cobas TaqScreen DPX Test
Contents
1. Mol CD Arvai AS Slupphaug G Kavli B Krokan HE Mosbaugh DW Tainer J mutational analysis of human uracil DNA glycosylase structural basis for speci 1995 80 869 878 Higuchi R Dollinger G Walsh PS Griffith R Simultaneous amplific a letection of specific DNA sequences Biotechnology NY 1992 10 413 417 Heid CA Stevens J Livak JK Williams PM Real time quantitative9PCR Genome Res 1996 6 986 994 Richmond J Y and McKinney R W eds 1999 Biosafety in Microbiological and Biomedical Laboratories HHS Publication Number CDC 93 8395 Clinical and Laboratory Standards Institute CL boc of Laboratory Workers from Occupationally Acquired Infections Approved Guideline Third Ei gt Document M29 A3 Wayne PA CLSI 2005 nternational Air Transport Association Dafigero s Goods Regulations 41st ed Quebec Canada 2000 Saldanha J Heath A Lelie N sehen Y and the Collaborative Study Group A World Health Organization International Stand titis A virus RNA nucleic acid amplification technology assays Vox Sanguinis 2005 89 52 58 Q Q ge 05509238001 02EN 25 Doc Rev 2 0 The following symbols are now used in labeling for Roche PCR diagnostic products Ancillary Software For in vitro diagnostic use For IVD Performance Evaluation only m Sl Authorized representative amp BARCODE Barcode Data Sheet LL Lower Limit of A Batch code padl TTE Biological Risk Potentially CoN dark2. Next Review control results on the Controls Review tab Refer to the Quality Control Section for control validity criteria 05509238001 02EN 14 Doc Rev 2 0 5 Review pool results on the Pools Review tab for the selected batch Pools with results of non reactive for HAV and lt cutoff for B19V can be invalidated manually by the user if required Donor specimens in an invalid pool must be retested 6 Review and release donors on the Donor Review tab for the selected batch 7 Print reports and send to Laboratory Information System LIS if applicable QUALITY CONTROL 1 One Negative Control DPX C and one of each of the two Positive Controls DPX D C and DPX H C must be processed with each batch 2 control within a batch is invalid the entire batch is invalid The invalidation of results based ntrol failures is performed automatically by the PDM software a Negative Control V Validity of the Negative Control is verified according to the test performed Batch Status A Batch Status of Complete Valid is assigned when the batch controls are aN any DPX Test HAV and B19V For the Negative Control DPX C to be val nterpreted result must be non reactive for both HAV and B19V and the associated QS must be valid If the interpreted result for the Negative Control is invalid the entire batch i id and must be repeated erpreted result must be non i reted result for the Negative Va
3. SD lev Lot Site Day Run Reactive Reactive Reactive Reactive D vaia vania vaia P vaia 0 5xLOD 37 9 1 35 3 6 1 49 60 _ 81 7 1 40 59 678 1 25 35 62 87 _ 71 3 2 40 56 714 2 46 60 767 2 27 35 67 89 75 3 3 40 60 66 7 3 43 57 _ 754 3 23 36 4 25 34 5 29 36 1 0xLOD 37 4 1 26 3 4 1 59 60 98 3 1 56 60 93 3 1 35 36 8 94 4 2 56 59 949 2 60 60 100 0 2 34 36 89 90 8 9 3 58 60 96 7 3 57 59 96 6 3 34 36 V 4 35 35 5 35 36 3 0x LOD 35 9 0 87 2 4 1 60 60 100 0 1 60 60 100 0 1 36 36 88 88 100 0 2 58 58 100 0 2 60 60 100 0 2 35 35 90 90 100 0 3 60 60 _ 100 0 3 58 58 _ 100 0 3 36 4 3 5 Cycle threshold The study generated one false Reactive result in 177 valid DPX testSyperformed on negative panel members The false Reactive was due to a Reactive B19V below the loWer limi quantitation Table 5 provides the analytical specificity estimated using the negative panel member Analytical Specificity Estimate Negative Panel Members No of Tests Reactive Result sults Specificity 95 CI 177 1 99 4 96 9 100 0 Note The result for B19V must be Ti etected and the result for HAV must be Non reactive to produce an overall negative sult 95 exact confidence interval Clinical Pool Resolution Pooling resolution fo and 5 pools of 480 e DNA and HAV RNA identification was assessed separately for 5
4. 22 microorganisms including 16 viral isolates 5 rains and 1 yeast isolate Table 15 The microorganisms were added to normal human plas ested with and without HAV at 3X the limit of detection and B19V at 5X the lower limit of quanti of the cobas TaqScreen DPX Test The potential cross reactivity and interference by other micro organisms with the cobas TagScreen DPX Agy Non reactive results were obtained with the cobas TaqScreen DPX Test for all of the organism samples tested without HAV and B19V The microorganisms tested do not cross react withthe cobas TaqScreen DPX Test Reactive results were obtained for all of the microorganism samplesste h HAV and B19V The microorganisms tested do not interfere with the cobas TaqScreen DPX EA Table 15 Microorganisms Tested Analytical Specificity Microorganisms Tested i Coagulase negative Adenovirus 5 Candida awen y Staphylococcus epidermidis Cytomegalovirus Epstein B i Escherichia coli Hepatitis B virus Hepatitis Cvi Hepatitis G virus Human immunodeficiency virus Herpes simplex virus type 1 X virus type 2 type 1 group M Human immunodeficiency virus Human T cell lymphotropic virus Human T cell lymphotropic virus type 2 pi type Il Influenza A virus luenza B virus Propionibacterium acnes Staphylococcus aureus Streptococcus viridans Varicella Zoster West Nile virus Potentially reactive and Interfering Other Dis
5. Mortimer PP Detection of antibodies and antigen of human parvovirus B18 by enzyme linked immunosorbent assay Journal of Clinical Microbiology 1986 24 522 526 3 Cohen BJ Buckley MM The prevalence of antibody to human parvovirus B19 in England and Wales Journal of Medical Microbiology 1988 25 151 153 4 Young NS Brown KE Mechanisms of disease parvovirus B19 New England Journal of Medicine 2004 350 586 597 5 Yoto Y Kudoh T Haseyama K Suzuki N Oda T Katoh T Takahashi T Sekiguchi S Chiba S Inciden e of human parvovirus B19 DNA detection in blood donors British Journal of Haematology 1995 9 1017 1018 6 McOmish F Yap PL Jordan A Hart H Cohen BJ Simmonds P Detection of parvovi donated blood a model system for screening by polymerase chain reaction Journal Microbiology 1993 31 323 328 7 Wu CG Mason B Jong J Erdman D McKernan L Oakley M Soucie M Bvatt B Yu MY Parvovirus B19 transmission by a high purity factor VIII concentrate Transfusion 2005 45 10 8 Saldanha J Minor P Detection of human parvovirus B19 in plasm blood products derived from these pools implications for efficiency and consistency of removah o DNA during manufacture gt British Journal of Haematology 1996 93 714 719 9 Eis Hubinger AM Sasowski U Brackmann HH Parvovii 9 DNA contamination in coagulation factor VIII products Thrombosis and Haemostasis 1999 81 476 10 Schmidt Blumel J Seitz H Wilkommen H Lowef J Parvovirus B19 DNA
6. SQ or Hamilton Disinfectant Spray Kit P N 06254250001 ICROLAB Detergent Kit P N 06254268001 Cy able gloves powderless 05509238001 02EN 5 Doc Rev 2 0 REAGENTS cobas TaqScreen DPX Test P N 05509211 190 DPX CS1 MGP Magnetic Glass Particles Magnetic glass particles 93 Isopropanol Xi 93 w w lsopropanol Irritant 93 w w lsopropanol Highly Flammable DPX CS2 LYS Lysis Reagent Sodium citrate dihydrate 42 5 Guanidine thiocyanate lt 14 Polydocanol 0 9 Dithiothreitol Xn 42 5 w w Guanidine thiocyai Harmful A DPX CS3 Pase Proteinase Solution K TRIS buffer lt 0 05 EDTA Calcium chlori Calcium ace lt 7 8 Pri EB Elution Buffer TRIS buffer 0 2 Methylparaben preservative 05509238001 02EN 96 Tests 2 x 48 Tests 2x7 0 mL 9 2 x 48 Tests 2x78 mL 2 x 48 Tests 2x3 8 mL 2x7 0 mL Doc Rev 2 0 DPX CS4 DPX MMX R1 DPX Master Mix Reagent 1 Potassium acetate Manganese acetate Glycerol 14 4 Dimethyl sulfoxide 0 08 Sodium azide Acetic Acid DPX MMX R2 DPX Master Mix Reagent 2 Tricine buffer Potassium acetate Potassium hydroxide 4 1 Dimethyl sulfoxide Glycerol Tween 20 lt 0 09 dATP dGTP dCTP dUTP dTTP lt 0 01 Upstream and downstream B19V and HAV primers lt 0 01 Fluorescent labeled B19V and HAV probes lt 0 01 Fluorescent labeled B19V QS and HAV IC probes lt 0 01 Oligonucleotide aptamer lt
7. and PCR amplification to occur in the same reaction mixture During PCR amplification the intermittent high temperature during the cycling denatures the target and IC QS amplicon to form single stranded DNA Z05 DNA Polymerase extends the annealed primers along the target templates to produce double stranded DNA amplicon This process is repeated for multiple cycles with each cycle doubling the amount of amplicon Amplification occurs only in the region of the target genomes between the primers the entire genomes are not amplified Prevention of Carry over Contamination Amplicon carry over contamination is prevented by the use of AmpErase uracil N glycosylase Nand deoxyuridine triphosphate dUTP Deoxyuridine is not present in naturally occurring DN is always present in amplicon because of the use of a deoxyuridine triphosphate thymidine triphosphate mi as one of the dNTPs in the Master Mix reagent therefore only amplicon contains deoxyuridine Ai ecognizes and catalyzes the destruction of DNA strands containing deoxyuridine by opening ibose chain at the C1 position thereby rendering the DNA non amplifiable DNA containing d midine or RNA containing ribouridine 7 is not affected During the initial reverse transcription step ase catalyzes the cleavage of carry over amplicon at the deoxyuridine residues AmpErase is ina tive for a prolonged period of time once exposed to temperatures above 55 C and therefore does not ly f rme
8. biohazardous material Bae amp Catalogue Number Temperature limitation Store At Test Definition File Consult instructions Contains sufficient for lt n gt tests Upper Limit of Assigned Range g AA Ofitents of kit T Use by last day of month Distributed by Epe US Customer Technical Support 1 800 428 2336 05509238001 02EN 27 Doc Rev 2 0 Document Revision Information Doc Rev 2 0 AMPLILINK Software v3 3 2 and PDM Software v3 0 27 were added to the 07 2011 Instrumentation and Software for cobas s 201 system section The INSTRUCTIONS FOR USE section was revised to indicate that SPU racks should be loaded into COBAS AmpliPrep Instrument in positions J K or L Please contact your local Roche Representative if you have any questions 4 Roche Molecular Systems Inc Branchburg NJ 08876 USA V A Member of the Roche Group U S Device Master File No 14454 O Roche Diagnostics 9115 Hague Road Indianapolis IN 46250 0457 USA Sy Distributed by For Technical Assistance call the Roche Response Center toll free 1 800 526 1247 ROCHE COBAS COBAS S TAQMAN TAQS AMPLILINK AMPERASE and AMPLIPREP are trademarks of Roche ROCHE RESPONSE CENTER is a service oche All other product names and tri s ate the property of their respective owners Armored RNA is a pateated technology developed by Ambion Inc and Cenetron Diagnostics LLC U S patents 45 677 124 5 afid
9. damaged the system will not be able to recognize the specimens or controls 3 Uncap the control tubes and load the specimens consumables and controls onto the Hamilton MICROLAB STAR STARIet IVD Pipettor When the specimens consumables and controls hav been loaded the instrument transfers controls and specimens into S tubes 4 When the pipetting run is completed review alarms print the pooling report s In pools and Deep Well Plate wells Invalidate pools and or wells if red blood cell contamination i d or if volumes are inconsistent 5 Cap the S tubes and transfer the SK24 rack s to the COBAS AmpliPrep Ins it for nucleic acid extraction Once transferred into S tubes all viral targets and controls are stab 6 hours in the S Tube 6 Cap and store the Deep Well Plates if plates were created durin ipetting run 7 Remove and store the donor tubes Refer to Specimen Colle ge and Pooling Section for conditions 8 Remove and discard the control tubes Control tubes are single use only 9 Test orders are automatically created and trans networked AMPLILINK Data Stations B Preparation and Loading of cobas TaqScree est Reagents Note Use caution to not damage ette labels The barcode reader on the COBAS AmpliPrep Instrument automatic ds the barcode label of each cassette when the reagent racks are loaded trument 1 Equilibrate reagents for 30yfinutes in the COBAS AmpliPrep Instrument before the first specimen
10. in plasma pools and plasma derivatives Vox Sanguinis 2001 81 228 235 11 Azzi A Moefini M Mannucci PM The trafisfusi n associated transmission of parvovirus B19 Transfusion Medicine Reviews 1999 13 194 204 12 Yee TT Cohen BJ Pasi KL Le mission of symptomatic parvovirus B19 infection by clotting factor concentrate British Journal matology 1996 93 457 459 13 Bl mel J Schmid l Effenber eitz H Willkommen H Brackmann H H Lower J Eis Hiibinger AM Parvovirus B19 transmissi y heat treated clotting factor concentrates Transfusion 2002 42 1473 1481 14 Francki RIB Fauque Khudson DL Brown F Classification and nomenclature of viruses Archives of Virology 1991 Sd 326 15 Gust ID 16 Shapiro 1 1 Global impact of hepatitis A virus infection changing patterns In Hollinger FB Lemon SM and largolis HS eds Viral Hepatitis and Liver Disease Baltimore Williams amp Wilkins 1991 14 20 18 Robertson BH Jansen RE Khanna B Totsuka A Nainan OV Siegl G Widell A Margolis H lsomura S Ito K Ishizu T Moritsugu Y Lemon SM Genetic relatedness of hepatitis A virus strains recovered from different geographical regions Journal of General Virology 1992 73 1365 1377 19 Costa Mattioli M Cristina J Romero H Perez Bercof R Casane D Colina R Garcia L Vega Glikman G Romanowsky V Castello A Nicand E Gassin M Billaudel S Ferre V Molecular evolution of hepatitis A virus a new classification based on t
11. including erythema infectiosium fifth disease in child rthropathy in adults However B19V may cause severe disease such as transient aplastic anemia i with hematological disorders and hydrops congenital anemia or fetal death in pregnant wome lence of B19V in blood and plasma donors can vary from 0 003 0 6 depending on whether the done during an epidemic or non epidemic period nging t6 the genus Erythrovirus of the family own as B19 A6 and V9 respectively Although B19V is normally transi e respiratory route transmission by plasma products can occur due to the size of plasma pool ce of acute inapparent B19V infections the high levels of virus in a viremic donation up to 1 jl and the resistance of B19V to most of the commonly used viral inactivation removal stepsfsuch as solvent detergent S D treatment or pasteurization The presence of B19V DNA in plasma pools a plasma products have been reported 10 and there are many reports in the literature of transmi by administration of plasma products especially coagulation factors 7 Hepatitis A vi HAV is a small non enveloped RNA virus which belongs to the Hepatovirus group of the Picornaviride ily HAV which has a global distribution is transmitted by the oral fecal route primarily by close perso t Epidemics are common in developing countries where the infection is acquired early in life a in a large proportion of the population having pro
12. kit contains sufficient material for processing a rat oat tests 3 All controls are single use only 4 The system will prevent the use of reagents from different lots rea which have exceeded the allowed hours on instruments reagents which have expired orgffike ettes from a set of four cassettes previously used on the system Do not load mixed on the COBAS AmpliPrep Instrument C Specimen Processing 1 Avoid contaminating gloves when handling specimel d controls 2 Care should be used to avoid contamination ofca s and Negative Controls with Positive Control material INSTRUCTIONS FOR USE The cobas s 201 system includes four ra esses Specimen and Control Pipetting on the Hamilton MICROLAB STAR STARIet IVD ee en preparation on the COBAS AmpliPrep Instrument e using the cobas TaqScreen DP mplification Detection on the COBAS TaqMan Analyzer and Data Management Each cobas TaqScreen DP kit ontains eight cassettes two DPX CS1 cassettes with Magnetic Glass Particles two DPX CS2 oasset with Lysis Reagent two DPX CS3 cassettes with Proteinase and Elution Buffer and two DPX sett s with IC MMX Reagent 1 and MMX Reagent 2 This test kit is to be used in conjunction with aqScreen DPX Control Kit and the cobas TaqScreen Wash Reagent Note Do not open the cassettes Note Do agents from different lots or from different bottles of the same lot Note Dom ix cassettes from different lots on the COBAS Ampl
13. s 201 system 2 Heparin has been shown to inhibit PCR Do not use heparinized plasma with this procedure 3 Reliable results are dependent on adequate specimen collection and proper transport procedures 4 Only the Hamilton MICROLAB STAR STARIet IVD Pipettor has been validated for use with the cobas TaqScreen DPX Test for the automated preparation of plasma pools Adhere to the hardware instructions and safety precautions outlined in the cobas s 207 system Operator s Manual and the User Manual for the Hamilton MICROLAB STAR STARIet IVD Pipettor 05509238001 02EN 16 Doc Rev 2 0 5 Detection of HAV RNA and quantitation of B19V DNA 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 6 Though rare mutations within the highly conserved regions of the viral genomes covered by the cobas TaqScreen DPX Test primers and or probes may result in failure to detect HAV or correctly quantitate B19V 7 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 Users should follow their own specific policies procedures PERFORMANCE CHARACTERISTICS Clinical Reproducibility Study The reproducibility of the cobas TaqScre
14. system must be installed and used as a complete system configuration Individual cobas s 201 system components cannot be used as stand alone devices nor may other components be substituted The cobas s 201 system utilizes the components listed below Instrumentation and Software for cobas s 201 system Hamilton MICROLAB STAR STARIet IVD Pipettor optional Pooling Manager Workstation and software COBAS AmpliPrep Instrument COBAS TaqMan Analyzer AMPLILINK Data Station and software v3 3 1 or v3 3 2 Roche PDM Data Manager Server Data Manager workstation and software v3 0 23 WY DPX Test Definition File for the cobas TaqScreen DPX Test B19 Test Definition File for the cobas TaqScreen DPX Test O Racks and Disposables COBAS AmpliPrep Sample Racks SK24 P N 28122172001 KQ COBAS AmpliPrep SPU racks P N 05471664001 COBAS AmpliPrep Reagent Racks P N 28122199001 Sample Processing Units SPU P N 03755525001 Sample Input Tubes S tubes with Barcode Clips 03137040001 Racks of K tips P N 03287343001 K tube Box of 12 x 96 P N 03137082001 COBAS TaqMan K carrier P N 281 High Volume CO RE Tips 1000 uL 04639642001 Deep Well Plates with Barcode 04639634001 Deep Well Plate Sealing Mi 04789288001 Sample Carrier for 24 Test Tubes P N 04639502001 Sample Carrier for t S P N 04639529001 Tip Carrier P 45001 Deep Well P N 04639553001 SK24 Rai P N 04639600001 Mi ide
15. there is any evidence of leakage do not use that material for testing 05509238001 02EN 9 Doc Rev 2 0 J Dispose of all materials that have come in contact with specimens and reagents in accordance with country federal state and local regulations K Do not use a cobas TaqScreen DPX Test kit cobas TaqScreen DPX Control Kit or cobas TaqScreen Wash Reagent after its expiration date Do not interchange mix or combine reagents from different kits or different lots Do not load mixed reagent lots on the COBAS AmpliPrep Instrument L Material Safety Data Sheets MSDS are available on request from your local Roche office M Avoid contact of reagents with skin eyes or mucous membranes If contact does occur immediately wash with large amounts of water otherwise burns can occur If these reagents are spilled dilute with water before wiping dry N Do not allow LYS which contains guanidine thiocyanate to contact sodium hypochlorite bleach solution This mixture can produce a highly toxic gas O Closely follow procedures and guidelines provided to ensure that the test is performe ectly Any deviation from the procedures and guidelines may affect optimal test performance P The use of excessively hemolyzed specimens should be avoided Q Red blood cell contamination of plasma specimens gt 5 may inhibit the col Q DPX Test R Do not use any component with damaged barcode labels at any phase of CONTROL AND REAGEN
16. 0 01 Z05 DNA Polymerase microbial lt 0 01 AmpErase uracil N glycosylase enzyme micr 0 08 Sodium azide DPX IC QS DPX Internal Control and Quantitation Standard cobas TaqScreen DPX P N 05509190 190 DPX D C Xi TRIS buffer lt 0 002 Poly rA RNA synthetic EDTA 0 05 Sodium azide lt 0 001 Non infectious syntheti in MS2 bacteriophage coat pro lt 0 001 Non infectious syn in Lambda bacteriophage g C ncapsulated 19V DNA encapsulated us synthetic HAV RNA encapsulated riophage coat protein Non ihfectious synthetic B19V DNA encapsulated iophage Lambda coat protein Human Plasma non reactive by US FDA licensed tests BT9V IgG and IgM Ab HBsAg HBV core Ab HCV Ab 1 2 Ab and HIV 1 RNA Non reactive by NAT for HAV RNA and B19V DNA lt 5 IU mL by PCR methods 0 1 ProClin 300 preservative 8 1 mixture of 5 Chloro 2 methyl 2H isothiazol 3 one and 2 Methyl 2H isothiazol 3 one Irritant R43 May cause sensitization by skin contact 05509238001 02EN 2 x 48 Tests 2x3 0 mL 2x 2 5 mL vy O Q o 2x13 mL 12 Sets 12x 1 6 mL Doc Rev 2 0 DPX H C 12x 1 6 mL DPX High Positive Control lt 0 001 Non infectious synthetic B19V DNA encapsulated in bacteriophage Lambda coat protein Negative Human Plasma non reactive by US FDA licensed tests for B19V IgG and IgM Ab HBsAg HBV core Ab HCV Ab HIV 1 2 Ab and HIV 1 RNA Non reactive by NA
17. 0 37 N A IIA 73 6 53 72 97 2 70 72 100 0 72 72 0 80 0 59 1 56 IIB 43 8 21 48 91 7 44 48 100 0 48 48 1 25 0 91 2 27 Eleven clinical specimens 10 genotype IA 1 genotype IB and 9 transcripts 2 genotype IB 1 genotype IIA 1 genotype IIB 3 genotype IIIA and 2 genotype IIIB were used PROBIT analysis confidence intervals could not be generated B19V Clinical specimens and plasmids of different B19V genotypes diluted to approximately 0 3X 1X and 3X the Limit of Detection LOD of the cobas TaqScreen DPX Test were tested PROBIT analysis of the results demonstrated that the LOD for each genotype sample was equivalent to or better than the LOD for the WHO Standard 99 800 Table 14 05509238001 02EN 21 Doc Rev 2 0 Table 14 B19V Genotype Inclusivity Genotype Reactive Rate Reactive Rate Reactive Rate LOD 95 C I at 0 3 x LOD at 1x LOD at 3 x LOD IU mL 1 15 6 15 96 90 6 87 96 100 0 97 97 13 57 11 39 17 27 2 88 5 85 96 100 0 96 96 99 0 95 96 6 07 N A 3A 83 3 40 48 100 0 48 48 100 0 48 48 3 94 N A 3B 91 7 44 48 97 9 47 48 100 0 48 48 5 31 N A Three clinical specimens genotypes 1A 3A and 3B and 2 plasmids genotype 2 were used PROBIT analysis confidence intervals could not be generated Potentially Cross reactive and Interfering Microorganisms Test was evaluated by testing a panel of
18. 5 939 262 and patents pending ARMORED RNA is a trademark of Ambion and Cenetron The cyanini this product are subject to patent rights of GE Healthcare Bio Sciences Corp and Carnegie Mellon Un d are licensed to Roche solely for incorporation into components of research and in vitro diag ny use of such kits for purposes other than research or in vitro diagnostics requires sub om GE Healthcare Bio Sciences Corp Piscataway New Jersey U S A and Carnegie Mellon burgh Pennsylvania U S A 2011 Roche Molecular Systems Inc All rights reserved 07 2011 05509238001 02 Doc Rev 2 0 05509238001 02EN 28 Doc Rev 2 0
19. Standard QS directly traceable to the WHO B19V International Standard The QS together with an Internal Control IC for the HAV target is co extracted and co amplified with each sample The cobas TaqScreen DPX Test uses a generic nucleic acid preparation technique on the COBAS AmpliPrep Instrument B19V DNA HAV RNA the QS and IC are amplified and detected in a single tube using automated real time PCR on the COBAS TaqMan Analyzer Discrimination of the viral target the QS and the IC is achieved by the use of fluorescently labeled probes which are detected in separate channels of the COBAS TaqMan Analyzer The test incorporates the AmpErase uracil N glycosylase enzyme to reduce potential contamination by previously amplified material amplicon PRINCIPLES OF THE PROCEDURE The cobas TaqScreen DPX Test for use on the cobas s 201 system is based on 4 major en 1 Automated Specimen Pooling and Control Pipetting using the Hamilton MICROLAB ST Oy IVD Pipettor optional 2 Automated Specimen Preparation using the COBAS AmpliPrep Instrument of oducts using the 3 Automated Amplification of Nucleic Acid and Real Time Automated Detecti COBAS TaqMan Analyzer 4 Automated Data Management using the Pooling and Data Managem oftware Automated Specimen Pooling and Pipetting using the Hamilto OLAB STAR STARIet IVD Pipettor The Hamilton MICROLAB STAR STARIet IVD Pipettor automat s pipetting of individual donor
20. T PREPARATION A Equilibrate the cobas TaqScreen DPX Control Kit to room aye for 30 minutes prior to loading onto the Hamilton MICROLAB STAR STARIet IVD Pipettor priol ise Equilibrate cobas TaqScreen DPX Test reagents in the COBAS AmpliPrep Instrumentfor 30 minutes prior to use SPECIMEN COLLECTION STORAGE and POOLING Note Handle all specimens as if they are infectie C2 Specimens A Specimens collected using EDTA sa 1 CP2D ACD A 4 Citrate and BD Vacutainer Plasma Preparation Tube PPT may bi the cobas TaqScreen DPX Test Follow the sample tube manufacturer s instructions B Blood collected in EDTA CP 1 CP2D ACD A 4 Citrate and PPT may be stored for up to 72 hours at 2 30 C folloy up to 48 hours at 2 8 C prior to plasma separation For storage longer than 5 days the plasma should be removed from the red blood cells and stored at 2 8 C for up to 9 days Figure 1 Whole Blood Stability lt Whole Blood O 2 C 30 C J D 24 2 g o a E 104 E Whole Blood Plasma 2 C 8 C 2 C 8 C 0 r r r r r r r r r r r r 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 05509238001 02EN 10 Doc Rev 2 0 Apheresis plasma in EDTA CPD CPDA 1 CP2D ACD A or 4 sodium citrate may be stored for up to 10 days at 2 30 C followed by storage for up to 46 days at 2 8 C The following plasma volume guidelines are based on pipetting from 13 x 100 mm glass or plastic donor tubes o
21. T for HAV RNA and B19V DNA lt 5 IU mL by PCR methods 0 1 ProClin 300 preservative Xi 38 1 mixture of 5 Chloro 2 methyl 2H isothiazol 3 one and 2 Methyl 2H isothiazol 3 one Irritant R43 May cause sensitization by skin contact V DPX C x 1 6 mL DPX Negative Control Sodium phosphate buffer EDTA O 0 002 Poly rA RNA synthetic Amaranth dye 0 1 ProClin 300 preservative n Xi 3 1 mixture of 5 Chloro 2 methyl 2H isothiazol and 2 Methyl 2H isothiazol 3 one Irritant Q R43 May cause sensitization by skin contact cobas TaqScreen Wash Reagent TS WR 5 1L P N 04404220 190 x TS WR cobas TaqScreen Wash Reage Sodium citrate dihydrate 0 1 Methylparaben preserva Qe Q O 05509238001 02EN 8 Doc Rev 2 0 STORAGE AND HANDLING REQUIREMENTS A B C Room temperature is defined as 15 to 30 C Do not freeze reagents or controls Store DPX CS1 DPX CS2 DPX CS3 and DPX CS4 at 2 to 8 C Unused reagents are stable until the expiration date indicated After initial use reagents are stable for 30 days at 2 to 8 C or until the expiration date whichever comes first Reagents can be used for up to 6 instrument runs and up to a maximum of 48 cumulative hours in the COBAS AmpliPrep Instrument Reagents must be stored at 2 to 8 C between uses The AMPLILINK software monitors the cumulative hours of the reagent cassettes on the COBAS AmpliPrep Instrument and blocks the cassettes from being
22. cobas TaqScreen DPX Test 7 for use on the cobas s 201 system CO bas cobas TaqScreen DPX Test 96 Tests P N 05509211 190 cobas TaqScreen DPX Control Kit 12 Sets P N 05509190 190 cobas TaqScreen Wash Reagent 5 1L P N 04404220 190 INDICATION FOR USE The cobas TaqScreen DPX Test for use on the cobas s 201 system is an in vitro nucleic ai awe test for the direct quantitation of human parvovirus B19 B19V DNA genotypes 1 2 a e direct qualitative detection of hepatitis A virus HAV RNA genotypes Il and II in human SUMMARY AND EXPLANATION OF THE TEST The cobas TaqScreen DPX Test incorporates the use of multiple dyes whichfalloWs for the simultaneous detection of individual targets without the use of discriminatory tests B Test Definition File TDF installed the cobas TaqScreen DPX Test will provide either both th titative B19V and qualitative HAV results or only the quantitative B19V result B19V is a small non enveloped single stranded DNA virus Parvoviridae and is grouped into genotypes 1 2 and 3 ori B19V has a world wide distribution and serological studi dicated that at least 50 of adults have circulating B19V IgG indicating a past infection The us isassociated with clinical illness the manifestation and severity of which are dependent on the immu nd hematological status of the individual In immunocompetent individuals the infection is tomatic or may result in a mild illness
23. d target amplicon Detection of PCR Products The cobas TaqScreen DPX MMX contains detection probes wing Yet for HAV IC B19V or QS nucleic acid Each detection probe is labeled with 1 one of fourluoresi dyes which act as a reporter and 2 another dye which acts as a quencher Each specific repo is associated with a corresponding target each measured at a defined wavelength A single type r dye is used in all probes This system permits detection of all the amplified targets at different Wavel Before PCR amplification begins the probes are intact e reporter dye fluorescence is suppressed by the quencher dye due to F rster type energy tran PCR amplification the probes hybridize to specific single stranded DNA sequences and are cle 5 to 3 nuclease activity of the Z05 DNA Polymerase at the same time that amplification is occusti the reporter and quencher dyes are separated by this cleavage the fluorescent activity of the po e is unmasked With each PCR cycle increasing amounts of cleaved probes are generated and the ci ative signal of the reporter dye is concomitantly increased Real time detection of PCR prodtic ag omplished by measuring the fluorescence of released reporter dyes representing the viral targets tl S independently Data Management usii M Software The Roche PDM s results for the coba ws the user to review and report results The Roche PDM software assigns test aqScreen DPX Test as non reac
24. e wi omplete cobas s 201 system to be installed without a Hamilton MICROLAB STAR ST ipettor by requiring the manual entry of the specimen barcode ID SK24 rack and position bafcode IDs and the s clip barcode ID Refer to the cobas s 207 System Operator s Manual for instrugti n manually entering this information obas TaqScreen DPX Test as being reactive for HAV or gt cutoff for B19V R STARIet IVD Pipettor is used to pipette from either Deep Well Plates or ate subdivided pools for secondary testing If pools are detected the Hamilton MICR original specime ge 05509238001 02EN 11 Doc Rev 2 0 PROCEDURAL NOTES A Equipment 1 Prepare the cobas s 201 system for use according to instructions in the cobas s 201 System Operator s Manual 2 Perform recommended maintenance on instruments to ensure proper functioning B Reagents 1 The cobas TaqScreen DPX Test reagents must be equilibrated for 30 minutes in the COBAS AmpliPrep Instrument prior to use The cobas TaqScreen DPX Control Kit and cobas TaqScreen Wash Reagent must be at room temperature before use See Storage and Handling Requir ments Section for reagent storage conditions which are recommended to be run in batches consisting of up to 24 tests per One Negative Control and one of each Positive Control must be processed with each 4 rack Controls are processed in the same way as specimens with the cobas OC Test 2 Each cobas TaqScreen DPX Test
25. ease State Clinical Specimens The potential Cross reactivity and interference by other disease state clinical specimens with the cobas e est was evaluated by testing Cytomegalovirus hepatitis B virus Hepatitis C virus and human ency virus type 1 group M clinical specimens The disease state clinical specimens were tested out HAV at 3X the limit of detection and B19V at 5X the lower limit of quantitation of the cobas TaqScreen DPX Test Non reactive results were obtained with the cobas TaqScreen DPX Test for all of the other disease state clinical specimens tested without HAV and B19V The other disease state clinical specimens tested do not cross react with the cobas TaqScreen DPX Test Reactive results were obtained for all of the other disease state clinical specimens tested with HAV and B19V The other disease state clinical specimens tested do not interfere with the cobas TaqScreen DPX Test 05509238001 02EN 22 Doc Rev 2 0 Potentially Interfering Substances Endogenous Interfering Substances Plasma specimens with abnormally high levels of triglycerides 33 g L hemoglobin 2 g L unconjugated bilirubin 200 mg dL albumin 60 g L human DNA 4 mg L or red blood cells 5 v v were tested with and without HAV at 3X the limit of detection and B19V at 5X the lower limit of quantitation of the cobas TaqScreen DPX Test These endogenous substances and red blood cells 5 v v did not interfere with the sensitiv
26. en DPX Test for use on the cobas s 201 system wa ee for B19V DNA and HAV RNA across 3 external sites In addition to the site the following vai ors were included in the assessment reagent lot day run and within run results A panel d of different concentrations of the 2 target viruses was used to evaluate the assay range Factors affecting the precision for B19V are shown in Table 3 Table 4 providestesults for HAV positive panel members summarized by lot site day and run Factors Affecting the Precision ara DNA Quani a 10 IU mL Expected Mean Ss Total SD of B19V Conc B19V Conc Logio B19V logio 1U mL logio IU ML No of Tests Lot Si ay Run Within run Conc 2 60 2 48 179 0 009 os 0 065 0 035 0 168 0 201 3 00 3 05 175 04 054 0 043 0 000 0 138 0 160 4 00 3 87 178 0 156 0 030 0 022 0 083 0 182 5 00 474 18l 0 026 0 057 0 034 0 039 0 059 0 101 6 00 5 75 1 0 072 0 085 0 000 0 051 0 097 0 156 7 00 6 84 Gr 0 057 0 258 0 042 0 076 0 075 0 288 Number of valid tests pee sts panel member were planned Invalid tests were not repeated lt 05509238001 02EN 17 Doc Rev 2 0 Table 4 Results for HAV Positive Panel Members Summarized by Lot Site Day and Run Number of Positive Tests Total Number of Valid Results HAV Mean Ct Ct P Conc ce
27. f Number of 95 Lower Concentranon Reactives Individual Tests Reactives Confidence Bound IU mL one sided 75 00 189 189 100 0 98 1 50 00 188 188 100 0 98 1 28 28 189 189 100 0 98 1 20 00 187 189 98 9 96 2 14 14 189 189 100 0 98 1 10 00 170 189 89 9 84 7 7 07 142 189 75 1 0 00 0 189 0 0 PROBIT analysis of the combined data from all replicates tested for each virus was Q to estimate the 95 Limit of Detection LOD and two sided 95 fiducial confidence intervals le 11 Virus LOD IU mL IU mL HAV 1 06 0 94 1 24 B19V O 10 56 12 91 Linearity of B19V Quantitation Table 11 95 LOD by ETR 9 Screen DPX Test was determined by testing a serial brated against the WHO Standard 99 800 The study was ge was determined to be between 75 and 3 0 x 10 IU mL The linearity of B19V quantitation of the dilution of a B19V genotype 1 sample th performed using 2 lots of reagents TI based on the CLSI guideline EP6 A Figu Figure 2 Lit ofthe cobas TaqScreen DPX Test for B19V 8 a 5 y 1 060x 0 442 g 4 R 0 996 E y3 v E 2 oe 1 1 2 3 4 5 6 7 8 Parvovirus B19 Nominal Concentration log IU mL 05509238001 02EN 20 Doc Rev 2 0 Linearity of B19V Quantitation by Genotype The linearity of the cobas TaqScreen DPX Test was tested for each genotype of B19V using 5 to 6 dilutions of a clinical sample for each B19V geno
28. he complete VP1 protein Journal of Virology 2002 76 9516 9525 20 Bower WA Nainan OV Han X Margolis HS Duration of viremia in hepatitis A virus infection Journal of Infectious Diseases 2000 182 12 17 05509238001 02EN 24 Doc Rev 2 0 21 22 23 24 25 26 27 28 29 30 31 32 33 Gowland P Fontana S Niederhauser C Manouri Taleghani B Molecular and serologic tracing of a transfusion transmitted hepatitis A virus Transfusion 2004 44 1555 1561 Soucie JM Robertson BH Bell BP McCaustland KA Evatt BL Hepatitis A virus infections associated with clotting factor concentrate in the United States Transfusion 1998 38 573 579 Chudy M Budek I Keller Stanislawski B McCaustland KA Neidhold S Robertson BH Niibling CM Seitz R L wer J A new cluster of hepatitis A infection in haemophiliacs traced to a contaminated plasma pool Journal of Medical Virology 1999 57 91 99 Saldanha J Lelie N Yu M Y Heath A and the B19 Collaborative Study group Establishment of the first World Health Organization international standard for human parvovirus B19 DNA nucleic acid amplification techniques Vox Sanguinis 2002 82 24 31 Longo MC Berninger MS Hartley JL Use of uracil DNA glycosylase to control carry over comin in polymerase chain reactions Gene 1990 93 125 128 Sawa R McAuley Hecht K Brown T Pearl L The structural basis of specific base excision fea DNA glycosylase Nature 1995 373 487 493
29. iPrep Instrument tef Do not separate control tubes from adapters Perform all required maintenance as described in the cobas s 201 system Operator s Manual The cobas TaqScreen DPX Test offers an alternate workflow that preserves specimens in a Library Plate LP so that testing can be performed at a later date The DPX LP workflow requires three pooling runs During the first pooling run Library Plate Run single specimen aliquots are pipetted into a Library Plate which is then frozen After the Quarantine Interval configured at installation has elapsed the Library Plate can be thawed and used to prepare 12 specimen intermediate pools Intermediate Plate Run During the third pooling run Batch Run aliquots are pipetted from the Intermediate Plate wells into S tubes to create the Primary Pools Refer to the cobas s 201 system Operator s Manual for detailed instructions for use 05509238001 02EN 12 Doc Rev 2 0 A Pipetting Controls and Specimens on the Hamilton MICROLAB STAR STARIet IVD Pipettor Note Avoid contaminating gloves when preparing the specimens and controls Note Mix controls by gentle inversion three times avoiding the creation of bubbles 1 Perform startup procedures on the Hamilton MICROLAB STAR STARIet IVD Pipettor then start the Roche PDM Pooling Wizard following the on screen instructions 2 Use caution not to damage the identifier barcode on specimen tubes and control tube adapters If
30. is to be processed No otl ent preparation is required 2 Prior to start a suffici umber of all cassettes must be loaded to accommodate the total number of specimens that cessed during continuous operation of the COBAS AmpliPrep Instrument Each cassette ci nough reagents for 48 tests Refer to the cobas s 201 system Operator s Manual fo regarding loading of reagents for continuous operation 3 Place the DI S1 cassette into a reagent rack ensuring the cassette barcode is in line with the rack located to the right side of the rack DPX CS1 cassettes must be loaded together on a the reagent rack containing DPX CS1 into rack position A sliding up to the stop pin on the GOBAS AmpliPrep Instrument then wait for the reagent rack LED to turn green before sliding the rack to the back of the instrument to its final seated position Do not load mixed reagent lots on the instrument Place one set of DPX CS2 DPX CS3 and DPX CS4 cassettes for each DPX CS1 cassette into a reagent rack s ensuring the cassette barcodes are in line with the rack barcode located to the right side of the rack Load the reagent rack s into rack position B C D or E sliding up to the pin on the COBAS AmpliPrep Instrument then wait for the reagent rack LED to turn green before sliding the rack to the back of the instrument to its final seated position 05509238001 02EN 13 Doc Rev 2 0 7 LED lights on the COBAS AmpliPrep Instrument Status bar wi
31. ity or specificity of the cobas TaqScreen DPX Test Exogenous Interfering Substances Normal human plasma specimens containing abnormally high concentrations of acetaminophen 1324 pmol L acetylsalicylic acid 3 62 mmol L ascorbic acid 342 pmol L atorvastatin 600 Eq L fluoxetine 11 2 pmol L ibuprofen 2425 pmol L loratadine 0 78 pmol L nadolol 3 88 pmol L naproxen 217 L paroxetine 3 04 pmol L phenylephrine HCI 491 pmol L and sertraline 1 96 pmol L were tested with and without HAV at 3X the limit of detection and B19V at 5X the lower limit of quantitation ofthe c bas TaqScreen DPX Test These exogenous substances did not interfere with the sensitivity or spi ity of the cobas TaqScreen DPX Test Specificity Five hundred thirty eight individual B19V sero negative plasma specimens were tested with one of two lots of cobas TaqScreen DPX Test kits The observed specificity for B19V an as 96 3 and 99 8 respectively The lower specificity for B19V gan be expected due to th ity of B19V in the general population and the sensitivity of the cobas TaqScreen DPX O 05509238001 02EN 23 Doc Rev 2 0 REFERENCES i Servant A Laperche S Lallemand F Marinho V De Saint Maur G Meritet JF Garbarg Chenon A Genetic diversity within human Erythroviruses identification of three genotypes Journal of Virology 2002 76 9124 34 2 Anderson MJ Tsou C Parker RA Chorba TL Wolfe H Tattersal P
32. ized Check AMPLILINK software to ensure adequate _re tsyand consumables are loaded for desired specimen preparation Press Start on AMPLILINK software worl o begin the COBAS AmpliPrep Instrument Specimen Preparation Procedure Any unused K tips and K tubes wi fainslocked within the COBAS AmpliPrep Instrument for use in the next run D Amplification and Detectio 1 completely filled k must be transferred within 1 hour of completion of specimen preparation on that rack Th C TaqMan Analyzer will automatically start amplification and detection A batch of a completly filled rack not transferred within 1 hour will be invalid Partially filled SK24 racks 1 hour may provide a valid batch The AMPLILINK software tracks the allowed aSter Mix addition and start of amplification detection for each sample and will tire rack if the first processed sample exceeds the time limit Transfer the SK24 me ing processed specimens to the COBAS TaqMan Analyzer A ic time betwe invalidate the e amplification and detection is completed on the COBAS TaqMan Analyzer the analyzed ns are automatically disposed of in the waste bin results are automatically accepted and transferred to the PDM software Qy and Releasing Results 1 2 Start the Roche PDM Workstation Review unevaluated batches on the Review Batches tab at the Data Manager workstation Review Alarms by highlighting a batch and then clicking
33. lidity of the DPX Dual Positive Control is rd ae g to the test performed B19V only test For the Negative Control DPX C to be vali reactive for B19V and the associated QS must be valid If tl Control is invalid the entire batch is invalid and must be ref b DPX Dual Positive Control DPX Test HAV and B19V For the Dual Positi U DPX D C to be valid the interpreted result for the HAV must be Reactive 9V concentration must be between 1 2 x 10 and 1 2 x 10 IU mL and the associated IC QS must be valid If the interpreted result for HAV is invalid or the B19V concentration side of the accepted range the entire batch is invalid and must be repeated B19V only test For the Dual Positive Control DPX D C fo be valid the interpreted result for the B19V concentration m etween 1 2 x 10 and 1 2 x 10 IU mL and the associated QS must be valid If the interpreted resu the B19V concentration is outside of the accepted range the entire batch is invalid ani be repeated ositive Control DPX H C to be valid the B19V concentration must be between 4 x 10 IU mL and the associated QS must be valid If the B19V concentration is be valid otherwise the non reactive result is invalid and the donor specimen must be retested C yi Donor Specimens X or a donor specimen to have a valid non reactive test result for HAV the associated IC must 4 b A valid HAV reactive donor specimen test result may have eithe
34. ll turn green when all required kit components are loaded and recognized by the system C Extraction of Nucleic Acids from the Pipetted Specimens and Controls Note Perform the following steps on a clean bench surface 1 2 10 Remove the wrap from Sample Processing Unit SPU bundle leaving tape and plastic cover intact With the large tab of the SPU rack facing toward the operator insert SPU bundle flush with the right side of the SPU rack Remove tape and plastic cover from SPUs seated in the rack Ensure all SPUs are pressed down level and fully seated in rack Elevated SPUs may cause an instrument failure Do not apply pressure to the S tip in the SPU Slide loaded SPU racks into COBAS AmpliPrep Instrument SPU positions J K or L until the rack is inserted completely and recognized The instrument will hold up to 72 SPUs at a d the number of SPUs needed for the run or insert more as needed Remove cellophane wrapping from manufacturer loaded K tube and K tip ra ng careful not to tip the racks Ensure that all are properly seated Slide at least the required number of K tube and K tip racks into th AS AmpliPrep Instrument positions M N O or P Load SK24 racks containing specimens pipetted by the Hai ROLAB STAR STARlet IVD Pipettor and controls into COBAS AmpliPrep Instrument Positidns F G or H Slide in until rack is locked Check system status Sample window to ensure all spi s on each rack were recogn
35. mL n 20 ferent Kit Lots B19V Panel Members IU mL Reagent Lot 1 Reagent Li 1 and 2 Combined 10 0 073 01 0 088 10 0 051 83 0 070 10 0 071 0 0 070 10 0 218 0 112 0 176 Over all 10 10 0 123 094 0 110 Analytical Sensitivity WHO International Stand The 95 Limits of Detection LOD of the as qScreen DPX Test for both HAV RNA and B19V DNA were determined using the WHO Internati tandards for HAV NIBSC code 00 560 and B19V NIBSC code 99 800 Three independent dilution series cC viral standard were prepared with normal virus negative human plasma Each dilution series wase using three different lots of the cobas TaqScreen DPX Test kit with 21 replicates per lot for a totalgof licates per concentration Tables 9 and 10 show the reactive rate analyses for HAV and B19Vresp ctively Table 9 V International Standard 00 560 Reactive Rate Summary HAV RNA Concentr tion Number of Number of 95 Lower Confidence al Reactives Individual Tests Reactives Bound one sided 189 189 100 0 98 1 0 188 188 100 0 98 1 1 41 186 188 98 9 96 2 1 00 176 189 93 1 88 5 0 71 162 189 85 7 79 9 0 50 142 189 75 1 68 3 0 35 128 189 67 7 60 6 0 00 0 189 0 0 0 0 05509238001 02EN 19 Doc Rev 2 0 Table 10 B19V WHO International Standard 99 800 Reactive Rate Summary BI9V DNA Number o
36. n the Hamilton MICROLAB STAR STARIet IVD Pipettor The listed volumes are for plasma on top of packed red blood cells and are for use when running the cobas TaqScreen DPX Test Table 1 Volume Guideline for Hamilton MICROLAB STAR STARIet IVD Pipettor Pool Type Minimum Plasma Volume Primary Pool 3mL Repeat Pool 1 5 mL Resolution Pool 2mL Includes creation of Deep Well Plate Library Plate Do not freeze whole blood V Covered Deep Well Plates may be stored at lt 18 C for up to 150 days followed b up to 30 days Plasma in EDTA CPD CPD A CP2D ACD A and 4 sodium citrate may be st up to 6 months at lt 18 C followed by up to 15 days at 2 8 C No adverse effect on t erformance was observed when plasma specimens were subjected to 3 freeze thaw cycles Equilibrate pooled or individual donor specimens to room temperature using The user must validate other collection and storage conditions ffispecimens are to be shipped they should be packaged and labeled in compliance with applicable federal an international regulations covering the transport of specimens and etiologic agents SPECIMEN POOLING AND PIPETTING A The cobas s 201 system utilizes the Hamilton MI STAR STARIet IVD Pipettor for all pipetting and pooling activities The Hamilton MICROLAB ARlet IVD Pipettor performs barcode scanning and pooling operations from specimens to fort pi PDM Software version 3 or high
37. pools of 96 lefined combinations of positive and negative HAV and B19V samples All known positive and negative samples were identified correctly for both studies demonstrating 100 sensitivity pecificity e hy reement of results and known samples for pools of 96 and 480 are shown in Tables 6 and 7 re Table 6 Agreement of Pool Resolution Results for Pools of 96 Positive Negative Total Reactive 27 0 27 Pool Resolution Result Non Reactive 0 453 453 Total 27 453 480 05509238001 02EN 18 Doc Rev 2 0 Table 7 Agreement of Pool Resolution Results for Pools of 480 Positive Negative Total Reactive 27 0 27 Pool Resolution Result Non Reactive 0 2 370 2 370 Total 27 2 370 2 397 There were 3 out of 2400 results that could not be completely resolved due to insufficient volume Precision of B19V Quantitation The precision of the cobas TaqScreen DPX Test was also evaluated internally for B19V by testing a semigmber panel composed of B19V positive plasma samples with concentrations of 10 10 10 and 10 IU mL All valid data were evaluated by calculating the standard deviation of the B19V titers log yor each panel member The data from 2 kit lots were analyzed separately as well as combined his study demonstrated consistent performance of the cobas TaqScreen DPX Test ai Table 8 Table 8 Standard Deviation of the B19V Titers logo IU
38. purities such as process controls are simulta processed The cobas TagScreen DPX Test contains reagents that P c denatured ellular debris and potential PCR inhibitors such as hemoglobin etc and reduces the salt conce j urified nucleic acids are released from the Magnetic Glass Particles at elevated temperature with El r ated Amplification and Detection of Nucleic Acid using the COBAS TaqMan Analyzer After isolation of the purified nucleic acids from human plasma during automated specimen preparation cobas TaqScreen DPX Master Mix MMX is used for the amplification and detection of HAV RNA and IC RNA and amplification and quantitation of B19V DNA and QS DNA Once activated by the addition of manganese acetate the cobas TaqScreen DPX Master Mix permits reverse transcription for RNA targets followed by PCR amplification of highly conserved regions of HAV RNA IC RNA B19V DNA and QS DNA using specific primers The amplicon are detected by hybridization of specific oligonucleotide probes Amplification hybridization and detection occur simultaneously 05509238001 02EN 2 Doc Rev 2 0 Reverse Transcription and PCR Amplification Reverse transcription and amplification reactions are performed with a thermostable recombinant enzyme Z05 DNA Polymerase In the presence of manganese Mn the Z05 DNA Polymerase has reverse transcriptase and DNA polymerase activities This allows both reverse transcription
39. r a valid or invalid associated IC result QS for Donor Specimens a For a donor specimen to have a valid test result for B19V the associated QS must be valid otherwise the result is invalid and the donor specimen must be retested 05509238001 02EN 15 Doc Rev 2 0 RESULTS 1 Specimen results are valid only if the batch containing them is valid See Quality Control Section for acceptance criteria Four parameters are measured for each specimen one for the HAV viral target one for B19V viral target one for the IC and one for the QS B19V only test only two parameters are measured for each specimen the B19V viral target and the QS 2 The final test results are non reactive reactive or invalid for the HAV target and lt cutoff gt cutoff or invalid for the B19V target 3 The final donor status for the cobas TaqScreen DPX Test is reported by the PDM software as follows Table 2 Final Donor Status Description Final Donor Status Description Completed The final result for each target was determined Accepted The completed donor was accepted Completed Unresolved The viability time limit expired before the donor wa Accepted Unresolved Completed unresolved donor was accepted Repeat Test for Individual Specimen Donor tubes with a final result of invalid for one target require repeat Ces of the final result for the other target However the user has the option of selecting theaForci resolve b
40. specimens pooling of multiple donor specimens and pipetting of Test Control ol s 201 system resolves reactive pools into individual component specimen results The cobas s 201 Syste lesigned to process specimens in batches A batch is defined as a collection of specimens and controls that are pipetted extracted amplified and detected together When the pipetting of a batch is completed_on ilton MICROLAB STAR STARIet IVD Pipettor the entire sample rack is transferred into the COBAS Instrument for the next step of the process Automated Specimen Preparation usi As AmpliPrep Instrument Nucleic acids from the specimens Armored HAV RNA IC molecules and lambda encapsulated B19V DNA QS molecules whichgSe specimen preparation and amplification detection quantitation accomplish five sequential ol e COBAS AmpliPrep Instrument The Proteinase Solution digests proteins to promote lysis te nucleases and facilitate the release of RNA and DNA from viral particles Addition of Lysis Reag he Specimen results in viral lysis and nuclease inactivation by denaturation of proteins RNA and DNAya ised and simultaneously protected from nucleases The released nucleic acids bind to the silica s face added Magnetic Glass Particles This is mainly due to the net positive charge on the glass particle surface and net negative charge of the nucleic acids due to the chaotropic salt concentration i e Lysis reaction Wash Reagent removes unbound substances and im
41. surance that products derived from hi will not transmit infectious agents All human blood sourced materials should o sidered potentially infectious and should be handled with universal precautions If urs immediately disinfect with a freshly prepared solution of 0 5 sodium hypochlorite dil r follow appropriate site procedures Use routine laboral work areas Wear di ions Do not pipette by mouth Do not eat drink or smoke in designated abl gloves laboratory coats and eye protection when handling specimens and kit oughly after handling specimens and kit reagents s tl R1 BPX MMX R2 and DPX IC QS contain sodium azide as a preservative If solutions zide are disposed of in a plumbing system they should be diluted and flushed with generous iG arin has been shown to inhibit PCR Do not use heparinized plasma with this procedure F e use of sterile disposable pipettes and nuclease free pipette tips is recommended False positive results may occur if cross contamination of specimens is not prevented during specimen handling and processing Use only supplied or specified required disposables to ensure optimal test performance Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of specimens or controls Before use visually inspect each reagent cassette control tube and Wash Reagent to ensure that there are no signs of leakage If
42. tective antibodies to HAV while in ountries the decline in the incidence rate of the virus has led to a shift towards infection in IAV infections in humans range from asymptomatic infections mainly seen in young children to fi ant hepatitis which in some cases may lead to death In Northern Europe Japan Canada and the USA the prevalence in the general population is very low about 0 01 and outbreaks are associated mainly with risk groups such as travelers to endemic regions HAV can be grouped into different genotypes I VI with genotypes Il and III being found in humans Although infectious HAV can be found in blood during the serology window period the risk of transfusion transmission of HAV is very low Nevertheless like B19V HAV is not easily inactivated by S D treatment or pasteurization and there have been some reports on the transmission of HAV through plasma products mainly coagulation factors The Document Revision Information Section is located at the end of this document 05509238001 02EN 1 Doc Rev 2 0 The cobas TaqScreen DPX Test is a duplex test for the simultaneous detection of B19V and HAV in individual samples or pooled plasma samples of human origin The use of multi dye technology enables the identification of each viral target without the need for further discriminatory testing In addition the test provides a quantitative value in IU mL for the B19V target through the use of a Quantitation
43. tive reactive or invalid for the HAV target and lt cutoff e B19V target In addition to retrieving and examining PCR results the Roche PDM gt cutoff or i id for software all perator to print reports search for results accept donor results and optionally transmit lt O 05509238001 02EN 3 Doc Rev 2 0 MATERIALS PROVIDED BY ROCHE Three kits are required and provided for the detection of HAV RNA and quantitation of B19V DNA in plasma specimens 1 cobas TaqScreen DPX Test 2 cobas TaqScreen DPX Control Kit and 3 cobas TaqScreen Wash Reagent Material Safety Data Sheets IMSDS are available on request from your local Roche office cobas TaqScreen DPX Test 96 Tests P N 05509211 190 DPX CS1 DPX Magnetic Glass Particles Reagent Cassette DPX CS2 DPX Lysis Reagent Cassette DPX CS3 DPX Multi Reagent Cassette UV DPX CS4 DPX Test Specific Reagent Cassette O cobas TaqScreen DPX Control Kit P N 05509190 190 DPX GIL DPX D C HAV Positive and B19V Low Positive Dual Control Sy DPX H C B19V High Positive Control 12 Sets DPX C cobas TaqScreen DPX Negative Control cobas TaqScreen Wash Reagent TS WR P N 04404220 190 lt JS TS WR cobas TaqScreen Wash Reag XY amp Qe lt O 05509238001 02EN Doc Rev 2 0 OTHER MATERIALS REQUIRED BUT SOLD SEPARATELY MAY BE PURCHASED FROM ROCHE This test must be run on the cobas s 201 system The cobas s 201
44. type Using one lot of reagent between 4 and 6 replicates were performed for each specimen and dilution point Table 12 Table 12 B19V Genotype Quantitation Mean a Genotype Input B19V Conc Observed ee S10 B19V Conc log Input B19V IU mL logio IU ML logio IU mL 5x107 2 70 2 49 0 21 1x10 4 00 3 94 1 1x10 5 00 477 1x 10 6 00 5 73 1x10 7 00 6 84 5x10 2 70 3 00 1x10 4 00 414 3 1x 10 5 00 4 96 3x10 5 48 5 32 0 16 1x10 6 00 5 72 0 28 1x10 7 00 64 0 56 5x107 2 70 04 0 34 1x10 4 00 4 0 24 as 1x 10 5 00 03 0 03 3x10 5 48 5 44 0 04 1x10 6 00 O 583 0 17 1x10 Z 6 53 0 47 Genotype Inclusivity The performance of the cobas TaqSc st was determined for different genotypes of HAV and B19V HAV Clinical specimens and RN 3X the Limit of Detection LOD of demonstrated that the LOD of different HAV genotypes diluted to approximately 0 3X 1X and s TaqScreen DPX Test were tested PROBIT analysis of the results jenotype sample was equivalent to the LOD of the WHO Standard 00 560 Table 13 Table 13 Q HAV Genotype Inclusivity mone fresco mine mario im l 65 8 158 240 96 3 231 240 100 0 240 240 0 92 0 77 1 18 41 7 30 72 86 1 62 72 100 0 72 72 1 58 1 18 2 51 I 79 2 19 24 91 7 22 24 100 0 24 24 1 18 0 61 257 IB 79 2 19 24 100 0 24 24 100 0 24 24
45. used once the 48 cumulative hours are reached The AMPLILINK software does not monitor the number of instrument runs the cassettes been used for It is the user s responsibility to discard the reagent cassettes once 6 instrument runs_are Store DPX D C DPX H C and DPX C at 2 to 8 C The controls are si il the expiration date indicated Once opened any unused portion must be discarded Store TS WR at 15 to 30 C Unopened TS WR is stable until the expiration date indicated Once opened this reagent is stable for 30 days at 15 to 30 C or until the expiration date Whichever comes first PRECAUTIONS A Specimens may be infectious Use Universal precautions whi ing the test Only personnel proficient in the use of the cobas TaqScreen DPX Test and d in handling infectious materials should perform this procedure Thoroughly clean and disi all laboratory work surfaces with a freshly prepared solution of 0 5 sodium hypochlorite in distil ionized water diluted bleach Follow by wiping the surface with 70 Ethanol CAUTION DPX D C and DPX H C contaii an plasma derived from human blood The plasma used has been tested and fo eactive for IgG and IgM antibodies to B19V HBsAg HBV core Ab HCV Ab HIV 1 2 Ab non reactive for HIV 1 RNA by NAT The plasma used has also been tested by the cobas n DPX Test and shown to be non reactive for HAV RNA and to contain lt 5 IU mL o known test method can offer complete as
46. utton to complete the workflow for a donor The Force Unresolve function assigns an Accepted Unresolved donor status to donors not having a final result of reactive for HAV or gt cutoff foryB19V or assigns an Accepted donor status to donors having a final result of reactive for HAV or cutoff fe Secondary Pooling Test Donor tubes in a multi specimen pool with a fin f invalid for one target require repeat testing if the final result for the other target is non reactive of invalid for HAV or lt cutoff or invalid for B19V When a multi specimen pool is reportedya ive for HAV or cutoff for B19V the cobas s 201 system h less donors or with a single donor and further tested with the e resolution process to identify the HAV reactive or B19V gt cutoff e cobas s 201 system Operator s Manual for specific information on original donor tubes in subdivi cobas TaqScreen DPX Test_a pai individual donor specimen performing resolution te When a subdivide individual specime ported as Completed with a final result of HAV non reactive and B19V lt cutoff Note As f an overall quality assurance program the user may wish to conduct additional testi termine the cause of the initial reactivity of the specimen v L LIMITATIONS 1 is test has been evaluated only for use in combination with the cobas TaqScreen DPX Test kit cobas aqScreen DPX Control Kit the cobas TaqScreen Wash Reagent and the cobas
Download Pdf Manuals
Related Search