Ratio and as a copy number range Table 8 Cervista HPV HR Test Analytical Sensitivity Summary LoD LoD HPV DNA Type Copy Number Reaction FAM FOZ Ratio SD 16 1250 2500 1 34 0 08 18 1250 2500 1 34 0 08 31 1250 2500 1 30 0 06 33 2500 5000 1 31 0 07 35 5000 75
17. TJ 2002 Effects of age and human papilloma viral load on colposcopy triage data from the randomized Atypical Squamous Cells of Undetermined Significance Low grade Squamous Intraepithelial Lesion Triage Study ALTS Jour Nat Can Inst 94 2 102 107 Davey DD Neal MH Wilbur DC Colgan TJ Styer PE and Mody DR 2004 Bethesda 2001 implementation and reporting rates 2003 practices of participants in the college of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology Arch Path Lab Med 128 1224 1229 Wright TC Jr Cox JT Massad LS Twiggs LB Wilkinson EJ 2001 Consensus Guidelines for the management of women with cervical cytological abnormalities JAMA 2002 287 2120 2129 Solomon D Schiffman M and Tarone R 2001 Comparison of three management strategies for patients with atypical squamous cells of undetermined significance baseline results from a randomized trial Jour Nat Can Inst 93 4 293 299 Mayrand MH E Duarte Franco Rodrigues SD Walter J Hanley 2007 A Ferenczy S Ratnam F Coutl e EL Franco Human Papillomavirus DNA versus Papanicolau Screening Tests for Cervical Cancer N Engl J Med 357 16 1579 1588 Wheeler CM WC Hunt M Schiffman PE Castle 2006 Human papillomavirus genotypes and the cumulative 2 Year risk of cervical cancer J Infect Dis 194 1291 1299 10 Saslow D Runowicz CD Solomon D Moscicki A B Smith RA Eyre HJ Cohen C American Cancer So
18. bles 5 and 6 11 Table 5 Cervista HPV HR versus Colposcopy Consensus Histology results CIN2 among women with ASC US cytology Cervista HPV Colposcopy Histology HR Positive Negative Total Positive 64 705 769 Negative 5 573 578 Total 69 1278 1347 Includes indeterminate results P CIN2 Histology No CIN or CIN1 by Central Histology or Colposcopy without Central Histology Table 6 Cervista HPV HR versus Colposcopy Consensus Histology results CIN3 among women with ASC US cytology Cervista HPV Colposcopy Histology HR Positiv Negative Total Positive 22 705 727 Negative 0 573 573 Total 22 1278 1300 Includes indeterminate results P CIN3 including one adenocarcinoma in situ No CIN CIN1 or CIN2 by Central Histology or Colposcopy without Central Histology Among women with ASC US cytology the clinical sensitivity of the test for CIN2 was 92 8 95 Cl 83 9 97 6 and the negative predictive value was 99 1 95 Cl 98 0 99 7 The clinical sensitivity and the negative predictive values of the test for CIN 3 are both 100 95 Cl 84 6 100 and 99 4 100 There are a number of key variables that are known to influence the performance characteristics of any HPV test in a clinical study These include but are limited to cervical sampling techniques the quality of the cytology results age of the population tested d
19. ciety guideline for the early detection of cervical neoplasia and cancer CA Can Jour Clin 2002 53 342 362 11 Wright TC Jr Schiffman M Solomon D Cox JT Garcia F Goldie S Hatch K Noller KL Roach N Runowicz C Saslow D 2004 Interim guidance for the use of human papillomavirus DNA testing as an adjunct to cervical cytology for screening Obstet Gynecol 103 304 309 12 Hall JG Eis PS Law SM Reynaldo LP Prudent JR Marshall DJ Allawi HT Mast AL Dahlberg JE Kwiatkowski RW de Arruda M Neri BP and Lyamichev VI 2000 Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction PNAS 97 15 8272 8277 TROUBLE SHOOTING GUIDE MANUAL TEST PROCEDURE FOR CERVISTA HPV HR Table 17 Troubleshooting Guide Problem Potential Cause Possible Solution Insufficient volume made for reaction mixes Number of samples entered in Assay Selection tab of software is less than samples added to the plate Manually recalculate the required amount of reaction mix needed to complete the entire plate Recreate software printouts using correct number of samples Excess reaction mix volume added to 96 well microplate Verify the correct reaction mix volumes were added to each well Verify the calibration information on equipment is current No Target Control displays the following results e Increase gain for scan 1 e Increase gain for scan 2 e Increase
20. consensus primers for the HPV L1 gene A portion of the human beta globin gene was also amplified as an internal control Purified amplicons were used as templates in multiple sequencing reactions for 14 high risk types of HPV 16 18 31 33 35 39 45 51 52 56 58 59 66 and 68 The sequencing data was analyzed using various sequence alignment software A comparison of the Cervista HPV HR test with the PCR Sequencing method among both ASC US and WNL subjects resulted in an overall 86 1 agreement between the two methods 95 Cl 84 9 87 3 The positive percent agreement between the two methods was 91 8 89 7 93 6 and the negative percent agreement was 84 2 95 Cl 82 7 85 7 The clinical performance of the Cervista HPV HR test was measured against colposcopy and histology results Biopsy samples were collected from women with ASC US cytology as warranted by standard of care guidelines at each participating clinical site Consensus histology results provided by a central review panel served as the gold standard for determining the presence or absence of disease In the absence of histology data the lack of colposcopically visible cervical lesions and no biopsy equated to the absence of disease There were 1347 ASC US subjects with known disease status central histology or negative colposcopy and Cervista HPV HR results A comparison of the Cervista HPV HR results with Colposcopy Central Histology is shown in Ta
21. e Cervista HPV HR test The recommended procedure for DNA extraction from cervical specimens in PreservCyt Solution or SurePath Preservative Fluid is included in the Genfind DNA Extraction Kit instructions for use Laboratories performing the Cervista HPV HR test with any extraction method other than that provided in the validated Genfind DNA Extraction Kit are responsible for their own validation of that method DNA samples can be stored at 2 to 8 C for up to four weeks For storage greater than four weeks place the samples in a freezer between 30 C and 15 C TEST PROCEDURE FOR CERVISTA MTA SYSTEM Refer to the Cervista MTA Operator s Manual Part Number MAN 02378 002 for the use of the automated system to perform the Cervista HPV HR test MANUAL TEST PROCEDURE FOR CERVISTA HPV HR Reaction Procedure 1 Add 10 pL of each control and sample DNA to three wells of a 96 well plate as indicated in the test Oh Ser plate layout see Figure 2 Mix 1 Mix 2 Mix 3 Mix 1 Mix 2 Mix 3 Mix 1 Mix 2 Mix 3 Mix 1 Mix 2 Mix3 1 2 3 4 5 6 7 8 9 10 11 12 A C1 C1 C1 S5 S5 S5 S13 S13 S13 S21 S21 S21 B C2 C2 C2 S6 S6 S6 S14 S14 S14 S22 S22 S22 C C3 C3 C3 S7 S7 S7 S15 S15 S15 S23 S23 S23 D NTC NTC NTC S8 S8 S8 S16 S16 S16 S24 S24 S24 E S1 S1 S1 S9 S9 S9 S17 S17 S17 S25 S25 S25 F S2 S2 S2 S10 S10 S10 S18 S18 S18 S26 S26 S26 G S3 S3 S3 S11 S11 S11 S19 S19 S19 S27 S27 S27 H S4 S
22. e plate reader Remove plate sealing tape 3 Define the plate type to set up the coordinates and probe height for the specific type of plate Save the settings 4 Read the entire plate Two separate scans are required FAM Excitation 485 nm Emission 530 nm and Red Excitation 560 nm Emission 612 nm To detect the HPV signal the instrument should be set to detect the FAM dye first To detect the sample genomic DNA the instrument should be set to detect the Red dye 5 Adjust the gain of the fluorescence plate reader to be in the linear dynamic range of the reader according to the manufacturer s instructions The gain should be set so that the No Target Control NTC yields values that are in the background range of the reader with a minimum RFU of 600 The NTC values do not have to be identical for the FAM and Red reads PROCEDURAL NOTES AND PRECAUTIONS 1 Laboratories should use good laboratory practices and comply with all applicable federal state and local regulatory requirements 2 Mix the samples reagents and reaction mixes thoroughly and consistently 3 Use nuclease free sterile disposable aerosol barrier pipette tips for each addition and transfer to avoid cross contamination 4 Use nuclease free disposable polypropylene tubes for preparing the reaction mixes 5 Verify that the 96 well plate type is compatible with the specific thermal cycler and fluorescence plate reader to be used before starting the tes
23. eans to determine the relative quantity of sample DNA in each reaction Human HIST2H2BE is measured by a Red fluorescent signal that lies above an empirically derived cut off value in each reaction The measure of this target serves as a quality control mechanism to confirm that a negative result is not due to insufficient sample 1b HIST2H2BE oligos form invasive 1a HPVoligos form invasive structure on genomic DNA structure on HPV DNA 4 N kiita Jal i 3 TARGET 5 TARGET 5 2 Cleavase enzyme recognizes structure and cleaves probe oligos 3a Flaps from HPV probe oligos form invasive structure on FAM FREToligos 3b Flaps from HIST2H2BE probe oligos form invasive structure on Red FRET oligos 4 Cleavase enzyme recognizes structure and releases fluorophores from FRET Oligos creating fluorescence signal FAM Red e N Fluorescence a Fluorescence STT TT ri A S B rs n Figure 1 A graphic representation of the Invader chemistry in Cervista HPV HR REAGENTS PROVIDED Table 1 Cervista HPV HR Contents Vial Quantity Vial Quantity R nt Vial Label amp Reagent amp Reagent Combonent Desciibtion ong Abbreviation Volume Volume REF PT cece REF 92 011 PRD 01560 01 Oligonucleotides with affinity to HPV Oligo HPV types 51 56 and 66 Mix 1 Blue cap and 1 1400 aL ae suspended in water and MOPS blue stripe buffer pH 7 5 02 Oligonucleotides with affinity to
24. ed by No Target Control signal gDNA Genomic DNA HIST2H2BE Human histone 2 gene H2be gene HPV Human papillomavirus HR High risk Max Maximum Min Minimum MTA Medium Throughput Automation NTC No Target Control Oligo Oligonucleotide Pap Papanicolau cervical cytology test Red Redmond red dye RFU Relative Fluorescence Unit SUMMARY AND EXPLANATION OF THE TEST Approximately 11 000 new U S cases of invasive cervical cancer and over 3 500 deaths are projected annually For the earliest stage of cervical cancer the 5 year relative survival rate is 92 and for all stages of cervical cancer the 5 year survival rate is about 72 Cervical cancer is caused by persistent infection with human papillomavirus HPV Cervical cancer has previously been shown to be highly preventable when cytological and HPV screening programs are employed to facilitate the detection and treatment of pre cancerous lesions Over 100 HPV types have been documented in the literature approximately 40 of which infect the anogenital area and are transmitted sexually Of the sexually transmitted types of HPV 14 oncogenic genotypes HPV16 18 31 33 35 39 45 51 52 56 58 59 66 and 68 referred to as high risk HR types are now recognized as the cause of almost all cervical cancers The presence of high risk HPV DNA in conjunction with an equivocal or ambiguous cytology result ASC US places a woman at increased risk for having an underlyin
25. ely 95 Cl 82 5 94 8 and 85 8 97 9 Table 9 Detection of HPV DNA Comparing Cervista HPV HR Test to PCR with Type Specific Sequencing PCR Sequencing Negative 74 n 74 11 Cervista HPV HR test 79 Reproducibility In this investigational study overall reproducibility of the Cervista HPV HR test was assessed at three sites using a panel of HPV positive and negative cultured cells and HPV positive and negative cervical specimens DNA was extracted from 2 mL of cervical specimen or cultured cells suspended in PreservCyt Solution The DNA was extracted using the Genfind DNA Extraction Kit Sixteen samples were tested at three locations on five non consecutive days within a two week time period Two lots of Cervista HPV HR kits and three lots of Genfind DNA Extraction Kits were used for the study Within day site agreement was assessed by calculating the between run percent agreement for the three possible pairings within each of the days sites The average percent agreement and one sided exact 95 confidence interval is presented first for each site intra site reproducibility then across all three sites inter site reproducibility Between day within site agreement was assessed by calculating the between run agreement for any two runs done on two separate days within a site for all possible pairings The average percent agreement and one sided exact 95 confidence interval is presented first for each site intra s
26. ent number of cells in specimen Suspected error during DNA extraction e Mix the specimen and repeat DNA extraction e Verify the correct sample volume was added to each well e Verify that proper procedure was followed for Sample displays 7 DNA extraction IND Low gDNA Insufficient amount of result DNA was used in the assay Repeat DNA extraction from the specimen DNA sample inhibition Refer to Instructions for Use Performance Characteristics Interfering Substances section The DNA sample s Verify that the sample was denatured at the may not have been correct temperature and for an appropriate completely denatured amount of time Suspected error e Repeat DNA extraction from the specimen Sample displays during DNA extraction Verify that proper procedure was followed for IND Low HPV DNA extraction FOZ result e Refer to Instructions for Use Performance DNA sample inhibition Characteristics Interfering Substances section Insufficient Insufficient elution Repeat DNA extraction from the specimen Sample DNA volume during DNA volume Extraction Verify that proper procedure was followed for DNA extraction High number of DNA samples with positive FAM FOZ values in all three reaction mixes Suspected error during DNA extraction Suspected DNA extraction reagent contamination e Repeat DNA extraction from the specimen e Verify that proper procedure was
27. f Hologic Except as otherwise stated in this paragraph no other license is granted expressly impliedly or by estoppel For information concerning the availability of additional licenses to practice the patented methodologies contact Legal Department Hologic Inc 502 South Rosa Rd Madison WI 53719 608 273 8933 The Cervista HPV HR test uses a proprietary Invader chemistry and specific components covered under U S Patent Nos 5 614 402 5 795 763 5 846 717 5 985 557 5 994 069 6 001 567 6 090 543 6 090 606 6 348 314 6 458 535 6 555 357 6 562 611 6 635 463 6 673 616 6 759 226 6 872 816 6 875 572 6 913 881 7 060 436 7 067 643 7 087 381 Canadian Patent Nos 2 163 015 2 203 627 Australian Patent Nos 694 736 731 062 737 449 738 849 744 369 779 443 781 188 Japanese Patent No 3 665 648 European Patent No 711 361 All U S patents and foreign patents where applicable that have or may hereafter issue in respect of such applications for patents and all U S and foreign patent applications and 23 patents issuing thereon where applicable whose subject matter in whole or in part is entitled to the benefit of the filing date s of any of the foregoing patents patent applications listed in this product insert LIMITED PRODUCT WARRANTY WARRANTIES Equipment Supplies and Software are warranted to the original Customer to perform substantially in accordance with published Product Specifications for one 1 year
28. ff For each copy of target the combined primary and secondary reactions result in 10 10 fold signal amplification per hour The flap sequences and FRET oligonucleotides are universal since they are not complementary to the targeted sequence The reagents for this assay are provided as three oligonucleotide mixtures which detect the 14 types of HPV grouped according to phylogenetic relatedness i e viral types with similar DNA sequences Oligonucleotides that bind to the human histone 2 gene H2be HIST2H2BE are also present in these three oligonucleotide mixtures HIST2H2BE serves as an internal control producing a semi quantitative signal from genomic DNA present in the sample The format of the Cervista HPV HR test allows simultaneous detection of HPV DNA sequences and HIST2H2BE in a single well by utilizing two different 5 flap sequences on the probes as well as two different FRET oligonucleotides each with a spectrally distinct fluorophore FAM and Red By design the released 5 flaps bind only to their respective FRET oligonucleotides to generate target specific signal see Figure 1 A positive result indicates that at least one of the 14 high risk types is present in the DNA sample This result is represented by a FAM fluorescent signal that lies above an empirically derived cut off value For each reaction a negative result is represented by a FAM fluorescent signal that lies below an empirically derived cut off value As a m
29. followed for DNA extraction 22 TROUBLESHOOTING FOR CERVISTA MTA SYSTEM Refer to the Troubleshooting section of the Cervista MTA Operator s Manual Part Number MAN 02378 002 for Cervista MTA systems Contact Information Manufacturer Hologic Inc 502 S Rosa Road Madison WI 53719 USA Phone 608 273 8933 Website www hologic com Technical Support Access routes for Technical Support outside of the US will be advised by your local representative Distributors Contact Hologic for a list of worldwide suppliers and distributors Phone 608 273 8933 Website www hologic com ec rer Authorized Representative for the European Community Hologic UK Ltd Link 10 Napier Way Crawley West Sussex RH10 9RA 44 0 1293 522 080 NOTICE TO RECIPIENT ABOUT LIMITED LICENSE The receipt of Product from Hologic or its authorized distributor includes a limited non exclusive non transferable license under certain intellectual property rights held by Hologic This license is only for the purpose of using the Product in the methods for which they were intended This limited license does not include a license to use the Product for new product research or development product manufacture reverse engineering improvements to the Product technology or any other commercial purpose Customer is not authorized to transfer this Product to any third party for any purpose whatsoever without the express written consent o
30. g cervical intraepithelial neoplasia 2 or 3 CIN 2 or CIN 3 CIN 3 while occurring in only approximately 5 of ASC US cases is an immediate precursor to cervical cancer and consequently its detection is very important for patient management Therefore the identification of those women with ASC US cytology in conjunction with a high risk HPV infection is a useful aid for clinicians to decide who should be monitored or treated more aggressively 2 Beginning in 2002 patient management guidelines have been published by various groups of U S healthcare professionals that recommend how women should be screened for cervical cancer according to age the presence of cytological abnormalities in a Pap test sample and other factors These patient management guidelines recommend testing for the presence of high risk types of HPV as a regular screening tool in combination with cytology in specific instances Principal recommendations of the most recent professional practice guidelines the 2006 Consensus Guidelines for the Management of Women with Abnormal Cervical Cancer Screening Tests include 1 screening women 30 years of age and over in conjunction with cytology or other screening methods and 2 management of women over age 20 with ASC US In all cases patient management decisions reflect patients overall cytology history and other risk factors in addition to the presence or absence of high risk HPV types PRINCIPLES
31. gain for both scans Fluorescence microplate reader gain settings are too low causing the raw fluorescent signal values to fall below the minimum requirement Increase the fluorometer gain settings for the designated scan s so that the No Target Control produces a minimum signal of 600 RFU and re read the plate Errors occur during data import Check FAM amp Red gain settings and read the whole plate again Partial plate reads are not allowed Check FAM gain setting and read the whole plate again Partial plate reads are not allowed Check Red gain setting and read the whole plate again Partial plate reads are not allowed No Target Control displays the following results High CV HPV NTC Fluorometer issues See Troubleshooting Guide in the Invader Call Reporter Software User Manual for fluorometer issues that may contribute to this error Incubation period was longer than the specified length of time recommended Insufficient or inconsistent mixing of reagents Confirm that the incubation was performed for the specified length of time and at the specified temperature e Be sure all samples reagents and reaction mixes are mixed thoroughly e When adding reaction mix to each well place tips at the bottom of the well beneath mineral oil and slowly pipette up and down 19 High CV gDNA NTC Incorrect preparation of reaction mixes Incon
32. in recommended time period Use reaction mixes within 30 minutes of preparation Sample displays IND High CV result Insufficient or inconsistent mixing of samples Inconsistent addition of reaction mix Inconsistent addition of sample Suspected contamination during sample addition e Be sure all samples and reagents are mixed thoroughly e When adding reaction mix to each well place tips at the bottom of the well beneath the mineral oil and slowly pipette up and down 3 4 times e Verify all liquid is expelled from the pipette tip during additions e Verify the correct sample was added to each well e Verify the correct sample volume was added to each well e Verify the calibration information on equipment is current e Visually inspect plate for consistent volumes between wells Use nuclease free aerosol barrier tips and sterile tubes during set up Wear gloves when setting up the test Make sure that pipette tips touch only the solution being dispensed Do not touch pipette tips with hands 21 Clean lab surfaces using appropriate materials Sample evaporation Verify mineral oil addition to each well Bubbles in the reaction wells If possible spin down plates prior to fluorescence scanning Prepared reaction mixes were not used within recommended time period Use reaction mixes within 30 minutes of preparation Insuffici
33. ing the Genfind DNA Extraction Kit DNA containing three levels 0 5 and 10 each of 70 ethanol or Genfind magnetic beads was tested to assess interference caused by the introduced substances Interference was observed when 10 of the DNA sample volume contained either 70 ethanol or the magnetic beads Cross Reactivity A panel of bacteria fungi and viruses commonly found in the female anogenital tract as well as several cloned Human papillomavirus types of low or undetermined risk were tested with the Cervista HPV HR test to assess potential cross reactivity see Tables 12 14 Table 12 The organisms listed below were added to PreservCyt Solution at concentrations of approximately 1x10 cfu mL and 1x10 cfu mL DNA from these organisms and a negative cell line Jurkat 1x10 cells mL was extracted using the Genfind DNA Extraction Kit All samples yielded negative results with the Cervista HPV HR test Candida albicans Proteus vulgaris Corynebacterium pseudodiptheriticum Staphylococcus aureus Enterococcus faecalis Staphylococcus epideridis Escherichia coli Streptococcus mitis Lactobacillus acidophilus Streptococcus pyogenes Table 13 Purified DNA obtained from the organisms listed below was tested at concentrations of 1x10 copies reaction and 1x10 copies reaction using the Cervista HPV HR test All samples yielded negative results Herpes simplex virus type 1 HSV 1 Chlamydia trachoma
34. is determined by dividing the highest HPV FOZ of the three reaction mixes by the lowest HPV FOZ of the three normalized to 1 0 if lower than 1 0 HPV Control 1 should yield a positive HPV FOZ value 21 525 for only HPV Oligo Mix 1 HPV Control 2 should yield a positive HPV FOZ value 21 525 for only HPV Oligo Mix 2 and HPV Control 3 should yield a positive HPV FOZ value 21 525 for only HPV Oligo Mix 3 3 The Mean gDNA FOZ of all three mixes must be greater than or equal to 1 50 21 50 or the control is invalid for low gDNA 4 The CV of the Mean gDNA FOZ from all three mixes should be less than 25 0 lt 25 0 Table 4 HPV Control and Sample Criteria Control HPV FOZ Positive Average CV Ratio FOZ Mix gDNA FOZ gDNA FOZ HPV Control 1 Valid Control gt 1 525 Mix 1 only lt 25 0 HPV Control 2 Valid Control gt 1 525 Mix 2 only lt 25 0 HPV Control 3 Valid Control gt 1 525 Mix 3 only lt 25 0 Test Verification 1 Sample results are valid when both positive and negative controls yield correct results If the No Target Control negative control is invalid and or any result for positive control s is invalid all sample results on that plate are invalid and must be repeated Refer to the Troubleshooting sections located in the Instruction For Use and in the Software User Manual For Invader Call Reporter software Refer to the Troubleshooting section of the Cervista MTA Operators Manual Part Number MAN
35. isease prevalence disease ascertainment methods and methods for histological interpretation Given the number of variables present during routine HPV testing across multiple Clinical sites it is noteworthy that many of the results obtained from the Hologic clinical trial are similar to those seen under the controlled trial conditions described in the ASC US LSIL Triage Study ALTS A comparison of the study design disease prevalence and clinical performance characteristics for the Hologic study and ALTS is shown in Table 7 The difference in CIN2 rates observed between the two studies may reflect population differences as well as disease ascertainment differences Table 7 Comparison of Hologic Clinical Trial and ALTS Criterion ALTS Hologic Number of Enrollment Sites States 4 4 89 22 Mean Age of Subjects 29 33 Subjects with colposcopy completed 1149 1347 Subjects with no lesion no biopsy performed 25 28 Subjects with no pathologic lesion on biopsy 49 53 Subjects with CIN1 15 14 Subjects with CIN2 11 5 Detection rate for CIN2 96 93 Detection rate for CIN3 96 100 Negative Predictive Value for CIN2 98 9 99 1 Negative Predictive Value for CIN3 99 5 100 0 Referral rate to colposcopy 57 57 PCR concordance 82 7 86 1 Immediate colposcopy arm of ALTS P Number of subjects with known disease status and Cervista HPV HR results
36. ite inter run reproducibility then across all three sites inter site inter run reproducibility Between site agreement was assessed by calculating the between run percent agreement for any two runs done by two different sites for all possible pairing n 3 sites 1 and 2 sites 1 and 3 sites 2 and 3 The average percent agreement and one sided 95 confidence interval are presented in Tables 10 and 11 Table 10 HPV HR Molecular Assay Between Day Within Site Percent Agreement Number of Number of Percent 1 sided 95 Confidence Site ae Comparisons Agreements Agreement Lower Limit Site 1 200 200 100 0 96 3 Site 2 200 193 96 5 90 8 Site 3 200 200 100 0 96 3 Across All 3 Sites 600 593 98 8 96 9 Table 11 HPV HR Molecular Assay Between Site Percent Agreement Number of Number of Percent 1 sided 95 Confidence Sites ae Comparisons Agreements Agreement Lower Limit Site 1 vs Site 2 500 490 98 0 96 6 Site 1 vs Site 3 500 500 100 0 99 4 Site 2 vs Site 3 500 490 98 0 96 6 All Site Pairs 1500 1480 98 7 97 9 14 Interfering Substances Four cervical specimens one HPV negative three HPV positive and three cell line samples one HPV negative two HPV positive were tested with added substances that potentially could be present in the cervical specimen The substances that were added to the specimens included PreservCyt Solution two
37. operty All claims shall be initiated by contacting Hologic within the applicable warranty period and thirty 30 days after discovery of the breach or non conformity Hologic must be given reasonable access and an opportunity to inspect all associated materials If Hologic and Customer are unable to settle any claim and Customer has not notified Hologic within one 1 year after the claim arises Customer shall be barred from instituting any legal action thereafter These remedies shall comprise Hologic s entire liability and Customer s exclusive remedy for breach of warranty and are in lieu of any other remedies at law or equity LIMIT OF LIABILITY HOLOGIC SHALL NOT BE LIABLE FOR ANY SPECIAL INCIDENTAL PUNITIVE EXEMPLARY OR CONSEQUENTIAL LOSSES DAMAGES OR EXPENSES INCLUDING BUT NOT LIMITED TO LOSS OF PROFITS DATA OR USE DIRECTLY OR INDIRECTLY ARISING FROM THE SALE HANDLING SERVICE OR USE OF PRODUCT ORDERED OR FURNISHED OR FROM ANY CAUSE RELATING THERETO UNLESS EXPRESSLY AGREED TO BY THE PARTIES IN WRITING 24 EXCEPT FOR PERSONAL INJURY OR DEATH TO THE EXTENT RESULTING FROM HOLOGIC S NEGLIGENT OR INTENTIONALLY WRONGFUL ACTS OR OMISSIONS IN NO EVENT SHALL HOLOGIC BE LIABLE UNDER ANY LEGAL THEORY OR FOR ANY CAUSE WHATSOEVER WHETHER BASED UPON WARRANTY CONTRACT TORT NEGLIGENCE OR OTHER THEORY EVEN IF ADVISED OF THE POSSIBILITY THEREOF FOR ANY AMOUNT IN EXCESS OF THE PRICE FEE OR CHARGE THEREFORE RECEIVED BY HOLOGIC Hologic
38. r fluids Specimens should be disposed according to local requirements Do not pool reagents from different lots or from different vials of the same lot Do not use reagents after their expiration date Product components product residuals packaging can be considered as laboratory waste Dispose of unused reagents and waste in accordance with applicable federal state and local regulations STORAGE AND HANDLING REQUIREMENTS e Store all reagents between 30 C and 15 C e Do not use reagents past expiration date indicated on outside of package e Do not store in a frost free freezer e Protect from light e Prior to use remove reagents from freezer and allow them to thaw at least 30 minutes at room temperature or until visual inspection indicates that no frozen material is present e Vortex reagents prior to each use e Hologic recommends no more than six 6 freeze thaw cycles for all Cervista HPV HR test reagents e Prepare reaction mixes prior to each use Prepared reaction mixture should be used within 30 minutes ADDITIONAL REAGENTS AND MATERIALS Invader Call Reporter software is a required component of this IVD test This software is provided once with the initial order of the Cervista HPV HR test and afterwards when incremental updates to the software are released Contact your local representative if additional copies are required The Genfind DNA Extraction Kit is an accessory of the Cervista HPV HR test
39. sistent addition of the No Target Control or reaction mix to the microplate 3 4 times e Verify all liquid is expelled from the pipette tip during additions e Verify the correct reagent was added to each well e Verify the correct reagent volumes were added to each well e Verify the calibration information on equipment is current e Visually inspect plate for consistent volumes between wells Suspected contamination during sample addition or reaction mix preparation Sample evaporation e Use nuclease free aerosol barrier tips and sterile tubes when making the reaction mixes e Wear gloves when setting up the test e Make sure that pipette tips touch only the solution being dispensed e Do not touch pipette tips with hands e Clean lab surfaces using appropriate materials Verify mineral oil addition to each well Bubbles in the reaction plate wells Prepared reaction mixes were not used within recommended time period If possible spin down plates prior to fluorescence scanning Use reaction mixes within 30 minutes of preparation Control s displays Invalid Control result Insufficient or inconsistent mixing of controls Inconsistent addition of reaction mix Insufficient or Inconsistent addition of control e Be sure all controls and reagents are mixed thoroughly and consistently e When adding reaction mix to each well place tips at the bottom of the well beneath
40. starting from the date of Installation if applicable or from the date of Delivery whichever occurs first After sale options and accessories are warranted for six 6 months and x ray tubes are warranted on a straight line prorated basis as stated in the applicable Product Specification Warranty Period Replacement parts are warranted for the remainder of the Warranty Period or ninety 90 days from Delivery whichever is longer Consumable Supplies are warranted to conform to published specifications for a period ending on the expiration date shown on their respective packages Services are warranted to be supplied in a workman like manner Hologic does not warrant that use of Products will be uninterrupted or error free or that Products will operate with non Hologic authorized third party products HOLOGIC S ENTIRE WARRANTY RESPONSIBILITY IS EXPRESSLY LIMITED TO REPAIR OR REPLACEMENT AT HOLOGIC S OPTION AND IN THE FORM ORIGINALLY SHIPPED OF PRODUCT OR CORRECTION OF SERVICE SUBJECT TO ANY CLAIM OR AT HOLOGIC S ELECTION REPAYMENT OF OR CREDITING CUSTOMER WITH AN AMOUNT EQUAL TO THE HOLOGIC PRICE FEE OR CHARGE THEREFORE THE FOREGOING WARRANTIES ARE IN LIEU OF AND EXCLUDE ALL OTHER WARRANTIES NOT EXPRESSLY SET FORTH HEREIN WHETHER EXPRESS OR IMPLIED BY OPERATION OF LAW OR OTHERWISE INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE SUCH LIMITED WARRANTY IS GIVEN SOLELY TO THE ORIGINAL
41. t 6 Use only calibrated equipment 7 Controls must be added to the designated positions on the test plate layout shown in Figure 2 in order for the Invader Call Reporter software to function properly 8 Use fresh mineral oil for each reaction setup do not transfer these reagents back to the original container once they have been dispensed 9 Refer to the test plate layout to ensure that the correct mix is added to the appropriate column 10 Always place the pipette tip near the bottom of the well to ensure that the reaction mix is added below the mineral oil Mix by carefully filling and emptying the pipette tip 3 5 times Procedural Notes 5 9 and 10 do not apply to the Cervista MTA system INTERPRETATION OF RESULTS A signal to noise value sample signal measured against signal from a No Target Control reaction well is generated for each of the three reactions This signal to noise value is referred to as FOZ Fold Over Zero A final positive or negative or indeterminate result for any particular sample is generated based on the analysis of three separate reaction wells The ratio between HPV FOZ values generated by the three reaction mixtures determines whether a sample is positive The HPV FOZ ratio is calculated by dividing the highest HPV FOZ value from any one of the three reaction mixtures by the lowest HPV FOZ value of the three When any FOZ value is less than 1 it is rounded up to 1 for the ratio calculation
42. th contraceptive jelly and or anti fungal creams at levels of 0 5 and the personal lubricant ASTROGLIDE at levels of 0 5 when DNA was isolated with the Genfind DNA Extraction Kit Under these conditions false negative results may be obtained The potential interference of the personal lubricant ASTROGLIDE was not tested in cervical specimens collected into PreservCyt Solution PERFORMANCE CHARACTERISTICS Clinical Trial Performance A multi center cross sectional and prospective clinical study was conducted to evaluate the performance of the Cervista HPV HR test for the detection of human papillomavirus and cervical intraepithelial neoplasia grade 2 or higher CIN2 in liquid cytology samples Residual ThinPrep cytology specimens were collected from 3 540 women undergoing routine cervical cancer screening This study included 2 026 women age 30 and over with normal cytology results WNL and 1 514 women age 18 and over with ASC US results Cytology samples were collected from 89 clinical sites across the United States DNA was extracted from residual ThinPrep cervical samples that remained after routine cervical cancer screening procedures were completed The DNA was subsequently tested using the Cervista HPV HR test The analytical performance of the test was measured against PCR Sequencing results Residual DNA samples from both ASC US and WNL subjects were used for PCR amplification and sequencing DNA samples were amplified using
43. tis Herpes simplex virus type 2 HSV 2 Neisseria gonorrhoeae Human Immunodeficiency Virus type 1 Neisseria meningitides HIV 1 pol and env regions Mycoplasma hominis 15 Table 14 Purified cloned DNA or PCR amplicon samples for the following HPV types were tested at concentrations of 1x10 copies reaction and 1x10 copies reaction unless noted using the Cervista HPV HR test All samples yielded negative results Human papillomavirus type 1a Human papillomavirus type 44 Human papillomavirus type 6 Human papillomavirus type 53 Human papillomavirus type 11 Human papillomavirus type 67 Human papillomavirus type 42 Human papillomavirus type 70 Human papillomavirus type 43 Human Internal Control gene Human papillomavirus types 67 and 70 yielded positive results with the Cervista HPV HR test at 1x10 and 1x10 copies reaction Upon further titration of these samples negative results were obtained with the Cervista HPV HR test at 1x10 copies reaction and 1x104 copies reaction respectively In addition DNA extracted from a panel of twelve cervical specimens that were stored in PreservCyt Solution and previously confirmed to contain HPV types of low or undetermined risk HPV Types 6 42 43 44 53 or 70 by PCR sequencing was also tested and yielded negative results with the Cervista HPV HR test Precision Repeatability and within laboratory precision of the Cervista HPV H
44. xes calculated by dividing the Red signal of the sample by the Red signal of the No Target Control CV_gDNA FOZ coefficient of variation for the gDNA FOZ values generated by the three HPV Oligo Mixes Criteria Result Generated ACV gDNA lt 25 xe IND High CV YES One or more HPV FOZ gt 0 70 NO YES YES One or more HPV FOZ lt 1 93 NO pos YES YES Figure 3 Sample Call Criteria Ordered Top to Bottom QUALITY CONTROL Negative Control 1 The No Target Control must yield the appropriate results in order for the samples on that plate to be valid If it does not meet these criteria the samples and controls on that plate are invalid and must be repeated see Table 3 2 The minimum signal for each of the three mixes must be greater than or equal to 600 RFU 2 600 9 3 The CV of the average HPV signal from all three mixes must be less than 25 0 lt 25 0 or the samples and controls on that plate are invalid and must be repeated See Table 3 4 The CV of the average gDNA signal from all three mixes must be less than 25 0 lt 25 0 Table 3 No Target Control Criteria Min HPV Min gDNA Max CV Signal Signal HPV and gDNA 24 9 HPV Controls 1 HPV controls HPV Controls 1 3 must yield the appropriate results for the test to be valid If controls do not meet these criteria the samples on that plate are also invalid and must be repeated See Table 4 2 A HPV FOZ Ratio
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