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COBAS Ampliscreen HBV Test

1. 5 Only the Hamilton MICROLAB AT plus 2 Pipettor has been validated for use with the COBAS AmpliScreen HBV Test for the automated prepa ration of plasma pools Adhere to the hardware instructions and safety precautions outlined in the User Manual for the Hamilton MICROLAB AT plus 2 Pipettor 6 Though rare mutations within the highly conserved regions of the viral genomes covered by the COBAS AmpliScreen HBV Test primers and or probes may result in failure to detect a virus 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 PERFORMANCE CHARACTERISTICS Reproducibility The reproducibility of the COBAS AmpliScreen HBV Test was established by testing two 6 member EDTA plasma panels with known concen trations of HBV Panel One which was tested by using the Multiprep Specimen Processing Procedure was composed of HBV DNA positive specimens at concentrations of 25 90 150 and 25 000 copies mL and two HBV negative specimens Panel Two which was tested by using the Standard Specimen Processing Procedure was composed of HBV positive specimens at concentrations of 75 300 500 and 25 000 copies mL and two HBV negative specimens Testing was performed at three sites with two operators at each site using three COBAS AmpliScreen HBV Test kit lots and analyzed in 5 differen
2. COBAS AmpliScreen HBV Test FOR IN VITRO DIAGNOSTIC USE COBAS AmpliScreen HBV Test HBV 96 Tests P N 03599779 190 COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit 96 Tests P N 03302555 018 COBAS AMPLICOR Wash Buffer WB 500 Tests P N 20759899 123 ART 07 5989 9 US 83314 INTENDED USE The COBAS AmpliScreen HBV Test is a qualitative in vitro test for the direct detection of Hepatitis B Virus HBV DNA in human plasma The COBAS AmpliScreen HBV Test is intended to be used to screen donors for HBV DNA in addition to the currently recommended serology tests This product is intended for use as a donor screening test to detect HBV in plasma samples 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 HBV DNA in blood specimens from cadaveric non heart beating organ and tissue donors This test is not intended for use on samples of cord blood Plasma from all donors may be screened as individual samples For donations of whole blood and blood components plasma may be tested in pools comprised of equal aliquots of not more than 24 individual donations in conjunction with licensed tests for detecting hepatitis B surface antigen HBsAg and antibody to hepatitis B core antigen anti HBc For donations of hematopoieti
3. Cw HBV AMP lt 0 001 Non infectious in vitro transcribed RNA microbial containing HIV 1 sequences O a lt 0 001 HBV 104UB and HBV 104D HW016TBB1 primers HBV 104UB is biotinylated lt 0 001 AmpErase uracil N glycosylase enzyme microbial lt 0 5 Tween 20 surfactant 0 09 Sodium azide HBV Mg2 HBV Magnesium Solution lt 1 Magnesium chloride Amaranth dye 0 05 Sodium azide 03599833001 07EN 8 x 9 0 mL 8 x 4 8 mL 8 x 0 1 mL 16 x 1 6 mL 96 Tests 8 x 0 7 mL 8 x 0 1 mL Doc Rev 70 COBAS AmpliScreen HBV Detection Reagents HBV DK BH PS1 HBV Probe Suspension 1 MES buffer lt 0 4 Suspension of Dynabeads paramagnetic particles coated with HBV specific oligonucleotide HBV DET capture probe 0 09 Sodium azide BH4 HBV Probe Suspension 2 Sodium phosphate buffer 24 9 Sodium thiocyanate 0 2 Solubilizer Xn 24 9 w w Sodium thiocyanate Harmful BIPS1 HBV IC Probe Suspension 1 MES buffer lt 0 4 Suspension of Dynabeads paramagnetic particles coated with HBV IC specific oligonucleotide capture probe SK535 0 09 Sodium azide BI4 HBV IC Probe Suspension 2 Sodium phosphate buffer 24 9 Sodium thiocyanate 0 2 Solubilizer Xn 24 9 w w Sodium thiocyanate Harmful DN4 Denaturation Solution 1 6 Sodium hydroxide EDTA Thymol blue Xi 1 6 w w Sodium hydroxide S Avidin Horseradish Peroxidase Conjugate Tris HCI buffer lt 0 0
4. MP C containing Working Lysis Reagent and NHP Cap the tube and vortex briefly b Positive Control Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 uL MP C to the tube labeled MP C containing Working Lysis Reagent and NHP Cap the tube and vortex briefly B11 Incubate all tubes for 10 to 15 minutes at room temperature after adding Working Lysis Reagent to the last tube After the incubation period briefly vortex all tubes B12 Pipette 700 uL of isopropanol into each tube Cap the tubes and vortex briefly B13 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 15 20 minutes at room temperature B14 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet B15 Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly B16 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with that will form Centrifuge at 14 250 1750 x g for 5 10 minutes at room temperature B17 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette relee much liquid as possible without dis turbing the pellet Use a new transfer pipette for each tube B18 Using a new transfer pipette for each tube repeat Step B17 to remove as much of the r mai ing supernatant
5. Of these 578 673 were also HBV DNA negative 21 spec imens were false positive The specificity of the COBAS AmpliScreen HBV Test in this study was 578 673 578 694 or 99 9964 with 95 confi dence limits of 99 9948 to 99 9979 There were 105 specimens that were determined to be HBV serology status positive HBsAg confirmed positive Of these 105 HBsAg specimens 87 specimens 82 9 were also HBV DNA positive when tested in primary mini pools using the Multiprep Specimen Processing Procedure and 18 specimens 17 1 were negative by this procedure Of these 18 HBV DNA negative specimens 17 specimens were also tested individually by the Standard Specimen Processing Procedure and 10 of these specimens were positive by this procedure and 7 specimens were negative It should be noted that three of the specimens with quantitative detection values of 1200 copies mL 2600 copies mL and 5900 copies mL of DNA that were also positive for HBsAg and anti HBc were negative on mini pool NAT COBAS AmpliScreen HBV Test Results in HBsAg Repeatedly Reactive Donations From November 11 2002 until J une 13 2007 903 349 donations were tested There were 212 HBsAg Repeatedly Reactive specimens of these 109 were also positive by the COBAS AmpliScreen HBV Test None of these were negative by neutralization Table 19 HBsAg Neutralization and COBAS AmpliScreen HBV Test Results for HBsAg Repeatedly Reactive Donations COBAS AmpliScreen HBV T
6. doephedrine HClI 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 HBV Test using either the Multiprep or Standard Specimen Processing Procedures CLINICAL PERFORMANCE Detection of HBV DNA in Seropositive Specimens A total of 1177 known HBV seropositive HBsAg positive specimens were tested by the COBAS AmpliScreen HBV Test These HBV seropositive specimens included the following specimens and sources 723 HBV seropositive specimens including 49 acute patients HBsAg and HBeAg posi tive obtained from commercial vendors and blood banks in the US J apan and China 100 chronic HBV patient specimens HBsAg positive for at least 6 months obtained from a commercial vendor 60 HBsAg seropositive specimens collected from patients at high risk for hepatitis and 189 HBsAg positive specimens from 40 seroconversion panels also described in Seroconversion Panels These specimens were tested using both the Multiprep Specimen Processing Procedure specimens diluted 1 24 in negative human plasma and Standard Specimen Processing Procedure spec imens tested undiluted An additional 105 HBsAg positive specimens were tested in the Clinical Performance study These specimens were initially tested in primary mini pools of 24 specimens using the Multiprep Specimen Processing Pro
7. All of the genotypes tested positive by the COBAS AmpliScreen HBV Test As a result of limited availability only one Genotype G was evaluated Therefore performance of the COBAS AmpliScreen HBV Test with this one Genotype G specimen may not be representative of all Genotype G specimens Data are pro vided in Table 7 03599833001 07EN Doc Rev 7 0 12 Table 7 HBV Genotypes Tested eoms f on E S A 18 18 18 18 18 B 24 24 24 24 24 C 18 D 10 E 18 F 25 G 1 AIC 3 A D 2 C D 2 D E 2 AIE T B C 1 C E T D F 1 E F T Seroconversion Panels Forty commercially available HBV seroconversion panels were tested with the COBAS AmpliScreen HBV Test Each panel member was tested undi luted with the Standard Specimen Processing Procedure and diluted 1 24 with HBV Negative Human Plasma for testing With the Multiprep Specimen Processing Procedure The COBAS AmpliScreen HBV Test results were compared to the results from an FDA licens V surface antigen HBsAg assay Ortho HBsAg ELISA Test System 3 Of the 40 panels tested one was removed from the analysis which was detected by the COBAS AmpliScreen HBV Test 108 days prior to the Ortho HBsAg ELISA Test System 3 In addition 6 panels tested wi Multiprep Specimen Processing Procedure and 3 panels tested by the Standard Specimen Processing Procedure that were intermittently HBV itive up to 100 days to the Ortho test were removed resultin
8. B36 Using a new transfer pipette for each tube repeat Step B35 to remove as much of the remaining supernatant as possible without disturbing he pellet Residual ethanol can inhibit amplification B37 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 he tip and scraping the sides and base of the tube in an up down motion for at least 10 seconds and dispensing 30 40 uL Cap the tubes and vortex briefly to resuspend the extracted DNA Note that some insoluble material may remain B38 At this point amplification of the processed specimens and controls must be started within 2 hours If not the processed specimens and con rols 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 B39 Create an A ring worklist record for each A ring to identify the A tube with the appropriate control or specimen to be pipetted B40 If processed specimens and controls were stored frozen thaw at room temperature before proceeding Briefly vortex the processed speci mens and controls B41 Pipette 50 uL of each processed specimen and control into the appropriate A tube containing HBV 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
9. HBV Patients Results for Standard Specimen Processing Procedure Specimens Undiluted HBV DNA Results 95 95 Standard Procedure a Total 100 All HBV DNA negative specimens were tested by alternate HBV DNA tests and contained lt 300 copies mL HBV DNA 03599833001 07EN Doc Rev 7 0 14 High Risk Patients There were 60 HBV seropositive specimens from patients at high risk for liver disease tested with the COBAS AmpliScreen HBV Test There were 59 specimens 98 3 which tested HBV DNA positive and 1 specimen 1 7 HBV DNA negative using the Multiprep Specimen Processing Procedure All 60 specimens 100 were HBV DNA positive using the Standard Specimen Processing Procedure See Table 14 and Table 15 Table 14 HBV Seropositive Specimens from Patients at High Risk for Liver Disease Results for Multiprep Specimen Processing Procedure Specimens Diluted 1 24 HBV DNA Results 59 98 3 MultiPrep Procedure T Total 60 The HBV DNA negative specimen was tested by alternate HBV DNA tests and contained lt 300 copies mL HBV DNA Table 15 HBV Seropositive Specimens from Patients at High Risk for Liver Disease Results for Standard Specimen Processing Procedure Specimens Undiluted HBV DNA Results 60 100 Standard Procedure ji Total 60 Seropositive Specimens from Seroconversion Panels For the 40 HBV seroconversion panels each panel was consistentl
10. HBV Test The specificity ce interval of 97 6 to 98 9 mined to be false positives Based on these results all 27 were presumed to be false positiv of the COBAS AmpliScreen HBV Test in this study was 98 4 1625 1652 with a 95 eon Table 20 N Paired Specimen Results for Individual Samples ssigned HBV Status HbsAg Anti HBc COBAS Result Result on es gai Total Negative NR Negati Negative 1625 Negative RR ative Unknown 12 Negative 4 Negative 27 Positive Positive Negative Negative Positive Unknown Positive Negative Positive Positive y Positive Positive 87 Total 1754 Status Positive re lassified due to follow up results Performance Characteristics lasma were reactive with the AmpliScreen HBV Test for an initial reactive rate of 0 74 Of the 8 reactive pools there were 3 identified HBV DNA positive pools and 2 po ere positive due to apparent window period cases The remaining 3 pools were reactive for HBV DNA but were not con Clinical Performance A total of 1 080 TE d in the 96 member mini pool format representing 103 680 specimens from 40 230 donors revealed that 8 pools firmed The data are prese ted in Table 21 Table 21 Pool Reactivity in Source Plasma Donors Category No of Pools Percentage Pools tested 1080 100 Non Reactive pools 1072 99 26 Initially Reactive pools 8 0 74 Initially pools containing a reactive individual
11. Specimen Processing Procedure correctly detected 98 3 59 60 pre mortem EDTA plasma specimens with a 95 confidence interval of 91 1 to 99 9 and 96 6 56 58 of cadaveric specimens with a 95 confidence interval of 88 1 to 99 6 that were spiked with HBV DNA at 3X the LOD of the COBAS AmpliScreen HBW Test The summary of the final test results of this study is presented in Table 23 below Table 23 Summary of Sensitivity Test Results AV Pre Mortem EDTA Post M orte Plasma Specimen Plasma Specime Replicates 60 a 59 6 Sensitivity 98 3 96 6 95 Confidence Upper 99 9 SS 99 6 Interval Lower 91 2 88 1 Specificity Study Sixty pre mortem and 58 post mortem specimens that were negative for HBV D ere divided into three groups diluted 1 5 in MP DIL processed using the Multiprep Specimen Processing Procedure and tested using e COBAS AmpliScreen HBV Test The COBAS AmpliScreen HBV Test using samples diluted 1 5 an iprep Specimen Processing Procedure yielded negative results on 100 60 60 of the pre mortem EDTA plasma specimens with a 95 confidence interval of 94 to 100 and 100 58 58 of the post mortem EDTA plasma specimens with a 95 confidence interval of 93 8 to b The summary of the specificity test results is presented in Table 24 below able 24 of Specificity Test Results Test Results S Pre Mortem EDTA Post Mortem EDTA
12. WHO HBV International Standard 97 746 The WHO HBV International Standard was serially diluted in Negative Human Plasma to final concentrations of 100 30 10 5 4 and 3 IU mL for the Multiprep Specimen Processing Procedure and 300 100 30 20 15 and 10 IU mL for the Standard Specimen Processing Procedure Each dilution was tested using two lots of COBAS AmpliScreen HBV Test at 60 replicates per lot When evaluated using PROBIT analysis the combined data from all specimens tested with the Multiprep Sample Processing Procedure indicate an average 95 LOD of 4 41 IU mL with lower and upper 95 confidence limits of 3 56 IU mL and 6 13 IU mL respectively The LOD of 4 41 IU mL corresponds to approximately 22 copies mL When evaluated using PROBIT analysis the combined data from all specimens tested with the Standard Sample P sing Procedure indicate an average 95 LOD of 15 99 IU mL with lower and upper 95 confidence limits of 13 78 IU mL and 20 06 IU mL respectively The LOD of 15 99 IU mL corresponds to approximately 80 copies mL WZ Table 5 and Table 6 summarize the overall results for the Multiprep and Standard Specimen Processing Proced wespectively Serial Dilution Testing Summary for Multiprep Procedure with HBV DNA WHO inane Standard 97 746 Table 5 Combined Input Values with Lower 95 Confidence Limit On ded HBV DNA 95 Lower O 0 Number of Individual Tests Number of Concentration Positives Confidenc
13. 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 C Reagents 1 All reagents except HBV MMX and HBV Mg must be at room temperature before use Visually examine reagents for sufficient volume before beginning the test procedure See section Reagent Preparation for specific reagent storage conditions 2 Add all reagents using a pipettor capable of delivering specified volume with 3 accuracy and a precision of 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 laborat
14. as possible without disturbing the pellet Residual ethanol can inhibit amplification B19 Pipette 200 uL MP DIL into each tube Use a pipette tip to break apart the pelle Koc e 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 tel seconds and dispensing 30 40 uL Cap the tubes and vortex briefly to resuspend the extracted DNA Note that some insoluble ay remain B20 At this point amplification of the processed specimens and controls must b started within 2 hours If not the processed specimens and con trols can be stored at 70 C or colder for up to one month Thawing s be completed within one hour at room temperature B21 Proceed to step B39 Loading the A ring Standard Specimen Processing Procedure Individual Specimens adaveric and Source Plasma Minipools B22 Pipette 200 uL of each specimen into an appropriately label ap tube using the COBAS AmpliScreen Pooling System a hand held pipettor or other user validated method Cap the tubes B23 Vortex NHP briefly B24 For each Negative and Positive Control pipette 200 o appropriately labeled screw cap tubes Cap the tubes B25 Use a permanent marker to make an orientation mark on each tube B26 Prepare a Working Lysis Reagent bottle for e specimens and controls to be processed B27 Pipette 600 uL Working Lysis Reagent into tube Cap and vortex tubes briefly B28 Prepare Controls as follows a Nega
15. each individual test as wel pErase uracil N glycosylase enzyme to reduce potential contamination by previously amplified material amplicon PRINCIPLES OF THE PROCEDURE The COBAS AmpliScreen HBV Test is Qe major processes I Sample Processing 2 PCR amplification of targe using HBV specific complementary primers 3 Hybridization of the a roducts to oligonucleotide probes specific to the target s 4 Detection of the ngakoni amplified products by colorimetric determination Specimen Processin Two specimen processing procedures are used with the COBAS AmpliScreen HBV Test as follows Multiprep Specimen Processing Procedure for preparation of mini pool specimens and individual cadaveric specimens Standard Specimen Processing Procedure for preparation of individual specimens 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 NOTE In order to detect 2 Intemational Units IU mL 2IU 10 copies triplicate testing using the Multiprep Specimen Processing Procedure should be performed i e three amplification detections are performed requiring 50 pL each of the 200 pL of eluate following a single extraction A positive result in one or more of the replicates indicates that the specimen is positive and contains at least 2 IU mL of HBV DNA In the Standard Specimen Processing Procedure HBV DNA is
16. isolated directly from plasma by lysis of the virus particles with Multiprep Lysis Reagent followed by precipitation of the DNA with alcohol In the Multiprep Specimen Processing Procedure HBV 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 DNA with alcohol The Multiprep Internal Control MP IC containing the HBV Internal Control is introduced into each specimen with the Multiprep Lysis Reagent and serves as an extraction and amplification control for each processed specimen and control The HBV Internal Control is a DNA plasmid with primer binding regions identical to those of the HBV target sequence a randomized internal sequence of similar length and base composition as the HBV target sequence and a unique probe binding region that differentiates the HBV Internal Control amplicon from target amplicon These features were selected to ensure equivalent amplification of the HBV Internal Control and the HBV target DNA PCR Amplification The amplification reactions are performed with the thermostable recombinant enzyme Thermus aquaticus DNA Polymerase Taq pol The reaction mixture is heated to separate double stranded DNA As the mixture cools primers anneal to the target DNA and in the presence of Mg and excess deoxynucleotide triphosphates dNTPs the Taq pol extends the annealed primers al
17. positive individual specimen testing 85 0 33 and with concordant serology TR Positive pools due to window period case 2 0 008 nitially reactive pools with negative serology and negative indi 51 0 2 vidual specimen AmpliScreen Testing false positive nee nitially reactive pools with positive individual specimen testing 9 0 03 and without concordant serology priate nitially reactive pools with 2 positive units by individual testing one concordant with serology and one without concordant 3 0 001 serology 03599833001 07EN Doc Rev 70 15 Of the 25 845 pools a total of 581 790 specimens were tested from 5 geographically divergent sites The results from these specimens were used to determine the specificity and sensitivity of COBAS AmpliScreen HBV Test The HBV serology status of each specimen was determined using each specimens antigen and antibody results HBV serology status negative included specimens that were HBsAg and anti HBc negative unless the subject was enrolled in the follow up study and had test results that changed this assessment HBV serology status positive included those speci mens that were HBsAg positive regardless of the anti HBc results unless the subject was enrolled in the follow up study and had test results that changed this assessment HBV serology status unknown included those specimens that were anti HBc positive and HBsAg negative There were 578 694 specimens that were determined to be HBV serology status negative
18. specimen and control processed For example mix 11 7 mL 90 ethanol and 3 3 mL of dis tilled or deionized water for every 12 specimens and controls to be processed SPECIMEN COLLECTION STORAGE AND POOLING NOTE Handle all specimens as if they are potentially infectious agents Living Donor Specimens A EDTA CPD CPDA 1 CP2D ACD A and 4 Sodium Citrate may be used with the COBAS AmpliScreen HBV Test 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 remove the plasma from the red blood cells by centrifugation at 800 1600 x g for 20 minutes Following removal plasma may be stored at 2 8 C for an additional seven days Alternatively plasma may be stored at lt 18 C for up to one month 2 to 30T gt 2 g f E 0 Q o 5 _ 2 to 8C V Plasma f w 0 O onn Days Post i G Blood collected in CPD CPDA 1 or CP2D may be stored for up to 72 hours at ag one 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 itional 7 days from the date the plasma was removed from the red blood cells Plasma separated from the cells may be store 18 C for up to one month D ACD A or 4 sodium citrate anticoagulated apheresis plasma can be C 8C for up to 6 hours followed by subsequent storag
19. specimen with concordant serology 3 0 28 Positive pools due to window period case 2 0 18 Initially Reactive pools with negative resolution COBAS 3 0 28 AmpliScreen Testing false positive 1 Two HBsAg negative specimens were in one 96 member mini pool There were 1075 pools used to determine the specificity of the COBAS AmpliScreen HBV Test Of these pools 1072 were HBV DNA negative and 3 were initially Reactive with negative resolution COBAS AmpliScreen Testing false positive The specificity of the COBAS AmpliScreen HBV Test in this study was 1072 1075 or 99 7209 with 95 confidence limits of 99 19 to 99 94 HBV Seroconversion Panels Ten commercially available HBV seroconversion panels were tested using the Multiprep Specimen Processing Procedure and compared to results obtained with licensed HBsAg tests Blinded panel members were diluted 1 96 with HBV negative human plasma In two panels COBAS AmpliScreen HBV Test detected HBV DNA on the same day as HBsAg was detected by the Ortho Antibody to HBsAg ELISA Test System 2 In three panels COBAS AmpliScreen HBV Test detected HBV DNA on the same day as HBsAg was detected by the Ortho HBsAg Test System 3 In two panels COBAS AmpliScreen HBV Test detected HBV DNA on the same day as HBsAg was detected by the Abbott Auszyme Test Data are presented in Table 22 03599833001 07EN Doc Rev 7 0 16 Table 22 Summary of Pre Seroconversion Detec
20. to the unborn child Skin contact inhalation of fumes and ingestio Id_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 otl Guide and the Operator s Manuals for the AMPLILINK Software Closely follow procedures and guidelines provided to ensure tl from the given procedures and guidelines may affect optimal AmpliScreen products COBAS AmpliScreen Pooling System AS AMPLICOR Analyzer pecimen and control preparation is performed correctly Any deviation performance i The use of excessively hemolyzed cadaveric specime IdgBe avoided REAGENT PREPARATION on A MP IC MP C MP C MP DIL and NHP 1 Warm MP IC MP C MP C MP DI a to room temperature before use by using a 37 C incubator or on laboratory bench top Working Lysis Reagent 1 Warm MP LYS to 25 37 C to di precipitate maximum 30 minutes Mix thoroughly until the crystals are dissolved Prior to use visually verify that crystals s d and examine each bottle of MP LYS against a white background for the appearance of a yellow color or leakage If tl y 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 e 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 vor fly The pink color confirm
21. was opened dentify 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 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 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 Place the D cup rack on the D cup platform Two D cups are required for each A tube and two D cups are required for each Working Substrate cassette to allow for blanking by the COBAS AMPLICOR Analyzer as described in the Operator s Manual for the COBAS AMPLICOR Analyzer Place the A ring into the thermal cycler segment of the COBAS AMPLICOR Analyzer and close the Ya thermal cycler segment Load the A ring into the COBAS AMPLICOR Analyzer using the COBAS AMPLICOR Analyzer b scanner for the AMPLILINK software as described in the Operator s Manual for AMPLILINK software Create an A ring order using the AMPLILINK software as described in the Operator s Man 0 PLILINK software Use the A ring worklist record cre
22. 01 Avidin horseradish peroxidase conjugate Bovine serum albumin mammalian Emulsit 25 Dai ichi Kogyo Seiyaku Co Ltd 0 1 Phenol 1 ProClin 150 preservative SB3 Substrate A X Citrate solution 0 01 Hydrogen peroxide 0 1 ProClin 150 preservative SB Substrate B 0 1 3 3 5 5 Tetrame nzidine TMB 40 Dimethylformami T 40 Dimethylformamide DMF see OXIC R 61 20 21 36 May cause harm to the unborn 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 COBAS AMPLICOR Wash Buffer Irritant CN4 O P N 20759899 123 ART 07 5989 9 US 83314 WB WB 10X Wash Concentrate lt 2 Phosphate buffer lt 9 0 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 HBV MMX HBV Mg2 BH PS1 BH4 BI PS1 B14 CN4 SB3 SB D Store DN4 at 2 25 C Store WB at 2 30 C DN4 and WB are stable until the expiration dates indicated 03599833001 07EN 96 Tests 1 x 100 Tests 1 x 100 Tests 1 x 100 Tests 1 x 100 Te
23. 20 120 120 ERI 100 100 F 120 120 119 120 Sitea 100 99 2 120 120 119 120 SIERS 100 99 2 03599833001 07EN Doc Rev 70 17 REFERENCES 1 Fagan EA and Harrison TJ 2000 Viral Hepatitis A Handbook for Clinicians and Scientists Springer Verlag New York Inc 89 130 2 Beasley RP 1982 Hepatitis B virus as etiologic agent in hepatocellular carcinoma epidemiological considerations Hepatology 2 Suppl 21S 26S 3 Feitelson M 1992 Hepatitis B virus infection and primary hepatocellular carcinoma Clin Microbiol Rev 14 257 301 4 Mast EE and Later MJ 1993 Epidemiology of viral hepatitis an overview Semin Virol 4 273 283 S Robinson WS Clayton DA and Greenman RL 1974 DNA of a human hepatitis B virus candidate J Virol 14 384 391 6 Galibert F Mandart E Fitoussi F Tiollais P and Charney P 1979 Nucleotide sequence of the hepatitis B virus genome subtype ayw cloned in E coli Nature 281 646 650 Te Kitchen A 1998 Hepatitis B and blood safety Vaccine 16 534 s37 8 Schreiber GB Busch MP Kleinman SH and Korelitz J J 1996 The risk of transfusion transmitted viral infections N Engl Med 334 1685 1690 9 Zuckerman AJ 1999 More than third of the world s population has been infected with hepatitis B virus letter Br Med J 318 1213 10 McQuillan GM Coleman PJ Kruszon Moran D Moyer LA Lambert SB and Margolis HS 1999 Prevalence of hepatitis B virus infection in the United States The National Health
24. 8 C between instrument cycles Remove from refrigerator 30 minutes before use on the COBAS AMPLICOR Analyzer DN4 Denaturation Reagent and CN4 Conjugate Reagent 1 Once opened DN4 and CN4 are stable for 30 days at 2 8 C or until the expiration date whichever comes first Both DN4 and CN4 can be used for a maximum of ten instrument cycles 12 hours per cycle 2 Store DN4 and CN4 at 2 8 C between instrument cycles Remove from refrigerator 30 minutes before use on the COBAS AMPLICOR Analyzer Working Substrate Reagent 1 Working Substrate must be prepared each day by pipetting 5 mL SB into one SB3 cassette Pipette up and down at least 5 times to mix 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 03599833001 07EN Doc Rev 7 0 G 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 dis tilled or deionized water Mix well Keep a minimum of 3 4 liters of Working Wash Buffer 1X in the Wash Buffer Reservoir of the COBAS AMPLICOR Analyzer at all times 2 Working Wash Buffer 1X should be stored at 2 25 C in the COBAS AMPLICOR Wash Buffer Reservoir and is stable for 2 weeks from the date of preparation H 70 Ethanol 1 Prepare 70 ethanol fresh daily 2 One mL 70 ethanol is needed for each
25. 89 88 90 90 90 1 84 99 98 100 Site 3 0 179 79 90 90 90 90 90 90 90 0 88 100 100 100 Table 3 Operator Variability Data Multiprep Specimen Processing Procedure Results By Operator Instrument Positive Tested Operator Negative 25 c mL 90 c mL 150 c mL 1 0 90 39 44 45 45 45 45 0 88 6 100 100 2 0 90 41 45 45 45 45 45 0 91 1 100 100 3 0 90 44 45 45 45 45 45 0 97 8 100 100 4 0 90 32 45 39 42 43 45 0 71 1 92 9 95 6 5 0 90 35 45 45 45 45 45 0 77 8 100 100 6 1 90 35 45 45 45 45 45 1 1 77 8 100 100 03599833001 07EN Doc Rev 70 11 Operator Variability Data Table 4 Standard Specimen Processing Procedure Results By Operator Instrument Positive Tested Operator Negative 75 c mL 300 c mL 500 c mL_ 25 000 c mL 1 0 90 39 44 45 45 45 45 45 45 0 88 6 100 100 100 2 0 90 33 45 44 44 45 45 45 45 0 73 3 100 100 100 3 0 90 41 45 45 45 45 45 45 45 0 91 1 100 100 100 4 1 89 35 45 43 44 43 45 45 45 1 1 77 8 97 7 95 6 100 5 0 89 41 45 45 45 45 45 45 45 0 91 1 100 100 100 6 0 90 38 45 45 45 45 45 45 45 0 84 4 100 100 100 Analytical Sensitivity WHO HBV International Standard The Limit of Detection of the COBAS AmpliScreen HBV Test was determined by using the
26. DNA nate NAT 11 of 18 were i tive NAT and 7 of poe glad Table 17 H opositive Donor Specimens Results for Stan pe cimen Processing Procedure Specimens Undiluted HBV DNA Results 0 Qe Procedure 97 93 3 7 Total 104 4or7 specimens were negative for HBV DNA by alternate NAT 1 was not ested by alternate NAT and the remaining 2 quantified by alternate NAT had HBV DNA lt 100 copies mL Pool Reactivity in Whole Blood A random selection of 25 845 pools revealed that 150 Primary Pools were reactive for an initial reactive rate of 0 58 There were 85 150 56 7 positive pools that were concordant with confirmed positive serology status Two of these pools were identified as having a window period case There were 51 25 845 0 197 pools that were initially reactive but determined to be HBV DNA negative upon resolution testing A total of 9 25 845 0 035 pools were found positive but were not confirmed positive by serology or by subsequent testing of individual specimens by the COBAS AmpliScreen HBV Test There were 3 pools with two positive units one with concordant serology and one without concordant serology Results are summarized in the Table 18 Table 18 Pool Reactivity in Whole Blood Category No of Pools Percentage Pools tested 25 845 100 Non Reactive pools 25 695 99 42 nitially Reactive pools 150 0 58 nitially reactive pools with
27. Haas Company Copyright 2010 Roche Molecular Systems Inc All rights reserved ea 1 2010 A lt amp O 03599833001 07EN Doc Rev 7 0 20
28. IC should be greater than or equal to 0 2 for the Negative Control to be valid lf 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 rifig is inyalid and the entire test procedure for that A ring specimen and control preparation ampli fication and detection must p b Positive Control The absorbance for th 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 66 e Positive Control to be valid If the absorbance value for the MP C is less than 1 0 and or its asso ration amplificati tection must be repeated ciated MP IC is 0 2 the entire A ring is invalid and the entire test procedure for that A ring specimen and control prepa Summary of Control Acceptance Criteria HBV Result IC Result A660 Comment A660 Comment Negative Control lt 0 2 Negative gt 0 2 Valid Positive Control 21 0 Positive 20 2 Valid 2 Flags and comments may be generated by the COBAS AMPLICOR Analyzer during a run The Operator must check the run printout s for flags and comments to verify that the run is valid Refer to the Operator s Manual for the AMPLILINK software and the Operator s Manual for the COBAS AMPLICOR Analyzer for interpretation of flags and comments 3 External Control INTERPRETATION OF RESULTS If an External Control i e an additional run control other than th
29. 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 B Specimen and Control Preparation Performed in Pre Amplification Specimen and Control Preparation Area Multiprep Specimen Processing Procedure Pooled Specimens Individual Cadaveric Specimens and For Testing Individual Specimens in Triplicate Bl For pooled specimens pipette 1000 uL of each pool into an appropriately labeled screw cap tube using the COBAS AmpliScreen Pooling System a hand held pipettor or other user validated method Cap the tubes Proceed to Step B2 For individual cadaveric specimens pipette 200 uL into an appropriately labeled screw cap tube and add 800 uL Multiprep Diluent MP DIL using a hand held pipettor or other user validated method Cap the tubes Vortex each specimen tube briefly Proceed to Step B2 For testing in triplicate pipette 1000 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 Proceed to Step B2 B2 Vortex NHP briefly 03599833001 07EN Doc Rev 7 0 B3 B4 B5 B6 For each Negative and Positive Control pipette 1000 uL NHP into an appropriately labeled screw cap tube Cap the tub
30. Plasma Specimen Plasma Specimen Replicates 60 58 0 0 60 58 Inhib 0 0 Final Specificity 100 100 Confidence Upper 100 100 nterval Lower 94 93 8 Reproducibility sua Twenty pre mortem EDTA plasma and 20 individual cadaveric specimens were spiked with HBV viral target using a secondary standard to a final con centration of 3X the LOD Each of the 20 pre and post mortem specimens were tested using three different COBAS AmpliScreen HBV Test 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 dif ferent kit lots total of six valid test results for each specimen at each site There were a total of 18 valid test results six results per site x 3 testing sites for each specimen All valid reproducibility data for post mortem and pre mortem specimens were evaluated by calculating the percentage of correct results for each assay The data were analyzed by lot and by testing site The summary of results of the reproducibility study test is presented in Table 25 below Table 25 Summary of Reproducibility Study Test Results Post Mortem versus Pre Mortem Post Mortem Pre Mortem Results by Lot Positive Tested Percent Hit Rate 120 120 120 120 hotel 100 100 120 120 119 120 pons 100 99 2 120 120 119 120 roles 100 99 2 Results by Site Positive Tested Percent Hit Rate F 120 1
31. R Wash Buffer 500 Tests P N 20759899 123 ART 07 5989 9 US 83314 WB WB 10X Wash Concentrate OTHER MATERIALS REQUIRED BUT SOLD SEPARATELY MAY BE PURCHASED FROM ROCHE COBAS AMPLICOR Analyzer with software version 0022B Printer and Operator s Manual for the COBAS AMPLICOR Analyzer COBAS AMPLICOR A rings COBAS AMPLICOR D cups e AMPLILINK Software version 1 4 and Operator s Manual for the AMPLILINK Software 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 AmpliScreen Pooling System Guide are validated to prepare pools of equal aliquots of not more than 24 individual plasma donations using the Hamilton MICROLAB AT plus 2 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 specimens NOTE The user must validate all pooling algorithms and equipment other than those supplied by Roche Sarstedt 1 5 mL tube Barcode Labels e Hamilton Archive and Intermediate Plate Barcode Labels Refrigerated high speed centrifuge with fixed angle rotor 45 degrees capacity for at least 24 x 1 5 mL tubes with an RCF of 23 600 x g Heraeus Centrifuge 17RS or Biofuge 28RS with HFA 22 1 rotor H
32. amethylbenzidine TMB to each D cup In the presence of hydrogen peroxide the particle bound horseradish peroxidase catalyzes the oxidation of TMB to form a colored complex The absorbance is measured by the COBAS AMPLICOR Analyzer at a wavelength of 660 nm MATERIALS PROVIDED BY ROCHE The COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit and the COBAS AMPLICOR Wash Buffer are provided as stand alone kits to be used in conjunction with the COBAS AmpliScreen HBV Test as well as the COBAS AmpliScreen HIV 1 Test v1 5 and the COBAS AmpliScreen HCV Test COBAS AmpliScreen Multiprep Specimen Preparation and Control Kit 96 Tests P N 03302555 018 MULTIPREP CTL MP C Multiprep Negative Control MP C Multiprep Positive Control MP LYS V Multiprep Lysis Reagent MP DIL Multiprep Specimen Diluent MP IC Multiprep Internal Control NHP 5 Negative Plasma Human COBAS AmpliScreen HBV Test P N 03599779 190 BV COBAS AmpliScreen HBV Amplification Reagents HBV MMX HBV Master Mix HBV Mg2 HBV Magnesium Solution i A COBAS AmpliScreen HBV Detection Reagents K HEV DK BH PS1 HBV Probe Suspension 1 BH4 K 96 Tests AMP 4 HBV Probe Suspension 2 BIPS1 HBV IC Probe Suspension 1 BI4 HBV IC Probe Suspension 2 Qe DN4 Denaturation Solution CN4 Avidin Horseradish P Conjugate SB3 Substrate A SB Substrate B COBAS AMPLICO
33. and Nutrition Examination Survey 1976 through 1994 Am J Pub Health 89 14 18 11 Van DP and Vellinga A 1998 Epidemiology of hepatitis B and C in Europe Acta Gastroenterol Belg 61 175 182 12 Maddrey WC 2000 Hepatitis B An important public health issue J Med Viol 61 362 366 13 Larsen J Hetland G and Skaug K 1990 Posttransfusion hepatitis B transmitted by blood from a hepatitis B surface antigen negative hepatitis B virus carrier Transfusion 30 431 14 Saraswat S Banerjee K Chaudhury N Mahant T Khandekar P Gupta RK and Naik S 1996 Post transfusion hepatitis type B following multiple transfusions of HBsAg negative blood J of Hepatol 25 639 643 15 Yotsuyanagi H Yasuda K lino S Moriya K Shintani Y Fujie H Tsutsumi T Kimura S and Koike K 1998 Persistent Viremia After Recovery From Self Limited Acute Hepatitis B Hepatology 27 1377 1382 16 Michalak TI Pasquinelli C Guilhot S and Chisari FV 1994 Hepatitis B virus persistence after recovery from acute viral hepatitis J Clin Invest 93 230 239 17 Cabrerizo M Bartolom J Caramelo C Barril G and Carre o V 2000 Molecular Analysis of Hepatitis B Vir A in Serum and Peripheral Blood Mononuclear Cells From Hepatitis B Surface Antigen Negative Cases Hepatology 32 116 123 18 Busch MP Stramer SL Kleinman SH 1997 Evolving applications of nucleic acid amplification assay reyention of virus transmission by blood components and derivatives In Garratty G ed Applicati
34. ated for specimen processing to assist in entering the A ring order Repeat steps h through j above to load a second A ring on the COBAS AMPLI Analyzer Start the COBAS AMPLICOR Analyzer as described in the Operator s Manualfor AM PIALINK software m Wait for the COBAS AMPLICOR Analyzer to indicate that the load check ha NOTE The required quantity of each detection reagent is automatically ca d by the COBAS AMPLICOR Analyzer during n Run Log with the selected A ring worklist to accountgfo follow up investigation QUALITY CONTROL PROCEDURES the Load Check to determine if sufficient reagents are available the requested tests The COBAS AMPLICOR Analyzer automatically performs 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 Rep d the Run Log may be printed e g daily weekly or monthly and retained along with the respective A ring worklist A se n of A ring worklist records should be periodically compared with the AMPLILINK A ring Results Report to verify that the A ring ID ment serial number and specimen IDs are identical Reconcile the ing IDs associated with the run If there are discrepancies perform E At least one Multiprep Control and one Multip Control must be processed with each A ring a Negative Control The absorbance for the MP C sho than 0 2 at 660 nm and its associated MP
35. ation to pre splashing and potential cross contamination of spec imens and controls Do not use snap cap tubes 2 Avoid contaminating gloves when manipulating specimens 3 Specimens and controls should be prepared in a laminar flow hood Failure to do so may result in specimen contamination Specimen and control preparation area must be cleaned and disinfec ordance with methods outlined in Precautions Item A L Decontamination gt 1 Thoroughly clean and disinfect all work surfaces ge ly prepared solution of 0 5 sodium hypochlorite in distilled or deion ized water Follow by wiping down the surface wi ethanol INSTRUCTIONS FOR USE The Multiprep Specimen Processing Procedure is used 0r extracting nucleic acid from pooled specimens and from individual cadaveric specimens and for testing individual specimens in triplicate The K men Processing Procedure is used for extracting nucleic acid from individual specimens The Multiprep and the Standard Specimen Pro Procedures are generic nucleic acid extraction procedures and can be used for the extraction of HIV 1 RNA HCV RNA and or HBV DNA Aingle 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 Detect Amplification hybridization and detecti detection are to be done on a sub Specimen Processing Procedure begin with Step A Reagent P nt day perf
36. btype E HTLV I Staphylococcus aureus HIV Subtype F HTLV II Staphylococcus epidermidis HIV Subtype G Up to 25 individual patient plasma specimens from each of the following disease categories were spiked with HBV positive plasma to a level of 90 copies mL for the Multiprep Specimen Processing Procedure and 300 copies mL for the Standard Specimen Processing Procedure or non spiked specimens that are serologically positive for HIV 1 HIV 2 HAV HCV autoimmune disease EBV CMV and negative human plasma spiked with Candida albicans No false negative test results were observed with the HBV spiked specimens and no cross reactivity was observed with the non spiked specimens using both the Multiprep and Standard Specimen Processing Procedures 03599833001 07EN Doc Rev 7 0 13 Potentially Interfering Substances Endogenous Interfering Substances HBV spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of bilirubin up to 20 mg dL triglyc erides up to 3000 mg dL hemoglobin up to 5 0 g dL and albumin up to 6 g dL were tested These endogenous substances did not interfere with the sensitivity or specificity of the COBAS AmpliScreen HBV Test using either the Multiprep or Standard Specimen Processing Procedures Exogenous Interfering Substances HBV spiked and non spiked plasma samples derived from whole blood containing abnormally high concentrations of aspirin up to 50 mg dL pseu
37. c stem progenitor cells HPCs peripheral blood or cord blood and donor lymphocytes for infusion DLI plasma may be tested in pools compris than 24 individual donor specimens For donations of source plasma plasma samples of the donations may ww ools comprised of equal aliquots of not more than 96 individual donations The COBAS AmpliScreen HBV Test can be considered a supplemental test that confirms HBV infection f i s that are repeatedly reactive on a licensed donor screening test for hepatitis B surface antigen and reactive on the COBAS Amplis BV Test This assay is not intended for use as an aid in diagnosis C SUMMARY AND EXPLANATION OF THE TEST Hepatitis B Virus is considered to be one of the major etiologic agents that cause chronic an te hepatitis cirrhosis and hepatocellular carci noma 1 4 HBV is one of the most infectious disease causing agents with about 350 millio 0 patitis B carriers worldwide The prevalence of HBV infection in the US is approximately 200 300 thousand and in Europe it is over 1 millioff 40 11 In the US there has been a steady decline in hepatitis B rates with the implementation of a national vaccination strategy 2 The glob lence of chronic HBV infection as determined by immunoserology ranges from lt 2 in western countries to gt 8 in Asian and Africafcountries 12 HBV is a partially double stranded circular DNA virus with a genome of approximately 3 2 ases that contains four overlapping open
38. cedure and tested individually using the Standard Specimen Processing Procedure Seropositive Specimens Including 49 Acute Patients from Commercial Vendors A total of 723 HBV seropositive specimens including 49 acute patients HBsAg and HBeAg positive obtained from commercial vendors and blood banks in the US J apan and China were tested with the COBAS AmpliScreen HBV Test Of the 723 HBsAg positive specimens that were tested using the Multiprep Specimen Processing Procedure 694 specimens 96 0 tested HBV DNA positive and 29 specimens 4 0 were HBV DNA negative Of the 723 HBsAg positive specimens that were tested individually using the Standard Specimen Processing Procedure 708 specimens 97 9 tested HBV DNA positive and 15 specimens 2 1 tested HBV DNA negative The spec imens discordant with serology when tested individually using the Standard Specimen Processing Procedure were a subset of the specimens that were discordant with serology when tested using the Multiprep Specimen Processing Procedure See Table 10 and 11 Table 10 HBV Seropositive Specimens Including 49 Acute Patients from Commercial Nye Results for Multiprep Specimen Processing Procedure Specimens Dil HBV DNA Res MultiPrep Procedure Total 22 of the 29 HBV DNA negative specimens were tes alternate HBV DNA tests and were negative lt 300 copies mL Includes 7 Specimens which were not retested by alternate HBV DNA tests
39. dividualSpecimens for follow up testing in the event a Primary Pool tests positive If less than 24 specimens are avail able testing erformed using the individual specimens 2 For Source Plasma the Hamilton MICROLAB AT plus 2 Pipettor performs barcode scanning and pooling operations that combine aliquots from 96 individual specimens 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 specimens for columns 1 12 and from the 12 indi vidual specimens for row 1 8 The positive specimen is identified by the intersection of the positive column and positive row Confirmatory testing is conducted on the implicated specimens using Standard Specimen Processing Procedure Hamilton MICROLAB AT plus 2 Pipettor with SUNRISE PLUS v3 3 software was used to prepare pools of up to 96 equal aliquots of plasma during the 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 instruction for cadaveric donor specimens or the instruction for living donor blood specimens O For collection storage and han
40. dling of specimens from deceased donors follow general standards and or regulations Cadaveric samples may be stored for up to 72 hours at refrigerated conditions 2 8 C or up to 48 hours at ambient temperature 15 30 C Other storage and han dling conditions must be validated by the user NOTE Cadaveric samples should be placed at 2 8 C as soon 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 which may be performed separately or simultaneously At least one preparation of the COBAS AmpliScreen Multiprep Negative Control and one preparation of the COBAS AmpliScreen Multiprep Positive Control must be included in each run 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 03599833001 07EN Doc Rev 7 0 B Equipment 1 Prepare the COBAS AMPLICOR Analyzer and AMPLILINK Data Station for use according to instructions in the Operator s Manual for the AMPLILINK software
41. e Limit IU mL one sided 97 5 Serial Dilution Testing Summary Cains Hed aih HBV DNA WHO International Standard 97 746 Combined In with Lower 95 Confidence Limit One Sided ea mber of Number of Positive Benfaenee Limi U mL ives Individual Tests one sided 300 120 120 100 0 97 5 10 120 120 100 0 97 5 O 118 119 99 2 96 1 116 120 96 7 92 5 15 115 120 95 8 91 4 10 101 120 84 2 77 6 Genotype Sensitivity and Inclusivity One hundred fifteen specimens were diluted to 60 copies mL and 200 copies mL with HBV Negative Human Plasma 16 Genotype A 22 Genotype B 16 Genotype C 8 Genotype D 16 Genotype E 22 Genotype F 1 Genotype G 3 Genotype A C 2 Genotype A D 2 Genotype C D 2 Genotype D E and 1 each of Genotypes A E B C C E D F and E F All the genotypes tested positive by the COBAS AmpliScreen HBV Test at 60 copies mL with the Multiprep Specimen Processing Procedure and at 200 copies mL with the Standard Specimen Processing Procedure An additional 13 spec imens 2 Genotype A 2 Genotype B 2 Genotype C 2 Genotype D 2 Genotype E and 3 Genotype F were diluted in HBV Negative Human Plasma and tested in replicates of 22 with the COBAS AmpliScreen HBV Test at a level which resulted in a gt 95 detection rate 2 to 100 copies mL for the Multiprep Specimen Processing Procedure and 15 to 400 copies mL for the Standard Specimen Processing Procedure
42. e Multiprep Control or Multiprep Control is required by the laboratory the External Control should meet regulatory requirements for such controls The absorbance of the HBV 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 HBV External Control does not meet the above criterion the negative results for specimens in the associated run may be invalidated However positive results for specimens in such a run should not be invalidated solely on the basis of the results obtained for an External Control those positive results should remain the test of record The laboratory should follow its established Standard Operating Procedure for the appropriate action 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 HBV target and one for the internal control MP IC For a specimen 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 HBV target is greater than or equal t
43. e at lt 182C for up to one month E Do not freeze whole blood F Heparin has been shown to inhibit PCR Use of heparini ens is not recommended G Warm pooled or individual specimens to room temperati ore using H Covered Archive Plates may be stored at 2 8 C for 7 from the date the plasma was removed from the red blood cells l No adverse effect on assay performance was obs rved when plasma specimens were subjected to three freeze thaw cycles J Thaw frozen specimens at room temperature using K The user should validate other collection orage 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 c ntamination of specimens is not adequately controlled during specimen handling and processing M SPECIMEN POOLING NOTE Pooling of specimens should only be performed on individual whole blood and source plasma donations or on plasma specimens from donors of hematopoietic stem progenitor cells or donor lymphocytes for infusion Cadaveric specimens must be tested indi vidually and not as part offa pool 1 The COBAS Anip Pooling System performs barcode scanning and pooling operations that combine aliquots from 24 indi vidual speci single Primary Pool that is used for testing The pooling algorithm requires preparation of Secondary Pools as well as in
44. e panels tested by the S pecimen Processing Procedure that were intermittently HBV DNA positive days prior to the Ortho test The results of these studies provi jective evidence that the COBAS AmpliScreen HBV Test using either the Multiprep or Standard Specimen Processing Procedures demons ater sensitivity than observed with current HBsAg serology assays in detecting early HBV infection Analytical Specificity P ly Cross Reactive and Interfering Microorganisms The analytical specificity AS AmpliScreen HBV Test was evaluated by testing a panel of 38 microorganisms including 33 viral isolates and 5 bacterial strains Using t ultiprep Specimen Processing Procedure No cross reactivity was observed with the COBAS AmpliScreen HBV Test Table 9 provides a of organisms tested and the level at which they were tested Table 9 Analytical Specificity Microorganisms Tested Adenovirus Human Type 2 Chlamydia trachomatis HCV la Adenovirus Human Type 3 Neisseria gonorrhoeae HCV 1b Adenovirus Human Type 7 Epstein Barr Virus Burkitt s lymphoma HCV 2a 2c F Epstein Barr Virus Cytomegalovirus 3 strains IRAJ I Human Burkitt s lymphoma HCV 2b Herpes Simplex type 1 Echovirus 1 HCV 3a Herpes Simplex type 2 Echovirus 5 HIV Subtype A Hepatitis A Coxsackievirus B1 HIV Subtype B Human Papilloma Virus Type 6a Varicella Zoster HIV Subtype C Human Papilloma Virus Type 16 Varicella HIV Subtype D Human Papilloma Virus Type 18 Propionibacterium acnes HIV Su
45. ed 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 repeat entire test procedure in single on the remaining replicate tubes The test result for the pool or indi vidual specimen is based only on the repeat valid test result If the last available replicate of a pooled specimen gives an invalid result each indi vidual specimen in that pool should be tested If an individual specimen gives an invalid result the test result for that individual specimen should be considered invalid for HBV DNA 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 Specimens The testing algorithm for testing of pooled specimens for the COBAS AmpliScreen HBV Test requires a single level of testing for Primary Pools that are negative for HBV DNA and three levels of testing Primary Pool Secondary Pool and tertiary resolution for Primary Po Pore positive for HBV DNA Negative Primar
46. eraeus Biofuge Stratos with the 3331 rotor or equivalent 03599833001 07EN Doc Rev 7 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 Combitips Reservoir sterile or equivalent Eppendorf Multipette pipette or equivalent Ethanol 90 or 95 reagent grade for Molecular Biology or Histology use e Distilled or deionized water Powderless disposable gloves Isopropyl alcohol reagent grade Disposable Sterile Polystyrene Pipettes 5 mL 10 mL and 25 mL Sterile RNase free fine tip transfer pipettes e Pipettors capacity 20 uL to 1000 uL capable of providing 3 accuracy and precision lt 5 with aerosol barrier or positive displace ment RNase free tips Tube racks Sarstedt P N 93 1428 or equivalent e 1 5 mL sterile non siliconized conical polypropylene screw cap tubes Sarstedt 72 692 105 or equivalent e 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 P N 03302555 018 MP C Multiprep Negative Control lt 0 005 Poly rA RNA synthetic EDTA 0 05 Sodium azide MP C Multiprep Positive Control Tris HCI buffer MULTIPREP CTL 4 96 Tests 8 x 0 1 mL 8 x 0 1 mL lt 0 001 Non infectious linea
47. es For cadaveric testing pipette 200 uL NHP into an appropriately labeled screw cap tube and add 800 uL Multiprep Diluent MP DIL using a hand held pipettor or other user validated method Cap the tubes Vortex each specimen tube briefly Use a permanent marker to make an orientation mark on each tube Place the specimen and control tubes into the pre cooled high speed centrifuge with the orientation marks facing outward so that the orien tation marks will align with the pellets formed during centrifugation Centrifuge specimens and control tubes at 23 000 24 000 x g for 60 4 minutes at 2 8 C The pellet will form on the outer wall as indi cated by the orientation mark NOTE The 60 4 minutes begins 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 supernatant and pipette tip appropriately Use a fresh pipette tip for each tube B8 Prepare a Working Lysis Reagent bottle for every batch of 12 specimens and controls to be processed B9 Pipette 600 uL Working Lysis Reagent into each specimen and control tube Cap and vortex tubes briefly B10 Prepare Controls as follows a Negative Control Vortex MP C briefly Tap vial to collect the solution in the base Pipette 20 uL MP C to the tube labeled
48. est Result HBsAg Neutralization Positive Negative Positive 109 29 Negative 0 74 Detection of Window Period Cases A confirmed window period case is defined as an enrolled individual from whom the index donation was positive in tecobase AmpliScreen HBV Test but tested negative by HBsAg and anti HBc and a follow up specimen tested positive either by COBAS AmpliScreen HBV Test HBsAg or anti HBc Two window period cases were found during the clinical trial for a detection rate of 1 290 895 with exact 95 confidence imits of 1 11 497 826 to 1 52 083 Single Donation Testing Performance A total of 1754 specimens for which serology results were available were tested individually in the COB results are shown in Table 20 There were 89 1754 classified as HBV status positive 87 were HBsAg HBV window period cases based on follow up testing In the absence of follow up testing 13 1754 we HBsAg negative anti HBc positive and HBV DNA negative and 1 was HBsAg negative anti 1652 1754 classified as HBV status negative and of these 1625 1652 were COBAS AmpliSefee mens that tested positive by the COBAS AmpliScreen HBV Test and negative by serology Sor id AS AmpliScreen HBV Test clinical trial The anti F and HBV DNA positive and 2 were sified as HBV status unknown 12 were positive and HBV DNA positive There were BV Test negative Of the remaining 27 speci 2Were enrolled in the follow up study and deter OBAS AmpliScreen
49. g in a total of 33 panels tested with the Multiprep Specimen Processing and 36 panels tested with the Standard Specimen Processing Procedure in the final analysis When compared to the Ortho HBsAg ELISA Test System 3 assay HBV DNA was consistently detected m A BsAg by the Ortho Test in all 33 panels using the Multiprep Specimen Processing Procedure specimens diluted 1 24 With the Standard Specimen Pro ing Procedure specimens undiluted HBV DNA was consistently detected earlier than HBsAg by the Ortho Test in all 36 panels In no instance was there a specimen that showed HBsAg reactivity yet was negative for HBV DNA The COBAS AmpliScreen HBV Test detected HBV DNA a mean of 1 rigfto detection of HBsAg by the Ortho HBsAg test using the Multiprep Specimen Processing Procedure and 22 days when using the Standard Spe i essing procedure see Table 8 Table 8 Summary of the Pre Seroconversion Detection of HBV DNA vs Ortho HBsAg Days Prior t o HBsAg Test System 3 MultiP rep i Standard Specimen Processing Procedure Processing Procedure Mean 22 Median K 4 19 Maximum 33 53 Minimum 7 7 One seroconversion as not included in the calculations which was detected by the COBAS 89AmpliScreen HBV Test 108 days prior to the Ortho HBsAg ELISA q Test System a ion the calculations do not included the results for six panels ested witl tirep Specimen Processing Procedure and thre
50. ive the negative cadaveric specimen is reported as HBV DNA 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 HBV DNA Positive If both duplicate specimens were negative or if one duplicate is negative and one is invalid the specimen is reported as HBV DNA 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 ort the results for all associated individual specimens in that Primary Pool as HBV DNA Negative 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 Heparin inhibits PCR specimens collected using heparin as the anticoagulant should not be used with the COBAS AmpliScreen HBV Test 03599833001 07EN Doc Rev 70 10 3 Reliable results are dependent on adequate specimen collection and proper transport procedures 4 Detection of HBV 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
51. l 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 rent 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 b other activities or moved between areas Fresh clean gloves must be worn in each area and must be changed before leaving area Equipment and supplies used for reagent preparation must not be used for specimen preparation activities or for pipetting KA th amplified DNA or other sources of target DNA Post amplification supplies and equipment must remain in the Post Amplification t all times Avoid contact of MP LYS HBV MMX HBV Mg2 BH4 B14 DN4 CN4 SB3 SB ifig Substrate mixed SB3 and SB reagent with the skin eyes or mucous membranes If contact does occur immediately wash wi amounts of water otherwise burns can occur If these reagents are spilled dilute with water before wiping dry Do not allow MP which contains guanidine thiocyanate or BH4 and B14 which contain sodium thiocyanate to contact sodium hypochlorite bleach solution This mixture can produce a highly toxic gas SB and Working Substrate contain dimethylformamide which has b reported to be toxic in high oral doses and may be harmful
52. m the positive subpool using the Standard Specimen Processing Procedure Results of Triplicate Testing with the Multiprep Specimen Processing Procedure Detection of 2 IU mL of HBV DNA If an individual specimen is tested in triplicate following the Multiprep Specimen Processing Procedure a positive result for one or more replicates indicates that the specimen is HBV DNA positive e Ifan individual specimen is tested in triplicate following the Multiprep Specimen Processing Procedure a negative result for all of the repli cates indicates that the specimen is HBV DNA negative NOTE The 95 hit rate using the Multiprep Specimen Processing Procedure can be improved to 99 99 by testing in triplicate and consid ering the test result positive if at least one of the three replicates is positive Dilutions of the WHO HBV International Standard 97 746 were prepared at concentrations of 30 10 3 and 1 copies mL 1IU 5 copies respectively Forty replicates of each dilution were tested For each replicate the sample was tested in triplicate and the result for the particular replicate was considered positive if at least one or more of the replicates was positive All 40 replicates of the 10 copies mL 2 IU mL dilution were detected with this algorithm Results of Individual Cadaveric Specimens If an individual cadaveric specimen is positive the positive cadaveric specimen is reported as HBV DNA Positive If an individual cadaveric specimen is negat
53. nd 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 HBV amplicon and the HBV Multiprep 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 HBV amplicon HBV DET or HBV Internal Control amplicon is added to the individual D cups The biotin labeled HBV and HBV Internal Control amplicon are hybridized to the target specific oligonucleotide probes bound to the magnetic particles This hybridization of amplicon to the target specific probe increases the overall speci ficity of the COBAS AmpliScreen HBV Test 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 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 tetr
54. o 0 2 the MP IC result is disregarded and the test result is valid and positive 03599833001 07EN Doc Rev 7 0 3 For a valid run results are interpreted as follows HBV Result IC Result A660 Comment Aseo Comment Interpretation lt 0 2 NEGATIVE 20 2 VALID Specimen is negative for HBV DNA lt 0 2 NEGATIVE lt 0 2 INVALID Invalid result Repeat entire test procedure for invalid specimen 20 2 POSITIVE ANY VALID Specimen is positive for HBV DNA 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 denatur
55. ong the target templates to produce a double stranded DNA molecule termed an amplicon The COBAS AMPLICOR Analyzer automatically 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 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 enzyme is added to the COBAS AmpliScreen HBV Test The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands The Document Revision Information section is located at the end of this document 03599833001 07EN Doc Rev 7 0 1 containing deoxyuridine23 but not DNA containing deoxythymidine Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon because of the use of deoxyuridine triphosphate in place of thymidine triphosphate as one of the dNTPs in the Master Mix reagent there fore only amplicon contain deoxyuridine Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme before ampli fication of the target DNA AmpErase enzyme which is included in the Master Mix reagent catalyzes the cleavage of deoxyuridine containing DNA rendering the DNA non amplifiable The AmpErase enzyme is inactive at temperatures above 55 C i e throughout the thermal cycling steps a
56. ons of molecular biology Bethes erican Association of Blood Banks 1997 123 76 presented at a workshop during the 50th Annual Meeting of the AABB Octo 7 Denver CO 19 Mortimer J 1997 Intersecting pools and their potential application in testing donated blood By enomes Vox Sanguinis 73 93 96 20 Kanemitsu K Tomono T Murozuka T Emura H Yugi H Miyamoto and Nishioka K 2000 National NAT screening of HBV HCV and HIV for blood transfusion Vox Sanguinis 78 1 0081 21 Yang Y Xu D Mendoza M Lamendola M Wu Y Yeh S Kung K 2000 A prototy epatitis B virus HBV PCR assay with a semi automated instrument system Transfusion 40 No 10S SP207 C 22 Rosenstraus M Gutekunst K Herman S et al 1998 Improved COBAS AMPL iral assays as a basis for mini pool screening of viruses in blood or plasma Infusionsther Tranfusionsmed 25 153 9 23 Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 24 Richmond J Y and McKinney R W eds 1999 Biosafety in RAS nd Biomedical Laboratories HHS Publication Number CDC 93 8395 25 Clinical and Laboratory Standards Institute CLSI1 Protecti atory Workers from Occupationally Acquired Infections Approved Guideline Third Edition CLSI Document M29 A3 Wayne 5 5 26 International Air Transport Association Dangerous Goo tions 41st Edition 2000 704 pp 27 Centers for Disease Cont
57. orm the Multiprep Specimen Processing Procedure described in steps B1 through B21 or the Standard in steps B22 through B38 Store the processed specimens and controls as indicated On the subsequent day Working Master Mix thaw processed specimens and controls at room temperature and continue with Step B39 ored Denatured Amplicon Hybridization and Detectio 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 Om 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 Al Determine the appropriate number of A ring s needed for specimen and control testing A2 Place the A ring s on the A ring holders A3 For each A ring prepare one Working Master Mix A4 Pipette 50 uL Working Master Mix into each A tube Discard unused Working Master Mix Do not close the covers of the A tubes at this time A5 Place the A ring containing Working Master Mix in a sealable bag and seal the plastic bag Record the assay name HBV and the time the Working Master
58. ory areas becoming contaminated with amplicon the laboratory area should be separated into several distinct areas organized around Pre Amplification and Post Amplification Personnel should use proper anti contamination safeguards when moving between areas 2 The Pre Amplification Area should have a template free area for preparation of Working Master Mix and an amplicon free area for specimen and control preparation 3 The Post Amplification Area should have a COBAS AMPLICOR Analyzer s and AMPLILINK Data Station s with additional area for preparing Working Amplification and Detection Reagents 4 Pipettors and other supplies should be dedicated to a specific area Specimens equipment and reagents should not be returned to the area where a previous step was performed E Temperature x 1 Room temperature is defined as 152 to 30 C F Vortexing UV 1 Proper vortexing during sample preparation is important to ensure homogeneous mixture aft ns 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 contfol Use aerosol barrier or positive displacement RNase free tips 3 Confirm that all pipettors are correctly set to dispense the specified volumes G ce with the specimen preparation procedures and guidelines H Specimen Processing 1 Screw cap tubes must be used for specimen and control prepar
59. reading frames encoding for all viral proteins 1 5 6 The current risk of transfusion transmitted i9 higher than that of HCV and HIV 7 8 The presence of HBV antigens or antibodies in patients infected with sci o the development of immunoserological tests that are spe cific for these antigens or antibodies Implementation of these tests has redu but not completely eliminated the incidence of post transfu sion hepatitis B Currently all blood units are screened for HBsAg HBc However it has been reported that some blood units from HBsAg and anti HBc negative donors were associated with pos sion hepatitis B in the recipients 43 14 HBV DNA was prospectively detected in HBsAg and anti HBc negative blood units by Polym raSe Chain Reaction PCR 13 Screening of blood donations for HBV DNA will further reduce the residual transmission risk PCR tests can alSodeteet viremic units donated by carriers who are in the window period early acute infection or late resolving infection that may not be y the existing immunological assays 3 15 17 A number of proposals have been made for performing nucleic acid tests on mini po ed of small aliquots from many individual units 18 20 The COBAS AmpliScreen HBV Test uses a generic imen preparation technique in a mini pool testing format HBV DNA in blood dona tions is detected by automated amplification by PCR COBAS AMPLICOR Analyzer The assay incorporates an Internal Control for monitoring assay performance in
60. rized plasmid DNA microbial containing HBV sequences V lt 0 001 Non infectious in vitro transcribed RNA microbial containing HCV sequences lt 0 005 Poly rA RNA synthetic EDTA 0 05 Sodium azide MP LYS Multiprep Lysis Reagent Tris HCI buffer 60 Guanidine thiocyanate Harmful MP DIL Multiprep Specimen Diluent Tris HCI buffer lt 0 005 Poly rA RNA synthetic EDTA 0 05 Sodium azide MP IC X Multiprep Internal Control Tris HCI buffer lt 0 001 Non infectious and a unique probe bj lt 0 001 Non infectio binding sequen nd lt 0 001 Non infi binding sequ n d a unique probe binding region lt 0 005 Po R synthetic EDTA lt 0 1 Amaranth dye 0 05 Sodium azide NHP Negative Plasma Human Human plasma non reactive by US FDA licensed tests for antibody antibody to HCV HBsAg HCV RNA HIV 1 RNA and HBV DNA 0 1 ProClin 300 preservative COBAS AmpliScreen HBV Test P N 03599779 190 COBAS AmpliScreen HBV Amplification Reagents HBV MMX HBV Master Mix Tris HCI buffer Glycerol lt 0 001 AmpliTaq DNA Polymerase microbial Ammonium sulfate 0 05 dATP dCTP dGTP dUTP g region unique probe binding region 3 Dithiothreitol lt 1 Glycogen Xn 60 w w Guanidine thiocyanate QO las DNA containing HBV primer binding sequences o transcribed RNA microbial containing HCV primer vitro transcribed RNA microbial containing HIV 1 primer to HIV 1 2
61. rol and Prevention Sum lable Diseases United States 2000 Morbidity and Mortality Weekly Report 2002 49 53 xy 03599833001 07EN Doc Rev 70 18 Document Revision Information Doc Rev 7 0 The PRINCIPLES OF THE PROCEDURE section and the RESULTS section have been updated to indicate international 1 2010 Units mL and the approximate equivalent copies mL Corrections were made to the sero conversion panel testing data in the PERFORMANCE CHARACTERISTICS section In the Distributed by section addresses have been updated Trademarks have been updated throughout Please contact your local Roche Representative if you have any questions Roche Molecular Systems Inc Branchburg NJ 08876 USA U S License No 1636 D A Member of the Roche Group Distributed by Roche Diagnostics 9115 Hague Road Indianapolis IN 46250 0457 USA For Technical Assistance call the Roche Response Center Toll free 1 800 526 1247 a ROCHE AMPERASE COBAS AMPLICOR AMPLISCREEN AMPLILINK and AMPLITAQ are trademarks C ROCHE RESPONSE CENTER is a service mark of Roche DYNABEADS paramagnetic particles are licensed under patents owned by Dynal Biotech ASA Oslo Norway Dynabeads is a trademark of Dynal h Biotech ASA Oslo Norway licensed to Roche EPPENDORF EPPENDORF MULTIPETTE and EPPENDORF COMBITIPS are trademarks E rf Netheler Hinz GmbH Hamburg Germany MICROLAB is a trademark of Hamilton Company PROCLIN is a trademark of Rohm and
62. s positive nee is reported as HBV DNA positive e If an individual specimen is negative tl cimen is reported as HBV DNA negative Results of Pooled Source Plasma cepa ools of up to 96 Individual Specimens be reported as HBV DNA Negative al testing to determine the cause of the initial positivity pecimen s is reported as HBV DNA positive and the remaining neg re reported as HBV DNA negative the specimens in the Secondary Pool may be reported as HBV DNA negative ish to conduct additional testing to determine the cause of the initial positivity The testing algorithm for testing of poole ns for the COBAS AmpliScreen HBV Test requires a single level of testing for Primary Pools that are negative for HBV DNA and three levels of testing Primary Pool Minipool and confirmatory testing for Primary Pools that are positive for HBV DNA Negative Primary Pools When the Primary Pool is neg Positive Primary Pools Testing Positive Primary Pools are traced to the positive individual If using an overlapping pool testing matrix minipools are prepared from the eight individual specimens for columns 1 12 and from the 12 indi vidual specimens for row 1 8 The 20 minipools are tested using the Standard Specimen Processing Procedure The positive specimen is iden tified by the intersection of the positive column and positive row Confirmatory testing is conducted on the implicated specimens fro
63. s that the MP IC has been added to the MP LYS Discard the remaining MP IC 3 Store Working gent at room temperature Use within 4 hours of preparation Working Amplification Master Mix 1 Prepare Working Master Mix in a template free area e g in a dead air box Reagent preparation area must be clean and disin fected in accordance with methods outlined in Precautions Item A Failure to do so may result in reagent contamination 2 Pipette 100 uL HBV Mg2 into 1 bottle HBV MMX Recap HBV MMX bottle and mix well by inverting 10 15 times The pink color confirms that the HBV Mg2 has been added to the HBV MMX Discard the remaining HBV Mg2 Do not vortex the Working 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 HBV Probe Suspension Mix BH PS1 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL BH PS1 into one BH4 cassette 2 Prepare Working IC Probe Suspension Mix BI PS1 well by vortexing briefly to suspend the microparticles Pipette 2 5 mL BI PS1 into one BI4 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
64. sts 1 x 100 Tests 2 x 100 Tests 10 x 75 Tests 10 x 75 Tests 10 x 5 mL 500 Tests 2 x 250 Tests Doc Rev 7 0 E Do not expose SB3 SB or Working Substrate to metals oxidizing agents or direct sunlight F The following reagents are one time use Discard any unused portion MP IC MP C MP C MP DIL and NHP HBV Mg2 and SB PRECAUTIONS FOR IN VITRO DIAGNOSTIC USE A R S Specimens may be infectious Use Universal Precautions when performing the assay 24 25 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 with a freshly prepared solution of 0 5 sodium hypochlorite in distilled or deionized water Follow by wiping down the surface with 70 ethanol CAUTION The Negative Human Plasma of this kit contains human blood products non reactive by US FDA licensed tests for antibody to HIV 1 2 antibody to HCV HBsAg HCV RNA HIV 1 RNA and HBV DNA No known test method can offer complete assurance that products derived from human blood will not transmit infectious agents All human blood sourced materials should be considered potentially 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 ro
65. t days 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 panel 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 HBV Test demonstrated consistency by lot and site for both the Multiprep and Standard Specimen Processing Procedures as seen in Table 1 and Table 2 below The reproducibility by operator is shown in Table 3 and Table 4 below Table 1 Reproducibility Results Multiprep Specimen Processing Procedure Results By Lot Positive Tested Negative 25 c mL 90 c mL 150 c mL 89 90 89 90 99 99 87 88 99 able 2 ucibility Results Standar ecimen Processing Procedure Q By Lot Positive Tested e 75 c mL 300 c mL 500 c mL 25 000 c mL Lot 1 9 76 89 89 89 90 90 90 90 85 100 100 100 Lot 2 1 179 73 90 88 89 88 90 90 90 a 1 81 99 98 100 ot 0 180 78 90 90 90 90 90 90 90 0 87 100 100 100 Results By Site Positive Tested 0 180 72 89 89 89 90 90 90 90 0 81 100 100 100 Site 2 1 179 76 90 88
66. the correct A tube position for each A ring B42 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 03599833001 07EN Doc Rev 70 NOTE Amplification must begin within 45 minutes from when the first specimen or control in the A ring is added to the Working Master Mix C Amplification and Detection Performed in Post Amplification Amplification Detection Area C1 Perform Daily Instrument Maintenance as outlined in the Operator s Manual for the COBAS AMPLICOR Analyzer including a b Wipe D cup handler tip with a lint free moist cloth and dry Wipe initialization post with a lint free moist cloth and dry C2 Before each run a b C d Check waste container and empty if necessary Check Wash Buffer Reservoir and add prepared Wash Buffer if necessary Replace used D cup racks Prime the COBAS AMPLICOR Analyzer C3 Instrument Loading and System Operation a b i j k Prepare enough of the following detection reagent cassettes to complete the workload Working HBV Probe Suspension Reagent BH4 Working IC Probe Suspension Reagent B14 Working Substrate SB3 Denaturation Reagent DN4 and Conjugate Reagent C N4 Place the BH4 and BI4 cassettes in the test specific reagent rack Place DN4 CN4 and SB3 cassettes in the generic reagent rack Record on the cassette the date when each cassette
67. tion of HBV DNA vs FDA Licensed Serology Tests Multiprep Specimen Processing Procedure Specimen diluted 1 96 Days Before Ortho Days Before Ortho Days Before Abbott HBsAg ELISA Test HBsAg ELISA Test Auszyme overnight System 2 System 3 for HBsAg 8 panels tested 9 panels tested 9 panels tested Mean 8 8 8 3 11 0 Median 8 7 9 Maximum 27 27 27 Minimum 0 0 0 One seroconversion panel was not included in the calculations which was detected by the COBAS AmpliScreen HBV Test 108 days prior to detection of HBsAg by the Ortho HBsAg ELISA Test System 2 101 days prior to the Ortho HBsAg ELISA Test System 3 and 87 days prior to the Abbott Auszyme test The seroconversion study demonstrates the COBAS AmpliScreen HBV Test used with the Multiprep Specimen Processing Procedure and pools of 96 specimens identified HBV infected specimens at the same time or earlier than the U S FDA licensed HBsAg tests NON CLINICAL PERFORMANCE CHARACTERISTICS FOR CADAVERIC SPECIMENS Sensitivity Study Sixty pre mortem EDTA plasma and 58 cadaveric specimens non reactive for HBV were divided into 5 groups Specimens within each group were spiked with HBV viral target to a concentration of 3X the LOD using a different clinical viral isolate for each group The spiked specimens were equally divided and tested with three COBAS AmpliScreen HBV Test kit lots The COBAS AmpliScreen HBV Test using samples diluted 1 5 and the Multiprep
68. tive Control Vortex MP C briefly Tap lect the solution in the base Pipette 20 uL MP C into the tube labeled MP C containing Working Lysis Reagent and Cap the tube and vortex briefly b Positive Control Vortex MP C brieflyfap vial to collect the solution in the base Pipette 20 uL MP C into the tube labeled MP C containing Working Lysis Rea and NHP Cap the tube and vortex briefly B29 Incubate all tube 5 minutes at room temperature after adding Working Lysis Reagent to the last tube After the incubation period briefly vortex all tubes B30 Pipette 800 uL of isopropanol into each tube Cap the tubes and vortex briefly B31 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 15 20 minutes at room temperature B32 Slowly aspirate the supernatant from each tube Remove as much liquid as possible without disturbing the pellet B33 Pipette 1 0 mL of 70 ethanol into each tube Cap the tubes and vortex briefly B34 Place the tubes into a microcentrifuge with the orientation marks facing outward to align with the pellets that will form Centrifuge at 14 250 1750 x g for 5 10 minutes at room temperature B35 Slowly aspirate the supernatant from each tube using a fine tip disposable transfer pipette Remove as much liquid as possible without dis urbing the pellet Use a new transfer pipette for each tube
69. utine 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 for specimen and control preparation to prevent splashing and potential cross contamination of specimens and controls Do not use snap cap tubes Adequately vortex where specified to ensure optimal assay performance Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of 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 al
70. y Pools V When the Primary Pool is negative report the results for all associated individual specimens in that P rima BV DNA Negative Positive Primary Pools Secondary Pool Testing When the Primary pool is positive each of the 4 Secondary Pools prepared from the Archive Plate usi D imens comprising the Primary Pool is tested by the COBAS AmpliScreen HBV Test using the Multiprep Specimen Processing Procedure If one or more of the Secondary Pools tests positive report the results for the speci n the negative Secondary Pools as HBV DNA negative For positive Secondary Pools proceed to the section entitled Positi Pool Positive Secondary Pools Tertiary Resolution Testing If all 4 Secondary Pools are negative the individual specimens in that hic As part of an overall Quality Assurance program you may wish to conduct a of the Primary Pool Positive Primary Pool Positive Secondary Pools Tertiary Resolution Tes For a positive Secondary Pool test each of the individual specimens in that Sec ry Pool The individual specimens must be processed using the Standard Specimen Processing procedure If one or more of the individual specimens is positive th ative specimens associated with the positive Seconda e Ifall of the individual specimens in that Secondary Pool As part of an overall Quality Assurance program of the Primary and Secondary Pools Results of Individual Specimens e Ifan individual specimen i
71. y positive by the COBAS AmpliScreen HBV Test y both the Multiprep and Standard Specimen Processing Procedures Combining all 40 panels a total of 189 HBsAg positive specimens wer ed and shown to be 100 concordant HBV DNA positive with serologic results Seropositive Specimens from Blood Donors During the clinical studies there were 105 HBV donor specimens confirmed positive for HBsAg which were als6 HBV Test There were 87 specimens 82 9 HBV DNA positive and 18 specimens 17 1 HBV D ne Processing Procedure Of these 105 HBsAg positive specimens 104 specimens were tested individua g the Standard Specimen Processing Procedure and 97 specimens 93 3 tested DNA positive and 7 specimens 6 7 tested DNA nega e specimens discordant with serology when tested individually using the Standard Specimen Processing Procedure were a subset of ecimens which were discordant with serology when tested using the Multiprep Specimen Processing Procedure See Table 16 and Table 17 Table 16 HBV Seropositive Specimen Results for Multiprep Specimen Processing Procedure ested with the COBAS AmpliScreen gative using the Multiprep Specimen imens Diluted 1 24 NA Results 87 82 9 MultiPrep Procedure Total 6 of 18 specimens were n for HBV DNA by alternate qualitative NAT and specimens from amp o re not available for testing by alter d for HBV DNA by alternate quantita ed lt 300 copies mL HBV
72. ydue to ficient volume Table HBV Seropositive Specimens Including 49 Acute ents from Commercial Vendors Results for Standard Specimen Processing Procedure Specimens Undiluted HBV DNA Results 708 97 9 Standard Procedu e 723 Total All HBV DNA P were tested by alternate HBV DNA tests and Da opies mL HBV DNA Chronic HBV Patients A total of 100 HBV seropositive specime m Hronic HBV patients were tested with the COBAS AmpliScreen HBV Test Of the 100 HBsAg pos itive specimens there were 78 specime 70 HBV DNA positive and 22 specimens 22 0 HBV DNA negative with the Multiprep Specimen Processing Procedure and 95 specimens 95 0 HBV DNA positive and 5 specimens 5 0 HBV DNA negative with the Standard Specimen Processing Procedure The specin discordant with serology when tested individually using the Standard Specimen Processing Procedure were a subset of the specimens which V diScordant with serology when tested using the Multiprep Specimen Processing Procedure See Table 12 and Table 13 Table 12 HBV Seropositive Specimens from Chronic HBV Patients Results for Multiprep Specimen Processing Procedure Specimens Diluted 1 24 HBV DNA Results 78 78 0 MultiPrep Procedure 22 Total 100 All HBV DNA negative specimens were tested by alternate HBV DNA tests and contained lt 300 copies mL HBV DNA Table 13 HBV Seropositive Specimens from Chronic

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