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COBAS® AmpliScreen HIV

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1. 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 HIV 1 MMX v1 5 and HIV 1 Mn v1 5 iH PS1 v1 5 IH4 v1 5 if PS1 v1 5 and 114 v1 5 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 Do not expose SBS 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 HIV 1 Mn v1 5 and SB PRECAUTIONS FOR IN VITRO DIAGNOSTIG USE A Specimens may be infectious Use Universal Precautions when performing the assay 30 31 Only personnel proficient in the use of the COBAS AmpliScreen System and trained in handling infectious materials should perform this procedure Thoroughly clean and disinfect all work surfaces m 05120705001 02EN 4 Doc Rev 2 0 I pmm rA Zz R S 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 Negati
2. arenes A y Z COBAS AmpliScreen HIV 1 Test version 1 5 FOR IN VITRO DIAGNOSTIC USE COBAS AmpliScreen HIV 1 Test version 1 5 96 Tests P N 03322114 018 COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit MULTIPREP CTL 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 HIV 1 Test version 1 5 v1 5 is a qualitative in vitro test for the direct detection of Human Immunodeficiency Virus Type 1 HIV 1 RNA in human plasma The COBAS AmpliScreen HIV 1 Test v1 5 is intended to be used for detection of HIV 1 RNA in conjunction with licensed tests for detecting anti bodies to HIV 1 This product is intended for use as a donor screening test to detect HIV 1 RNA 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 specimens are obtained while the donor s heart is still beating and to detect HIV 1 RNA in blood specimens from cadaveric non heart beating organ and tissue donors This test is not intended for use on samples of cord blood This test is not intended for use as an aid in diagnosis 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 individu
3. Results By Lot Positive Tested a neoe oom asom T5 cml 00cm 1 57 86 96 100 100 0 52 80 92 98 100 co 2 56 30 99 98 100 com cox 0 50 87 93 0 57 82 _ 92 100 100 Results By Site Positive Tested 2 48 89 95 99 100 0 55 73 91 100 Site 3 0 149 90 150 139 150 146 150 150 150 150 150 Table 2 Reproducibility Results Standard Specimen Processing Procedure Results By Lot Positive Tested Teste soom toot 260 250 emt 25000 et 0 49 84 93 97 100 tos 0 56 82 93 100 0 44 81 85 96 100 T 1 54 67 80 94 Lot 5 0 89 37 90 65 89 76 88 85 89 89 89 Results By Site Positive Tested Site 1 0 150 134 150 145 150 450 150 0 49 78 89 97 100 Site 3 1 150 82 150 117 149 135 150 144 150 149 149 Analytical Sensitivity Dilutional Panels The analytical sensitivity of the COBAS AmpliScreen HIV 1 Test v1 5 was determined by testing 10 HIV 1 seropositive clinical specimens The titer of each Specimen was quantitated with a commercially available assay using a secondary standard calibrated against the WHO Intemational Standard These specimens were diluted in norma human plasma to 150 50 and 16 7 copies mL for the Multiprep Specimen Processing Procedure and 300 1
4. Avoid contact of MP LYS HIV 1 MMX v1 5 HIV 1 Mn v1 5 IH4 v1 5 114 v1 5 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 oth erwise burns can occur If these reagents are spilled dilute with water before wiping dry Do not alow MP LYS which contains guanidine thiocyanate or IH4 v1 5 and ll4 v1 5 which contain sodium thiocyanate to contact sodium hypochlorite bleach solution This mix ture 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 contro preparation is performed correctly Any deviation from the given procedures and guidelines may affect optimal assay performance The use of excessively hemolyzed cadaveric specimens should be avoided REAGENT PREPARATION A B MP IC MP C MP C MP DIL and
5. Al A2 A3 A4 B1 B3 B4 BS B6 B7 B8 Cevieamt samo asmo imo wo S ammo 30 amow w i so smo 0 Multiprep 100 100 100 100 400 100 100 0 100 Method Standard 100 100 100 100 100 0 100 o 100 75 100 0 Prep Method Group Subtype Detectability One hundred culture specimens representing 20 each of HIV 1 Group M subtypes A through E 3 culture specimens of Subtype F 4 culture speci mens of Subtype G 8 culture specimens of Group O and 1 culture specimen of Group N were tested The Group M specimens were tested at 400 copies mL using the Standard Specimen Processing Procedure and at 200 copies mL using the Multiprep Specimen Processing Procedure The Group O and N specimens were diluted 5 25 125 625 and 3125 fold and tested using the Multiprep and Standard Specimen Processing Procedures Data are provided in Table 8 Group O specimens were only evaluated as diluted samples due to limited specimen volume Table 8 HIV 1 Group Subtype Tested Subtype Quantity Reactive Total Reactive Total Multiprep Standard Prep 20 20 20 20 Due to limited volume specimens were only tested diluted and the actual HIV 1 RNA Group O and Group N copy numbers were not determined l Seroconversion Panels Forty one commercially available anti HIV seroconversion panels were tested undiluted using the Standard Specimen Processing Procedure and diluted 1 24 using the Multiprep
6. 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 pipettor with Hamilton SUNPLUS and RUNENDE Software e Additional MP DIL from the COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit is required for testing of cadaveric speci mens NOTE The user must validate all pooling algorithms and equipment other than those supplied by Roche e Sarstedt 1 5 mL tube Barcode Labels e Hamilton Archive and Intermediate Plate Barcode Labels e 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 05120705001 02EN 2 Doc Rev 2 0 MATERIALS REQUIRED BUT NOT PROVIDED BY ROCHE Microcentrifuge max RCF 16 000 x g min RCF 12 500 x g Eppendorf 5415C HERMLE Z230M or equivalent Eppendorf 1 25 mL Combitip Reservoir sterile or equivalent Eppendorf Muttipette pipette or equivalent Ethanol 90 or 95 reagent grade for Molecular Biology or Histology use Distilled or deionized water Powderless disposable gloves lsopropyl alcohol reagent grade Disposable Sterile Polystyrene pipettes 6 mL 10 mL and 25 mL Sterile RNase free fine tip transfer pipettes Pipettors capacity 20 pL to 1000 pL capable of providing
7. Of the 39 COBAS AmpliScreen HIV 1 Test v1 5 positive specimens none were negative by Western Blot or IFA 05120705001 02EN 15 Doc Rev 2 0 Table 17 COBAS AmpliScreen HIV 1 Test v1 5 Results for EIA Repeatedly Reactive Specimens RR 463 Western Blot IFA Western BIot IFA Detection of Window Period Cases From November 11 1999 to December 31 2001 approximately 8 million donations were tested During this period there were 2 confirmed window period cases detected A confirmed window period case is defined as an enrolled individual from whom the index donation was positive in the COBAS AmpliScreen HIV 1 Test v1 5 but non reactive by EIA for HIV 1 2 and a follow up specimen was shown to be anti HIV 1 EIA repeatedly reactive and or HIV 1 RNA positive The detection rate of such window period cases was 0 0000002 1 in 4 000 000 There was one additional specimen that was anti HIV 1 EIA negative HIV 1 p24 antigen positive and HIV 1 RNA positive however this donor was not enrolled in the follow up study Single Donation Testing Performance A total of 587 specimens were tested individually in the COBAS AmpliScreen HIV 1 Test v1 5 clinical trial The HIV 1 status of these samples was based upon EIA and supplemental test results as described above Of the 587 specimens 271 specimens had available HIV 1 antibody test data Of these 271 specimens 271 were classified as HIV 1 status nega tive there were no HIV 1 status posi
8. 239 61 0 616 Gaines H von Sydow M A von Stedingk LV 1990 Immunological changes in primary HIV 1 infection AIDS 4 995 999 Tindall B and Cooper D A 1991 Primary HIV 1 infection host responses and intervention strategies AIDS 5 1 14 Daar E S Moudgil T Meyer R D et al 1991 Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection New England Journal of Medicine 324 961 964 Clark S J Saag M S Decker W D 1991 High titers of cytopathic virus in plasma of patients with symptomatic primary HIV 1 infection New England Journal of Medicine 324 954 960 Albert J Abrahamsson B Nagy K 1990 Rapid development of isolate specific neutralizing antibodies after primary HIV 1 infection and con sequent emergence of virus variants which resist neutralization by autologous sera AIDS 4 107 112 Hornsburgh C R Jason J Longini I M et al 1989 Duration of human immunodeficiency virus infection before detection of antibody Lancet 26 637 640 AIDS epidemic update December 1998 UNAIDS Joint United Nations Programme on HIV 1 AIDS Dodd R Y 1994 Adverse consequences of blood transfusion quantitative risk estimates In Nance ST ed Blood supply risks perceptions and prospects for the future Bethesda American Association of Blood Banks 1 24 Schreiber GB Busch MP Kleinman SH Korelitz JJ 1996 The risk of transfusion transmitted viral infections The Retrovirus Ep
9. 3 accuracy and precision lt 5 with aerosol barrier or positive displace ment RNase free tips Tube racks Sarstedt P N 93 1428 or equivalent 1 5 mL sterile non siliconized conical polypropylene screw cap tubes Sarstedt 72 692 105 or equivalent Vortex mixer 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 3 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 EDTA 0 05 Sodium azide MP IC 8 x 0 1 mL Multiprep Internal Control Tris HCI buffer lt 0 001 Non infectious plasmid DNA containing HBV primer binding sequences and a unique probe b
10. LYS Multiprep Lysis Reagent MP DIL Multiprep Specimen Diluent MP IC Multiprep Internal Control NHP Negative Plasma Human COBAS AmpliScreen HIV 1 Test version 1 5 96 Tests P N 03322114 018 COBAS AmpliScreen HIV 1 Amplification Reagents version 1 5 HIV 1 MMX v1 5 HIV 1 Master Mix version 1 5 HIV 1 Mn2 v1 5 HIV 1 Manganese Solution version 1 5 COBAS AmpliScreen HIV 1 Detection Reagents version 1 5 IH PS1 v1 5 HIV 1 Probe Suspension 1 version 1 5 1H4 v1 5 HiV 1 Probe Suspension 2 version 1 5 il PS1 v1 5 IC Probe Suspension 1 114 v1 5 IC Probe Suspension 2 DN4 Denaturation Solution CN4 Avidin Horseradish Peroxidase Conjugate SBS 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 00228 Printer and Operator s Manual for the COBAS 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 the Roche Pooling Methods Software version 1 3 the COBAS AmpliScreen Pooling System Guide Roche Pooling Methods Software version 1 3 and the COBAS
11. 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 disinfected in accordance with methods outlined in Precautions Item A Failure to do so may result in reagent contamination 2 Pipette 100 uL HIV 1 Mn2 v1 5 into 1 bottle HIV 1 MMX v1 5 Recap HIV 1 MMX v1 5 bottle and mix well by inverting 10 15 times The pink color confirms that the HIV 1 Mn v1 5 has been added to the HIV 1 MMX v1 5 Discard the remaining HIV 1 Mn2 v1 5 Do not vortex the Working
12. at least 5 times to mix 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 Wash Buffer Reagent 1 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 distilled 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 70 Ethanol 1 Prepare 70 ethanol fresh daily 05120705001 02EN 3 Doc Rev 2 0 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 distilled 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 GPD CPDA 1 CP2D ACD A and 4 Sodium Citrate may be used with the COBAS AmpliScreen HIV 1 Test v1 5 Follow sample tube manufacturer s instructions B Blood collected in EDTA may 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 remov
13. for specimen and control preparation to prevent splashing and potentia cross contamination of specimens and controls Do not use snap cap tubes Adequately vortex where specified to ensure optimal assay performance Handie all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of spec imens or controls 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 regulations 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 activities 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
14. invalid specimen POSITIVE Specimen is positive for HIV 1 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 speci mens 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 perform repeat testing in single on the remaining rep
15. occur in the same reaction mixture Reverse transcription using rTth pol produces a cDNA copy of the HIV 1 target and the HIV 1 Internal Control RNA PCR Amplification Following reverse transcription using rTth pol a second DNA strand is produced from the cDNA copy thereby yielding a double stranded DNA copy of the HIV 1 target and HIV 1 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 in the presence of Mn and excess deoxynucleotide triphosphates dNTPs the r7th pol extends the annealed primers along the target templates to produce a double stranded DNA molecule termed an amplicon The COBAS AMPLICOR Analyzer automat ically repeats this process for a designated 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 05120705001 02EN 4 Doc Rev 2 0 om Sf a Selective Amplification To ensure selective amplification of nucleic acid target in the sample and prevent amplification of pre existing amplicon the AmpErase uracil N gly cosylase anzyme is added to the COBAS AmpliScreen HIV 1 Test v1 5 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine2 but not DNA containing deoxythymidine Deoxyuridin
16. the tube and vortex briefly 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 Pipette 800 uL of isopropanol into each tube Cap the tubes and vortex briefly 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 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly 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 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette Remove as much liquid as possible without disturbing the pellet Use a new transfer pipette for each tube Using a new transfer pipette for each tube repeat Step B35 to remove as much of the remaining supernatant as possible without dis turbing the pellet Residual ethanol can inhibit amplification 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 pL of the diluent in the tip and scraping the sides and base of the tube in an up down motion for at l
17. the tubes and vortex briefly 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 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly 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 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette Remove as much liquid as possible without disturbing the pellet Use a new transfer pipette for each tube Using a new transfer pipette for each tube repeat Step B17 to remove as much of the remaining supernatant as possible without dis turbing the pellet Residual ethanol can inhibit amplification 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 pL 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 hou
18. total of 463 specimens were repeatedly reactive by EIA and of those 39 were also COBAS AmpliScreen HIV 1 Test v1 5 positive Of the 39 COBAS AmpliScreen HIV 1 Test v1 5 positive specimens none were negative by Western Blot or IFA e Table 17 COBAS AmpliScreen HIV 1 Test v1 5 Results for EIA Repeatedly Reactive Specimens Please contact your local Roche Representative if you have any questions 05120705001 02EN 19 Doc Rev 2 0 Roche Molecular Systems Inc Branchburg NJ 08876 USA U S License No 1636 A Member of the Roche Group 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 Response Center Pour toute assistance technique toll free 1 800 526 1247 appeler le 1 877 273 3433 ROCHE AMPLICOR AMPLICOR HIV 1 MONITOR 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 registered trademark of Dynal Biotech ASA Oslo Norway licensed to Roche Diagnostics Corporation Indianapolis Indiana Eppendorf Eppendorf Multipette and Eppendorf Combitip are registered trademarks of Eppendorf Netheler Hinz GmbH Hamburg Germany MICROLAB is a register
19. 0 Primary Pools that contained on average 10 HIV 1 antibody EIA Repeatedly Reactive and 14 HIV 1 antibody EIA non Reactive samples In addition 600 HIV 1 antibody EIA non Reactive samples were used to create 25 negative Primary Pools This resulted in 125 panels each representing a Primary Pool comprised of 24 sample tubes 20 panels containing AIDS samples 80 panels containing asymptomatic samples and 25 negative panels These panels were pooled using the Hamitton MICROLAB AT plus and tested with the COBAS AmpliScreen HIV 1 Test v1 5 Primary Secondary and Tertiary testing was performed at the clinical sites If discordant results between the Primary Pool test result and either the Secondary or Tertiary testing were observed at the sites resolution testing was performed at Roche A summary of the testing performed at the clinical sites is provided in Tables 12 and 13 There were a total of 23 HIV 1 antibody EIA Repeatedly Reactive specimens that resulted in one or more HIV 1 RNA positive primary pools All were found to be negative in either the secondary or tertiary testing at the clinical sites Of these 23 specimens 9 tested negative at the secondary pool level in 5 different secondary pools at a single clinical Site and 14 tested negative by tertiary testing The results of the resolution testing performed at Roche yielded 21 of 23 specimens that were resolved as HIV 1 RNA positive with the COBAS AmpliScreen HIV 1 Test v1 5 A summary of the te
20. 00 and 33 3 copies mL for the Standard Specimen Processing Procedure The COBAS AmpliScreen HIV 1 Test v1 5 detected 50 copies mL HIV 1 RNA at a frequency greater than 98 with a lower 95 confidence limit of 98 5 using the Multiprep Specimen Processing Procedure The assay detected 100 copies mL HIV 1 RNA at a frequency greater than 98 with 8 8 z 3 05120705001 02EN 11 Doc Rev 2 0 a lower 95 confidence limit of 96 5 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 39 2 copies mL with the lower and upper 95 confidence limits of 34 0 copies mL and 48 3 copies mL respectively The LOD of 39 2 copies mL corresponds to approximately 61 25 IU 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 96 2 copies mL with the lower and upper 95 confidence limit of 83 3 copies mL and 116 7 copies mL respectively The LOD of 96 2 copies mL corresponds to approximately 150 3 IU mL Table 3 Multiprep Procedure Testing Summary for AH Clinical Samples Combined Input Values with 95 One tailed Lower Confidence Limit Multiprep Sample Processing Procedure HIV 1 RNA Nu
21. 000 copies mL were negative for HIV 1 p24 antigen All 25 samples tested with COBAS AmpliScreen HIV 1 Test v1 5 at the 1 24 dilution of the 5 000 copies mL 208 copies mL and all 25 samples tested 05120705001 02EN 13 Doc Rev 2 0 at 100 copies mL were positive for HIV 1 RNA Dilutional Sensitivity with Weakly Reactive HIV 1 Antibody Positive Samples Twenty five known HIV 1 seropositive specimens were diluted to Signal Cutoff S CO levels between 1 and 5 and tested using a licensed HIV 1 anti body assay Abbott HIVAB HIV 1 HIV 2 rDNA EIA These weakly reactive seropositive samples were then singly introduced into pools with 23 neg ative plasma samples in random fashion An additional 144 negative plasma tubes were used to make six negative pools and randomly distributed as discrete sets among the 25 positive pools for testing A total of 744 samples were tested according to the COBAS AmpliScreen test algorithm NAT positive specimens were deconstructed and resolved to the individual sample Of the 25 weakly reactive serologically positive samples a total of 19 were concordant positive and six were discordant negative in the COBAS AmpliScreen HIV 1 Test v1 5 Each of the six discordant NAT negative samples was subject to viral load determination by Roche s quantitative PCR assay AMPLICOR HIV 1 MONITOR Test v1 5 Five of the six discordant NAT negative samples were observed to have less than 100 copies mL HIV 1 RNA and one had a mean titer
22. 4 IU mL with lower and upper 95 confidence limits of 284 9 IU mL and 387 3 IU mL respectively Tables 5 and 6 summarize the overall results for the Multiprep and Standard Specimen Processing Procedures respectively Table 5 Serial Dilution Testing Summary for Multiprep Method with HIV 1 RNA WHO International Standard 97 656 Combined Input Values with Lower 95 Confidence Limit One Sided HIV 1 RNA Number of Number of 95 Lower Concentration Positives Individual Tests Confidence Limit One sided 120 58 3 Table 6 Serial Dilution Testing Summary for Standard Method with HIV 1 RNA WHO International Standard 97 656 Combined Input Values with Lower 95 Confidence Limit One Sided Concentration Confidence Limit Positive IU mL One sided so C w f omn o o e o e on e Analytical Sensitivity CBER HIV 1 Panel The FDA GBER HIV 1 Panel Members were processed using the Multiprep and Standard Specimen Processing Procedures The Multiprep Specimen Processing Procedure detected 100 of all positive members ranging from 10 250 000 copies mL The Standard Specimen Processing Procedure detected 100 of all positive members ranging from 100 250 000 copies mL The data are shown in Table 7 HIV 1 RNA Number of 95 Lower Individual Tests Number of Positives 05120705001 02EN 12 Doc Rev 2 0 Table 7 FDA CBER HiV 1 RNA Panel Results CBER HIV 1 Panel Test Results CBER HIV 1
23. CTERISTICS Reproducibility The reproducibility of the COBAS AmpliScreen HIV 1 Test v1 5 was established by testing two six member EDTA plasma panels with known con centrations of HIV 1 Panel One was tested using the Multiprep Specimen Processing Procedure Panel One was comprised of HIV 1 RNA positive samples at concentrations of 10 25 50 75 and 25 000 copies mL and one HIV 1 negative sample Panel Two was tested using the Standard Specimen Processing Procedure Panel Two was comprised of HIV 1 positive samples at concentrations of 50 100 150 250 and 25 000 copies mL and one HIV 1 negative sample Testing was performed at three sites with two operators at each site using five COBAS AmpliScreen HIV 1 Test v1 5 kit lots Each operator used a dedicated COBAS AMPLICOR Analyzer throughout the study Each operator was provided panel sets that had been randomized and labeled in blinded fashion 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 HIV 1 Test version 1 5 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 Muttiprep Specimen Processing Procedure
24. F Smith F C et al ASM Washington D C Longo M C Berninger M S Hartley J L 1990 Use of uracil DNA glycosylase to contro carry over contamination in polymerase chain reac tions Gene 93 125 128 ha J Y and McKinney R W eds 1999 Biosafety in Microbiological and Biomedical Laboratories HHS Publication Number CDC 93 Clinical and Laboratory Standards Institute CLSI Protection of Laboratory Workers from Occupationally Acquired Infections Approved Guideline Third Edition CLSI Document M29 A3 Wayne PA CLSI 2005 International Air Transport Association Dangerous Goods Regulations 41st Edition 2000 704 pp 05120705001 02EN 18 Doc Rev 2 0 Document Revision Information foe Lin 2 0 The part number of the COBAS AmpliScreen HIV 1 Test v1 5 package insert has been updated to 05120705001 The PROCEDURAL LIMITATIONS section has been modified to include the following statements e Though rare mutations within the highly conserved region of the viral genome covered by the COBAS AmpliScreen HIV 1 Test v1 5 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 technology to the next users perform method correlation studies in their laboratory to qualify technology differences The CLINICAL PERFORMANCE Pool Reactivity in Volunteer Blood Donors section has been modified to add the following gt A
25. 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 ae are required for each A tube and two D cups are required for each Working Substrate cassette to allow for blanking by the COB 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 AMPLILINK software m Wait for the COBAS AMPLICOR Analyzer to indicate that the load check has passed NOTE The required quantity of ea
26. Master Mix These reagents do 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 HIV 1 Probe Suspension Mix IH PS1 v1 5 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL IH PS1 v1 5 into one IH4 v1 5 cassette 2 Prepare Working IC Probe Suspension Mix II PS1 v1 5 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL il PS1 v1 5 into one 114 v1 5 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 AMPLIGOR 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 paat DN4 and CN4 at 2 8 C between instrument cycles Remove from refrigerator 30 minutes before use on the COBAS AMPLICOR er Working Substrate Reagent 1 Working Substrate must be prepared each day by pipetting 5 mL SB into one SB3 cassette Pipette up and down
27. Secondary Pools Results of Individual Donor Samples If an individual donor specimen is positive the positive donor specimen is reported as HIV 1 RNA Positive If an individual donor specimen is negative the negative donor specimen is reported as HIV 1 RNA Negative Results of Pooled Source Plasma Specimens Pools of up to 96 Individual Donations The testing algorithm for testing of pooled samples for the COBAS AmpliScreen HIV 1 Test v1 5 requires a single level of testing for Primary Pools that are negative for HIV 1 RNA and three levels of testing Primary Pool Minipool and confirmatory testing for Primary Pools that are positive for HIV 1 RNA Negative Primary Pools When the Primary Pool is negative report the results for all associated individual donor specimens in that Primary Pool as HIV 1 RNA Negative Positive Primary Pools Minipool 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 p
28. Specimen Processing Procedure The COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA earlier than Abbott HIV 1 2 antibody test in 39 of the 41 panels using both the Multiprep and Standard Specimen Processing Procedures The COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA a mean of 12 8 days median 11 days minimum 0 days and maximum of 89 days before HIV 1 2 antibody using the Multiprep Specimen Processing procedure and a mean of 14 2 days median 12 days minimum 0 days and max imum of 89 days before HIV 1 2 antibody when using the Standard Specimen Processing Procedure The data are presented in Tables 9 and 10 The COBAS AmpliScreen HIV 1 Test v1 5 was also compared to the licensed HIV 1 p24 antigen assays Abbott and Coulter Forty of the 41 panels contained specimens collected before the antigenemia ramp up phase and were used to assess the effectiveness of the COBAS AmpliScreen HIV 1 Test v1 5 in closing the pre seroconversion window period as compared to licensed Abbott HIV 1 p24 antigen assays due to limited volume only 38 panels were tested with the licensed Coulter HIV 1 p24 antigen test In every instance where HIV 1 p24 antigen is detected HIV 1 RNA was also detected in the same specimen time point In some panels HIV 1 RNA was detected before HIV 1 p24 antigen COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA a mean of 4 4 to 6 8 days before the licensed HIV 1 p24 antigen tests using the Multiprep Speci
29. al donations For donations of hematopoietic stem progenitor cells HPCs sourced from bone marrow peripheral blood or cord blood and donor lymphocytes for infusion DLI 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 This assay may be used as an alternative to licensed HIV 1 p24 antigen tests for screening human plasma from donors The COBAS AmpliScreen HIV 1 Test v1 5 can be considered a supplemental test that confirms HIV 1 infection for specimens that are repeatedly reac tive on a licensed donor screening test for antibodies to HIV 1 and reactive on the COBAS AmpliScreen HIV 1 Test v1 5 SUMMARY AND EXPLANATION OF THE TEST Human Immunodeficiency Virus HIV 1 is the etiologic agent of Acquired immunodeficiency Syndrome AIDS 3 HIV 1 infection can be transmitted by sexual contact exposure to infected blood or blood products or by an infected mother to the fetus4 Within three to six weeks of exposure to HIV 1 infected individuals generally develop a brief acute syndrome characterized by flu like symptoms and associated with high levels of viremia in the peripheral blood gt 8 In most infected individuals this is followed by an HIV 1 specific immune response and a decline of plasma viremia usu ally within four to six weeks of the onset of symptom
30. amplification and detec tion 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 associated MP IC is less than 0 2 the entire A ring is invalid and the entire test procedure for that A ring specimen and contro preparation ampli fication and detection must be repeated p o Summary of Control Acceptance Criteria HIV 1 Result IC Result neo common haw commer neste Con pose como 210 Posie zoz Vaid 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 lt y External Control if an External Control i e an additional run contro other than the Multiprep Control or Multiprep Control is required by the laboratory the External Contro should meet regulatory requirements for such controls The absorbance of the HIV 1 External Control should be equal to or greater than 0 2 at 660 nm irrespective of the MP IC absorbance If the absorbance of the HIV 1 External Control does not m
31. cessing 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 HIV 1 RNA Negative For positive Secondary Pools proceed to the section entitled Positive Primary Pool Positive Secondary Pools Tertiary Resolution Testing e ifall four Secondary Pools are negative the individual donor specimens in that Primary Poo may be reported as HIV 1 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 positivity 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 HIV 1 RNA Positive and the remaining negative donor specimens associated with the positive Secondary Pool are reported as HIV 1 RNA Negative e ifall of the individual donor specimens in that Secondary Pool test negative the donor specimens in the Secondary Pool may be reported as HIV 1 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 and
32. ch 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 2 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 2 and or its associated 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
33. e 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 HIV 1 target amplicon and the HIV 1 Internal Control amplicon to form single stranded DNA Aliquots of denatured amplicon are then transferred to two detection cups D cups A suspension of magnetic particles coated with an oligonucleotide probe specific for HIV 1 target amplicon or HIV 1 Internal Control amplicon is added to the individual D cups The biotin labeled HIV 1 target and HIV 1 Internal Control amplicon are hybridized to the target specific oligonuc
34. e 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 18 C for up to one month 2 to 30 C gt EEL E A Plasma 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Temperature C Time 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 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 Do not freeze whole blood Heparin has been shown to inhibit PCR Use of heparinized specimens is not recommended Warm pooled or individual donor specimens to room temperature before using 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 No adverse effect on assay performance was observed when plasma specimens were subjected to three freeze thaw cycles Thaw frozen specimens at room temperature before using The user should validate other collection and
35. east 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 controls 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 ring 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 specimens and controls Pipette 50 uL of each processed specimen and control into the appropriate A tube containing HIV 1 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 rier must begin within 45 minutes from when the first specimen or control in the A ring is added to the Working aster Mix C Reverse Transcription Amplification and Detection Performed in Post Amplification Amplification Detecti
36. ed trademark of the Hamilton Company ProClin is a registered trademark of Rohm and Haas Company Gopyright 2009 Roche Molecular Systems Inc All rights reserved 10 2009 05120705001 02 Doc Rev 2 0 05120705001 02EN 20 Doc Rev 2 0
37. eet 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 1 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 HIV 1 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 HIV 1 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 05120705001 02EN 9 Doc Rev 2 0 NEGATIVE Specimen is negative for HIV 1 RNA lt 0 2 NEGATIVE INVALID Invalid result Repeat entire test procedure for
38. ese pools 1082 were HIV 1 RNA negative The specificity of the COBAS AmpliScreen HIV 1 Test v1 5 in this study was 1082 1085 or 99 7235 with 95 confidence level of 99 19 to 99 94 NON CLINICAL PERFORMANCE Analysis of HIV 1 p24 Positive Antibody Negative Samples Twenty samples were selected from commercially available seroconversion panels that met the criterion of positive for HIV 1 p24 antigen and nega tive for anti HIV 1 2 using licensed tests The selected samples were diluted 1 96 in Normal Human Plasma that was found negative for HIV 1 RNA using the COBAS AmpliScreen HIV 1 Test v1 5 Each sample was processed diluted 1 96 using the Multiprep Specimen Processing Procedure to simulate Primary Plasma Pools The COBAS AmpliScreen HIV 1 Test v1 5 successfully detected HIV 1 RNA in all 20 samples that were positive by HIV 1 p24 antigen and negative for anti HIV 1 2 The results demonstrate that the test has sufficient sensitivity to detect HIV 1 yield samples in a 96 sample minipool format Results are summarized in Table 19 Table 19 Summary of Test Results US FDA Licensed HIV 1 p24 Antigen ron o o Messe ed COBAS AmpliScreen HIV 1 Test v1 5 j HIV 1 Seroconversion Panels Ten commercially available HIV 1 seroconversion panels were tested using the Multiprep Specimen Processing Procedure Blinded pane members e diluted an i HIV 1 negative human plasrna COBAS AmpliScreen HIV 1 Test v1 5 results were c
39. h may be performed separately or simultaneously At least one prepa ration 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 Perform manufacturer recommended maintenance and calibration on all instruments including pipettors to ensure proper functioning 9 romm zer 05120705001 02EN 6 Doc Rev 2 0 C Reagents 1 All reagents except HIV 1 MMX
40. idemiology Donor Study New England Journal of Medicine 334 1685 90 Holland PV 1996 Viral infections and the blood supply editorial New England Journal of Medicine 334 1734 35 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 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 Banks121 73 Presented at a workshop during the 50th Annual Meeting of the AABB October 1997 Denver CO Busch MP Lee LL Satten FA et al 1995 Time course of detection of viral and serologic markers preceding human immunodeficiency virus type 1 seroconversion implications for screening of blood and tissue donors Transfusion 35 91 7 Henrard DR Phillips J Windsor I et al Detection of human immunodeficiency virus type 1 p24 antigen and plasma RNA relevance to inde terminate serologic test Transfusion 1994 34 376 80 Soriano V Dronda F Gonzalez Lopez A et al 1994 HIV 1 causing AIDS and death in a seronegative individual letter Vox Sang 67 410 11 Centers for Disease Control and Prevention Persistent lack of detectable HIV 1 antibody in a person with HIV 1
41. imen and Control Preparation Performed in Pre Amplification Specimen and Control Preparation Area Multiprep Specimen Processing Procedure Pooled Specimens and Individual Cadaveric Specimens B1 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 pL into an appropriately labeled screw cap tube and add 800 uL Multiprep Diluent al mr using a hand held pipettor or other user validated method Cap the tubes Vortex each specimen tube briefly Proceed to tep 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 pL 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 the orientation marks facing outward so that the ori entation 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 wi
42. inding 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 HIV 1 Test version 1 5 96 Tests COBAS AmpliScreen HIV 1 Amplification Reagents version 1 5 HIV 1 MMX v1 5 8 x 0 7 mL HIV 1 Master Mix version 1 5 Bicine buffer Glycerol lt 0 01 rTth DNA Polymerase 7th pol microbial Potassium acetate lt 0 07 dATP dCTP dGTP dUTP dTTP lt 0 001 SKCC1B and SK145 biotinylated primers lt 0 01 AmpErase uracil N glycosylase enzyme microbial 0 05 Sodium azide HIV 1 Mn2 v1 5 8 x 0 1 mL HIV 1 Manganese Solution version 1 5 lt 2 Manganese Acetic acid Amaranth dye 0 05 Sodium azide 05120705001 02EN 3 Doc Rev 2 0 COBAS AmpliScreen HIV 1 Detection Reagents version 1 5 IH PS1 v1 5 HIV 1 Probe Suspension 1 version 1 5 MES buffer lt 0 01 Suspension of Dynabeads paramagnetic particles coated with HIV 1 specific oligonucleotide capture probe SK102 0 09 Sodiu
43. infection Utah 1995 MMWR 1996 45 181 85 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B Globin genomic sequences and restriction site analysis for diag nosis of Sickle Cell Anemia Science 230 1350 1354 Saiki R K Gelfand D H Stoffel S et al 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 487 491 Mullis K B Faloona FA 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods in Enzymology 155 335 350 Mulder J McKinney N Christopherson C et al 1994 Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma Application to acute retroviral infection Journal of Clinical Microbiology 32 292 300 Mortimer J 1997 intersecting pools and their potential application in testing donated blood for viral genomes Vox Sanguinis 73 93 6 Yerly S Pedrocchi M and Perrin L 1998 The use of polymerase chain reaction in plasma pools for the concomitant detection of hepatitis C and HIV 1 type 1 RNA Transfusion 10 908 914 Myers T W and Gelfand D H 1991 Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase Biochemistry 30 7661 7666 Kwok S Sninsky J J 1993 PCR detection of human immunodeficiency virus type 1 proviral DNA sequences In Diagnostic Molecular Biology Principles and Applications Eds Persing D H Smith T
44. ith 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 Standard Specimen Processing Procedure may also be used for testing of Source Plasma minipools The Multiprep and the Standard Specimen Processing Procedures are generic nucleic acid extraction procedures and can be used for the extraction of HIV 1 RNA HCV 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 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 tem
45. l samples were found to be negative No false positive test results were observed Potentially Interfering Substances Endogenous Interfering Substances HIV 1 spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of bilirubin up to 20 mg mL triglycerides 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 not inter fere with the sensitivity or specificity of the COBAS AmpliScreen HIV 1 Test v1 5 using either the Multiprep or Standard Specimen Processing Procedures Exogenous Interfering Substances HIV 1 spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of aspirin up to 50 mg mL pseu doephedrine HCI 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 of the COBAS AmpliScreen HIV 1 Test v1 5 using either the Multiprep or Standard Specimen Processing Procedures CLINICAL PERFORMANCE AIDS and HIV 1 Asymptomatic Populations HIV 1 antibody EIA Repeatedly Reactive samples from 217 patients diagnosed with AIDS and HIV 1 antibody EIA Repeatedly Reactive samples from 784 HIV 1 asymptomatic patients were randomly intermixed with 1 399 HIV 1 antibody EIA non Reactive plasma samples These 2 400 samples were used to create 10
46. leotide probes bound to the magnetic particles This hybridization of amplicon to the target specific probe increases the overall specificity of the COBAS AmpliScreen HIV 1 Test v1 5 Detection Reaction Following the hybridization reaction the COBAS AMPLICOR Analyzer washes the magnetic particles in the D cups to remove unbound material and then adds avidin horseradish 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 cat alyzes the oxidation of TMB to form a colored complex The absorbance is measured by the COBAS AMPLICOR Analyzer at a wavelength of 660 nm MATERIALS PROVIDED BY ROCHE The COBAS AmpliScreen 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 HIV 1 Test v1 5 as well as the COBAS AmpliScreen HCV Test v2 0 and the COBAS AmpliScreen HBV Test COBAS 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
47. licate tube s The test result for the pool or individual doner 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 donor specimen in that pool should be tested If an individual donor specimen gives an invalid result the test result for that individual donor specimen should be considered invalid for HIV 1 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 The testing algorithm for testing of pooled samples for the COBAS AmpliScreen HIV 1 Test v1 5 requires a single level of testing for Primary Pools that are negative for HIV 1 RNA and three levels of testing Primary Pool Secondary Pool and tertiary resolution for Primary Pools that are positive for HIV 1 RNA Negative Primary Pools When the Primary Pool is negative report the results for all associated individual donor specimens in that Primary Pool as HIV 1 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 Specimen Pro
48. ll form on the outer wall as indi RBR 05120705001 02EN 7 Doc Rev 2 0 cated by the orientation mark NOTE The 60 4 minutes begins when 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 supematant 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 Bi0 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 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 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 Incubate all tubes for 10 to 15 minutes at room temperature after adding Working Lysis Reagent to the last tube After the incubation period briefly vortex all tubes Pipette 700 uL of isopropanol into each tube Cap
49. m azide 1H4 vi 5 HIV 1 Probe Suspension 2 version 1 5 Sodium phosphate buffer 24 9 Sodium thiocyanate 0 2 Solubilizer ll PS1 v1 5 IC Probe Suspension 1 MES buffer lt 0 01 Suspension of Dynabeads paramagnetic particles coated with HIV 1 IC specific oligonucleotide capture probe CP35 0 09 Sodium azide Il 4 v1 5 IC Probe Suspension 2 Sodium phosphate buffer 24 9 Sodium thiocyanate lt 0 2 Solubilizer 1 x 100 Tests 1 x 100 Tests 1 x 100 Tests 1 x 100 Tests DN4 1 x 100 Tests Denaturation Solution 1 6 Sodium hydroxide EDTA Thymol blue Xi bd 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 SB 10 x 75 Tests Substrate B 10 x 5 mL 0 1 3 3 5 5 Tetramethyibenzidine TMB 40 Dimethylformamide DMF T 40 w w Dimethylformamide DMF Toxic R 61 20 21 36 May cause harm to the unbom child Harmful by inhalation and in contact with skin 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
50. ma by lysis of the virus particles with Multiprep Lysis Reagent followed by precipitation of the RNA with alcohol In the Multiprep Specimen Processing Procedure HIV 1 viral particles are first pelleted from the plasma sample by high speed centrifugation followed by lysis of the pelleted virus with a chaotropic agent Multiprep Lysis Reagent and precipita tion of the RNA with alcohol The Multiprep Internal Control MP IC containing the HIV 1 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 HIV 1 Internal Control is an RNA transcript with primer binding regions identical to those of the HIV 1 target sequence a randomized internal sequence of similar length and base composition as the HIV 1 target sequence and a unique probe binding region that differentiates the HIV 1 Internal Control amplicon from target amplicon These features were selected to ensure equivalent amplification of the HIV 1 Internal Control and the HIV 1 target RNA Reverse Transcription The reverse transcription and amplification reactions are performed with the thermostable recombinant enzyme Thermus thermophilus DNA Polymerase rTth pol In the presence of manganese Mn and under the appropriate buffer conditions rTth pol has both reverse transcriptase and DNA poly merase activity 8 This allows both reverse transcription and PCR amplification to
51. mber of Number of Positive 95 Lower Concentration Positives individual Tests Confidence Limit c mL One Tailed 100 0 98 6 wo m ar e 5 Table 4 wo Standard Procedure Testing Summary for All Clinical Samples Combined Input Values with 95 One tailed Lower Confidence Limit Standard Sample Processing Procedure HIV 1 RNA Number of Number of Positive 95 Lower Concentration Positives Individual Tests Confidence Limit c mL One Tailed Analytical Sensitivity WHO HIV 1 International Standard The analytical sensitivity of the COBAS AmpliScreen HIV 1 Test v1 5 was also determined using the WHO HIV 1 International Standard 97 656 The WHO HIV 1 International Standard was serially diluted in HIV 1 negative plasma to final concentrations of 140 100 70 50 35 and 25 IU mL for the Multiprep Specimen Processing Procedure and 800 560 400 280 200 and 140 IU mL for the Standard Specimen Processing Procedure Each dilution was tested using two lots of COBAS AmpliScreen HIV 1 Test v1 5 When evaluated using PROBIT analysis the combined data from all samples using the Multiprep Sample Processing Procedure indicate an average 95 LOD of 78 4 IU mL with lower and upper 95 confidence limits of 68 4 IU mL and 94 4 IU mL respectively When evaluated using PROBIT analysis the combined data from all samples tested using the Standard Sample Processing Procedure indicate an average 95 LOD of 323
52. men Processing procedure and a mean of 5 8 to 8 3 days before the licensed HIV 1 p24 antigen tests when using the Standard Specimen Processing Procedure The data are presented in Tables 9 and 10 Table 9 Summary of the Pre Seroconversion Detection of HIV 1 RNA vs HIV 1 2 Antibody and HIV 1 p24 Antigen Assays Multiprep Specimen Processing Procedure Days Before HIV 1 2 Days Before Abbott Days Before Coulter Antibody p24 Antigen p24 Antigen 41 Panels Tested 40 Panels Tested 38 Panels Tested For one panel the time interval between sampling was 80 days Table 10 Summary of the Pre Seroconversion Detection of HIV 1 RNA vs HIV 1 2 Antibody and HIV 1 p24 Antigen Assays Standard Specimen Processing Procedure p24 Antigen p24 Antigen 40 Panels Tested 38 Panels Tested For one panel the time interval between sampling was 80 days Dilutional Sensitivity with Weakly Reactive HIV 1 p24 Antigen Samples Twenty five HIV 4 p24 antigen weakly positive S CO 1 00 to 3 7 using a licensed HIV 1 p24 EIA samples were evaluated These were diluted with HIV 1 negative plasma to 5 000 copies mL and further diluted 1 24 to penne the Primary Pool The HIV 1 RNA copy numbers were determined by a commercially available HIV 1 quantitative assay Roche s AMPLICOR HIV 1 MONITOR Test The final viral concentration was approximately 208 copies mL In addition another set was diluted to 100 copies mL All 25 samples tested at 5
53. mns 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 Hamilton MICROLAB AT plus 2 Pipettor with SUNRISE PLUS v3 3 soft ware was used to prepare pools of up to 96 equal aliquots of plasma during clinical trials NOTE The user must validate other 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 for 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 as possible after collection The use of excessively hemolyzed cadaveric specimens should be avoided PROCEDURAL NOTES A Run Size 1 Each kit contains reagents sufficient for eight 12 specimen runs whic
54. mples The high sensitivity of PCR has demonstrated that potentially infectious donations contained within mini pools can be detected even if the mini pool contains a single viremic donor 25 26 The assay incorporates an Internal Control for monitoring assay performance in each individual test as well as the AmpErase enzyme uracil N gly cosylase to reduce potential contamination by previously amplified material amplicon PRINCIPLES OF THE PROCEDURE The COBAS AmpliScreen HIV 1 Test v1 5 is based on five major processes Sample Processing Reverse transcription of target RNA to generate complementary DNA cDNA 27 PCR amplification of target cDNA using HIV specific complementary primers Hybridization of the amplified products to oligonucleotide probes specific to the target s Detection of the probe bound amplified products by colorimetric determination Sample Processing Two specimen processing procedures are used with the COBAS AmpliScreen HIV 1 Test v1 5 as follows e Multiprep Specimen Processing Procedure for preparation of mini pool specimens and individual cadaveric specimens e 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 HIV 1 RNA is isolated directly from plas
55. n HIV 1 Test v1 5 using both the Standard Sample Processing Procedure and the Multiprep Sample Processing Procedure Specimens for the Multiprep Procedure were diluted 1 24 with Normal Human Plasma Samples for the Standard Procedure were tested without dilution Of 374 specimens tested 55 were found positive for HIV 1 RNA when tested using the Standard procedure and 54 were found positive when tested using the Multiprep procedure One sample was found to be positive when tested using the Standard Sample Processing Procedure but negative when diluted 1 24 with NHP and tested using the Multiprep Sample Processing Procedure This sample was negative when tested by both HIV 1 p24 antigen and HIV 1 antibody tests indicating that this sample may be a window period specimen Fifty four of the 55 specimens were positive either by a quantitative HIV 1 RNA test or p24 Antigen test Samples were judged to be NAT serology concor dant if the NAT result was 1 positive and at least one serologic assay is positive or 2 negative and serologic assays are both negative A total of 316 of the 374 samples were negative for HIV 1 antibody There were three antibody positive specimens that were negative for HIV 1 RNA using the COBAS AmpliScreen HIV 1 Test v1 5 However these specimens were negative by HIV 1 p24 antigen EIA and when tested with a quantitative assay AMPLICOR HIV 1 MONITOR Test v1 5 the titer was below the assay detectable limit The data are presented i
56. n Tables 14 and 15 Table 14 Clinical Sensitivity in a High Risk Population with Standard Prep HIV 1 Antibody Reactive HIV 1 by EIA HIV 1 Antibody Antibody HIV 1 RNA 2 100 c mL Negative Negative RNA 2 100 c mL Western Blot Western Blot NT Neg Neg Ind Ind 5 a 8 EAA HIV 1 Antibody Reactive by EIA o ee a al P Def e fe e Table 15 Clinical Sensitivity in a High Risk Population with Multiprep HIV 1 Antibody Reactive by EIA HIV 1 RNA 2 100 c mL HIV 1 Antibody Reactive by EIA HIV 1 Antibody Negative RNA 2 100 c mL HIV 1 Antibody Negative Pool Reactivity in Volunteer Blood Donors A random selection of 10 727 primary pools revealed that 26 primary pools were reactive with the COBAS AmpliScreen HIV 1 Test v1 5 for an ini tially reactive rate of 0 24 There were 11 reactive pools with at least 1 confirmed anti HIV positive specimen and 0 pools were positive due to con firmed window period cases A total of 15 pools were reactive but were not confirmed Results are summarized in Table 16 Table 16 Pool Reactivity in Volunteer Blood Donors Category Initial reactive pools with negative serology and negative individual donation AmpliScreen Testing false positive A total of 792 055 specimens were selected from geographically divergent sites The results from these specimens were used to determine the speci ficity and sensitivi
57. nalyzer 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 i 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 1 Screw cap tubes must be used for specimen and control preparation to prevent splashing and potential cross contamination of speci mens 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 contro 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 w
58. of 100 copies mL Because each of these samples when diluted 24 fold would not be expected to be reliably detected in 24 membered mini pools they were removed from the sensitivity calculation Therefore the overall observed sensitivity of the COBAS AmpliScreen HIV 1 Test v1 5 in this study was 100 0 Analytical Specificity Potentially Cross Reactive and Interfering Microorganisms The analytical specificity of the COBAS AmpliScreen HIV 1 Test v1 5 was evaluated by testing a pane of microorganisms and other disease states including 21 viral isolates five bacterial strains and one yeast isolate No cross reactivity was observed with the COBAS AmpliScreen HIV 1 Test v1 5 Table 11 below summarizes the microorganisms studied Table 11 Analytical Specificity Microorganisms and Disease States Tested l Neisseria gonorrhoeae e Up to 25 individual patient plasma specimens from each of the following disease categories were spiked with low levels of HIV 1 positive plasma HAV HBV HCV HIV 2 autoimmune disease EBV CMV and Candida albicans No false negative test results were observed Analytical Specificity Non HIV 1 Samples Up to 25 individual patient plasma specimens all HIV 1 negative from each of the following disease categories HAV HBV HCV HIV 2 autoimmune disease EBV CMV and Candida albicans were tested with COBAS AmpliScreen HIV 1 Test v1 5 by using both Multiprep and Standard Specimen Processing Procedures Al
59. ompared to HIV 1 2 antibody and HIV 1 p24 antigen results In two panels the COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA on the same bleed as HIV 1 p24 antigen In the remaining 8 panels COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA 2 to 12 days earlier Data are presented in Table 20 Table 20 Summary of the Pre Seroconversion Detection of HIV 1 RNA vs HIV 1 2 Antibody and HIV 1 p24 Antigen Assays Multiprep Specimen Processing Procedure Days Before Days Before Days Before HIV 1 2 Antibody Abbott HIV 1 Coulter HIV 1 10 panels tested p24 Antigen p24 Antigen 10 panels tested 10 panels tested Men 12 12 i i 05120705001 02EN 16 Doc Rev 2 0 In 100 of the HIV 1 seroconversion panels tested COBAS AmpliScreen HIV 1 Test v1 5 detected HIV 1 RNA prior to anti HIV 1 2 reactivity range 9 to 15 days Positive Pooled Deconstruction This study was performed with 10 pools Each pool had 1 to 10 positive sample s intermixed among the 96 members and was used to evaluate the ability of the resolution algorithm to correctly identify the positive member or members and to evaluate the pooling dilution effect The algorithm includes the following three testing levels Level 1 96 Member Mini pool Testing Level 2 Column and Row Mini pool Testing and Level 3 Single sample testing The results of this study demonstrate that the 96 sample pooling strategy in combination with the COBAS AmpliScreen HIV 1 Test v1 5 i
60. on Area C1 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 05120705001 02EN 8 Doc Rev 2 0 b Wipe initialization post with a lint free moist cloth and dry C2 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 C3 Instrument Loading and System Operation a Prepare enough of the following detection reagent cassettes to complete the workload Working HIV 1 Probe Suspension Reagent IH4 v1 5 Working IC Probe Suspension Reagent II PS1 v1 5 Working Substrate SB3 Denaturation Reagent DN4 and Conjugate Reagent CN4 Place the IH4 v1 5 and II PS1 v1 5 cassettes in the test specific reagent rack Place DN4 GN4 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 using the COBAS AMPLICOR Analyzer barcode scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software f
61. or detecting antibodies to HIV 1 The COBAS AmpliScreen HIV 1 Test v1 5 may not be used to replace HIV 1 antibody detection tests such as EIA or Western Blot See Performance Characteristics section Tables 12 and 13 4 aparin oe PCR specimens collected using heparin as the anticoagulant should not be used with the COBAS AmpliScreen a esi V1 3 5 Reliable results are dependent on adequate specimen collection and proper transport procedures 05120705001 02EN 10 Doc Rev 2 0 6 Detection of HIV 1 RNA is dependent on the number of virus particles present in the specimen and may be affected by specimen collection methods patient factors i e age presence of symptoms and or stage of infection and pool size 7 Only the Hamitton MICROLAB AT plus 2 Pipettor has been validated for use with the COBAS AmpliScreen HIV 1 Test v1 5 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 8 Though rare mutations within the highly conserved region of the viral genome covered by the COBAS AmpliScreen HIV 1 Test v1 5 primers and or probe may result in the failure to detect the virus 9 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 CHARA
62. ositive the positive cadaveric specimen is reported as HIV 1 RNA Positive if an individual cadaveric specimen is negative the negative cadaveric specimen is reported as HIV 1 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 HIV 1 RNA Positive If both duplicate specimens are negative or if one duplicate is negative and one is invalid the specimen is reported as HIV 1 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 AmpliScreen Multiprep Specimen Preparation and Control Kit COBAS AMPLICOR Analyzer and the Hamilton MICROLAB AT plus 2 Pipettor for the automated preparation of plasma pools 2 Eight Group O culture specimens were only evaluated as diluted samples due to limited specimen volume All HIV 1 Group O specimens tested were found to be HIV 1 p24 antigen positive however only five 63 were detected by the COBAS AmpliScreen HIV 1 Test v1 5 These data indicate that the COBAS AmpliScreen HIV 1 Test v1 5 will not consistently detect HIV 1 RNA in all Group O specimens 3 This COBAS AmpliScreen HIV 1 Test v1 5 is intended to be used in conjunction with licensed tests f
63. ots of the COBAS AmpliScreen HIV 1 Test v1 5 The COBAS AmpliScreen HIV 1 Test v1 5 using samples diluted 1 5 and the Multiprep Specimen Processing Procedure yielded negative results on 96 7 58 60 of the pre mortem EDTA plasma specimens and 100 57 57 of the post mortem EDTA plasma specimens One post mortem EDTA plasma specimen exhibited inhibition on initial and repeat testing The summary of the specificity study results is presented in Table 22 below Table 22 Summary of Specificity Test Results Pre Mortem EDTA Plasma Specimen Total Specimens Tested GO 2 5 100 95 Confidence Interval Two pre mortem specimen found initially reactive were negative upon repeat testing Reproducibility Study Twenty pre mortem EDTA plasma and 20 individual cadaveric specimens were spiked with HIV 1 viral target using a secondary standard to a final concentration of 3X the LOD Each of the 20 pre and post mortem specimens were tested using three different COBAS AmpliScreen HIV 1 Test v1 5 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 percen
64. perature 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 Determine the appropriate number of A ring s needed for specimen and control testing Place the A ring s on the A ring holder s For each A ring prepare one Working Master Mix 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 AS Place the A ring containing Working Master Mix in a sealable bag and seal the plastic bag Record the assay name HIV 1 and the time the Working Master Mix was prepared A6 Store the A ring s containing 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 Item I B Spec
65. rs If not the processed specimens and controls 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 B23 B24 B25 B26 B27 B28 B29 B30 B31 B32 B33 B34 B35 B36 B37 B39 B41 B42 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 Vortex NHP briefly For each Negative and Positive Contro pipette 200 uL NHP into appropriately labeled screw cap tubes Cap the tubes Use a permanent marker to make an orientation mark on each tube Prepare a Working Lysis Reagent bottle for every 12 specimens and controls to be processed Pipette 600 uL Working Lysis Reagent into each tube Cap and vortex tubes briefly Prepare Controls as follows a Negative Control Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 pL MP C into the tube labeled MP C con taining 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 pL MP C into the tube labeled MP C con taining Working Lysis Reagent and NHP Cap
66. s 9_ The prevalence of HIV 1 infection is 1 1 overall in the world 0 56 in North America and 0 25 in West Europe Serological screening assays have greatly reduced but not completely eliminated the risk of transmitting viral infections by transfusion of blood prod ucts 2 15_ HIV 1 p24 antigen is the principal core protein of HIV 1 and is found in serum or plasma either free or bound by anti p24 antibody HIV 1 p24 antigen can be measured with commercially available enzyme immunoassays EIA which reduce the seroconversion window period i e the time between infection and the rise of antibodies to the virus by approximately 5 to 6 days17 18 Recent studies indicate that nucleic acid based amplification tests for HIV 1 RNA will further reduce the residual transmission risk by detecting HIV 1 RNA in donations made during the serocon version window period Nucleic acid based tests can detect viremic units donated by carriers who do not seroconvert or who lack antibodies to sero logical markers normally detected by immunological assays 6 19 20 HIV 1 RNA in plasma can be detected by nucleic acid amplification technologies such as the Polymerase Chain Reaction PCR 21 23 The COBAS AmpliScreen HIV 1 Test v1 5 uses PCR technology to achieve maximum sensitivity for the detection of HIV 1 RNA in plasma samples 4 A number of proposals have been made for performing nucleic acid tests on mini pools comprised of small aliquots from many individual sa
67. s capable of detecting and identifying HIV 1 positive donations in plasma minipools of 96 samples NON CLINICAL PERFORMANCE CHARACTERISTICS FOR CADAVERIC SPECIMENS Sensitivity Study Sixty pre mortem EDTA plasma and fifty eight cadaveric EDTA plasma specimens non reactive for HIV 1 were divided into 5 groups Specimens within each group were spiked with HIV 1 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 HIV 1 Test v1 5 kit lots The COBAS AmpliScreen HIV 1 Test v1 5 using samples diluted 1 5 and the Multiprep Specimen Processing Procedure correctly detected 100 60 60 pre mortem EDTA plasma specimens and 94 8 55 58 of cadaveric specimens spiked with HIV 1 RNA at 3X the LOD Two post mortem specimens negative on the initial test were repeated and found to be positive One specimen that exhibited inhibition on initial testing was negative on repeat testing The summary of the test results of this study is presented in Table 21 below Table 21 Summary of Sensitivity Test Results Pre Mortem EDTA Post Mortem EDTA Plasma Specimen Plasma Specimen 95 Confidence Interval Specificity Study Sixty pre mortem and 60 post mortem specimens which were negative for HIV 1 RNA were divided into three groups diluted 1 5 in MP DIL processed using the Multiprep Specimen Processing Procedure and tested using 3 l
68. sting after resolution at Roche is provided in Table 13 Sensitivity and specificity were based upon the final resolution status of all samples Individual specimens known to contain less than 100 copies mL were not included in the sensitivity calculation The sensitivity of the COBAS AmpliScreen HIV 1 Test v1 5 relative to HIV 1 antibody EIA Repeatedly Reactive status in this study was determined to be 99 7 with the 95 confidence interval ranging from 99 1 to 100 The specificity of the COBAS AmpliScreen HIV 1 Test v1 5 relative to HIV 1 antibody EIA non Reactive status in this study was determined to be 98 9 with the 95 confidence interval ranging from 98 3 to 99 3 Table 12 Results of HIV 1 Antibody EIA Repeatedly Reactive Specimens Tested at the Clinical Sites Discordant Specimens lt 100 Copies mL Removed HIV 1 Antibody EIA Total Repeatedly Non Reactive Reactive COBAS Ampiioree Hive Tea vi S Benli T 1977 Ooo O e i 05120705001 02EN 14 Doc Rev 2 0 Table 13 Results of HIV 1 Antibody EIA Repeatedly Reactive Specimens Discordant Specimens lt 100 Copies mL Removed Following Resolution Testing at Roche COBAS AmpliScreen HIV 1 Test v1 5 Result High Risk Population Specimens were prospectively collected from a population of patients being evaluated at AIDS clinics Specimens were tested in a blinded fashion in order to identify at least 50 HIV 1 RNA positives with the COBAS AmpliScree
69. 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 L False positive results may occur if cross contamination of specimens is not adequately controlled during specimen handling and processing M ECIMEN 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 individual samples into a single Primary Pool that is used for testing The pooling algorithm requires preparation of Secondary Pools as well as indi vidual specimens for follow up testing in the event a Primary Poo tests positive If less than 24 specimens are available testing is per formed using the individual specimens 2 For Source Piasma the Hamilton performs barcode scanning and pooling operations that combine aliquots from 96 individual samples into a single Primary Pool that is used for 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 colu
70. tage 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 23 below Table 23 Summary of Reproducibility Study Test Results Post Mortem versus Pre Mortem a Results by Lot Positive Tested Percent Hit Rate 118 120 119 120 ee 98 3 99 2 120 120 119 119 116 120 119 120 Lot ae 96 7 99 2 Results by Site Positive Tested Percent Hit Rate 114 120 118 120 it R 120 120 120 120 i 120 120 119 119 oe Ba 100 100 05120705001 02EN 17 Doc Rev 2 0 REFERENCES i 10 Fie 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Barre Sinoussi F Chermann J C Rey F 1983 Isolation of a T lymphotropic retrovirus from a patient at risk for acquired immunodeficiency syndrome Science 220 868 8771 Popovic M Sarngadharan M G Read E et al 1984 Detection isolation and continuous production of cytopathic retroviruses HTLV II from patients with AIDS and pre AIDS Science 224 497 500 Gallo R C Salahuddin S 2 Popovic M et al 1984 Frequent detection and isolation of cytopathic retroviruses HTLV II from patients with AIDS and at risk for AIDS Science 224 500 502 Curran J W Jaffe H W Hardy A M et al 1988 Epidemiology of HIV 1 Infection and AIDS in the United States Science
71. tive donors The specificity of the COBAS AmpliScreen HIV 1 Test v1 5 in this study was 100 271 271 with 95 confidence interval of 98 65 to 100 There were no HIV 1 RNA positive specimens detected by individual donation testing using the COBAS AmpliScreen HIV 1 Test v1 5 PERFORMANCE CHARACTERISTICS OF SOURCE PLASMA Clinical Performance A total of 104 448 donations from 35 905 donors were tested in the 96 member minipoo format in 1 088 pools Two donations from 2 donors were positive for HIV 1 RNA and negative by antibody to HIV 1 2 and HIV 1 p24 antigen One additional donation from one donor was positive for HIV 1 RNA and negative by antibody to HIV 1 2 but positive for HIV 1 p24 antigen Of the 2 eligible donors one was lost to follow up and the other refused enrollment Additional testing on the index donation was positive by both an alternate NAT procedure and a commercially available HIV quantitation assay The quantitation in these two samples were 4 116 and 74 428 copies mL The data are presented in Table 18 Table 18 Pool Reactivity in Source Plasma Donors Pools tested Non Reactive pools Initially Reactive pools Initial pools containing donation with concordant serology Positive pools due to window case Initially Reactive pools with negative resolution COBAS AmpliScreen Testing false positive There were 1085 pools that were used to determine the specificity of HIV 1 RNA Of th
72. ty of COBAS AmpliScreen HIV 1 Test v1 5 Using the antibody and antigen results the HIV 1 status of each specimen was deter mined HIV 1 status negative included either 1 anti HIV 1 EIA negative and HIV 1 p24 antigen negative EIA nonreactive or neutralization negative unless the subject was enrolled in the follow up study and had test results that changed this assessment or 2 anti HIV EIA repeatedly reactive WB IFA negative and HIV 1 p24 antigen negative or indeterminate HIV 1 status positive included either 1 anti HIV 1 EIA repeatedly reactive WB IFA positive or 2 follow up study test results of anti HIV 1 repeatedly reactive or HIV 1 RNA positive HIV status unknown included anti HIV 1 EIA repeat reactive WB IFA indeterminate or unknown HIV 1 p24 antigen negative or indeterminate There were 791 733 specimens that were determined to be HIV status negative Of these 791 732 were also HIV 1 RNA negative The specificity of the COBAS AmpliScreen HIV 1 Test v1 5 in this study was 791 732 791 733 or 99 9999 with 95 confidence limits of 99 99 to 100 00 There were 42 specimens that were determined as HIV 1 status positive Of these 38 were also HIV 1 RNA positive The sensitivity of the COBAS AmpliScreen HIV 1 Test v1 5 in this study was 38 42 or 90 48 with 95 confidence limits of 77 38 to 97 34 A total of 463 specimens were repeatedly reactive by EIA and of those 39 were also COBAS AmpliScreen HIV 1 Test v1 5 positive
73. v1 5 and HIV 4 Mn2 v1 5 must be at room temperature before use Visually examine reagents for suf ficient 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 disinfected 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 sev eral 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 free area for preparation of Working Master Mix and an amplicon free area for spec imen and contro preparation 3 The Post Amplification Area should have a COBAS AMPLICOR A
74. ve 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 nat 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 lab oratory 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 rec ommended to avoid accumulation of deposits in metal piping in which explosive conditions could develop Heparin has been shown to inhibit 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

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