Optiplex HPV Genotyping Kit

Transcription

1 Highend diagnostic products Optiplex HPV Genotyping Kit Fluorescent Bead Assay (FBA) for diagnostic determination of 24 Human Papillomaviruses (HPV) in Polymerase Chain Reaction (PCR) amplified samples Kit Content: 96 tests Cat. No.: IN0601 FOR IN VITRO DIAGNOSTIC USE Store components at indicated temperature immediately after arrival! Content I. Introduction 2 II. Intended Use 2 III. Test Principle 2 IV. Kit Content 2 V. Storage 3 VI. Materials required but not provided 3 VII. Laboratory organization 3 VIII. Sample DNA preparation 3 IX. Detailed Test Instructions 3 X. Read-Out Interpretation 5 XI. Troubleshooting 6 XII. Luminex Template Settings 7 XIII. Performance Characterization 8 XIV. References 11 Abbreviations 11 Quick Guide Hybridization Assay 12

2 I. Introduction More than 100 HPV types of the Alphapapillomavirus genus are known. 24 of the most common types have been divided into three groups based on their association to cervical cancer (1): 15 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), 3 putative high-risk types (26, 53, and 66), and 6 lowrisk types primarily found in genital warts and low-grade cervical lesions (6, 11, 42, 43, 44 and 70). II. Intended Use Multiplex In vitro test kit for the qualitative determination of Human Papillomavirus (HPV) genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82 in Polymerase Chain Reaction (PCR)-amplified samples of genomic DNA isolated from cervical smears. III. Test Principle Luminex Technology The Luminex suspension array technology is based on polystyrene beads, internally dyed with various ratios of two spectrally distinct red fluorophores. Each bead set in the bead mix is coupled to a specific oligonucleotide probe. Target sequences are indirectly labelled with a third fluorophore. The bead sets can be differentiated and bound fluorescence can be quantified by a Luminex analyzer. Hybridization Assay The sample DNA, extracted from cervical smear, is subjected to PCR amplification, using sets of biotinylated primers contained in the kit. Optionally, a pair of primers for the amplification of a ß-globin gene fragment can be added to the PCR as a control. PCR products are added to the bead mix containing 26 distinct bead populations coupled to 24 HPV, one ß-globin and one Hybridization Control specific oligonucleotide probe. The ß-globin control serves as quality control for genomic DNA in the PCR. The Hybridization Control should be spiked into one or more sample wells, confirming that proper hybridization conditions were applied. After thermal denaturation and hybridization of target sequences to the bead-bound probes, labeling of the hybridized biotinylated PCR products is achieved by R-Phycoerythrin labeled Streptavidin (reporter fluorescence). IV. Kit Content - Sufficient for 96 samples - Each kit consists of 2 separate boxes, which must be stored at different temperatures immediately. Table 1: Content Reagent Volume Storage at Box 1 of 2: 1. Bead Mix (BM) 4.0 ml 2-8 C 2. Assay Buffer (DIL) 100 ml 2-8 C 3. Conjugate (CON 10x) 0.75 ml 2-8 C 4. Staining Buffer (STB) 7.2 ml 2-8 C well Hybridization plate 2-8 C well Filtration plate 2-8 C Lock-well plate 2-8 C 8. Seal foil 2-8 C Box 2 of 2: 1. PCR Primer Set µl -20 C 2. PCR Primer Set 2 52 µl -20 C 3. Hybridization control (HYB) 120 µl -20 C Instruction sheet Notes for the User For professional use only. End-User Licence By purchasing this kit with fluorescent dye coated microspheres, which are authorized by the Luminex Corp., the customer acquires the right to use this product only with the Luminex analyzer. The use of Luminex microspheres is covered by US patents. Safety Precautions Treat the waste bottle with a suitable disinfectant, autoclave solid waste at 121 C for 30 minutes. Follow the rules of good laboratory practice. The Bead Mix contains sodium azide (0.09%) and the Assay Buffer ProClin (0.05%) as preservatives. Avoid contact with eyes, skin or mucous membranes. Bead-Mix, Conjugate and Staining Buffer contain Tetramethylammonium chloride (TMAC). Solutions can be harmful if swallowed and/or irritating to skin (R22, R38, S23, 36/37, 45, 60). See material safety data sheets. Waste Management Chemicals and mixtures containing chemicals and items contaminated by human specimen are, as a rule, considered hazardous and biohazardous waste (see safety data sheets). Damage in Transit If a test kit is considerably damaged, please contact the manufacturer or local distributor. Do not use considerably damaged components 2

3 V. Storage Frozen components may thaw during shipment and must be stored at -20 C immediately after arrival. Stability see indicated dates on reagent labels. After the first opening the reagents are stable for 6 months at the indicated storage temperatures. Avoid direct light irradiation! There is the possibility to use the kit sequentially up to four times, beyond this, aliquotation and storage immediately thereafter is recommended for Primer Set 1, Primer Set 2 and HYB at -20 C, just after arrival. VI. Materials Required but not Provided Reagents/Kit for the extraction of genomic DNA from biological samples Thermocycler PCR reagents (Polymerase, dntps, PCR buffer), except primers Incubation oven (95 C) Thermomixer with PCR plate top Horizontal shaker Adjustable single chanel pipettes (1-100 µl) Disposable sterile, nuclease free filter tips Luminex LX100 or LX200 Vacuum Wash Station Vortex 8-Channel Pipette, (5-100 µl) VII. Laboratory Organization In order to prevent contaminations of genomic DNA samples we highly recommend to perform the individual operations of the HPV genoptyping kit in separate laboratory areas (please ensure a 3-room-solution), as described below, each with its own set of supplies and pipettes which may not be interchanged. Personnel involved in specimen processing should wear clean laboratory coats, which must not be used outside of this particular area. Filter tips should be used for pipetting to prevent cross-contamination between specimens and disposable examitantion gloves should be used and exchanged frequently in all operation steps. 1. Preparation of samples (DNA isolation) on day 1 in laboratory 1; 2. Preparation and aliquoting of PCR mastermixes on day 2 in laboratory 2; 3. Perfomance of the Polymerase Chain Reaction on day 2 in laboratory 3; 4. Analysis of the biotinylated PCR products on day 2 in laboratory 3. Laboratory 1 for sample preparation (DNA isolation). This room and its equipment must be kept free of PCR products. Spin down sample containing vials before uncaping with care. Avoid opening more than one sample containing vial at the same time. Do not process high concentrations of plasmids in this area. After being involved in step 1, one should not participate in subsequent work for step 2, in lab 2 at the same day! Laboratory 2 for storage and preparation of PCR reagents (Master-Mix). This room and its equipment must be completely kept free of PCR products, plasmid DNA and genomic DNA samples. Laboratory 3 for amplification and PCR product detection. Personnel involved in steps 3 and 4 of this laboratory should not perform subsequently steps 1 and 2 in laboratory 1 and 2 at the same day! VIII. Sample DNA Preparation Specified test performance can be achieved with genomic DNA isolated from clinical specimens as verified for the test: Protocols for DNA isolation from cervical exfoliated cells: A. DNA was isolated from 2 ml of 20 ml total cervical swab suspension in PreservCyt Solution (CYTYC Corp.) using High Pure PCR Template Preparation Kit (Roche, Cat. No ) according to the manufacturers instructions. Elution was performed with 200 µl Elution Buffer out of which 10 µl were subjected to PCR amplification for HPV genotyping. B. DNA was isolated from 0.8 ml of 20 ml total cervical swab suspension in PreservCyt Solution (CYTYC Corp.) using QIAsymphony DSP Virus/Pathogen Midi Kit, version 1 (Qiagen, Cat. No ) with the Complex800 V5 DSP protocol according to the manufacturer s instructions. Elution was performed with 60 µl buffer out of which 10 µl were subjected to PCR amplification for HPV genotyping. IX. Detailed Test Instructions Step 1: Amplification of HPV and β-globin specific gene fragments by PCR. Following the isolation of genomic DNA from biological samples with an established method (section VIII) in laboratory 1 on day 1 a PCR mastermix for an adequate number of samples according to Table 2 is prepared in a pre-pcr area (laboratory 2 on day 2). Each sample will be amplified in a total volume of 50 µl. Primer Set 1 contains all HPV primers: 9 biotinylated forward and 3 reverse primers for amplifying the HPV types under investigation. Primer Set 2 (DNA quality control primers) contains primers for the amplification of a ß-globin gene fragment. It can be included optional in each individual PCR in order to verify the amount and the quality of human genomic sample DNA. A Negative Control containing no genomic DNA should be included to confirm the absence of a contamination in the amplification reactions. 3

4 Table 2: Pipetting scheme for PCR mastermix preparation 100 reactions per sample component 200 µl 2 µl Primer Set 1 (50 µl) (0.5 µl) (Primer Set 2) optional 2500 µl 25 µl 2x Multiplex PCR Master Mix* ad 4000 µl ad 40 µl PCR grade water (RNase-free) * The PCR protocol was optimized using the Multiplex PCR Kit (incl. 2x Multiplex PCR Master Mix, RNase-free water) from Qiagen (Cat. No , ). This DNA Polymerase / Buffer combination should be utilized by the user in order to achieve valid test results. PCR samples: Pipette 40 µl of PCR mastermix for each sample to PCR tubes, shut the tubes and place on ice. Transfer the PCR tubes containing the PCR mastermix to laboratory 1. In laboratory 1 add 10 µl of PCR proof water to one PCR mastermix aliquot and immediately shut the PCR tube. Add 10 µl of each individual genomic DNA sample to residual PCR mastermix aliquots and immediately shut the PCR tubes. Avoid opening more than one PCR tube at a time. Transfer PCR tubes to laboratory 3 and place the tubes in the thermocycler. Start the thermocycler with the following protocol: PCR protocol: (min:sec): 94 C 15:0 94 C 0:20 38 C 0:30 x 40 } 71 C 0:80 71 C 4:00 4 C Note: The PCR protocol was optimized using following ramping rates: 1.8 C/sec from 94 C to 38 C, 2.0 C/sec from 38 C to 71 C and 2.8 C/sec from 71 C to 94 C Note: PCR products are stable for 2 weeks when stored at 2-8 C. Optional: Analyze 10 µl of each PCR sample in a 2% Agarose Gel, stained with Ethidium bromide or SYBR Green. Under UV light a band of approximately 150 bp of the HPV specific PCR product and a band of 208 bp for the ß-globin PCR product should be visible. Due to its limited sensitivity, this visualisation modality can not be used for pre-screening of HPV positive samples. Step 2: Reagent Preparation and Denaturation of PCR product Equilibrate all test components at room temperature in laboratory 3. 4 Initialize the Luminex analyzer according to the Luminex manual. Use template settings according to Appendix. Pre-heat the incubation oven to 95 C. Pre-heat the thermomixer with 96-well PCR plate top to 41 C. Staining Solution: Prepare Staining Solution by diluting Conjugate 1:10 in Staining Buffer. For each well 75 µl Staining Solution should be prepared (e.g. for one reaction 7.5 µl Conjugate to 67.5 µl Staining Buffer). Bead Mix: Resuspend the beads by vortexing the Bead Mix for 20 sec. Pipette 40 µl of the Bead Mix to each required sample well of a 96 well Hybridization plate. Negative control: Pipette 10 µl water in well A1, as Negative control. Hybridization control: Pipette 9 µl water and 1 µl of the Hybridization control to well B1. Do not use the Hybridization control more than 12 hours after dilution. Optional: Add 1 µl of Hybridization control to each PCR product for internal control of hybridization efficiency or add 1 µl of Hybridization control per 40 µl of Bead Mix before applying to the Hybridization plate. Bead Mix PCR Samples: To each sample well, starting from C1, add 10 µl of PCR product. After addition of Negative control, Hybridization control and PCR samples to the wells, please, mix thoroughly by pipetting up and down several times. Cover the PCR plate with seal foil in order to prevent evaporation and incubate in an incubation oven at 95 C for 10 min. Step 3: Hybridization and Wash Immediately place the plate on ice for 1 min and transfer subsequently to the thermomixer. Incubate at 41 C for 30 min under agitation (500 rpm) and protected from light. Note: The hybridization incubation should be performed in the provided Denaturation / Hybridization Plates at exactly 41 C and is the most critical step to avoid false positive (temperature too low) or false negative/very weak signals (temperature too high). A 96-well thermomixer with inclined lid allows good control of temperature variations. Always close the lid of the thermomixer during incubation and seal the plate tightly. During hybridization, equilibrate each well of a Filtration plate by pipetting 100 µl Assay Buffer in each well and incubating the Filtration plate for 30 min at room temperature. Remove the Wash Buffer by vaccum filtration. Mix the Bead Mix PCR samples by pipetting up and down several times while the Hybridization plate

5 is still located in the Thermomixer and transfer the Bead Mix PCR samples to the Filtration plate with an 8-channel pipette. Transfer the Filtration plate to the vacuum manifold and remove the liquid by vacuum filtration. Wash once with 100 µl of Assay Buffer. Remove the liquid by vacuum filtration. Note: For optimal wash efficiency, allow the negative pressure to reach 12 inhg, do not allow the wash membrane to dry between the washing steps and do not exert more than 15 inhg negative pressure. Shut the Filtration plate with the corresponding lid and remove all residual liquid from the bottom side of the Filtration plate by multiple vigorous blotting on a clean paper towel! Note: Non-removed liquid on the bottom of the Filtration plate may lead to complete sample loss by capillary force upon contact with the shaker surface. Step 4: Staining of Hybridized PCR products Add 70 µl Staining Solution to each well of the Filtration plate and incubate protected from light at room temperature (18-22 C) for 30 min under slight agitation (250 rpm) on a horizontal shaker. Transfer the Filtration plate to the vacuum filtration manifold. Remove the liquid by vacuum filtration. Add 100 µl Assay Buffer to each well and remove the liquid by vacuum filtration. Repeat the wash step twice with 100 µl Assay Buffer per well each. Step 5: Resuspension of the Beads Shut the Filtration plate with the corresponding lid and remove all residual liquid from the bottom side of the Filtration plate by multiple vigorous blotting on a clean paper towel! Note: Non-removed liquid on the bottom of the Filtration plate may lead to complete sample loss by capillary force upon contact with the shaker surface. Pipette 100 µl Assay Buffer to the Filtration plate and incubate for 5 min on a horizontal shaker. Alternatively, in order to avoid vigorous blotting of the plate, resuspend the beads in 100 µl Assay Buffer by pipetting up and down several times. Transfer the resuspended beads to the 96 Lock-well plate supplied with the kit or any available microtiter plate, using a 8-chanel pipette. Note: Usage of different microtiter plates may require the adjustment of the Luminex probe height prior to the analysis. Step 6: Plate Read-Out Make sure the reservoir of the Luminex analyzer contains sheath fluid. Place the Filtration plate (or the 96 Lock-well plate) into the Luminex analyzer and select the appropriate batch created with the corresponding template. Note: For proper specimen analysis, it is important that the instrument is set-up, calibrated and maintained according to the manufacturer`s instructions. X. Read-Out Interpretation The Luminex instrument analyzes the reporter fluorescence signal of at least 70 beads of each individual bead set per well. The fluorescence signals are evaluated by the software of the Luminex analyzer and are saved in a folder named with the test run in the Output. csv file. The fluorescence intensities of each sample are calculated as the median fluorescence intensity (MFI) and are listed in tabular form under Data Type: Median. We suggest to evaluate the results following the guidelines below. A reasonable number of control samples should be used with every run. Calculation of background and Cut-off value for analytical sensitivity: For each run and each HPV type a background and Cut-off value is calculated based on the signal of the Negative Control in well A1 as follows: Background = 1.25 x Negative Control value (MFI) Cut-off value (c.o.) = Background + 15 MFI Exceptions: For HPV18, 59, 82 the c.o. is Background + 50 MFI For HPV 16 the c.o. is Background MFI, (see XI, case 3.2). Negative sample: individual measured value < Cut-off No PCR product of this HPV type detected. Positive sample: individual measured value Cut-off PCR products corresponding to the HPV type of this bead set detected. Interpretation of the genomic DNA quality control (Primer Set 2): The amount and quality of the genomic DNA can partly be assesed by including Primer Set 2 into the PCR. The Primer Set 2 targets a region of the ß-globin gene and is designed not to compete with the HPV primer binding site. A minimum of approx human cellular genome equivalents are necessary for the detection of the ß-globin gene in the Optiplex HPV Genotyping Kit. Note: The Primer Set 2 cannot specifically control for the presence of PCR inhibitors in the sample. Based on the detection of specific HPV and ß-globin PCR products and their combination, the following evaluation scheme should be applied (provided that usual signal values for the Hybrididization control of > 200 MFI are achieved). 5

6 Table 3: Evaluation scheme for HPV and ß-globin signals No. HPV signal (MFI) ß-globin signal (MFI) Interpretation 1. Cut-off Cut-off HPV-positive sample, sufficient DNA content < Cut-off HPV-positive, ß-globin signal potentially suppressed 3.* Cut-off but < 200 < Cut-off HPV-positive, DNA content too low or inefficient PCR 4. < Cut-off Cut-off HPV-negative but sufficient DNA content 5.* < Cut-off < Cut-off DNA content too low or inefficient PCR Explanations HPV and ß-globin signals indicate the presence of HPV and an appropriate amount of intact DNA in the sample. Large amounts of HPV in the sample may cause competition of the ß-globin amplification during PCR resulting in no ß-globin signal and a simultaneous high ( 200 MFI) HPV signal. A low (< 200 MFI) HPV signal accompanied by no ß-globin signal indicates insufficient cellular DNA or inefficient PCR. ß-globin signals only, indicates the absence of HPV in the sample. PCR failed or no adequate genomic DNA was present in the sample. * Repetition of the test is recommended according to section XI. Troubleshooting. XI. Troubleshooting Case 1: Low or no signals on HPV type beads and Hybridization Control beads Possible reasons: 1. Hybridization temperature was above 41.5 C. Check the temperature of your thermomixer during hybridization. Make sure that the hybridization is performed under agitation as otherwise beads will settle down resulting in reduced sensitivity. 2. Amount of Hybridization Control added was to low. Repeat the experiment with proper amount of fresh diluted Hybridization Control. 3. Bead Mix was not properly mixed. Repeat the experiment with proper mixing of Bead Mix. 4. An inappropriate amount of Conjugate was added Repeat the experiment with the proper amount of Conjugate. Case 2: Low signals on HPV type and ß-globin beads (< 50 MFI, after background subtraction) but high signals (> 400 MFI, after background subtraction) on Hybridization Control bead Possible reasons: 1. Denaturation was inefficient. Check the temperature of the incubation oven during denaturation with independed instrument. Repeat 6 the experiment with proper denaturation temperature (95 C). 2. The amount of specific HPV PCR products is too low due to inefficient PCR amplification. Check the presence of the amplified product on a 2% agarose gel. Follow instructions described in section IX. Detailed Test Instructions, PCR Protocol. Factors influencing the efficiency of the PCR amplification: Amount of genomic DNA added to the amplification reaction was too high. A smear of genomic DNA is visible in the upper part of the gel. Dilute the DNA preparation and repeat the amplification by adding 10 times less genomic DNA. Amount of genomic DNA added to the amplification reaction was too low. No PCR product band is visible in the gel. Repeat the amplification using the proper amount DNA. The quality of the DNA preparation does not allow an efficient amplification. Repeat the sample preparation procedure or try a different sample preparation method. PCR amplification was performed with an incorrect thermal profile. Weak or no PCR product bands are visible in the gel. Calibrate the thermal cycler re-run the PCR with the correct thermal profile.

7 Case 3: Signals above Cut-off value on unreasonably many types of beads (false-positive suspects) Possible reasons: 1. Hybridization temperature was below 40,5 C. Repeat the experiment with hybridization at 41 C ± 0.5 C 2. Depending on the amount of amplified product high concentrations of HPV 51 PCR products may show a weak cross-reaction with HPV 82 probe. Additionally, weak cross-reactivity may be observed with high amounts of HPV 66 and 56 PCR products and HPV 16 probe. Large amounts of HPV 33 PCR products may also cross-react with the HPV 59 probe. Weak crossreactivity may be observed with high amounts of HPV 44 products and the HPV 18 probe. 3. A reduced number of wash steps was performed. Repeat the experiment with proper amount of wash steps. 4. A contamination of the sample specimens has occurred if the same specific signal pattern is visible in all samples but not in water PCR. Repeat the experiment beginning with sample preparation using fresh extraction reagents/kits. 5. A contamination of the PCR reagents has occurred, if the same specific pattern is visible in water PCR. Repeat the experiment beginning with PCR amplification using freshly prepared solutions. 6. Conjugate was not properly diluted and/or the excess of conjugate was not washed away properly. Immediately after the read-out repeat the wash step three times and re-read the plate. XII. Luminex Template Settings Description: Multiplex HPV Genotyping Version: 1.0 Template Type: Data Collection Only Sample Vol (µl): 50 Sample Timeout (sec): 60 Doublet Discriminator Gate: Low Limit: 7000 High Limit: Table 4: Assignment of HPV types to bead populations Name (HPV type) Units Description Bead ID Min Beads 6 MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI MFI (ME180) MFI MFI MFI MFI ß-globin MFI Hybridization Control Template Command: Command Warm-up Prime Wash from reservoir Acquire MFI Sample Type Wash solution Test specimen 1 MFI, Median Fluorescence Intensity 7

8 XIII. Performance Characterization A. Detection limit The detection limit was evaluated on the basis of the WHO HPV genotyping proficiency panel (2013) which covers 16 of the 24 genotypes addressed by the Optiplex HPV Genotyping Kit. The panel contains cloned HPV genomes (human placenta DNA matrix) in different single- and multiple-type combinations with 10 to 1000 international units (IU), resp. genome equivalents (GE) of the individual genotypes. In all cases, the lowest genotype content of the panel (10 resp. 100 IU/GE) could be determined exactly for all single- and multiple-type combinations except for HPV 31 (Table 5). Table 5: Detection limit in dependence of the addressed HPV genotype HPV type A Detection limit (IU or GE) B for single-type infections Detection limit (IU or GE) B for multiple-type (n 4) infections HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV 68 (ME180) HPV HPV HPV A HPV genotypes 26, 42, 43, 44, 53, 70, 73, 82 are covered by the Optiplex HPV genotyping Kit but not by the WHO HPV genotyping proficiency panel. 8 B Lowest content of the panel: 10 IU for single-type infections HPV 16 and HPV 18; 100 IU/GE for all other genotypes and all multipletype infections (10 µl input). B. Analytical Specificity for HPV genotypes A high HPV genotype specificity of each bead-bound HPV-probe in the bead mix could be determined for all 24 addressed genotypes (HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82) of the Optiplex HPV Genotyping Test. After a saturated PCR, 4 µl (~ ng DNA) of PCR products, derived from bacterial colonies transformed with cloned HPV plasmid templates, were hybridized to the bead mix according to the manual guidelines and showed repeatable an absolute correct identification of the specific HPV genotype. None of the HPV probes cross-reacted with either ß-globin or the hybridization control probe. In rare cases very weak cross reactivity could be found with high amounts of PCR products for HPV 51 vs. HPV 82 probe, HPV 56/66 vs. HPV 16 probe, HPV 44 vs HPV 18 probe and HPV 33 vs. HPV 59 probe. In order to avoid false positive results due to cross-reactivity, elevated cut-offs are used for HPV 16, 18, 59 and 82. C. Analytical Specificity for non HPV microorganisms The cross-reactivity with other microorganisms (bacteria, virus, protozoan or yeast) which could be present in cervical specimen is very unlikely because of the high sequence specificity of the primer and probes. To assess the specificity of the Optiplex HPV Genotyping Kit for exclusion of non-hpv microorganisms, DNA extracted from PreservCyt material that has been tested positive for Chlamydia trachomatis, HSV2, Mycoplasma (M.) genitalium, M. hominis, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma (U.) urealyticum, U. parvum, Candida (C.) albicans, C. glabrata, Atopobium vaginae, Gardnerella vaginalis, Lactobacillus (L.) iners and L. crispaticus were tested with an equally constructed (RUO) multiplex HPV Genotyping assay at the DKFZ Heidelberg. Concentrations of microorganisms were unknown. The results indicated that the HPV Genotyping Kit did not cross-react with a variety of bacteria, protozoa, viruses and yeast that could be present in cervical specimens. D. Reproducibility The reproducibility was determined with an equally constructed (RUO) multiplex HPV Genotyping assay at the DKFZ Heidelberg (2,3). 21 genotype specific PCR products were combined in 16 mixtures to obtain double positives. The following hybridization to 10 distinct probe-coupled bead sets specific for the addressed genotypes was repeated twice, once on the same plate and once on a second plate. The intra-assay and inter-assay reproducibility of all 160 data points was very high (Table 6), with neither individual nor systematic variation. The inter-day assay reproducibility was determined by the genotyping of PCR products derived from 40 clinical specimens. The two test series were performed

9 within 2 weeks using different mixes of 22 type-specific probe-coupled bead sets from the same coupling batch. The concordance of all typing results (n = 880) is presented in Table 6. Table 6: Estimation of the intra- / inter-assay reproducibility (n = 160 data points) and inter-day assay reproducibility (n = 880 typing results) Intra-assay reproducibility individual variation: R 2 = systematic variation: slope of the regression line = median coefficient of variation (CV) for specific signals: 4.5% (range 0.4 to 13.2%) Inter-assay reproducibility individual variation: R 2 = systematic variation: slope of the regression line = median coefficient of variation (CV) for specific signals: 5.2% (range 0.4 to 14.7%) Inter-day assay reproducibility concordantly positive (5.9%, 52/880) concordantly negative (93.6%, 824/880) discordant (0.5%, 4/880) kappa value: 0.96 linear correlation coefficient (positive cases): R 2 = slope of the regression line (positive cases): median coefficient of variation (CV) for specific signals: 13.8% (range 0.4 to 44.8%) E. Diagnostic performance The diagnostic sensitivity and specificity of the Optiplex HPV Genotyping Kit was verified with the identically constructed (RUO) HPV Genotyping assay of the DKFZ in Heidelberg (4). According to the presence of high-risk (HR) or probably high-risk (phr) HPV DNA, the diagnostic performance was assessed in n = 1296 cervical specimens (999 consecutive collected BD-SurePath liquid-based cervical cytology (LBC) samples of a screened population and additional 297 samples with abnormal cervical cell outcomes ASC-US, LSIL, HSIL) according to the VAL- GENT protocol (5). The presence of CIN2+ and CIN3+, identified through the usual diagnostic work-up of screen positive women, was considered as thresholds of clinical disease outcome. The number of women from whom the continuous samples were taken with index NIL/M cytology and again NIL/M at the next screening was taken as the denominator for the computation of clinical specificity. The LBC samples of histological verifiable CIN2+ and CIN3+ in a 36 month follow up examination were analyzed for the determination of the diagnostic sensitivity with regard to the CIN2+ cases and the CIN3+ cases. The result was equal for the 18 (p)hr-hpv spectrum covered by the multiplex HPV Genotyping Test and the calculation of a reduced 13 HR-HPV spectrum theoretically covered by the Hybrid Capture 2 (HC2) screen test (Table 7). Table 7: Calculation of the diagnostic Sensitivity and Specificity HPV spectrum A Sensitivity [%] B for CIN2+ Sensitivity [%] B for CIN3+ Specificity [%] C 18 (p)hr types 100 (76/76) 100 (49/49) 78.3 (624/797) 13 HR types 100 (76/76) 100 (49/49) 80.3 (640/797) A Calculations according to the 18 (p)hr-hpv spectrum (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82) covered by the Optiplex HPV Genotyping test or the reduced spectrum of 13 HR-HPV (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) theoretically covered by the Hybrid Capture 2 (HC2) screen test B Sensitivity (%, number of true positives/number of women with disease) C Specificity (%, number of true negatives/number of women without disease) From 797 women with two consecutive negative cervical cytology results (NIL/M) in a 36 months follow-up examination (screened population mentioned above) 78.3 % showed true negative test cases for (p)hr- HPV. The indicated diagnostic specificity according to the 18 (p)hr-hpv spectrum covered by the Optiplex HPV Genotyping Kit and with regard to the 13 HR-HPV spectrum theoretically covered by the Hybrid Capture 2 (HC2) screen test is reported in Table 7. 9

10 F. Comparative evaluation In the context of an epidemiological prevalence study the performance of the Optiplex HPV Genotyping Kit (named as Multimetrix test in this study) was compared in HPV-genotyping with a line-blot assay which has been evaluated in several studies (6,7). The presence of 18 different high-risk (HR) or probably high risk (phr) HPV genotypes common for both assays was assessed by analyzing extracted DNA (QIAamp Media Kit, Qiagen Ltd.) of cervical specimens (liquid-based cytology samples, LBC) from a screened population (n = 1445) of young women (8). The detection of the 18 (p)hr HPV covered by both assays and the theoretical calculations to the reduced spectrum of 13 HR-genotypes according to the Hybrid Capture 2 (HC2) screen test demonstrate similar good agreements (Table 8). Table 8: Agreement between the Optiplex HPV Genotyping Kit and the line-blot assay on the basis of 1445 screening samples HPV spectrum A % Agreement κ Agreement Positive Negative Overall κ 95% CI 18 (p)hr types ( ) 13 HR types ( ) A Calculations according to the 18 (p)hr-hpv spectrum (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82) common for the Optiplex HPV Genotyping Kit and the line-blot assay or the reduced spectrum of 13 HR-HPV (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) theoretically covered by the Hybrid Capture 2 (HC2) screen test. The assay agreement of all samples classified to the 18 HPV genotypes common for both assays was also calculated and shows a good conformity in most cases (Table 9). Kappa values (κ) lower 0.5 found in few cases (HPV 35, 53, 68, 82) were associated with the low positivity of these genotypes in the screened population (8) but also with the different detection limits of the PCR primer systems used in the two assays (3). Table 9: Individual agreement of the Optiplex HPV Genotyping Kit and the line-blot assay according to the 18 HPV genotypes common for both assays HPV Type % Agreement A κ Agreement A Positive Negative Overall κ 95%CI ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) A Combined data (n = 2142) for LBCs and other sample types (urine, self-taken swab) according to this study. 10

11 XIV. References 1. Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ; International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med Feb 6;348(6): Schmitt M, Bravo IG, Snijders PJ, Gissmann L, Pawlita M, Waterboer T. Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol Feb;44(2): Schmitt M, Dondog B, Waterboer T, Pawlita M. Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers. J Clin Microbiol Mar;46(3): Schmitt M, Depuydt C, Benoy I, Bogers J, Antoine J, Pawlita M, Arbyn M; VALGENT study group. Viral load of high-risk human papillomaviruses as reliable clinical predictor for the presence of cervical lesions. Cancer Epidemiol Biomarkers Prev Mar;22(3): Arbyn M, Kaufmann A, Gissmann L, Schneider A. Highlights of the 27th International Papillomavirus Conference and Clinical Workshop (Berlin, September, 2011) part II. Future Virol 2012;7: Seme K, Lepej SZ, Lunar MM, Iscić-Bes J, Planinić A, Kocjan BJ, Vince A, Poljak M. Digene HPV Genotyping RH Test RUO: comparative evaluation with INNO-LiPA HPV Genotyping Extra Test for detection of 18 high-risk and probable high-risk human papillomavirus genotypes. J Clin Virol Oct;46(2): Geraets DT, Heideman DA, de Koning MN, Snijders PJ, Meijer CJ, van Doorn LJ, Quint WG. High genotyping concordance between the digene HPV Genotyping RH Test and the Reverse Line Blot genotyping assay on GP5+/6+-PCR products. J Clin Virol Nov;46 Suppl 3:S Cuschieri K, Kavanagh K, Sinka K, Robertson C, Cubie H, Moore C, Donaghy M. Effect of HPV assay choice on perceived prevalence in a population-based sample. Diagn Mol Pathol Jun;22(2): Abbreviations ASC-US CI atypical squamous cells of undetermined significance confidence interval CIN2+ cervical intraepithelial neoplasia grade 2+ CIN3+ cervical intraepithelial neoplasia grade 3+ DKFZ HPV HSIL LBC LSIL NIL/M (p)hr RUO German Cancer Research Center human papillomavirus high-grade squamous intraepithelial lesions liquid-based cytology (samples) low-grade squamous intraepithelial lesions no intraepithelial lesion or malignancy (probably) high-risk research use only VALGENT validation of HPV genotyping tests DiaMex GmbH, Siemensstraße 38, Heidelberg, Germany Tel. +49(0) , Fax +49(0) Valid as of: May V 1.1 Index of Symbols Consult instructions for use Tests per kit Lot Number Catalogue Number Temperature limitation Upper limit of temperature xn xi For in vitro diagnostic use only Use by Manufacturer European Conformity Harmful / Irritant 11

12 Quick Guide Hybridization Assay Add PCR samples and controls to the Bead Mix in a Hybridization plate Denaturation at 95 C for 10 min. Cool on ice for 1 min. Hybridization at 41 C for 30 min. (500 rpm) Transfer the beads to a Filtration plate Remove the liquid by vacuum filtration Wash once with 100 µl Assay Buffer Add 70 µl Staining Solution to the beads Incubate at room temperature for 30 min. (250 rpm) Remove the liquid by vacuum filtration Wash three times with 100 µl Assay Buffer Resuspend the beads in 100 µl Assay Buffer and transfer it to a Lock-well plate Luminex Lx100/200 Read-Out with the appropriate template settings Analyze the results DiaMex GmbH Siemensstraße Heidelberg, GERMANY Tel. +49(0) Fax +49(0)