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1. gt Mlle COBAS AmpliScreen HCV Test version 2 0 FOR IN VITRO DIAGNOSTIC USE COBAS AmpliScreen HCV Test version 2 0 96 Tests P N 03302563 018 COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit 96 Tests P N 03302555 018 COBAS AMPLICOR Wash Buffer 500 Tests P N 20759899 123 ART 07 5989 9 US 83314 INTENDED USE The COBAS AmpliScreen HCV Test version 2 0 v2 0 is a qualitative in vitro test for the direct detection of Hepatitis C Virus HCV RNA in human plasma The COBAS AmpliScreen HGV Test v2 0 is intended to be used for the detection of HCV RNA in conjunction with licensed tests for detecting anti bodies to HCV This product is intended for use as a donor screening test to detect HCV in plasma specimens from individual human donors including donors of whole blood and blood components source plasma and other living donors It is also intended for use to screen organ donors when speci mens are obtained while the donor s heart is still beating and to detect HCV RNA in blood specimens from cadaveric nort heart beating organ and tissue donors This test is not intended for use on sarnples of cord blood Plasma from all donors may be screened as individual specimens For donations of whole blood and blood components plasma may be tested in pools comprised of equal aliquots of not more than 24 individual donations For donations of hematopoietic stem progenitor cells HPCs sourced from bone marrow peripheral
2. Perform manufacturer recommended maintenance and calibration on all instruments including pipettors to ensure proper functioning C Reagents 1 All reagents except HCV MMX v2 0 and HCV Mn v2 0 must be at room temperature before use Visually examine reagents for sufficient volume before beginning the test procedure See section Reagent Preparation for specific reagent storage conditions 2 Add all reagents using a pipettor capable of delivering specified volume with 3 accuracy and a precision of lt 5 CV Check pipettor functionality and calibrate as recommended by pipettor manufacturer 3 Prepare Working Master Mix in a template free area e g in a dead air box Reagent preparation area must be clean and disin fected in accordance with methods outlined in Precautions Item A Failure to do so may result in reagent contamination 4 Prepare 70 ethanol fresh each day 5 Check expiration date of opened or Working Reagents before loading on the COBAS AMPLICOR Analyzer 6 Check to ensure that all reagents used are of the same master lot of kit reagents D Workflow 1 To minimize the possibility of laboratory areas becoming contaminated with amplicon the laboratory area should be separated into several distinct areas organized around Pre Amplification and Post Amplification Personnel should use proper anti contamination safeguards when moving between areas 2 The Pre Amplification Area should have a template fre
3. Genotypes 6a HCV RNA transcripts were included in the testing and all yielded positive results Seroconversion Panels 3 3 9 9 1 1 Nine anti HCV seroconversion panels were tested using both the Multiprep and the Standard Specimen Processing Procedures Each specimen in each panel was tested by the Ortho HCV version 3 0 ELISA Test system and all samples with reactive EIA results were also tested by Chiron RIBA HCV 3 0 SIA The HCV RNA test results were then compared to the EIA test results for each specimen to determine if HCV RNA testing detected the presence of HCV infection prior to seroconversion The COBAS AmpliScreen HCV Test v2 0 detected HCV infection an average of 32 days before seroconversion for the nine seroconversion panels The data are summarized in Table 9 Table 9 a HCV Seroconversion Study Day Positive Ortho 3 0 EIA and Chiron RIBA 3 0 oe Mean Days Earlier Detection specimen was RNA positive again 051207 13001 03EN 13 anh Spa nN e5 Difference Day Positive COBASO AmpliScreen COBAS AmpliScreen HCV Test v2 0 vs EIA oh SS 4 74 Specimen was RNA positive on Day 0 but negative on Days 22 and 24 Day 74 Doc Rev 2 1 Analytical Specificity Potentially Cross Reactive and Interfering Microorganisms The analytical specificity of the COBAS AmpliScreen HCV Test v2 0 was evaluated by testing a panel of microorganisms and other di
4. Irritating to eyes S 53 45 Avoid exposure obtain special instructions before use In case of accident or if you feel unwell seek medical advice immediately show the label where possible COBAS AMPLICOR Wash Buffer 500 Tests WB 2 x 250 Tests 10X Wash Concentrate lt 2 Phosphate buffer lt 9 Sodium chloride EDTA lt 2 Detergent 0 5 ProClin 300 preservative STORAGE INSTRUCTIONS A Room Temperature is defined as 15 30 C B Do not freeze reagents C Store the following reagents at 2 8 C Unopened these reagents are stable until the expiration date indicated MP LYS MP IC MP C MP C MP DIL and NHP HCV MMX v2 0 and HCV Mn v2 0 CH PS1 v2 0 CH4 v2 0 CI PS1 and Cl4 CN4 SB3 and SB D Store DN4 at 2 25 C Store WB at 2 30 C DN4 and WB are stable until the expiration dates indicated E Do not expose SB3 SB or Working Substrate to metals oxidizing agents or direct sunlight F The following reagents are one time use Discard any unused portion MP IC MP C MP C MP DIL and NHP HCV Mn v2 0 and SB 05120713001 03EN Doc Rev 2 1 PRECAUTIONS FOR IN VITRO DIAGNOSTIC USE A z pmm f R S Za rF A Specimens may be infectious Use Universal Precautions when performing the assay 12 13 Only personne proficient in the use of the COBAS AmpliScreen System and trained in handling infectious materials should perform this procedure Thoroughly clean and disinfect al
5. Single Donation Testing Performance A total of 2 515 blood donor specimens were tested individually in the COBAS AmpliScreen HCV Test v2 0 clinical trial Of the 2 515 specimens five were classified as HCV seropositive and were removed from the calculation of specificity Of the 2 510 specimens tested 2 508 were HCV RNA negative and two were HCV RNA positive No follow up was conducted on these two donors and they were presumed to be false positive The specificity of the COBAS AmpliScreen HCV Test v2 0 in this study was 99 92 2 508 2 510 with a 95 confidence interval of 99 71 to 99 99 PERFORMANCE CHARACTERISTICS OF SOURCE PLASMA Clinical Performance A total of 104 448 donations from 35 905 donors were tested in the 96 member minipool format in 1 088 pools Seven donations from 3 donors were positive for HCV RNA and negative by antibody to HCV EIA and RIBA Two donors each donated a HCV RNA positive amp anti HCV positive sample that was tested in one 96 member minipool The data are presented in Table 13 Table 13 Pool Reactivity in Source Plasma Donors Initially Reactive pools with negative resolution COBAS AmpliScreen Testing false positive 1Two HCV EIA positive donations in one 96 member minipool Of the 3 eligible donors one donor had been previously qualified but had been absent from the collection center for more than 6 months as was reclassified to Applicant status upon return The other 2 donors indicated the
6. and Secondary Pools Resu Individual Donor Samples If an individual donor specimen is positive the positive donor specimen is reported as HCV RNA Positive If an individual donor specimen is negative the negative donor specimen is reported as HCV RNA Negative esu P Source Plasma cimens Pools of u 96 Individual Donation The testing algorithm for testing of pooled samples for the COBAS AmpliScreen HCV Test v2 0 requires a single level of testing for Primary Pools that are negative for HCV RNA and three levels of testing Primary Pool Minipool and confirmatory testing for Primary Pools that are positive for HCV RNA Negative Primary Pools When the Primary Pool is negative report the results for all associated individual donor specimens in that Primary Pool as HCV RNA Negative 05120713001 03EN Doc Rev 2 1 10 Positive Primary Pools Minipoo l Testing Positive Primary pools are traced to the positive individual using an overlapping pool testing matrix Minipools are prepared from the eight individual donations for columns 1 12 and from the 12 individual donations for rows 1 8 The 20 minipools are tested using the Standard Specimen Processing Procedure The positive unit is identified by the intersection of the positive column and positive row Confirmatory testing is conducted on the impli cated unit using Standard Specimen Processing Procedure Results of Individual Cadaveric Specimens If an individual cadaveric specimen is
7. assay performance The use of excessively hemolyzed cadaveric specimens should be avoided REAGENT PREPARATION A MP IC MP C MP C MP DIL and NHP 1 Warm MP IC MP C MP C MP DIL and NHP to room temperature before use by using a 37 C incubator or on the laboratory bench top Working Lysis Reagent 1 Warm MP LYS to 25 37 C to dissolve precipitate maximum 30 minutes Mix thoroughly until the crystals are dissolved Prior to use examine each bottle of MP LYS against a white background for appearance of a yellow color or signs of leakage If there is any yellow color or signs of leakage do not use that bottle for testing Contact your local Roche office for replacement 2 Vortex MP IC briefly before use Tap vial to collect the solution in the base Pipette 100 uL MP IC into 1 bottle MP LYS Cap the MP LYS bottle and vortex briefly The pink color confirms that the MP IC has been added to the MP LYS Discard the remaining MP IC 3 Store Working Lysis Reagent at room temperature Use within 4 hours of preparation Working Amplification Master Mix 1 Prepare Working Master Mix in a template free area e g in a dead air box Reagent preparation area must be clean and disin fected in accordance with methods outlined in Precautions Item A Failure to do so may result in reagent contamination 2 Pipette 100 uL HCV Mn v2 0 into 1 bottle HCV MMX v2 0 Recap HCV MMX v2 0 bottle and mix well by inverting 10 15
8. blood or cord blood and donor lymphocytes for infusion DL plasma may be tested in pools comprised of equal aliquots of not more than 24 individual donor specimens For donations of source plasma plasma may be tested in pools comprised of equal aliquots of not more than 96 individual donations The COBAS AmpliScreen HCV Test v2 0 can be considered a supplemental test that confirms HCV infection for specimens that are repeatedly reac tive on a licensed donor screening test for antibodies to HCV and reactive on the COBAS AmpliScreen HCV Test v2 0 This test is not intended for use as an aid in diagnosis SUMMARY AND EXPLANATION OF THE TEST Hepatitis C Virus is considered to be the principal etiologic agent responsible for 90 95 of the cases of post transfusion non A and non B hepatitis 1 2 HCV is a single stranded positive sense RNA virus with a genome of approximately 10 000 nucleotides coding for 3 000 amino acids As a blood borne virus HCV can be transmitted by blood and blood products The global prevalence of HCV infection as determined by immunoserology ranges from 0 6 in Canada to 1 5 in Japan 2 Serological screening assays have greatly reduced but not completely eliminated the risk of transmitting viral infections by transfusion of blood prod ucts 5 Recent studies indicate that nucleic acid based amplification tests for HCV RNA will allow detection of HCV infection earlier than the current antibody based tests Nucleic ac
9. e i 100 Genotype Detectability Twenty individual plasma specimens Genotypes 1 and 4 sixteen plasma specimens of Genotype 2 nineteen plasma specimens of representing Genotype 3 four plasma specimens of Genotype 5 and eight plasma specimens of Genotype 6 have been tested As an additional measure of the ability of the COBAS AmpliScreen HCV Test v2 0 to identify HCV six Genotype 6a transcripts and one Genotype 5a HCV RNA tran genotypes script were diluted to 5 U PCR and directly tested by the COBAS AmpliScreen HCV Test v2 0 With the exception of one sample Genotype 2a 2c which was below the limit of quantitation by a quantitative assay each specimen was diluted to approximately 200 IU mL of HCV RNA in pooled negative human plasma Diluted samples were processed using both the Multiprep and Standard Sample Processing Procedures The COBAS AmpliScreen HCV Test v2 0 detected all Genotypes at 200 IU mL except the one sample that was not quantifiable This sample Genotype 2a 2c was detected using the Multiprep Specimen Processing Procedure but was negative when tested using the Standard Specimen Processing Procedure This result is consistent with HCV RNA levels below the detection limit of the assay Data are provided in Table 8 Table 8 HCV Genotype Samples Tested a One sample contained HCV RNA at a level below the Limit of Quantitation of a quantitative assay Sample was tested undiluted b One Genotype 5a and six
10. e Disposable Sterile Polystyrene pipettes 5 mL 10 mL and 25 mL e Sterile RNase free fine tip transfer pipettes e Pipettors capacity 20 uL to 1000 uL capable of providing 3 accuracy and precision lt 5 with aerosol barrier or positive dis placement RNase free tips e Tube racks Sarstedt P N 93 1428 or equivalent e 1 5 mL sterile non siliconized conical polypropylene screw cap tubes Sarstedt 72 692 105 or equivalent Vortex mixer e Hamilton Slotted Deepwell Archive Plate 2 2 mL and Sealing Capmat Hamilton Slotted Intermediate Plate REAGENTS COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit 96 Tests MP C 8 x 0 1 mL Multiprep Negative Control lt 0 005 Poly rA RNA synthetic EDTA 0 05 Sodium azide MP C 8 x 0 1 mL Multiprep Positive Control Tris HCl buffer lt 0 001 Non infectious linearized plasmid DNA microbial containing HBV sequences lt 0 001 Non infectious in vitro transcribed RNA microbial containing HCV sequences lt 0 001 Non infectious in vitro transcribed RNA microbial containing HIV 1 sequences lt 0 005 Poly rA RNA synthetic EDTA 0 05 Sodium azide MP LYS 8 x 9 0 mL Multiprep Lysis Reagent Tris HCI buffer 60 Guanidine thiocyanate 3 Dithiothreitol lt 1 Glycogen Xn 60 w w Guanidine thiocyanate Harmful MP DIL 8 x 4 8 mL Multiprep Specimen Diluent Tris HCI buffer lt 0 005 Poly rA RNA synthetic ED
11. perform repeat testing in single on the remaining replicate tube s The test result for the pool or individual donor specimen is based only on the repeat valid test result If the last available replicate of a pooled specimen gives an invalid result each indi vidual specimen in that pool should be tested If an individual donor specimen gives an invalid result the test result for that individual donor spec imen should be considered invalid for HCV RNA For cadaveric specimens that are invalid additional cadaveric specimen is diluted 1 5 with MP DIL reagent and retested in duplicate using the Multiprep Specimen Processing Procedure The test result for the cadaveric specimen is based on the repeat valid test results Results of Pooled Donor Specimens Pools of up to 24 Individual Donations Testing of pooled samples for the COBAS AmpliScreen HCV Test v2 0 requires a single level of testing for Primary Pools that are negative for HCV RNA and three levels of testing Primary Pool Secondary Pool and tertiary resolution for Primary Pools that are positive for HCV RNA Negative Primary Pools When the Primary Pool is negative report the results for all associated individual donor specimens in that Primary Pool as HCV RNA Negative Positive Primary Pools Secondary Pool Testing When the Primary Pool is positive prepare four Secondary Pools containing the associated donor specimens The Secondary Pools must be processed using the Multiprep Specime
12. prevent amplification of pre existing amplicon AmpErase uracil N glyco ee E the COBAS fomi caah HCV Test v2 0 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing deoxythymidine Deoxyuridine is not present in naturally occurring DNA but is always pre sent in amplicon because of the use of deoxyuridine triphosphate in place of deoxythymidine triphosphate as one of the dNTPs in the Master Mix reagent therefore only amplicon contain deoxyuridine Deoxyuridine renders contaminating amplicon susceptible to destruction by the AmpErase enzyme before amplification of the target DNA The AmpErase enzyme which is included in the Master Mix reagent catalyzes the cleavage of DNA thereby rendering the DNA non amplifiable The AmpErase enzyme is inactive at temperatures above 55 C i e throughout the thermal cycling steps and therefore does not destroy target amplicon Following amplification any residual enzyme is denatured by the addition of the Denaturation Solution thereby preventing the degradation of any target amplicon Hybridization Reaction Following PCR amplification the COBAS AMPLICOR Analyzer automatically adds Denaturation Solution to the A tubes to chemically denature the HCV amplicon and the HCV Internal Control amplicon to form single stranded DNA Aliquots of denatured amplicon are then transferred to two detec tion cups D cups A suspension of magnetic parti
13. the extracted RNA Note that some insoluble material may remain B20 At this point amplification of the processed specimens and controls must be started within 2 hours If not the processed specimens and con trols can be stored at 70 C or colder for up to one month Thawing should be completed within one hour at room temperature B21 Proceed to step B39 Loading the A ring Standard Specimen Processing Procedure Individual Specimens Non Cadaveric and Source Plasma Minipools B22 Pipette 200 uL of each specimen into an appropriately labeled screw cap tube using the COBAS AmpliScreen Pooling System a hand held pipettor or other user validated method Cap the tubes B23 Vortex NHP briefly B24 For each Negative and Positive Control pipette 200 uL NHP into appropriately labeled screw cap tubes Cap the tubes B25 Use a permanent marker to make an orientation mark on each tube B26 Prepare a Working Lysis Reagent bottle for every 12 specimens and controls to be processed B27 Pipette 600 uL Working Lysis Reagent into each tube Cap and vortex tubes briefly B28 Prepare Controls as follows a Negative Control Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 uL MP C into the tube labeled MP C con taining Working Lysis Reagent and NHP Cap the tube and vortex briefly b Positiv ntrol Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 uL MP C into the tube
14. times The pink color confirms that the HCV Mn v2 0 has been added to the HCV MMX v2 0 Discard the remaining HCV Mn v2 0 Do not vortex the Working Master Mix These reagents clo not need to be at room temperature before use 3 Store at 2 8 C and use within 4 hours of preparation Working Probe Suspension Detection Reagents 1 Prepare Working HCV Probe Suspension Mix CH PS1 v2 0 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL CH PS1 v2 0 into one CH4 v2 0 cassette 2 Prepare Working IC Probe Suspension Mix Cl PS1 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL Cl PS1 into one Cl4 cassette 3 Both Working Probe Suspension Detection Reagents are stable for 30 days at 2 8 C Working Reagents can be used for a maximum of ten instrument cycles 12 hours per cycle Mixing occurs automatically on the COBAS AMPLICOR Analyzer 4 Store Working Probe Suspension Detection Reagents at 2 8 C between instrument cycles Remove from refrigerator 30 minutes before use on the COBAS AMPLICOR Analyzer DN4 Denaturation Reagent and CN4 Conjugate Reagent 1 Once opened DN4 and CN4 are stable for 30 days at 2 8 C or until the expiration date whichever comes first Both DN4 and CN4 can be used for a maximum of ten instrument cycles 12 hours per cycle 2 oe DN4 and CN4 at 2 8 C between instrument cycles Remove from refrigerator 30 minutes before use on the COBAS AMPLICOR naly
15. 2011 Roche Molecular Systems Inc All rights reserved 3 2011 l 05120713001 03 Doc Rev 2 1 05120713001 03EN Doc Rev 2 1
16. All valid reproducibility data were evaluated by calculating the percentage of correct results for each pane member The data were analyzed by site lot testing day run and operator for each Specimen Processing Procedure Multiprep and Standard The reproducibility study for the COBAS AmpliScreen HCV Test version 2 0 demonstrated consistency by lot and site for both the Multiprep and Standard Specimen Processing Procedures as seen in Table 1 and 2 below Table 7 Reproducibility Results Multiprep Specimen Processing Procedure Results By Lot Number Positive Number Tested C me oum eum ou aou 72 89 164 177 88 90 a om em em oa eoo E a 93 er ea KAII Results By Site Number ae lt Cu om om om oie atl ele Table 2 Reproducibility Results Standard Specimen Processing Procedure Results By Lot Number Positive Number Tested Negative 25iuim soimi too nim 50 000 iwm m ot em eo em 63 92 99 wo a te om em ems 0 74 92 RENE 3 83 ae 100 Results By Site Saree Positive Number Tested i to 05120713001 03EN Doc Rev 2 1 11 Analytical Sensitivity Dilutional Panels The analytical sensitivity of the COBAS AmpliScreen HCV Test v2 0 was determined by testing 10 HCV seropositive clinical specimens The titer of each specimen was quantitated with a commercially available assay using a secondary st
17. D Invalid result Repeat entire test procedure for invalid specimen POSITIVE VALID Specimen is positive for HCV RNA Invalid Test Runs When invalid Positive or Negative Control results are obtained on an A ring that A ring is invalid Repeat the entire test procedure for the associated specimens including specimen and control preparation amplification and detection in the A ring by processing another aliquot of the original plasma specimens With the exception of instrument failures subsequent to denaturation of amplicon an instrument failure during a test run as indicated by system error messages also constitutes an invalid test run In such instances repeat the test procedure for the associated controls and specimens amplification and detection in the run by processing another aliquot of the processed specimen For instrument failures subsequent to successful denaturation of amplicon it is not necessary to repeat the entire test procedure for the associated specimens in such instances the denatured amplicon may be redetected by the COBAS AMPLICOR Analyzer The denatured amplicon may be left on the COBAS AMPLICOR Analyzer for not more than 24 hours before continuing with the hybridization and detection steps Alternatively the denatured amplicon may be stored at 2 8 C for not more than five days before continuing with the hybridization and detection steps Invalid Specimen Results For plasma specimen s that are invalid
18. LOD of the COBAS AmpliScreen HCV Test v2 0 The results reflect the retesting of two cadaveric specimens that were inhibited on initial testing In repeat testing both Pre Mortem EDTA Plasma Specimen resolved negative The summary of the test results of this study is presented in Table 15 below Post Mortem EDTA Plasma Specimen Total Specimens Tested 6e o 8 E ee vestResuts TP o S Table 15 Summary of Sensitivity Test Results Sensitivity 98 3 95 Confidence Interval Specificity Study Sixty pre mortem and fifty eight post mortem specimens which were negative for HCV RNA were divided into three groups diluted 1 5 in MP DIL processed using the Multiprep Specimen Processing Procedure and tested using 3 lots of the COBAS AmpliScreen HCV Test v2 0 The COBAS AmpliScreen HCV Test v2 0 using samples diluted 1 5 and the Multiprep Specimen Processing Procedure yielded negative results on 100 60 60 of the pre mortem EDTA plasma specimens and 100 58 58 of the post mortem EDTA plasma specimens The summary of results is presented in Table 16 Table 16 Summary of Specificity Test Results Pre Mortem EDTA Post Mortem EDTA Plasma Specimen Plasma Specimen Total Specimens Tested 60 Oeo eee eee ee stress o f wooo ooo Final Specificity a 95 Confidence interval Reproducibility Study Twenty pre mortem EDTA plasma and 20 individual cadaveric specimens were spiked with HCV vira
19. MICROLAB AT Plus 2 Pipettor has been validated for use with the COBAS AmpliScreen HCV Test v2 0 for the automated preparation of plasma pools Adhere to the hardware instructions and safety precautions outlined in the User Manual for the Hamilton MICROLAB AT Plus 2 Pipettor 6 AL A ee ae Ee ee ae and or probe may result in the failure to detect the virus rf Due to inherent differences between technologies it is recommended that prior to switching from one technology to the next users perform method correlation studies in their laboratory to qualify technology differences PERFORMANCE CHARACTERISTICS Reproducibility The reproducibility of the COBAS AmpliScreen HCV Test v2 0 was established by testing two six member EDTA plasma panels with known concentrations of HCV Panel One which was tested using the Multiprep Specimen Processing Procedure contained one HCV negative sample and HCV positive samples with HCV RNA concentrations of 10 25 50 and 50 000 IU mL Panel Two which was tested using the Standard Specimen Processing Procedure contained one HCV negative sample and HCV positive samples with concentrations of 25 50 100 and 50 000 IU mL Testing was performed at three sites with two operators at each site using three COBAS AmpliScreen HCV Test v2 0 kit lots Each operator used a dedi cated COBAS AMPLICOR Analyzer throughout the study Each operator was provided pane sets that had been randomized and labeled in blinded fashion
20. NK software m Wait for the COBAS AMPLICOR Analyzer to indicate that the load check has passed oF NOTE The required quantity of each detection reagent is automatically calculated by the COBAS AMPLICOR Analyzer during the Load Check to determine if sufficient reagents are available for the requested tests n The COBAS AMPLICOR Analyzer automatically performs reverse transcription amplification and detection Results are expressed as absorbance values at 660 nm and as positive or negative o As a Quality Control measure the AMPLILINK A ring Results Report and the Run Log may be printed e g daily weekly or monthly and retained along with the respective A ring worklist A selection of A ring worklist records should be periodically compared with the AMPLILINK A ring Results Report to verify that the A ring ID instrument serial number and specimen IDs are identical Reconcile the Run Log with the selected A ring worklist to account for all A ring IDs associated with the run If there are discrepancies perform follow up investigation QUALITY CONTROL PROCEDURES 1 At least one Multiprep Control and one Multiprep Control must be processed with each A ring a Negative Control The absorbance for the MP C should be less than 0 1 at 660 nm and its associated MP IC should be greater than or equal to 0 2 for the Negative Control to be valid If the absorbance value for the MP C is greater than or equal to 0 1 and or its a
21. S AmpliScreen Multiprep Specimen Preparation and Control Kit MULTIPREP CTL 96 Tests P N 03302555 018 MP C Multiprep Negative Control MP C Multiprep Positive Control MP LYS Multiprep Lysis Reagent MP DIL Multiprep Specimen Diluent MP IC Multiprep Internal Control NHP Negative Plasma Human COBAS AmpliScreen HCV Test version 2 0 96 Tests P N 03302563 018 COBAS AmpliScreen HCV Amplification Reagents version 2 0 HCV MMX v2 0 HCV Master Mix version 2 0 HCV Mn v2 0 HCV Manganese Solution version 2 0 COBAS AmpliScreen HCV Detection Reagents version 2 0 CH PS1 v2 0 HCV Probe Suspension 1 version 2 0 CH4 v2 0 HCV Probe Suspension 2 version 2 0 CI PS1 IC Probe Suspension 1 cl4 IC Probe Suspension 2 DN4 Denaturation Solution CN4 Avidin Horseradish Peroxidase Conjugate SB3 Substrate A SB Substrate B COBAS AMPLICOR Wash Buffer 500 Tests P N 20759899 123 ART 07 5989 9 US 83314 WB 10X Wash Concentrate OTHER MATERIALS REQUIRED BUT SOLD SEPARATELY MAY BE PURCHASED FROM ROCHE e COBAS AMPLICOR Analyzer with software version 0022B Printer and Operator s Manual for the GCOBAS AMPLICOR Analyzer e COBAS AMPLICOR A rings e COBAS AMPLICOR D cups e AMPLILINK Software version 1 4 and Operator s Manual for the AMPLILINK software e Hamilton MICROLAB AT Plus 2 Pipettor with Hamilton SUNPLUS and RUNENDE Software and
22. Store the processed specimens and controls as indicated On the subsequent day begin with Step A Reagent Preparation Working Master Mix thaw processed specimens and controls at room temperature and continue with Step B39 Hybridization and Detection of Stored Denatured Amplicon Hybridization and detection of the denatured amplicon may occur on the same day as amplification or on a subsequent day If hybridization and detection are to be done on a subsequent day the denatured amplicon may be left on board the COBAS AMPLICOR Analyzer for not more than 24 hours before starting the hybridization and detection steps Alternatively the denatured amplicon may be stored at 2 8 C for not more than five days before starting the hybridization and detection steps A Reagent Preparation Working Master Mix Performed in Pre Amplification Reagent Preparation Area e g dead air box A1 Determine the appropriate number of A ring s needed for specimen and control testing A2 Place the A ring s on the A ring holder s 05120713001 03EN Doc Rev 2 1 A3 For each A ring prepare one Working Master Mix A4 Pipette 50 uL Working Master Mix into each A tube Discard unused Working Master Mix Do not close the covers of the A tubes at this time A5 Place the A ring containing Working Master Mix in a sealable bag and seal the plastic bag Record the assay name HCV and the time the Working Master Mix was prepared A6 Store the A ring s conta
23. TA 0 05 Sodium azide MP IC 8 x 0 1 mL Multiprep Internal Control Tris HC buffer lt 0 001 Non infectious plasmid DNA containing HBV primer binding sequences and a unique probe binding region lt 0 001 Non infectious in vitro transcribed RNA microbial containing HCV primer binding sequences and a unique probe binding region lt 0 001 Non infectious in vitro transcribed RNA microbial containing HIV 1 primer binding sequences and a unique probe binding region lt 0 005 Poly rA RNA synthetic EDTA lt 0 1 Amaranth dye 0 05 Sodium azide NHP 16 x 1 6 mL Negative Plasma Human Human plasma non reactive by US FDA licensed tests for antibody to HCV antibody to HIV 1 2 HIV p24 antigen and HBsAg 0 1 ProClin 300 preservative COBAS AmpliScreen HCV Test version 2 0 cv 96 Tests COBAS AmpliScreen HCV Amplification Reagents HCV MMX v2 0 HCV Master Mix version 2 0 Bicine buffer 16 DMSO Glycerol lt 0 01 rTth DNA Polymerase rTth pol microbial Potassium acetate lt 0 001 dATP dCTP dGTP dUTP lt 0 005 KY78 and KY80 primers KY78 is biotinylated lt 0 01 AmpErase uracil N glycosylase enzyme microbial 0 05 Sodium azide HCV Mn v2 0 8 x 0 1 mL HCV Manganese Solution version 2 0 lt 2 Manganese Acetic acid Amaranth dye 0 05 Sodium azide 8 x 0 7 mL 05120713001 03EN Doc Rev 2 1 COBAS AmpliScreen HCV Detection Reagents version 2 0 CH PS1 v2 0 1 x 100 T
24. andard calibrated against the WHO International Standard These specimens were diluted in normal human plasma to 150 50 16 7 and 5 6 HCV RNA IU mL for the Multiprep Specimen Pracessing Procedure and 300 100 33 3 and 11 1 U ml for the Standard Specimen Processing Procedure The COBAS AmpliScreen HCV Test v2 0 detected 16 7 HCV RNA IU mL at a frequency greater than 90 with a lower 95 confidence limit of 86 4 using the Multiprep Specimen Processing Procedure The assay detected 33 3 HCV RNA IU mL at a frequency greater than 84 with a lower 95 confidence limit of 79 7 using the Standard Specimen Processing Procedure The data are presented in Tables 3 and 4 When evaluated using PROBIT analysis the combined data for all samples processed by the Multiprep Specimen Processing Procedure indicate an average 95 Limit of Detection LOD of 21 0 IU mL with lower and upper 95 confidence limits of 17 1 IU mL and 27 8 IU mL respectively The LOD of 21 0 IU mL corresponds to approximately 57 copies mL When evaluated using PROBIT analysis the combined data for all samples processed by the Standard Specimen Processing Procedure indicate an average 95 LOD of 54 1 IU mL with lower and upper 95 confidence limits of 44 1 U mL and 71 7 IU mL respectively The LOD of 54 1 IU mL corresponds to approximately 146 copies mL Table 3 Multiprep Procedure Testing Summary for All Clinical Samples Combined Input Values with 95 One tailed Lower Confidence Limit Mul
25. as possible after collection The use of excessively hemolyzed cadaveric specimens should be avoided 051207 13001 03EN Doc Rev 2 1 PROCEDURAL NOTES A Run Size 1 Each kit contains reagents sufficient for eight 12 specimen runs which may be performed separately or simultaneously At least one preparation of the COBAS AmpliScreen Multiprep Negative Control and one preparation of the COBAS AmpliScreen Multiprep Positive Control must be included in each A ring see Quality Control section 2 The Specimen Preparation and Amplification Reagents are packaged in eight single use bottles The Multiprep Negative and Multiprep Positive Controls are packaged in single use vials For the most efficient use of reagents specimens and controls should be processed in batches that are multiples of 12 3 The use of sterile gauze when uncapping sample tubes may reduce the potential for cross contamination between specimens B Equipment 1 Prepare the COBAS AMPLICOR Analyzer and the Data Station for the AMPLILINK Software for use according to instructions in the Operator s Manual for the AMPLILINK software and the Operator s Manual for the COBAS AMPLICOR Analyzer 2 Prepare the Hamilton MICROLAB AT Plus 2 System and SUNPLUS Data Station for use according to instructions in the Operator s Manuals 3 Pre cool the high speed centrifuge and rotor to 2 8 C See operating instructions for the high speed centrifuge for details 4
26. ce of the HCV External Control does not meet the above criterion the negative results for specimens in the associated run may be invalidated However positive results for specimens in such a run should not be invalidated solely on the basis of the results obtained for an External Control those positive results should remain the test of record The laboratory should follow its established Standard Operating Procedure for the appropriate action INTERPRETATION OF RESULTS he Flags and comments may be generated by the COBAS AMPLICOR Analyzer during a run The Operator must check the run printout s for flags and comments to verify that the run is valid Refer to the Operator s Manual for the AMPLILINK software and the Operator s Manual for the COBAS AMPLICOR Analyzer for interpretation of flags and comments 2 Specimen Results Two absorbance values are obtained for each specimen one for the HCV target and one for the internal control MP IC For a sample with an absorbance less than 0 2 the MP IC absorbance for that specimen must be greater than or equal to 0 2 at 660 nm for a valid negative specimen test result If the absorbance for the HCV target is greater than or equal to 0 2 the MP IC result is disregarded and the test result is valid and positive 3 For a valid run results are interpreted as follows HCV Result IC Result Aes Comment Aas Comont mteretation NEGATIVE VALID Specimen is negative for HCV RNA NEGATIVE INVALI
27. cimens were positive for HCV RNA Four specimens negative for HCV RNA were also negative for HCV antibody by both a licensed screening EIA and confirmatory assay and were excluded from the analysis The COBAS AmpliScreen HCV Test v2 0 detected 57 out of 58 HCV antibody positive specimens Pool Reactivity in Volunteer Blood Donors A random selection of 8 240 pools revealed that 117 Primary Pools were reactive for an initial reactive rate of 1 42 There were 106 117 90 6 positive pools that were concordant with confirmed positive serology status None of these pools were identified as having a window period case A total of 11 pools were found positive but were not confirmed positive by serology or by subsequent testing of individual donations by the COBAS AmpliScreen Test v2 0 Results are summarized in Table 11 Table 17 Pool Reactivity in Volunteer Blood Donors Percentage C dC postive pods aue to windowease 0d initial pools with negative serology and negative 0 13 individual donation AmpliScreen Testing false positive A random selection of approximately 250 000 specimens was selected from geographically divergent sites The results from these specimens were used to determine the specificity and sensitivity of COBAS AmpliScreen HCV Test v2 0 Using the antibody results the HCV status of each spec imen was determined HCV status negative included either 1 anti HCV EIA negative regardless of other results unl
28. cles coated with an oligonucleotide probe specific for HCV amplicon or HCV Internal Control amplicon is added to the individual D cups The biotin labeled HCV target and HCV Internal Control amplicon are hybridized to the target specific oligonucleotide probes bound to the magnetic particles This hybridization of amplicon to the target specific probe increases the overall specificity of the test Detection Reaction Following the hybridization reaction the COBAS AMPLICOR Analyzer washes the magnetic particles in the D cups to remove unbound material and Meade Car Taian peroxidase conjugate The avidin horseradish peroxidase conjugate binds to the hybridized biotin labeled amplicon The COBAS AMPLICOR Analyzer removes unbound conjugate by washing the magnetic particles and then adds a substrate solution containing hydrogen peroxide and 3 3 5 5 tetramethylbenzidine TMB to each D cup In the presence of hydrogen peroxide the particle bound horseradish peroxidase catalyzes the oxidation of TMB to form a colored complex The absorbance is measured by the COBAS AMPLICOR Analyzer at a wave length of 660 nm MATERIALS PROVIDED BY ROCHE The COBAS AmpliSereen Multiprep Specimen Preparation and Control Kit and the COBAS AMPLICOR Wash Buffer kit are provided as stand alone kits to be used in conjunction with the COBAS AmpliScreen HCV Test v2 0 as well as the COBAS AmpliScreen HIV 1 Test v1 5 and the COBAS AmpliScreen HBV Test COBA
29. ctivities or for pipetting or processing amplified DNA or other sources of target DNA Post amplification supplies and equipment must remain in the Post Amplification Area at all times Avoid contact of MP LYS HCV MMX v2 0 HCV Mn v2 0 CH4 v2 0 Cl4 DN4 CN4 SB3 SB and Working Substrate mixed SB3 and SB reagent with the skin eyes or mucous membranes if contact does occur immediately wash with large amounts of water otherwise burns can occur If these reagents are spilled dilute with water before wiping dry Do not allow MP LYS which contains guanidine thio cyanate or CH4 v2 0 and Ci4 which contain sodium thiocyanate to contact sodium hypochlorite bleach solution This mixture can produce a highly toxic gas SB and Working Substrate contain dimethylformamide which has been reported to be toxic in high oral doses and may be harmful to the unborn child Skin contact inhalation of fumes and ingestion should be avoided If skin contact occurs wash thoroughly with soap and water and seek medical advice immediately Refer to Precautions in the package inserts accompanying other COBAS AmpliScreen products the COBAS AmpliScreen Pooling System Guide and the Operator s Manuals for the AMPLILINK software and COBAS AMPLICOR Analyzer Closely follow procedures and guidelines provided to ensure that the specimen and control preparation is performed correctly Any deviation from the given procedures and guidelines may affect optimal
30. e area for preparation of Working Master Mix and an amplicon free area for specimen and control preparation 3 The Post Amplification Area should have a COBAS AMPLICOR Analyzer s and AMPLILINK Data Station s with additional area for preparing Working Amplification and Detection Reagents 4 Pipettors and other supplies should be dedicated to a specific area Samples equipment and reagents should not be returned to the area where a previous step was performed E Temperature Room temperature is defined as 15 to 30 C F Vortexing Proper vortexing during sample preparation is important to ensure homogeneous mixture after additions of reagents G Pipetting 1 Pooled or individual plasma specimens must be at room temperature before pipetting 2 Use a clean pipette tip or disposable transfer pipette with each specimen or control Use aerosol barrier or positive displacement RNase free tips 3 Confirm that all pipettors are correctly set to dispense the specified volumes in accordance with the specimen preparation procedures and guidelines H Specimen Processing Screw cap tubes must be used for specimen and control preparation to prevent splashing and potential cross contamination of spec imens and controls Do not use snap cap tubes 2 Avoid contaminating gloves when manipulating specimens 3 Specimens and controls should be prepared in a laminar flow hood Failure to do so may result in sample contamination Specimen and co
31. es before the horse clone therefore am The hepatitis C virus in current perspective Annals of Internal Medicine 2 1415 044 649 3 Dodd RY 1994 Adverse consequences of blood transfusion quantitative risk estimates In Nance ST ed Blood supply risks percep tions and prospects for the future Bethesda American Association of Blood Banks1 24 4 Schreiber GB Busch MP Kleinman SH Korelitz JJ 1996 The risk of transfusion transmitted viral infections The Retrovirus Epidemiology Donor Study N Engl J Med 334 1685 90 5 Holland PV 1996 Viral infections and the blood supply editorial N Engl J Med 334 1734 35 6 Kleinman SH Busch MP General overview of transfusion transmitted infections In Petz LD Swisher S Kleinman SH Spence R Strauss RG eds 1995 Clinical practice of transfusion medicine 3rd ed New York Churchill Livingstone 809 21 7 Busch MP Stramer SL Kleinman SH 1997 Evolving applications of nucleic acid amplification assays for prevention of virus transmission by blood components and derivatives In Garratty G ed Applications of molecular biology Bethesda American Association of Blood Banks 121 73 presented at a workshop during the 50th Annual Meeting of the AABB October 1997 Denver CO 8 Soriano V Dronda F Gonzalez Lopez A et al 1994 HIV 1 causing AIDS and death in a seronegative individual fletter Vox Sang 67 410 11 9 Centers for Disease Control and Prevention Persistent lack of detectab
32. ess the subject was enrolled in the follow up study and had test results that changed this assessment or 2 anti HCV EIA positive and RIBA negative HCV status positive included either 1 anti HCV EIA repeat reactive and RIBA positive or 2 anti HCV EIA repeat reactive or HCV RNA positive upon follow up HCV status unknown included anti HCV EIA repeat reactive with RIBA indeterminate or unknown There were 247 998 specimens that were determined to be HCV status negative Of these 247 990 were also HCV RNA negative The specificity of the COBAS AmpliScreen HCV Test v2 0 in this study was 247 990 247 998 or 99 997 with 95 confidence limits of 99 99 to 100 00 The negative predictive value obtained by summing all the cases determined to have HCV status negative among the 248 106 COBAS AmpliScreen HCV Test v2 0 negative donations is estimated in this study to be 99 95 with exact 95 confidence limits 99 94 99 96 There were 243 specimens that were determined to be HCV status positive Of these 203 were also HCV RNA positive The positive predictive value obtained by finding the percentage of specimens detected to be HCV status positive among 215 COBAS AmpliScreen HCY Test v2 0 positive dona tions Is estimated to be 94 42 with exact 95 confidence limits 90 45 97 08 All 243 samples in this population were included in the analysis ts prop ig of HCV RNA titers These data are consistent with previous reports that about 20 of HCV serop
33. ests HCV Probe Suspension 1 version 2 0 MES buffer lt 0 4 Suspension of Dynabeads paramagnetic particles coated with HCV specific oligonucleotide capture probe KY150 0 09 Sodium azide CH4 v2 0 HCV Probe Suspension 2 version 2 0 Sodium phosphate buffer 34 7 Sodium thiocyanate 0 2 Solubilizer Xn 3 34 7 w w Sodium thiocyanate Harmful CI PS1 IC Probe Suspension 1 MES buffer lt 0 4 Suspension of Dynabeads paramagnetic particles coated with IC specific oligonucleotide capture probe SK535 0 09 Sodium azide 1 x 100 Tests 1 x 100 Tests Ci4 1 x 100 Tests IC Probe Suspension 2 Sodium phosphate buffer 24 9 Sodium thiocyanate 0 2 Solubilizer DN4 Denaturation Solution 1 6 Sodium hydroxide EDTA Thymol biue Xi Rd 1 6 w w Sodium hydroxide Irritant CN4 2 x 100 Tests Avidin Horseradish Peroxidase Conjugate Tris HCI buffer lt 0 001 Avidin horseradish peroxidase conjugate Bovine serum albumin mammalian Emulsit 25 Dai ichi Kogyo Seiyaku Co Ltd 0 1 Phenol 1 ProClin 150 preservative SB3 10 x 75 Tests Substrate A Citrate solution 0 01 Hydrogen peroxide 0 1 ProClin 150 preservative 1 x 100 Tests SB 10 x 75 Tests Substrate B 10 x 5 mL 0 1 3 3 5 5 Tetramethylbenzidine TMB 40 Dimethylformamide DMF T 40 w w Dimethylformamide DMF Toxic R 61 20 21 36 May cause harm to the unborn child Harmful by inhalation and in contact with skin
34. he last tube After the incubation period briefly vortex all tubes B12 Pipette 700 uL of isopropanol into each tube Cap the tubes and vortex briefly B13 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 15 20 minutes at room temperature B14 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet B15 Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly B16 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 5 10 minutes at room temperature B17 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette Remove as much liquid as possible without dis turbing the pellet Use a new transfer pipette for each tube B18 Using a new transfer pipette for each tube repeat Step B17 to remove as much of the remaining supernatant as possible without disturbing the pellet Residual ethanol can inhibit amplification B19 Pipette 200 uL MP DIL into each tube Use a pipette tip to break apart the pellet This can be done by aspirating 30 40 uL of the diluent in the tip and scraping the sides and base of the tube in an up down motion for at least 10 seconds and dispensing 30 40 uL Cap the tubes and vortex briefly to resuspend
35. he orientation marks facing outward so that the orien tation marks will align with the pellets formed during centrifugation B6 Centrifuge specimens and control tubes at 23 000 24 000 x g for 60 4 minutes at 2 8 C The pellet will form on the outer wall as indi cated by the orientation mark NOTE The 60 4 minutes begins wher the centrifuge reaches 23 000 24 000 x g B7 Remove the tubes from the centrifuge and remove the caps Slowly aspirate 900 uL of the supernatant from each centrifuged tube leaving approximately 100 uL of supernatant Avoid contact with the pellet Discard the supernatant and pipette tip appropriately Use a fresh pipette tip for each tube B8 Prepare a Working Lysis Reagent bottle for every batch of 12 specimens and controls to be processed B9 Pipette 600 uL Working Lysis Reagent into each specimen and control tube Cap and vortex tubes briefly B10 Prepare Controls as follows a N ive Contro Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 yL MP C to the tube labeled MP C containing Working Lysis Reagent and NHP Cap the tube and vortex briefly b Positive Control Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 uL MP C to the tube labeled MP C containing Working Lysis Reagent and NHP Cap the tube and vortex briefly B11 Incubate all tubes for 10 to 15 minutes at room temperature after adding Working Lysis Reagent to t
36. hnology to the next users perform method correlation studies in their laboratory to qualify technology differences The Distributed by affiliate addresses have been updated Please contact your local Roche Representative if you have any questions Roche Molecular Systems Inc Branchburg NJ 08876 USA U S License No 1636 A Member of the Roche Group D Roche Diagnostics Roche Diagnostics 9115 Hague Road 201 boulevard Armand Frappier Indianapolis IN 46250 0457 USA H7V 4A2 Laval Qu bec Canada Distributed by For Technical Assistance call the For Technical Assistance call Roche nse Center Pour toute assistance technique toll free 1 800 526 1247 appeler le 1 877 273 3433 ROCHE AMPLICOR COBAS AMPLISCREEN AMPLILINK and AMPERASE are trademarks of Roche ROCHE RESPONSE CENTER is a service mark of Roche DYNABEADS paramagnetic particles are licensed under patents owned by Dynal Biotech ASA Oslo Norway DYNABEADS is a trademark of Dynal Biotech ASA Oslo Norway licensed to Roche Diagnostics Corporation Indianapolis Indiana EPPENDORF EPPENDORF MULTIPETTE and EPPENDORF COMBITIP are trademarks of Eppendorf Netheler Hinz GmbH Hamburg Germany MICROLAB is a trademark of the Hamilton Company PROCLIN is a trademark of Rohm and Haas Company ULTRAQUAL is a trademark of National Genetics Institute a subsidiary of Laboratory Corporation of America Holdings LabCorp VERSANT is a trademark of Bayer Corporation
37. id testing NAT of whole biood donations has been in place in the United States since 1999 under Investigational New Drug Application IND Nucleic acid based tests can detect viremic units donated by carriers who do not seroconvert or who lack antibodies to serological markers normally detected by immunological assays 9 The COBAS AmpliScreen HCV Test v2 0 uses a generic sample preparation technique in a mini pool testing format along with automated ampli fication and detection using Polymerase Chain Reaction PCR on the COBAS AMPLICOR Analyzer for the detection of HCV RNA in blood dona tions The assay incorporates an Internal Control for monitoring assay performance in each individual test as well as the AmpErase uracil N glycosylase enzyme to reduce potential contamination by previously amplified material amplicon PRINCIPLES OF THE PROCEDURE The COBAS AmpliScreen HCV Test v2 0 is based on five major processes 1 Sample Processing 2 Reverse transcription of target RNA to generate complementary DNA cDNA 10 3 PCR amplification of target cDNA using HCV specific complementary primers 4 Hybridization of the amplified products to oligonucleotide probes specific to the target s 5 Detection of the probe bound amplified products by colorimetric determination Sample Processing Two specimen processing procedures are used with the COBAS AmpliScreen HCV Test v2 0 as follows e Multiprep Specimen Processing Procedure for p
38. ining Working Master Mix at 2 8 C until specimen and control preparation is completed The A rings with Working Master Mix must be used within 4 hours of preparation A7 Decontaminate area See Procedural Notes ttem B Specimen and Control Preparation Performed in Pre Amplification Specimen and Control Preparation Area Muttiprep Specimen Processing Procedure Pooled Specimens and Individual Cadaveric Specimens Bi For pooled specimens pipette 1000 uL of each pool into an appropriately labeled screw cap tube using the COBAS AmpliScreen Pooling System a hand held pipettor or other user validated method Cap the tubes Proceed to Step B2 For individual cadaveric specimens pipette 200 uL into an appropriately labeled screw cap tube and add 800 uL Multiprep Diluent MP DIL using a hand held pipettor or other user validated method Cap the tubes Vortex each specimen tube briefly Proceed to Step B2 B2 Vortex NHP briefly B3 For each Negative and Positive Control pipette 1000 uL NHP into an appropriately labeled screw cap tube Cap the tubes For cadaveric testing pipette 200 uL NHP into an appropriately labeled screw cap tube and add 800 uL Multiprep Diluent MP DIL using a hand held pipettor or other user validated method Cap the tubes Vortex each specimen tube briefly B4 Use a permanent marker to make an orientation mark on each tube B5 Place the specimen and control tubes into the pre cooled high speed centrifuge with t
39. ir willingness to participate in the HCV follow up study All three donors are considered to be confirmed window cases due to subsequent donations testing positive for HCV RNA Additional testing on the index donation sample volume permitting was positive by both National Genetics Institute NGI HCV UltraQual reaction per primer pair and Bayer Versant HCV Quantitation The quantitation for one sample was 492 047 copies mL Both enrolled follow up study participants were anti HCV positive and HCV positive by the Roche COBAS AmpliScreen HCV Test v2 0 upon the first study samples collected Antibody was detected by RIBA for one of the study participants and sample was sent out for Ortho HCV EIA 3 0 analysis and yielded a reactive result The specimen from the other follow up study participant was reactive for HCV antibodies by the Abbott HCV EIA Test v2 0 There were 1080 pools that were used to determine the specificity of HCV RNA Of these pools 1077 were HCV RNA negative The specificity of the COBAS AmpliScreen HCV Test v2 0 in this study was 1077 1080 or 99 7222 with 95 confidence interval of 99 19 to 99 94 NON CLINICAL PERFORMANCE Ten commercially available HCV seroconversion panels were diluted 1 96 with HCV negative human plasma and tested using the Multiprep Specimen Processing Procedure Results were compared with test results from U S FDA licensed tests for anti HCV EIA and RIBA Five 5 of the 10 panels 50 were never p
40. it PCR Do not use heparinized plasma with this procedure Use only supplied or specified required disposables to ensure optimal assay performance Screw cap tubes must be used for specimen and control preparation to prevent splashing and potential cross contamination of specimens and controls Do not use snap cap tubes Adequately vortex where specified to ensure optimal assay performance Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of specimens or controls n Before use visually inspect each reagent bottle to ensure that there are no signs of leakage and or abnormal color If there is any evidence of leakage and or abnormal color do not use that bottle for testing Dispose of all materials that have come in contact with specimens and reagents in accordance with country federal state and local regula tions Do not use a kit after its expiration date DO NOT interchange mix or combine reagents from kits with different master lot numbers Do not use expired reagents Material Safety Data Sheets MSDS are available on request Supplies and equipment must be dedicated to each pre amplification activity and should not be used for other activities or moved between areas Fresh clean gloves must be worn in each area and must be changed before leaving that area Equipment and supplies used for reagent preparation must not be used for specimen preparation a
41. l target using a secondary standard to a final con centration of 3X the LOD Each of the 20 pre and post mortem specimens were tested using three different COBAS AmpliScreen HCV Test v2 0 kit lots at three different testing sites in this study At each testing site each specimen was tested singly in two separate runs using each of the three different kit lots total of six valid test results for each specimen at each site There were a total of 18 valid test results six results per site x 3 testing sites for each specimen All valid reproducibility data for post mortem and pre mortem specimens were evaluated by calculating the percentage of correct results for each assay The data were analyzed by lot and by testing site The summary of results of the reproducibility study test is presented in Table 17 below Table 17 Summary of Reproducibility Study Test Results Post Mortem versus Pre Mortem as Post Mortem Results by Lot Positive Tested Percent Hit Rate 120 120 119 120 100 99 2 117 120 117 119 97 5 98 3 Lot 3 119 120 120 120 99 2 100 Results by Site Positive Tested Percent Hit Rate 98 3 100 98 3 98 3 Site 3 120 120 118 119 100 99 2 051207 13001 03EN Doc Rev 2 1 16 REFERENCES i Choo Q L Weiner A J Overby L R et al 1990 Hepatitis C Virus The major causative agent of viral non a non b hepatitis British Medical Bulletin 46 423 441 Alter H 1991 Descart
42. l work surfaces with a freshly prepared solution of 0 5 sodium hypochlorite in distilled or deionized water Follow by wiping down the surface with 70 ethanol CAUTION The Negative Human Plasma NHP of this kit contains human blood products non reactive by US FDA licensed tests for antibody to HIV 1 2 antibody to HCV HIV 1 p24 antigen and HBsAg No known test method can offer complete assurance that prod ucts derived from human blood will not transmit infectious agents All human blood sourced materials should be considered poten tially infectious and should be handled with Universal Precautions If spillage occurs immediately disinfect then wipe up with a 0 5 final concentration sodium hypochlorite solution diluted bleach or follow appropriate site procedures Use routine laboratory precautions Do not pipette by mouth Do not eat drink or smoke in designated work areas Wear disposable gloves laboratory coats and eye protection when handling specimens and kit reagents Wash hands thoroughly after handling specimens and kit reagents This product contains sodium azide as a preservative Do not use metal tubing for reagent transfer If solutions containing azide compounds are disposed of in a plumbing system they should be diluted and flushed with generous amounts of running water These precautions are recommended to avoid accumulation of deposits in metal piping in which explosive conditions could develop Heparin has been shown to inhib
43. labeled MP C con taining Working Lysis Reagent and NHP Cap the tube and vortex briefly B29 Incubate all tubes for 10 15 minutes at room temperature after adding Working Lysis Reagent to the last tube After the incubation period briefly vortex all tubes B30 Pipette 800 uL of isopropanol into each tube Cap the tubes and vortex briefly B31 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 15 20 minutes at room temperature B32 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet B33 Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly B34 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 5 10 minutes at room temperature B35 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette Remove as much liquid as possible without dis turbing the pellet Use a new transfer pipette for each tube B36 Using a new transfer pipette for each tube repeat Step B35 to remove as much of the remaining supernatant as possible without disturbing the pellet Residual ethanol can inhibit amplification 05120713001 03EN Doc Rev 2 1 B37 B38 B39 B40 B42 Pipette 200 uL MP DIL into each tube Use a pi
44. le HIV 1 antibody in a person with HIV infection Utah 1995 MMWR 1996 45 181 85 10 Myers T W and Gelfand D H 1991 Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase Biochemistry 30 7661 76668 11 Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 12 Richmond J Y and McKinney R W eds 1999 Biosafety in Microbiological and Biomedical Laboratories HHS Publication Number CDC 93 8395 13 Clinical and Laboratory Standards Institute CLSI Protection of Laboratory Workers from Occupationally Acquired Infections Approved Guideline Third Edition CLS Document M29 A3 Wayne PA CLSI 2005 14 international Air Transport Association Dangerous Goods Regulations 41st Edition 2000 704 pp 05120713001 03EN Doc Rev 2 1 17 Document Revision information Added language abbreviation EN to material number in footer on English only Please contact your local Roche Representative if you have any questions PROCEDURAL LIMITATIONS section was modified with the following revisions Though rare mutations within the highly conserved region of the viral genome covered by the COBAS AmpliScreen HCV Test v2 0 primers and or probe may result in the failure to detect the virus e Due to inherent differences between technologies it is recommended that prior to switching from one tec
45. mance was observed when plasma specimens were subjected to three freeze thaw cycles J Thaw frozen specimens at room temperature before using The user should validate other collection and storage conditions If specimens are to be shipped they should be packaged and labeled in compliance with applicable federal and international regulations covering the transport of clinical specimens and etiologic agents 14 L False positive results may occur if cross contamination of specimens is not adequately controlled during specimen handling and processing M SPECIMEN POOLING NOTE Pooling of specimens should only be performed on individual whole blood and source plasma donations or on plasma speci mens from donors of hematopoietic progenitor cells or donor lymphocytes for infusion Cadaveric specimens must be tested individually and not as part of a pool 1 The COBAS AmpliScreen Pooling System performs barcode scanning and pooling operations that combine aliquots from 24 indi vidual samples into a single Primary Pool that is used for testing The pooling algorithm requires preparation of Secondary Pools as well as individual specimens for follow up testing in the event a Primary Pool tests positive If fewer than 24 specimens are available testing is performed using the individual specimens 2 For Source Plasma the Hamitton MICROLAB AT Plus 2 Pipettor performs barcode scanning and pooling operations that combine aliquots from 96 individual samples i
46. n Processing Procedure e If one or more of the Secondary Pools tests positive report the results for the donor specimens in the negative Secondary Pools as HCV RNA Negative For positive Secondary Pools proceed to the section entitled Positive Primary Pool Positive Secondary Pools Tertiary Resolution Testing e If all four Secondary Pools are negative the individual donor specimens in that Primary Pool may be reported as HCV RNA Negative e As part of an overall Quality Assurance program you may wish to conduct additional testing to determine the cause of the initial pos itivity of the Primary Pool Positive Primary Pool Positive Secondary Pools Tertiary Resolution Testing For a positive Secondary Pool test each of the individual donor specimens in that Secondary Pool The individual donor specimens must be processed using the Standard Specimen Processing procedure e If one or more of the individual donor specimens is positive the positive donor specimen s is are reported as HCV RNA Positive and the remaining negative donor specimens associated with the positive Secondary Pool are reported as HCV RNA Negative e If all of the individual donor specimens in that Secondary Pool test negative the donor specimens in the Secondary Pool may be reported as HCV RNA Negative e As part of an overall Quality Assurance program you may wish to conduct additional testing to determine the cause of the positivity of the Primary
47. n Processing Procedure indicate an average 95 LOD of 41 9 IU mL with lower and upper 95 confidence limits of 28 0 IU mL and 111 8 IU mL respectively Tables 5 and 6 summa rize the overall results for the Multiprep and Standard Specimen Processing Procedures respectively Table Serial Dilution Testing Summary for Multiprep Method Combined Input Values with Lower 95 Confidence Limit One Sided HCV RNA 95 Lower Concentration Confidence Limit One sided 97 76 97 76 95 31 Table 6 Serial Dilution Testing Summary for Standard Method Combined Input Values with Lower 95 Confidence Limit One Sided Number of Number of Positive 95 Lower Positives Individual Trials Confidence Limit One sided 05120713001 03EN Doc Rev 2 1 Analytical Sensitivity CBER HCV Panel The FDA CBER HCV Panel Members 1 10 were processed using the Multiprep and Standard Sample Processing Procedures Both specimen pro cessing methods detected HCV RNA at 50 copies mL The Multiprep Sample Processing Procedure detected 100 of all positive members ranging from 10 100 000 copies mL The Standard Sample Processing Procedure detected 100 of all positive members ranging from 50 to 100 000 copies mL Both negative members of the panel were negative by both methods The data are shown in Table 7 Table 7 CBER HCV RNA Panel Results CBER HCV Panel Member Test Results Percent Positive CBER HCV RNA Panel 2 3 Et wn T eta
48. not interfere with the sensitivity or specificity of the COBAS AmpliScreen HCV Test v2 0 using either the Standard or Multiprep Specimen Processing Procedure Exogenous Interfering Substances HCV spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of aspirin up to 50 mg mL pseu doephedrine HCl up to 3 mg dL ascorbic acid up to 20 mg dL acetaminophen up to 40 mg dL or ibuprofen up to 40 mg dL were tested These exogenous substances did not interfere with the sensitivity or specificity using either the Standard or Multiprep Specimen Processing Procedure CLINICAL PERFORMANCE Chronic HCV Population Fifty eight specimens were obtained from patients with a diagnosis of chronic HCV disease All specimens were confirmed to be serologically posi tive by a licensed anti HCV EIA followed by RIBA 3 0 The specimens were tested undiluted using the Standard Specimen Processing procedure and diluted 1 24 using the Multiprep Specimen Processing procedure All specimens were positive in the COBAS AmpliScreen HCV Test v2 0 by both specimen processing procedures High Risk Population Specimens were prospectively collected from a patient population being evaluated at hematology clinics for biochemical clinical and or histological evidence of liver disease and or evidence of HCV infection Specimens were tested in a blinded fashion with COBAS AmpliScreen HCV Test v2 0 Fifty seven of 62 total spe
49. nto a single Primary Pool that is used for testing Positive Primary pools are traced to the posi tive donor using an overlapping pool testing matrix Minipools are prepared from the eight individual donations for columns 1 12 and from the 12 individual donations for rows 1 8 The positive unit is identified by the intersection of the positive column and positive row Confirmatory testing is conducted on the implicated unit using Standard Specimen Processing Procedure Hamitton MICROLAB AT Plus 2 Pipettor with the SUNRISE PLUS v3 3 software was used to prepare pools of up to 96 equal aliquots of plasma during clinical trials NOTE The user must validate all pooling algorithms and equipment other than those supplied by Roche Cadaveric Blood Specimens N Cadaveric blood specimens can be collected in serum or EDTA anticoagulant tubes NOTE A serum or plasma specimen collected from a donor prior to death may be tested instead of a cadaveric blood specimen using either the instructions for cadaveric donor specimens or the instructions for living donor blood specimens O For collection storage and handling of specimens from deceased donors follow general standards and or regulations Cadaveric samples may be stored for up to 72 hours at refrigerated conditions 2 8 C or up to 48 hours at ambient temperature 15 30 C Other storage and han dling conditions must be validated by the user NOTE Cadaveric samples should be placed at 2 8 C as soon
50. ntrol preparation area must be cleaned and disinfected in accordance with methods outlined in Precautions Item A l Decontamination Thoroughly clean and disinfect all work surfaces with a freshly prepared solution of 0 5 sodium hypochlorite in distilled or deionized water Follow by wiping down the surface with 70 ethanol INSTRUCTIONS FOR USE The Multiprep Specimen Processing Procedure is used for extracting nucleic acid from pooled specimens and from individual cadaveric specimens The Standard Specimen Processing Procedure is used for extracting nucleic acid from individual specimens The Multiprep Specimen Processing Procedure is also used for testing minipools of source plasma The Multiprep and the Standard Specimen Processing Procedures are generic nucleic acid extraction procedures and can be used for the extraction of HCV RNA HIV 1 RNA and or HBV DNA A single extraction is sufficient for multiple assays Workflow can be performed on the same day or over multiple days under the following conditions Amplification Hybridization and Detection of Stored Processed Specimens Amplification hybridization and detection can occur on the same day as specimen processing or on a subsequent day If amplification hybridization and detection are to be done on a subsequent day perform the Multiprep Specimen Processing Procedure described in steps B1 through B21 or the Standard Specimen Processing Procedure described in steps B22 through B38
51. on The reverse transcription and amplification reactions are a n with the thermostable recombinant enzyme Thermus thermophilus DNA Polymerase rTth pol in the presence of manganese Mn and under the appropriate buffer conditions r7th pol has both reverse transcrip tase and DNA polymerase activity 1 This allows both reverse transcription and PCR amplification to occur in the same reaction mixture Reverse transcription using r7th pol produces a cDNA copy of the HCV target and the HCV Internal Control RNA PCR Amplification Following reverse transcription using r7th pol a second DNA strand is produced from the cDNA copy thereby yielding a double stranded DNA copy of the HCV target and HCV internal Control RNA The reaction mixture is heated to separate the resulting double stranded DNA As the mixture cools primers anneal to the target DNA and in the presence of Mn and excess deoxynucleotide triphosphates dNTPs the r7th pol extends the annealed os along a target produce a settee eigna spices molecule termed an amplicon The COBAS AMPLICOR Analyzer automati cally repeats this process for a des number of cycles each cycle effectively doubling the amount of amplicon DNA The required number of cycles is preprogrammed in the COBAS AMPLICOR Analyzer The Document Revision Information section is located at the end of this document 05120713001 03EN Doc Rev 2 1 1 ificati j ification of nucleic acid target in the sample and
52. ositive for EIA two 2 of the 10 20 panels were never positive for ELISA and seven 7 of the 10 panels 70 were never reac tive for RIBA The data are presented in Table 14 Table 14 Summary of Pre Seroconversion Detection of HCV RNA vs HCV FDA Licensed Tests Days Before Abbott Days Before Ortho Days Before Chiron HCV EIA 2 0 HCV 3 0 ELISA 10 panels tested 10 panels tested 10 panels tested in 100 10 10 of the HCV seroconversion panels tested the COBAS AmpliScreen HCV Test v2 0 used with the Multiprep Processing procedure and pools of 96 specimens identifies HCV RNA infected specimens earlier than did the U S FDA licensed HCV EIA ELISA and the RIBA assays 05120713001 03EN Doc Rev 2 1 15 NON CLINICAL PERFORMANCE CHARACTERISTICS FOR CADAVERIC SPECIMENS Sensitivity Study Sixty pre mortem EDTA plasma and fifty eight cadaveric EDTA plasma specimens non reactive for HCV were divided into 5 groups Specimens within each group were spiked with HCV viral target to a concentration of 3X the LOD using a different clinical viral isolate for each group The spiked spec imens were equally divided and tested with three COBAS AmpliScreen HCV Test version 2 0 kit lots The COBAS AmpliScreen HCV Test v2 0 using samples diluted 1 5 and the Multiprep Specimen Processing Procedure correctly detected 98 3 59 60 pre mortem EDTA plasma specimens and 94 8 55 58 of cadaveric specimens spiked with HCV RNA at 3X the
53. ositive samples will have undetectable A total of 416 specimens were repeatedly reactive by EIA and of these 204 were also COBAS AmpliScreen HCV Test v2 0 positive Of the 204 COBAS AmpliScreen HCV Test v2 0 positive specimens none were RIBA negative 051207 13001 03EN Doc Rev 2 1 14 Table 12 COBAS AmpliScreen HCV Test v2 0 Results for EIA Repeatedly Reactive Specimens Pets ee T jejeje ee Detection of Window Period Cases From April 8 1999 to December 31 2000 approximately 7 million donations were tested During this period there were 20 confirmed window period cases detected A confirmed window period case is defined as an enrolied individual from whom the index donation was positive with the COBAS AmpliScreen HCV Test v2 0 but non reactive by EIA for anti HCV and a follow up specimen was shown to be anti HCV EIA repeat reactive using the Abbott HCV EIA 2 0 assay and or the Ortho HCV Version 3 0 ELISA test system and or HCV RNA positive The detection rate of such window period cases was 0 00029 1 in 350 000 with a 95 confidence interval of 0 00017 to 0 00041 In addition four subjects with negative serology and no follow up specimens were presumed to be window period cases as a specimen from the plasma bag for each confirmed the index HCV RNA positive result If these four subjects are included the detection rate of window period cases is 0 00034 1 in 292 000 with a 95 confidence interval of 0 00021 to 0 00049
54. pette tip to break apart the pellet This can be done by aspirating 30 40 uL of the diluent in the tip and scraping the sides and base of the tube in an up down motion for at least 10 seconds and dispensing 30 40 uL Cap the tubes and vortex briefly to resuspend the extracted RNA Note that some insoluble material may remain At this point amplification of the processed specimens and controls must be started within 2 hours If not the processed specimens and con trols can be stored at 70 C or colder for up to one month Thawing should be completed within one hour at room temperature Loading the A ring Create an A ting worklist record for each A ring to identify the A tube with the appropriate control or specimen to be pipetted if processed specimens and controls were stored frozen thaw at room temperature before proceeding Briefly vortex the processed speci mens and controls Pipette 50 uL of each processed specimen and control into the appropriate A tube containing HCV Working Master Mix Immediately cap the A tube and repeat this step for all the 12 A tubes to complete the A ring loading Use the A ring worklist record to ensure the appropriate specimen or control is added to the correct A tube position for each A ring Transfer the A ring with sealed tubes containing the processed specimens and controls in Working Master Mix to the Amplification Detection Area Proceed to Part C NOTE Amplification must begin within 45 minutes from when
55. positive the positive cadaveric specimen is reported as HCV RNA Positive ff an individual cadaveric specimen is negative the negative cadaveric specimen is reported as HCV RNA Negative For cadaveric specimens that had an initial invalid result and were repeated in duplicate if either or both the duplicate samples are positive the spec imen is reported as HCV RNA Positive If both duplicate specimens are negative or if one duplicate is negative and one Is Invalid the specimen Is reported as HCV RNA Negative If both replicates are invalid it is most likely due to inhibitory substances in the specimen and the results should be marked as invalid or unresolved PROCEDURAL LIMITATIONS 1 This test has been evaluated only for use in combination with the COBAS Amp liScreen Multiprep Specimen Preparation and Control Kit COBAS AMPLICOR Analyzer and the Hamitton MICROLAB AT Plus 2 Pipettor for the automated preparation of plasma pools 2 Heparin inhibits PCR specimens collected using heparin as the anticoagulant should not be used with the COBAS AmpliScreen HCV Test v2 0 3 Reliable results are dependent on adequate specimen collection and proper transport procedures 4 Detection of HCV RNA is dependent on the number of virus particles present in the specimen and may be affected by specimen collection methods patient factors e age presence of symptoms and or stage of infection and pool size i 5 Only the Hamilton
56. reparation of mini pool specimens and individual cadaveric specimens Standard Sample Processing for preparation of individual donor samples NOTE For testing of cadaveric specimens the specimen should be first diluted 1 5 in Multiprep Specimen Diluent MP DIL prior to processing using the Multiprep Specimen Processing Procedure In the Standard Specimen Processing Procedure HCV RNA is isolated directly from plasma by lysis of the virus particles with Multiprep Lysis Reagent followed by precipitation of the RNA with alcohol In the Muttiprep Specimen Processing Procedure HCV viral particles are first pelleted from the sepia wise centrifugation followed by lysis of the pelleted virus with a chaotropic agent Multiprep Lysis Reagent and precipita ono alcohol The Multiprep internal Control MP IC containing the HCV Internal Control is introduced into each sample with the Multiprep Lysis Reagent and serves as an extraction and amplification control for each processed specimen and control The HCV Internal Control is an RNA transcript with primer binding regions identical to those of the HCV target sequence a randomized internal sequence of similar length and base composition as the HCV target sequence and a unique probe binding region that differentiates the HCV Internal Control amplicon from target amplicon These features were selected to ensure equivalent amplification of the HCV Internal Control and the HCV target RNA Reverse Transcripti
57. sease states including 23 viral isolates two bacterial strains and one yeast isolate No cross reactivity was observed with the COBAS AmpliScreen HCV Test v2 0 Table 10 summarizes the microorganisms studied Table 10 Analytical Specificity Microorganisms Tested Up to ten individual patient plasma specimens from each of the following disease categories were spiked with low levels of HCV positive plasma within 2 3X the 95 LOD HIV 1 HIV 2 autoimmune disease EBV CMV and Candida albicans No false negative test results were observed Analytical Specificity Non Hepatitis Samples Twenty five HAV and 25 HBV positive specimens all HCV negative were tested for cross reactivity with the COBAS AmpliScreen HCV Test v2 0 by using Doh Gee Sanik acl Whee Sample Processing Procedures All samples were found to be negative No false positive test results were observed These samples were also spiked with low levels of HCV positive plasma and tested using both the Standard and Multiprep Sample Processing Procedures All samples were found to be positive No false negative test results were observed Potentially Interfering Substances Endogenous Interfering Substances HCV spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of bilirubin up to 20 mg mL triglyc erides up to 3000 mg dL hemoglobin up to 1 0 g dL and albumin up to 6 g dL were tested These endogenous substances did
58. ssociated MP IC is less than 0 2 the entire A ring is invalid and the entire test procedure for that A ring sample and control preparation ampli fication and detection must be repeated b Positive Control The absorbance for the MP C should be greater than or equal to 1 0 at 660 nm and its associated MP IC should be greater than or equal to 0 2 at 660 nm for the Positive Control to be valid If the absorbance value for the MP C is less than 1 0 and or its asso ciated MP IC is less than 0 2 the entire A ring is invalid and the entire test procedure for that A ring specimen and control prepa ration amplification and detection must be repeated Summary of Control Acceptance Criteria 05120713001 03EN Doc Rev 2 1 2 Flags and comments may be generated by the COBAS AMPLICOR Analyzer during a run The Operator must check the run printout s for flags and comments to verify that the run is valid Refer to the Operator s Manual for the AMPLILINK software and the Operator s Manual for the COBAS AMPLICOR Analyzer for interpretation of flags and comments 3 External Control if an External Control i e an additional run control other than the Multiprep Control or Multiprep Control is required by the laboratory the External Control should meet regulatory requirements for such controls The absorbance of the HCV External Control should be equal to or greater than 0 2 at 660 nm irrespective of the MP IC absorbance If the absorban
59. the Roche Pooling Methods Software version 1 3 the COBAS AmpliScreen Pooling System Guide Roche Pooling Methods Software version 1 3 and the COBAS AmpliScreen Pooling System Guide are validated to prepare pools of equal aliquots of not more than 24 individual plasma donations using Hamilton MICROLAB AT Plus 2 Pipettor with Hamitton SUNPLUS and RUNENDE Software o Additional MP DIL from the COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit is required for testing of cadav eric specimens NOTE The user must validate all pooling algorithms and equipment other than those supplied by Roche Sarstedt 1 5 mL tube Barcode Labels e Hamilton Archive and Intermediate Plate Barcode Labels Refrigerated high speed centrifuge with fixed angle rotor 45 degrees capacity for at least 24 x 1 5 mL tubes with an RCF of 23 600 x g Heraeus Centrifuge 17RS or Biofuge 28RS with HFA 22 1 rotor Heraeus Biofuge Stratos with the 3331 rotor or equivalent 05120713001 03EN Doc Rev 2 1 MATERIALS REQUIRED BUT NOT PROVIDED BY ROCHE e Microcentrifuge max RCF 16 000 x g min RCF 12 500 x g Eppendorf 5415C HERMLE Z230M or equivalent Eppendorf 1 25 mL Eppendorf Combitip Reservoir sterile or equivalent e Eppendorf Multipette pipette or equivalent Ethanol 90 or 95 reagent grade for Molecular Biology or Histology use e Distilled or deionized water e Powderless disposable gloves e Isopropyl alcohol reagent grade
60. the first specimen or control in the A ring is added to the Working C C1 C2 C3 Master Mix Reverse Transcription Amplification and Detection Performed in Post Amplification Amplification Detection Area Perform Daily instrument Maintenance as outlined in the Operator s Manual for the COBAS AMPLICOR Analyzer including a Wipe D cup handler tip with a lint free moist cloth and dry b Wipe initialization post with a lint free moist cloth and dry Before each run a Check waste container and empty if necessary b Check Wash Buffer Reservoir and add prepared Wash Buffer if necessary c Replace used D cup racks d Prime the COBAS AMPLICOR Analyzer Instrument Loading and System Operation a Prepare enough of the following detection reagent cassettes to complete the workload Working HCV Probe Suspension Reagent CH4 v2 0 Working IC Probe Suspension Reagent CI PS1 Working Substrate SB3 Denaturation Reagent DN4 and Conjugate Reagent CN4 Place the CH4 v2 0 and CI PS1 cassettes in the test specific reagent rack Place DN4 CN4 and SB3 cassettes in the generic reagent rack Record on the cassette the date when each cassette was opened d Identify the reagent racks as generic or test specific using the COBAS AMPLICOR Analyzer barcode scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software e Configure the reagent racks by entering the reagent positions and lots
61. tiprep Sample Processing Procedure HCV RNA Number of Number of Positive 95 Lower Concentration Positives Individual Trials Confidence Limit IU mL One Tailed 100 0 28 6 o so 2 100 98 6 90 3 86 4 Table 4 Standard Procedure Testing Summary for All Clinical Samples Combined input Values with 95 Qne tailed Lower Confidence Limit Standard Sample Processing Procedure HCV RNA Number of Number of Positive 95 Lower Concentration Positives Individual Trials Confidence Limit IU mL One Tailed soos 2 w w w 98 6 o ss w 3 sm 77 62 1 52 7 Analytical Sensitivity WHO HCV International Standard The analytical sensitivity of the COBAS AmpliScreen HCV Test v2 0 was also determined using the WHO HCV International Standard 96 780 The WHO HCV International Standard was serially diluted in HCV negative plasma to final concentrations of 200 100 50 25 15 and 10 IU mL Each dilution was tested with two lots of the COBAS AmpliScreen HCV Test v2 0 using both the Multiprep and Standard Specimen Processing Procedures When evaluated using PROBIT analysis the combined data for all samples processed by the Multiprep Specimen Processing Procedure indicate an average 95 LOD of 28 8 IU mL with lower and upper 95 confidence limits of 20 5 IU mL and 85 8 IU mL respectively When evaluated using PROBIT analysis the combined data for all samples processed by the Standard Specime
62. using the COBAS AMPLICOR Analyzer barcode scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software f Load the reagent racks onto the COBAS AMPLICOR Analyzer using the COBAS AMPLICOR Analyzer barcode scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software Make sure that each reagent cassette is in its assigned position and that each cassette fits tightly into its rack g Place the D cup rack on the D cup platform Two D cups are required for each A tube and two D cups are required for each Workin Substrate cassette to allow for blanking by the COBAS AMPLICOR Analyzer as described in the Operator s Manual for the COBAS AMPLICOR Analyzer h Place the A ring into the thermal cycler segment of the COBAS AMPLICOR Analyzer and close the cover on the thermal cycler segment i Load the A ring into the COBAS AMPLICOR Analyzer using the COBAS AMPLICOR Analyzer barcode scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software j Create an A ring order using the AMPLILINK software as described in the Operator s Manual for AMPLILINK software Use the A ring worklist record created for specimen processing to assist in entering the A ring order k Repeat steps h through j above to load a second A ring on the COBAS AMPLICOR Analyzer Start the COBAS AMPLICOR Analyzer as described in the Operator s Manual for AMPLILI
63. y be stored at 2 30 C for up to 72 hours from time of draw followed by an additional two days at 2 8 C For storage longer than five days remove the plasma from the red blood cells by centrifugation at 800 1600 x g for 20 minutes Following removal plasma may be stored at 2 8 C for an additional seven days Alternatively plasma may be stored at lt 1 8 C for up to one month 2 to 30 C gt 30 200 are Plasma 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Temperature C oe M Days Post Collection C Blood collected in CPD CPDA 1 or CP2D may be stored for up to 72 hours at 1 24 C Following centrifugation of the CPD CPDA 1 or CP2D samples at 800 1600 x g for 20 minutes plasma may be stored at 1 6 C for an additional 7 days from the date the plasma was removed from the red blood cells Plasma separated from the cells may be stored at lt 18 C for up to one month D ACD A or 4 sodium citrate anticoagulated apheresis plasma can be stored at 1 6 C for up to 6 hours followed by subsequent storage at lt 18 C for up to one month E Do not freeze whole blood k Heparin has been shown to inhibit PCR Use of heparinized specimens is not recommended G Warm pooled or individual donor specimens to room temperature before using H Covered Archive Plates may be stored at 2 8 C for up to 7 days from the date the plasma was removed from the red blood cells l No adverse effect on assay perfor
64. zer Working Substrate Reagent 1 Working Substrate must be prepared each day by pipetting 5 mL SB into one SB3 cassette Pipette up and down at least 5 times to mix 05120713001 03EN Doc Rev 2 1 2 Working Substrate is stable on the COBAS AMPLICOR Analyzer for a maximum of 16 hours 3 Do not expose SB3 SB or Working Substrate to metals oxidizing agents or direct light G Wash Buffer Reagent 4 Examine WB before dilution and if necessary warm at 30 37 C to dissolve any precipitate Add 1 volume of WB to 9 volumes of dis tilled or deionized water Mix well Keep a minimum of 3 4 liters of Working Wash Buffer 1X in the Wash Buffer Reservoir of the COBAS AMPLICOR Analyzer at all times 2 Working Wash Buffer 1X should be stored at 2 25 C in the COBAS AMPLICOR Wash Buffer Reservoir and is stable for 2 weeks from the date of preparation H 70 Ethanol 1 Prepare 70 ethanol fresh daily 2 One mL 70 ethanol is needed for each specimen and control processed For example mix 11 7 mL 90 ethanol and 3 3 mL of dis tilled or deionized water for every 12 specimens and controls to be processed SPECIMEN COLLECTION STORAGE AND POOLING NOTE Handle all specimens as if they are potentially infectious agents Living Donor Specimens A EDTA CPD CPDA 1 CP2D ACD A and 4 Sodium Citrate may be used with the COBAS AmpliScreen HCV Test v2 0 Follow sample tube manufacturer s instructions B Blood collected in EDTA ma

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