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The Document Revision Information section is located at the end of this document.

05382874001-10EN 1 Doc Rev. 10.0

COBAS®

AmpliPrep/COBAS® TaqMan

®

HBV Test, version 2.0

FOR IN VITRO DIAGNOSTIC USE.

COBAS

®

AmpliPrep/COBAS

®

TaqMan

®

72 Tests P/N: 04894570 190HBV Test, v2.0

COBAS® AmpliPrep/COBAS

® TaqMan

® 5.1 Liters P/N: 03587797 190

Wash Reagent

INTENDED USE

The COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, version 2.0 (v2.0) is an in vitro nucleic acid

amplification test for the quantitation of Hepatitis B Virus (HBV) DNA in human plasma and serum, using

the COBAS® AmpliPrep Instrument for automated specimen processing and the COBAS

® TaqMan

®

Analyzer or COBAS®

TaqMan®

48 Analyzer for automated amplification and detection.

This test is intended for use as an aid in the management of patients with chronic HBV infection

undergoing anti-viral therapy. The assay can be used to measure HBV DNA levels at baseline and during

treatment to aid in assessing response to treatment. The results from the COBAS® AmpliPrep/COBAS

®

TaqMan® HBV Test, v2.0 must interpreted within the context of all relevant clinical and laboratory findings .


® TaqMan

® HBV Test, v2.0 is not intended for use as a screening

test for the presence of HBV in blood or blood products or as a diagnostic test to confirm the

presence of HBV infection.

SUMMARY AND EXPLANATION OF THE TEST

Hepatitis B Virus (HBV) is one of several viruses known to cause viral hepatitis. Over two billion people

throughout the world have been infected with HBV and over 350 million of them are chronically infected

carriers1,2

. Chronic carriers are at high risk of long term complications of infection, including chronic

hepatitis, cirrhosis and hepatocellular carcinoma3,4,5

. Serologic markers are commonly used as diagnostic

and/or prognostic indicators of acute or chronic HBV infection. The most common marker of HBV infection

is the presence of HBV surface antigen (HBsAg). Although carriers may clear HBsAg and develop antibody

to HBsAg, there appears to still be a risk of serious liver complications later in life6,7

. HBV e antigen

(HBeAg) is generally used as a secondary marker to indicate active HBV replication associated with

progressive liver disease. Failure to clear HBeAg appears to increase the risk of end stage liver disease8,9

.

Variant strains of HBV can either produce HBeAg that is not detectable in serum or the strain can lose the

ability to make HBeAg even when an active infection is present10. Therefore, using this marker to monitor

disease progression may be of limited utility11

. The ability to detect HBV DNA in serum has been reported

to have prognostic value for the outcome of acute and chronic HBV infections12-15

. The methodology can

allow the detection of HBV DNA after HBsAg clearance16

or detection of HBV lacking serologic markers17

.

However, a relationship between serologic markers and HBV DNA levels has not yet been established. The

efficacy of antiviral therapy used to treat patients with HBV can also be assessed by serologic markers or

by measurement of liver enzyme function. However, the most direct and reliable measurement of viral

replication is thought to be quantitation of HBV viral DNA in serum or plasma14,18-20

. A rapid and sustained

drop in HBV DNA levels in patients receiving treatment with alpha-interferon, Lamivudine, Ganciclovir or

Adefovir Dipivoxil has been shown to be a predictive factor for a favorable treatment outcome11,21-25

.

Monitoring of HBV DNA levels can predict the development of resistance to Lamivudine26

. Therefore, a

quantitative test for the measurement of HBV DNA is a valuable tool that can be used in conjunction with

other serological markers in the management of HBV infection.

HBV V2.0

PG WR

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HBV DNA in plasma and serum can be quantitated by nucleic acid amplification technologies, such as the

Polymerase Chain Reaction (PCR)27-29

. The COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 uses

PCR technology to achieve maximum sensitivity and dynamic range for the quantitative detection of HBV

DNA in EDTA anticoagulated plasma and serum.

PRINCIPLES OF THE PROCEDURE


® TaqMan

® HBV Test, v2.0 is a nucleic acid amplification test for the

quantitation of Hepatitis B Virus (HBV) DNA in human plasma and serum. The COBAS

®


® HBV Test, v2.0 is based on two major processes: (1) specimen preparation

to isolate HBV DNA and (2) simultaneous PCR amplification27

of target DNA and detection of cleaved

dual-labeled oligonucleotide detection probe specific to the target.


® TaqMan

® HBV Test, v2.0 permits automated specimen preparation followed

by automated PCR amplification and detection of HBV target DNA and HBV Quantitation Standard (QS) DNA.

The Master Mix reagent contains primer pairs and probes specific for both HBV DNA and HBV QS DNA. The

Master Mix has been developed to ensure equivalent quantitation of genotypes A through H of HBV. The

detection of amplified DNA is performed using a target-specific and a QS-specific dual-labeled oligonucleotide

probe that permit independent identification of HBV amplicon and HBV QS amplicon.

The quantitation of HBV viral DNA is performed using the HBV QS. It compensates for effects of inhibitionand controls the preparation and amplification processes, allowing a more accurate quantitation of HBV

DNA in each specimen. It is a non-infectious DNA construct that contains HBV sequences with identical

primer binding sites as the HBV target DNA and a unique probe binding region that allows HBV QS

amplicon to be distinguished from HBV amplicon.

The HBV QS is added to each specimen at a known copy number and is carried through the subsequent

steps of specimen preparation, simultaneous PCR amplification and detection of cleaved dual-labeled

oligonucleotide detection probes. The COBAS® TaqMan

® Analyzer or COBAS

® TaqMan

® 48 Analyzer

calculates the HBV DNA concentration in the test specimens by comparing the HBV signal to the HBV QS

signal for each specimen and control.

Target Selection

Selection of the target DNA sequence for HBV depends on identification of regions within the HBV genome

that show maximum sequence conservation among the various HBV genotypes. Generic silica-based specimen

preparation is used to capture the HBV DNA and HBV QS DNA and defined oligonucleotides are used as

primers in amplification of the HBV DNA and HBV QS DNA. A target-specific and a QS-specific dual-labeled

oligonucleotide probe permit independent identification of HBV amplicon and HBV QS amplicon. Accordingly,

the appropriate selection of the primers and the dual-labeled oligonucleotide probe is critical to the ability of

the test to amplify and detect the HBV genotypes. The COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

uses three amplification primers for PCR. A signal generating dual-labeled probe hybridizes to the anti-sense

strand and is cleaved by Z05 DNA polymerase during extension of the primers. The amplicon of genotypes A-F

and H consists of a sequence of 145 nucleotides. Genotype G amplicon, however, consists of a sequence of

181 nucleotides. A 36 base insertion within the single-stranded highly conserved pre-Core/Core region of the

HBV genome results in a larger amplicon for genotype G31

.

Specimen Preparation


® TaqMan

® HBV Test, v2.0 utilizes automated specimen preparation on the

COBAS® AmpliPrep Instrument by a generic silica-based capture technique. The procedure processes 500 μL

of plasma or serum. The HBV virus particles are lysed by incubation at elevated temperature with a protease

and chaotropic lysis/binding buffer that releases nucleic acids and protects the released HBV DNA from

DNases in plasma and serum. Protease and a known number of HBV QS DNA molecules are introduced into

each specimen along with the lysis reagent and magnetic glass particles. Subsequently, the mixture isincubated and the HBV DNA and HBV QS DNA are bound to the surface of the glass particles. Unbound

substances, such as salts, proteins and other cellular impurities, are removed by washing the magnetic glass

particles. After separating the beads and completing the washing steps, the adsorbed nucleic acids are eluted

at elevated temperature with an aqueous solution. The processed specimen, containing the magnetic glass

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particles as well as released HBV DNA and HBV QS DNA, is added to the amplification mixture and transferred

to the COBAS® TaqMan


® TaqMan

® 48 Analyzer.

PCR Amplification

The PCR amplification reaction is performed with the thermostable recombinant enzyme Thermus specie

Z05 DNA Polymerase (Z05). In the presence of manganese (Mn2+

) and under the appropriate buffer

conditions, Z05 has DNA polymerase activity32, 33

. This allows PCR amplification to occur together with

real-time detection of the amplicon.

Processed specimens are added to the amplification mixture in amplification tubes (K-tubes) in which PCR

amplification occurs. In the presence of Mn2+

and excess deoxynucleotide triphosphates (dNTPs),

including deoxyadenosine, deoxyguanosine, deoxycytidine and deoxyuridine triphosphates, Z05 DNA

polymerase extends the annealed primers forming a DNA strand.

Target Amplification

The Thermal Cycler in the COBAS® TaqMan


® TaqMan

® 48 Analyzer heats the

reaction mixture to denature the double-stranded DNA and expose the specific primer target sequences

on the HBV circular DNA genome and the HBV QS DNA. As the mixture cools, the primers anneal to the

target DNA. The thermostable Thermus specie Z05 DNA Polymerase (Z05) in the presence of Mn2+

and

excess deoxynucleotide triphosphates (dNTPs), including deoxyadenosine, deoxyguanosine, deoxycytidine

and deoxyuridine (in place of thymidine), extends the annealed primers along the target template to

produce a double-stranded DNA molecule termed an amplicon. The COBAS® TaqMan

® Analyzer or

COBAS® TaqMan

® 48 Analyzer automatically repeats this process for a designated number of cycles, with

each cycle intended to double the amount of amplicon DNA. The required number of cycles is

preprogrammed into the COBAS® TaqMan


® TaqMan

® 48 Analyzer. Amplification

occurs only in the region of the HBV genome between the primers; the entire HBV genome is not amplified.

Selective Amplification

Selective amplification of target nucleic acid from the specimen is achieved in the COBAS®

AmpliPrep/COBAS

®

TaqMan

®

HBV Test, v2.0 by the use of AmpErase (uracil-N-glycosylase) enzyme anddeoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA

strands containing deoxyuridine34

, but not DNA containing deoxythymidine. Deoxyuridine is not present in

naturally occurring DNA, but is always present in amplicon due to the use of deoxyuridine triphosphate as one

of the dNTPs in the Master Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders

contaminating amplicon susceptible to destruction by the AmpErase enzyme prior to amplification of the target

DNA. Also, any nonspecific product formed after initial activation of the Master Mix by manganese is destroyed

by the AmpErase enzyme. The AmpErase enzyme, which is included in the Master Mix reagent, catalyzes the

cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the

C1-position. When heated in the first thermal cycling step, the amplicon DNA chain breaks at the position of

the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme remains inactive for a

prolonged period of time once exposed to temperatures above 55ºC, i.e. throughout the thermal cycling steps,and therefore does not destroy target amplicon formed during amplification.

Detection of PCR Products in a COBAS® TaqMan

® Test


® TaqMan

® HBV Test, v2.0 utilizes real-time

27,28 PCR technology. The use

of dual-labeled fluorescent probes allows for real-time detection of PCR product accumulation by

monitoring of the emission intensity of fluorescent reporter dyes released during the amplification process.

The probes consist of HBV and HBV QS-specific oligonucleotide probes with a reporter dye and a

quencher dye. In the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 the HBV and HBV QS probes

are labeled with different fluorescent reporter dyes. When these probes are intact, the fluorescence of the

reporter dye is suppressed by the proximity of the quencher dye due to Förster-type energy transfer

effects. During PCR, the probe hybridizes to a target sequence and is cleaved by the 5' → 3' nucleaseactivity of the thermostable Z05 DNA polymerase. Once the reporter and quencher dyes are released and

separated, quenching no longer occurs, and the fluorescent activity of the reporter dye is increased. The

amplification of HBV DNA and HBV QS DNA are measured independently at different wavelengths. This

process is repeated for a designated number of cycles, each cycle effectively increasing the emission

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intensity of the individual reporter dyes, permitting independent identification of HBV and HBV QS DNA.

The PCR cycle where a growth curve starts exponential growth is related to the amount of starting material

at the beginning of the PCR.

Fundamentals of COBAS® TaqMan

® Test Quantitation


® TaqMan

® HBV Test, v2.0 is quantitative over a very wide dynamic range

since the monitoring of amplicon is performed during the exponential phase of amplification. The higher

the HBV titer of a specimen, the earlier the fluorescence of the reporter dye of the HBV probe rises abovethe baseline fluorescence level (see Figure 1). Since the amount of HBV QS DNA is constant between all

specimens, the fluorescence of the reporter dye of the HBV QS probe should appear at the same cycle for

all specimens (see Figure 2). In specimens where the QS fluorescence is affected, the concentration is

adjusted accordingly. The appearance of the specific fluorescent signals is reported as a critical threshold

value (Ct). The Ct is defined as the fractional cycle number where reporter dye fluorescence exceeds a

predetermined threshold (the Assigned Fluorescence Level), and starts the exponential growth phase of

this signal (see Figure 3). A higher Ct value indicates a lower titer of initial HBV target material. A 2-fold

increase in titer correlates with a decrease of 1 Ct for target HBV DNA, while a 10-fold increase in titer

correlates with a decrease of 3.3 Ct.

Figure 1 shows the target growth curves for a dilution series spanning a 5-log 10 range. As theconcentration of the virus increases, the growth curves shift to earlier cycles. Therefore, the leftmost

growth curve corresponds to the highest viral titer level, whereas, the rightmost growth curve corresponds

to the lowest viral titer level.

Figure 1

Target Growth Curves for a Dilution Series of Virus Spanning a 5-Log10 Range

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 10 20 30 40 50

Cycle Number

N o r m a l i z e d F l u o r e s c e n c e

Highest Titer

Lowest Titer

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Figure 2 shows the Quantitation Standard growth curves for specimens from a viral dilution series that

spans a 5-log10 range. The amount of Quantitation Standard added to each specimen is constant for each

reaction. The Ct value of the Quantitation Standard is similar regardless of the viral titer.

Figure 2

Quantitation Standard Growth Curves for a Dilution Series of Virus Spanning a 5-Log10 Range

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0 10 20 30 40 50

Cycle Number


Lowest Titer

Highest Titer

Figure 3 provides an example of how the fluorescence values at every cycle are normalized for each

growth curve. The fractional cycle number (Ct) is calculated where the fluorescence signal crosses the

Assigned Fluorescence Level.

Figure 3 Fluorescence Values at Every Cycle are Normalized for Each Growth Curve

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

19 20 21 22 23 24 25 26 27 28 29

Cycle Number


Assigned Fluorescence

Level

Ct Value = 27.1

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HBV DNA Quantitation


® TaqMan

® HBV Test, v2.0 quantitates HBV viral DNA by utilizing a second

target sequence (HBV Quantitation Standard) that is added to each test specimen at a known

concentration. The HBV QS is a non-infectious DNA construct, containing fragments of HBV sequences

with primer binding regions identical to those of the HBV target sequence. The HBV QS contains HBV

primer binding regions and generates an amplification product of the same length and base composition

as the HBV target DNA. The detection probe binding region of the HBV QS has been modified to

differentiate HBV QS amplicon from HBV target amplicon.

During the annealing phase of the PCR in the COBAS® TaqMan


® TaqMan

® 48

Analyzer, the specimens are illuminated and excited by filtered light and filtered emission fluorescence

data are collected for each specimen. The readings from each specimen are then corrected for

instrumental fluctuations. These fluorescence readings are sent by the instrument to the AMPLILINK

software and stored in a database. Pre-Checks are used to determine if the HBV DNA and HBV QS DNA

data represent sets that are valid, and flags are generated when the data lie outside the preset limits. After

all Pre-Checks are completed and passed, the fluorescence readings are processed to generate Ct values

for the HBV DNA and the HBV QS DNA. The lot-specific calibration constants provided with the COBAS®


® HBV Test, v2.0 are used to calculate the titer value for the specimens and

controls based upon the HBV DNA and HBV QS DNA Ct values. The COBAS

®

AmpliPrep/COBAS

®

TaqMan® HBV Test, v2.0 is standardized against the WHO International Standard for Hepatitis B Virus DNA

for Nucleic Acid Technology (NAT) Assays Testing (NIBSC 97/746)29

and titer results are reported in

International Units (IU/mL).

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REAGENTS


® TaqMan

®HBV Test, v2.0 72 Tests

(P/N: 04894570 190)

HBV2 CS1 1 x 72 Tests

(HBV Magnetic Glass Particles Reagent Cassette) 1 x 7.0 mL

Magnetic glass particles

93% Isopropanol

Xi 93% (w/w) Isopropanol

Irritant

F 93% (w/w) Isopropanol

Highly

Flammable


(HBV Lysis Reagent Cassette) 1x 78 mL

Sodium citrate dihydrate

42.5% Guanidine thiocyanate

< 5% Polydocanol

0.9% Dithiothreitol

Xn 42.5% (w/w) Guanidine thiocyanate

Harmful

N < 5% (w/w) Polydocanol

Dangerous For

The Environment


HBV Multi-Reagent Cassette containing:

Pase 1 x 3.8 mL

(Proteinase Solution)

Tris buffer

< 0.05% EDTA

Calcium chloride

Calcium acetate

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EB, v2.0 1 x 8.1 mL

(Elution Buffer, v2.0)

Tris-base buffer

0.2% Methylparaben


HBV Test-Specific Reagent Cassette containing:

HBV QS, v2.0 1 x 3.6 mL(HBV Quantitation Standard, v2.0)

Tris-HCl buffer

EDTA

< 0.005% Poly rA RNA (synthetic)

< 0.001% Non-infectious, linearized, double-stranded

plasmid DNA containing an insert with HBV primer

binding sequences and a unique probe binding region

0.05% Sodium azide

HBV MMX, v2.0 1 x 3.4 mL

(HBV Master Mix, v2.0)

Tricine buffer

Potassium acetate

Potassium hydroxide

0.09% Sodium azide

Glycerol

< 0.04% dATP, dCTP, dGTP, dUTP

< 0.003% Upstream and downstream HBV primers

< 0.003% Oligonucleotide aptamer

< 0.003% Fluorescent-labeled oligonucleotide probes

specific for HBV and the HBV Quantitation

Standard

< 0.05% Z05 DNA Polymerase (microbial)

< 0.1% AmpErase (uracil-N-glycosylase) enzyme

(microbial)

HBV Mn2+

1 x 19.8 mL

(HBV Manganese Solution)

< 0.5% Manganese acetate

Glacial acetic acid

0.09% Sodium azide

HBV H(+)C, v2.0 6 x 1.0 mL

(HBV High Positive Control, v2.0)< 0.001% Linearized, double-stranded plasmid DNA

containing HBV sequences

Negative Human Plasma, non-reactive by tests for

antibody to HCV, antibody to HIV-1/2, HIV p24

antigen and HBsAg; HIV-1 RNA, HCV RNA and HBV

DNA not detectable by PCR methods

0.1% ProClin 300 preservative

Xi (3:1) mixture of 5-Chloro-2-methyl-2H-isothiazol-3-one and

2-Methyl-2H-isothiazol-3-one

Irritant

R36/38: Irritating to eyes and skin

R43: May cause sensitization by skin contact

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HBV L(+)C, v2.0 6 x 1.0 mL

(HBV Low Positive Control, v2.0)

< 0.001% Linearized, double-stranded plasmid DNA

containing HBV sequences

Negative Human Plasma, non-reactive by tests for

antibody to HCV, antibody to HIV-1/2, HIV p24 antigen

and HBsAg; HIV-1 RNA, HCV RNA and HBV DNA not

detectable by PCR methods

0.1% ProClin® 300 preservative



Irritant



CTM (–) C 6 x 1.0 mL

[COBAS® TaqMan

® Negative Control (Human Plasma)]

Negative Human Plasma, non-reactive by tests forantibody to HCV, antibody to HIV-1/2, HIV p24 antigen

and HBsAg; HIV-1 RNA, HCV RNA and HBV DNA not

detectable by PCR methods

0.1% ProClin 300 preservative



Irritant



HBV H(+)C, v2.0 Clip 1 x 6 Clips

(HBV High Positive Control, v2.0 Barcode Clip)

HBV L(+)C, v2.0 Clip 1 x 6 Clips

(HBV Low Positive Control, v2.0 Barcode Clip)

HBV (–) C Clip 1 x 6 Clips

(HBV Negative Control Barcode Clip)


® TaqMan

® Wash Reagent 1 x 5.1 L

(P/N: 03587797 190)

PG WR

(COBAS® AmpliPrep/COBAS

® TaqMan

®Wash Reagent)

Sodium citrate dihydrate

< 0.1% N-Methylisothiazolone-HCl

WARNINGS AND PRECAUTIONS

A. FOR IN VITRO DIAGNOSTIC USE.

B. This test is for use with human serum or plasma collected in the anticoagulant EDTA.

C. Do not pipette by mouth.

PG WR

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D. Do not eat, drink or smoke in laboratory work areas. Wear protective disposable gloves, laboratory

coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after

handling specimens and test reagents.

E. Avoid microbial and ribonuclease contamination of reagents when removing aliquots from

control vials.

F. The use of sterile disposable pipettes and DNase-free pipette tips is recommended.

G. Do not pool controls from different lots or from different vials of the same lot.

H. Do not mix reagent cassettes or controls from different kits.

I. Do not open COBAS® AmpliPrep cassettes and exchange, mix, remove or add bottles.

J. Dispose of unused reagents, waste and specimens in accordance with country, federal, state and

local regulations.

K. Do not use a kit after its expiration date.

L. Material Safety Data Sheets (MSDS) are available on request from your local Roche office.

M. Specimens and controls should be handled as if infectious using safe laboratory procedures such

as those outlined in Biosafety in Microbiological and Biomedical Laboratories35

and in the CLSI

Document M29-A336

. Thoroughly clean and disinfect all work surfaces with a freshly prepared

solution of 0.5% sodium hypochlorite in deionized or distilled water.

Note: Commercial liquid household bleach typically contains sodium hypochlorite at aconcentration of 5.25%. A 1:10 dilution of household bleach will produce a 0.5%

sodium hypochlorite solution.

N. CAUTION: CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C, v2.0 contain Human Plasma derived

from human blood. The source material has been tested and found non-reactive for the presence ofHepatitis B Surface Antigen (HBsAg), antibodies to HIV-1/2 and HCV, and HIV p24 Antigen. Testing

of Negative Human Plasma by PCR methods showed no detectable HIV-1 RNA, HCV RNA or HBV

DNA. No known test methods can offer complete assurance that products derived from human

blood will not transmit infectious agents. Therefore, all human sourced material should be

considered potentially infectious. CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C, v2.0 should be

handled as if infectious using safe laboratory procedures such as those outlined in Biosafety in

Microbiological and Biomedical Laboratories35

and in the CLSI Document M29-A336

. Thoroughly

clean and disinfect all work surfaces with a freshly prepared solution of 0.5% sodium hypochlorite

in deionized or distilled water.

O. HBV QS, v2.0, HBV Mn2+

and HBV MMX, v2.0 contain sodium azide. Sodium azide may reactwith lead and copper plumbing to form highly explosive metal azides. While disposing of sodium

azide-containing solutions down laboratory sinks, flush the drains with a large volume of water to

prevent azide buildup.

P. Wear eye protection, laboratory coats and disposable gloves when handling any reagent. Avoid

contact of these materials with the skin, eyes or mucous membranes. If contact does occur,

immediately wash with large amounts of water. Burns can occur if left untreated. If spills of these

reagents occur, dilute with water before wiping dry.

Q. Do not allow HBV2 CS2 and liquid waste from the COBAS® AmpliPrep Instrument, which contain

guanidine thiocyanate, to contact sodium hypochlorite (bleach) solution. These mixtures can

produce a highly toxic gas.

R. When disposing of used COBAS® AmpliPrep Sample Processing Units (SPUs), which contain

guanidine thiocyanate, avoid any contact with sodium hypochlorite (bleach) solution. These

mixtures can produce a highly toxic gas.

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STORAGE AND HANDLING REQUIREMENTS

A. Do not freeze reagents or controls.

B. Store HBV2 CS1, HBV2 CS2, HBV2 CS3 and HBV2 CS4 at 2-8ºC. Unused, these reagents are

stable until the expiration date indicated. Once used, these reagents are stable for 63 days at 2-8°C

or until the expiration date, whichever comes first. HBV2 CS1, HBV2 CS2, HBV2 CS3 and HBV2

CS4 can be used for up to a maximum of 64 hours cumulative on board the COBAS® AmpliPrep

Instrument. Reagents must be stored at 2-8°C between instrument cycles.

C. Store HBV H(+)C, v2.0; HBV L(+)C, v2.0 and CTM (–) C at 2-8ºC. The controls are stable until

the expiration date indicated. Once opened, any unused portion must be discarded.

D. Store Barcode clips [HBV H(+)C, v2.0 Clip, HBV L(+)C, v2.0 Clip and HBV (–) C Clip] at 2-30°C.

E. Store PG WR at 2-30°C. PG WR is stable until the expiration date indicated. Once opened, this

reagent is stable for 28 days at 2-30°C or until the expiration date, whichever comes first.

MATERIALS PROVIDED

A. COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

(P/N: 04894570 190)

HBV2 CS1

(HBV Magnetic Glass Particles Reagent Cassette)

HBV2 CS2

(HBV Lysis Reagent Cassette)

HBV2 CS3

(HBV Multi-Reagent Cassette)

HBV2 CS4

(HBV Test-Specific Reagent Cassette)

HBV H(+)C, v2.0

(HBV High Positive Control, v2.0)

HBV L(+)C, v2.0

(HBV Low Positive Control, v2.0)

CTM (–) C

[COBAS® TaqMan

® Negative Control (Human Plasma)]

HBV H(+)C, v2.0 Clip (HBV High Positive Control, v2.0 Barcode Clip)

HBV L(+)C, v2.0 Clip

(HBV Low Positive Control, v2.0 Barcode Clip)

HBV (–) C Clip

(HBV Negative Control Barcode Clip)

B. COBAS® AmpliPrep/COBAS

® TaqMan

® Wash Reagent

(P/N: 03587797 190)

PG WR (COBAS

® AmpliPrep/COBAS

® TaqMan

® Wash Reagent)

HBV V2.0

PG WR

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MATERIALS REQUIRED BUT NOT PROVIDED

Instrumentation and Software

• COBAS® AmpliPrep Instrument

• COBAS® TaqMan


® TaqMan

® 48 Analyzer

• Optional: Docking Station

• Optional: cobas p 630 instrument

• AMPLILINK Software – revision 3.2 series or 3.3 series

• Data Station for the AMPLILINK software, with printer

AMPLILINK Software v3.2 or v3.3 Series Manuals:

– COBAS® AmpliPrep instrument Instrument Manual For use with the COBAS

® TaqMan

®

analyzer, COBAS® TaqMan

® 48 analyzer, COBAS

® AMPLICOR

® analyzer, or cobas p 630

instrument, and the AMPLILINK software, version 3.2 and 3.3 series

– COBAS® TaqMan

® analyzer (plus optional docking station) Instrument Manual For use with

the AMPLILINK software, version 3.2 and 3.3 series Application Manual

– COBAS

®

TaqMan

®

48 analyzer Instrument Manual For use with the AMPLILINK software,version 3.2 and 3.3 series Application Manual

– AMPLILINK Software Version 3.2 Series Application Manual For use with the COBAS®

AmpliPrep Instrument, COBAS® TaqMan

® Analyzer, COBAS

® TaqMan

® 48 Analyzer, and

COBAS® AMPLICOR

® Analyzer

or

– AMPLILINK software Version 3.3 Series Application Manual For use with COBAS®

AmpliPrep instrument, COBAS® TaqMan

® analyzer, COBAS

® TaqMan

® 48 analyzer,

COBAS® AMPLICOR

® analyzer, and cobas p 630 instrument

– Optional: cobas p 630 instrument Operator’s Manual Software Version 2.2

Disposables

• Sample processing units: SPUs

• Sample input tubes (S-tubes) with barcode clips

• Racks of K-tips

• K-tube Box of 12 x 96

OTHER MATERIALS REQUIRED BUT NOT PROVIDED

• Sample Rack (SK 24 rack)

• Reagent Rack• SPU rack

• K-carrier

• K-carrier Transporter

• K-carrier rack

• Pipettors with aerosol barrier or positive displacement DNase-free tips (capacity 1000 μL)*

• Disposable gloves, powderless

• Vortex mixer

* Pipettors should be accurate within 3% of stated volume. Aerosol barrier or positive displacementDNase-free tips must be used where specified to prevent specimen and amplicon cross-contamination.

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SPECIMEN COLLECTION, TRANSPORT AND STORAGE

Note: Handle all specimens and controls as if they are capable of transmitting infectious agents.

Note: This test has been validated for use with only human serum or plasma collected in EDTA

anticoagulant. Testing of other specimen types may result in inaccurate results.

A. Specimen Collection

The COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, v2.0 is for use with plasma or serum specimens.Blood should be collected in sterile tubes using EDTA (lavender top) as the anticoagulant or in SST

®

Serum Separation Tubes.

Store whole blood at 2-25°C for no longer than 24 hours. Separate plasma or serum from whole blood

within 24 hours of collection by centrifugation at 800-1600 x g for 20 minutes at room temperature

(15-30°C). Transfer plasma or serum to a sterile polypropylene tube. Figures 4 and 5 show specimen

stability data from specimen stability studies performed with the COBAS® AmpliPrep/COBAS

® TaqMan

®

HBV Test, v2.0.

Figure 4

HBV Stability in Whole Blood (Collected in EDTA Plasma Tubes)

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Figure 5

HBV Stability in Whole Blood (Collected in Serum Separator Tubes)

B. Specimen Transport

Transportation of whole blood, plasma or serum must comply with country, federal, state and local

regulations for the transport of etiologic agents37

. Whole blood must be transported at 2-25°C and

centrifuged within 24 hours of collection. Plasma or serum may be transported at 2-8°C or frozen at -20°C

to -80°C.

C. Specimen Storage

Plasma or serum specimens may be stored at 25-30°C for up to 3 days, at 2-8°C for up to 7 days or frozen

at -20°C to -80°C for at least six weeks. It is recommended that specimens be stored in 800-900 μLaliquots in sterile, 2.0 mL polypropylene screw-cap tubes (such as Sarstedt 72.694.006). Figures 6 and 7

show the specimen stability data from specimen storage studies performed with the COBAS®


® HBV Test, v2.0.

Figure 6

HBV Stability in EDTA-Plasma

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Figure 7 HBV Stability in Serum

Plasma and serum specimens may be frozen and thawed up to five times without a loss of HBV DNA.

Figures 8 and 9 show the data from a freeze-thaw study performed with the COBAS® AmpliPrep/COBAS

®

TaqMan® HBV Test, v2.0

Figure 8

HBV Results after up to Five Freeze-Thaw Cycles (EDTA Plasma)

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Figure 9

HBV Results after up to Five Freeze-Thaw Cycles (Serum)

INSTRUCTIONS FOR USE

Note: For detailed operating instructions, a detailed description of the possible configurations, printing results and interpreting flags, comments and error messages, refer to (1) the

COBAS ® AmpliPrep instrument Instrument Manual For use with the COBAS ® TaqMan®

analyzer, COBAS ® TaqMan

® 48 analyzer, COBAS

® AMPLICOR

® analyzer, or cobas p 630

instrument, and the AMPLILINK software, version 3.2 and 3.3 series; (2) the COBAS ®

TaqMan® analyzer (plus optional docking station) Instrument Manual For use with the

AMPLILINK software, version 3.2 and 3.3 series Application Manual; (3) the COBAS ®

TaqMan® 48 analyzer Instrument Manual For use with the AMPLILINK software, version 3.2

and 3.3 series Application Manual; (4) the AMPLILINK Software Version 3.2 Series

Application Manual For use with the COBAS ® AmpliPrep Instrument, COBAS ® TaqMan®

Analyzer, COBAS ® TaqMan

® 48 Analyzer, and COBAS

® AMPLICOR

® Analyzer or the

AMPLILINK software Version 3.3 Series Application Manual For use with COBAS ®

AmpliPrep instrument, COBAS ® TaqMan

® analyzer, COBAS

® TaqMan

® 48 analyzer,

COBAS ® AMPLICOR

® analyzer, and cobas p 630 instrument; (5) Optional: cobas p 630

instrument Operator’s Manual Software Version 2.2.

Batch Size

Each kit contains reagents sufficient for 72 tests, which may be performed in batches of 12 to 24 tests. At

least one of each control (CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C, v2.0) must be included in

each batch (see "Quality Control" section).

Workflow

The COBAS® TaqMan


® TaqMan

® 48 Analyzer run must be started within 120 minutes

following completion of specimen and control preparation.

Note: Do not freeze or store processed specimens and controls.

Specimen and Control Preparation

Note: If using frozen specimens, place the specimens at room temperature (15-30°C) untilcompletely thawed and vortex for 3-5 seconds before use. Controls should be removed

from 2-8°C storage and equilibrated to room temperature before use.

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COBAS ® AmpliPrep Instrument Set-up

Part A. Maintenance and Priming

A1. The COBAS® AmpliPrep Instrument is ready for operation in stand-by mode.

A2. Turn the Data Station for the AMPLILINK software ON. Prepare the Data Station as follows:

a. Log onto the Windows®

XP operating system.b. Double click the AMPLILINK software icon.

c. Log onto AMPLILINK software by entering the assigned User ID and password.

A3. Check the supply of PG WR using the Status Screen and replace if necessary.

A4. Perform all Maintenance that is listed in the Due Tab. The COBAS® AmpliPrep Instrument will

automatically prime the system.

Part B. Loading of Reagent Cassettes

Note:

All reagent cassettes should be removed from 2-8°C storage, immediately loaded onto theCOBAS

® AmpliPrep Instrument and allowed to equilibrate to ambient temperature on the

instrument for at least 30 minutes before the first specimen is to be processed. Do not let reagent

cassettes come to ambient temperature outside the instrument as condensation may form on the

barcode labels. Do not wipe off condensation if it appears on the barcode labels.

B1. Place HBV2 CS1 onto a reagent rack. Place HBV2 CS2, HBV2 CS3 and HBV2 CS4 onto a

separate reagent rack.

B2. Load the reagent rack containing HBV2 CS1 onto rack position A of the COBAS® AmpliPrep

Instrument.

B3. Load the reagent rack containing HBV2 CS2, HBV2 CS3 and HBV2 CS4 onto rack position B, C,

D or E of the COBAS® AmpliPrep Instrument. (See Table 1 for additional information).

Part C. Loading of Disposables

Note: Determine the number of COBAS ® AmpliPrep reagent cassettes, Sample Processing Units

(SPUs), Input Sample tubes (S-tubes), K-tips and K-tubes needed. One SPU, one Input S-

tube, one K-tip and one K-tube are needed for each specimen or control.

Multiple workflows for use of the COBAS®

AmpliPrep Instrument with the COBAS®

TaqMan®

Analyzer orCOBAS

® TaqMan

® 48 Analyzer are possible. For reference, see Table 1 below. Depending on the workflow

used, load the appropriate number of reagent cassette racks, sample racks with Input S-tubes, SPU racks,

K-tip racks, K-tube racks and K-carriers on K-carrier racks onto the respective rack positions of the

COBAS® AmpliPrep Instrument (see Table 1 for additional information).

C1. Place the SPUs in the SPU rack(s) and load the rack(s) onto rack position J, K or L of the COBAS®

AmpliPrep Instrument.

C2. Depending on the workflow used, load full K-tube rack(s) onto rack position M, N, O or P of the

COBAS® AmpliPrep Instrument.

C3. Load full K-tip rack(s) onto rack position M, N, O or P of the COBAS® AmpliPrep Instrument.

C4. For workflow 3 using the COBAS® TaqMan

® 48 Analyzer, load K-carriers on K-carrier rack(s) onto

rack position M & N, or O & P of the COBAS® AmpliPrep Instrument.

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Table 1

Possible Workflows for using COBAS ® AmpliPrep Instrument with

COBAS ® TaqMan


® TaqMan

® 48 Analyzer

Workflow

Transfer Mode to

COBAS® TaqMan®

Analyzer or COBAS®

TaqMan® 48 Analyzer

Racks, Carriers and

Disposables

Position on

COBAS®

AmpliPrep

Instrument

1

COBAS®

AmpliPrep

Instrument

plus

Docking Station

plus

COBAS® TaqMan®

Analyzer

Automated transfer of

K-carrier

K-tubes in full K-tube racks M – PK-tips in full K-tip racks M – P

Input S-tubes containing

specimens and controls on

sample racks

F – H

SPUs in SPU racks J – L

CS1 on Cassette rack A

CS2, CS3, CS4 on Cassette

rackB – E

2

COBAS®

AmpliPrep

Instrument

plus

COBAS® TaqMan®

Analyzer

Manual transfer of

K-tubes via sample

rack(s) onto COBAS®

TaqMan® Analyzer

K-tubes in full K-tube racks M – P

K-tips in full K-tip racks M – P

Input S-tubes containing


sample racks

F – H

SPUs in SPU racks J – L



rackB – E

After specimen processing is

finished:

K-tubes on sample racks

(ready for manual transfer)

Same as

above

(F – H)

3

COBAS®

AmpliPrep

Instrument

plus

COBAS® TaqMan®

48 Analyzer(s)

Manual transfer of

K-carrier via

K-carrier rack(s)

onto COBAS®

TaqMan® 48

Analyzer

K-tubes on sample racks F - H

K-tips in full K-tip racks M - PInput S-tubes containing


sample racks

F - H

SPUs in SPU racks J - L



rackB - E

Empty barcoded K-carrier on

K-carrier rackM - P

After specimen processing is

finished:

K-tubes in K-carrier on K-carrier rack

Same as

above(M – P)

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Part D. Ordering and Loading of Specimens

D1. Prepare sample racks as follows: Attach a barcode label clip to each sample rack position where a

specimen (S-tube) is to be placed. Attach one of the specific barcode label clips for the controls

[CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C, v2.0] to each sample rack position where the

controls (S-tube) are to be placed. The CTM (–) C must be placed in the sample rack position

following the last clinical specimen or positive control [HBV L(+)C, v2.0 or HBV H(+)C, v2.0] in

each batch of 12 to 24 tests. The barcode label clips for controls should have the same control lot

number as the lot number on the control vials in the kit. Take care in assigning the right control tothe position with the appropriate control barcode clip. Place one Input S-tube into each position

containing a barcode label clip.

D2. Using the AMPLILINK software, create specimen orders for each specimen and control in the

Orders window Sample folder. Select the appropriate test file and complete by saving.

D3. Assign specimen and control orders to sample rack positions in the Orders window Sample Rack

folder. The sample rack number must be for the rack prepared in Step D1.

D4. Print the Sample Rack Order report to use as a worksheet.

D5. Prepare specimen and control racks in the designated area for specimen and control addition as

follows: Vortex each specimen and control [CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C, v2.0]

for 3 to 5 seconds. Avoid contaminating gloves when manipulating the specimens and controls.

D6. Transfer 650 μL of each specimen and control [CTM (–) C, HBV L(+)C, v2.0 and HBV H(+)C,

v2.0] to the appropriate barcode labeled Input S-tube using a micropipettor with an aerosol barrier

or positive displacement DNase-free tip. Avoid transferring particulates and/or fibrin clots

from the original specimen to the Input S-tube. Specimens and controls should be transferredto tube positions as assigned and recorded on the worksheet in Step D4. The barcode label clips for

controls should have the same control lot number as the lot number on the control vials in the kit.

Assign the right control to the position with the appropriate control barcode clip. Avoid contami-

nating the upper part of the S-tubes with specimens or controls.

D7. If using the cobas p 630 instrument for preparation of specimens, refer to the cobas p 630

instrument Operators Manual.

D8. For workflows 1 and 2, load the sample rack(s) filled with Input S-tubes onto rack positions F, G or

H of the COBAS® AmpliPrep Instrument.

D9. For workflow 3 using the COBAS® TaqMan

® 48 Analyzer, load sample rack(s) with Input S-tubes

and K-tubes (one for each Input S-tube, loaded in the right position adjacent to Input S-tubes) onto

rack position F, G or H of the COBAS® AmpliPrep Instrument.

Part E. Start of COBAS® AmpliPrep Instrument Run

E1. Start the COBAS® AmpliPrep Instrument using the AMPLILINK software.

Part F. End of COBAS® AmpliPrep Instrument Run and Transfer to COBAS

® TaqMan

®

Analyzer or COBAS® TaqMan

® 48 Analyzer (for workflows 2 and 3 only)

F1. Check for flags or error messages.

F2. Remove processed specimens and controls from the COBAS® AmpliPrep Instrument on either

sample racks (for COBAS® TaqMan

® Analyzer without Docking Station) or K-carrier racks (for

COBAS® TaqMan

® 48 Analyzer), depending on the workflow (for further details see Part G).

F3. Remove waste from the COBAS® AmpliPrep Instrument.

Note: Processed specimens and controls should not be exposed to light after completion of

specimen and control preparation.

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Amplification and Detection

COBAS ® TaqMan


® TaqMan

® 48 Analyzer Set-up

The COBAS® TaqMan


® TaqMan

® 48 Analyzer run must be started within 120 minutes

following completion of specimen and control preparation.

Note: Do not freeze or store processed specimens and controls.

Part G. Loading Processed Specimens

G1. Depending on the workflow, perform the appropriate steps to transfer the K-tubes to the COBAS®

TaqMan® Analyzer or COBAS

® TaqMan

® 48 Analyzer:

Workflow 1: Automated transfer of K-carrier via docking station to COBAS®

TaqMan® Analyzer. Manual intervention is unnecessary.

Workflow 2: Manual transfer of K-tubes in sample rack(s) to COBAS® TaqMan

®

Analyzer.

Workflow 3: Manual transfer of K-carrier on K-carrier rack(s) to the COBAS®

TaqMan® 48 Analyzer. Manual transfer of K-carriers into COBAS

®

TaqMan® 48 Analyzer using the K-carrier Transporter.

Part H. Start of COBAS® TaqMan


® TaqMan

® 48 Analyzer Run

H1. Start the COBAS® TaqMan


® TaqMan

® 48 Analyzer by one of the options

below depending on the workflow used:

Workflow 1: No intervention necessary.

Workflow 2: Automatic start of the COBAS® TaqMan

® Analyzer after insertion of

sample rack(s).

Workflow 3: Fill K-carrier with empty K-tubes if there are fewer than 6 K-tubes onthe K-carrier. Filling is guided by the AMPLILINK software. Open

thermal cycler cover, load K-carrier into thermal cycler and close lid.

Start the COBAS® TaqMan

® 48 Analyzer run.

Part I. End of COBAS® TaqMan


® TaqMan

® 48 Analyzer Run

I1. At the completion of the COBAS® TaqMan


® TaqMan

® 48 Analyzer run, print

Results Report. Check for flags or error messages in the Result report. Specimens with flags and

comments are interpreted as described in the Results section. After acceptance, store data in archive.

I2. Remove used K-tubes from the COBAS® TaqMan


® TaqMan

® 48 Analyzer.

RESULTS

The COBAS® TaqMan

® Analyzer or the COBAS

® TaqMan

® 48 Analyzer automatically determines the HBV

DNA concentration for the specimens and controls. The HBV DNA concentration is expressed in

International Units (IU) /mL. The conversion factor between HBV copies/mL and HBV IU/mL is

5.82 copies/IU, using the WHO International Standard for Hepatitis B Virus DNA for Nucleic Acid

Technology (NAT) Assays Testing (NIBSC 97/746)29

.

If needed, results can be converted manually to copies/mL using the conversion calculation as

follows:

HBV DNA concentration in IU/mL x 5.82 copies/IU = HBV DNA in copies/mLExample: 1.23E+04 IU/mL x 5.82 copies/IU = 7.16E+04 copies/mL

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Note: The analytical measurement range of analyte values that can be directly measured on a

specimen without any dilution using the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test,

v2.0 is 20 to 1.7E+08 IU/mL.

Note: The clinical reportable range of analyte values that can be directly measured on a

specimen with a maximum dilution of one to ten using the COBAS ® AmpliPrep/COBAS

®

TaqMan® HBV Test, v2.0 is 20 to 1.7E+09 IU/mL.

AMPLILINK Software:

• Determines the Cycle Threshold value (Ct) for the HBV DNA and the HBV QS DNA.

• Determines the HBV DNA concentration based upon the Ct values for the HBV DNA and

HBV QS DNA and the lot-specific calibration coefficients provided on the cassette barcodes.

• Determines that the calculated IU/mL for HBV L(+)C, v2.0 and HBV H(+)C, v2.0 fall

within the fixed ranges.

Batch Validation – AMPLILINK Version 3.2 Series

Check AMPLILINK software results window or printout for flags and comments to ensure that the batch isvalid. For control orders, a check is made to determine if the IU/mL value for the control is within its

specified range. If the IU/mL value for the control lies outside of its range, a FLAG is generated to show the

control has failed.

The batch is valid if no flags appear for any of the controls [HBV L(+)C, v2.0; HBV H(+)C, v2.0 and

CTM (–) C].

The batch is not valid if any of the following flags appear for the HBV Controls:

Negative Control

Flag Result Interpretation

_N_NC_INVALID InvalidAn invalid result or a “valid” result that was not

negative for HBV target

HBV Low Positive Control, v2.0


_L_LPCINVALID 1.70E+08 IU/mL Control above range

_L_LPCINVALID Invalid An invalid result

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HBV High Positive Control, v2.0


_H_HPCINVALID 1.70E+08 IU/mL Control above range

_H_HPCINVALID Invalid An invalid result

If the batch is invalid, repeat the entire batch including specimen and control preparation, amplification

and detection.

Batch Validation – AMPLILINK Version 3.3 Series

Check AMPLILINK software results window or printout for flags and comments to ensure that the batch is

valid. For control orders, a check is made to determine if the IU/mL value for the control is within its specified range. If the IU/mL value for the control lies outside of its range, a FLAG is generated to show the

control has failed.

The batch is valid if no flags appear for any of the controls [HBV L(+)C, v2.0; HBV H(+)C, v2.0 and

CTM (–) C].

The batch is not valid if any of the following flags appear for the HBV Controls:

Negative Control:


NC_INVALID Invalid An invalid result or a “valid” result that was not negative for HBV target

HBV Low Positive Control, v2.0:


LPCINVALID Invalid An invalid result or a control out of range

HBV High Positive Control, v2.0:


HPCINVALID Invalid An invalid result or a control out of range

If the batch is invalid, repeat the entire batch including specimen and control preparation, amplification

and detection.

Interpretation of Results

For a valid batch, check each individual specimen for flags or comments on the result printout. Interpret

the results as follows:

• A valid batch may include both valid and invalid specimen results depending on whether

flags and/or comments are obtained for the individual specimens.

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Specimen results are interpreted as follows:

Titer Result Interpretation

Target Not Detected Report results as "HBV DNA not detected".

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PROCEDURAL PRECAUTIONS

As with any test procedure, good laboratory technique is essential to the proper performance of this assay.

PROCEDURAL LIMITATIONS

1. This test has been validated for use with human serum or plasma collected in EDTA anticoagulant.

Testing of other specimen types may result in inaccurate results.

2. Reliable results are dependent on adequate specimen collection, transport, storage and processingprocedures.

3. The presence of AmpErase enzyme in the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Master Mix,

v2.0 reduces the risk of amplicon contamination. However, contamination from HBV positive

controls and clinical specimens can be avoided only by good laboratory practices and careful

adherence to the procedures specified in this Package Insert.

4. Use of this product should be limited to personnel trained in the techniques of PCR.

5. This product can only be used with the COBAS® AmpliPrep Instrument and the COBAS

® TaqMan

®

Analyzer or COBAS

®

TaqMan

®

48 Analyzer.

6. Though rare, mutations within the highly conserved region of the viral genome covered by the


® TaqMan

® HBV Test, v2.0 primers and/or probe may result in the

under-quantitation of or failure to detect the virus.

7. Detection of HBV DNA is dependent on the number of virus particles present in the specimen and

may be affected by specimen collection methods and patient factors, (i.e., age, presence of

symptoms, and/or stage of the infection).

8. 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 quantify

technology differences.

INTERFERING SUBSTANCES

Elevated levels of triglycerides, bilirubin and albumin in specimens have been shown not to interfere with the

quantitation of HBV DNA by this test. Elevated levels of hemoglobin in specimens up to a concentration of

250 mg/dL have been shown not to interfere with the quantitation of HBV DNA by this test.

The following drug compounds tested at 3 times of the Peak Plasma Level (Cmax) have been shown not to

interfere with the quantitation of HBV DNA by the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0:

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Nucleotide DNA Polymerase Inhibitors

Tenofovir

Adefovir dipivoxil

Telbivudine

Nucleoside Reverse Transcriptase and

DNA Polymerase Inhibitors

Lamivudine

Zidovudine

Stavudine

Abacavir

Didanosine

Entecavir

HIV Protease Inhibitors

Indinavir

Saquinavir

Ritonavir

Amprenavir

Lopinavir/Ritonavir

Non-nucleoside HIV Reverse Transcriptase

Inhibitors

Nevirapine

Efavirenz

HIV Fusion Inhibitors

Enfurvitide

Immune Modulators

Interferon alpha-2a

Ribavirin

Interferon alpha-2b

Peginterferon alfa-2a

Peginterferon alfa-2a + Ribavirin

Interferon alpha-2b + Ribavirin

Peginterferon alfa-2b

Antidepressants

Paroxetine HCl

Fluoxetine

Sertraline

Compounds for Treatment of Herpes

Viruses

Ganciclovir

Valganciclovir HCl

Acyclovir

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NON-CLINICAL PERFORMANCE EVALUATION

A. Limit of Detection Using the WHO International Standard

The limit of detection of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was determined for

HBV DNA by using dilutions of the WHO International Standard for Hepatitis B Virus DNA for Nucleic Acid


, genotype A in HBV-negative human EDTA plasma or

serum. Three dilution series were analyzed for each matrix. A total of 72 replicates per concentration were

tested for each matrix type.

The concentration of HBV DNA that can be detected with a positivity rate of greater than 95% as determined

by PROBIT Analysis, is 9 IU/mL for EDTA plasma (see Table 2) and 19 IU/mL for serum (see Table 3).

Table 2

Limit of Detection in EDTA Plasma of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

using the WHO International Standard

Nominal Input

(HBV DNA IU/mL)

No. Replicates No. Positives Positivity Rate

30 72 72 100%

20 71 71 100%

10 72 68 94%

5 72 63 88%

2.5 72 38 53%

PROBIT 95% Hit Rate 9 IU/mL (95% confidence range: 6.8 – 12.1 IU/mL)

Table 3

Limit of Detection in Serum of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

using the WHO International Standard

Nominal Input

(HBV DNA IU/mL)

No. Replicates No. Positives Positivity Rate

30 72 72 100%

20 72 70 97%

10 72 58 81%

5 72 29 40%

2.5 72 24 33%

PROBIT 95% Hit Rate 19 IU/mL (95% confidence range: 11.0 – 107.1 IU/mL)

B. Limit of Detection Using Clinical Specimens Across All Genotypes

In addition, dilutions of clinical specimens in HBV-negative human EDTA plasma or serum representing

genotypes A-F and H and two quantified purified plasmid DNAs, one for genotype G and one for the pre-

core mutant were analyzed with two kit lots of COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

reagents. Twenty-four replicates per level, genotype, matrix and kit lot were tested.

The concentration of HBV DNA that can be detected with a hit rate of greater than 95% as determined by

PROBIT analysis, is 16.4 IU/mL combined for both matrices (see Table 4).

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Table 4

Limit of Detection in EDTA Plasma and Serum of the

COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 using

Genotype A-H and Pre-core Mutant

Genotype Matrix

95% PROBIT

hit rate

concentration

Lot 1

95% PROBIT

hit rate

concentration

Lot 2

95% PROBIT

hit rate

concentration

(max.)

95%

PROBIT

confidence

interval

APlasma 10.6

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C. Precision

The precision of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was determined by analysis of

serial dilutions of clinical HBV specimens (genotype A) in HBV-negative human EDTA plasma and serum.

The titer assignment of the clinical specimens (stock concentrations) was performed by a method that

ensures traceability to the WHO International Standard for Hepatitis B Virus DNA for Nucleic Acid


. 15 runs per matrix were performed, each consisting of

6 dilution levels and 3 replicates at each level. Each specimen was taken through the entire COBAS®

AmpliPrep/COBAS® TaqMan® HBV Test, v2.0 procedure, including specimen preparation, amplification anddetection. Therefore, the precision reported here represents all aspects of the test procedure. The study

was performed using three lots of COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 reagents. The

results are shown in Table 5 (EDTA plasma) and Table 6 (serum).

Table 5 Precision of the COBAS

® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 (EDTA Plasma Samples)

Titer

(IU/mL)

Lot 1 Lot 2 Lot 3

Total %CV Total SD

in log Total %CV

Total SD

in log Total %CV

Total SD

in log

1.00E+02 26 0.12 28 0.13 25 0.11

1.00E+03 17 0.07 17 0.07 20 0.09

1.00E+04 12 0.05 11 0.05 14 0.06

1.00E+05 8 0.04 8 0.04 13 0.06

1.00E+06 11 0.05 11 0.05 12 0.05

1.00E+07 14 0.06 12 0.06 18 0.08

Table 6

Precision of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 (Serum Samples)

Titer

(IU/mL)

Lot 1 Lot 2 Lot 3

Total %CV Total SD

in log Total %CV

Total SD

In log Total %CV

Total SD

In log

1.00E+02 27 0.13 22 0.11 28 0.13

1.00E+03 13 0.06 15 0.06 17 0.08

1.00E+04 11 0.05 10 0.04 14 0.06

1.00E+05 10 0.04 12 0.05 11 0.05

1.00E+06 11 0.05 12 0.05 11 0.05

1.00E+07 15 0.07 15 0.07 13 0.06

D. Linear Range

As shown in Figure 10 and Figure 11, the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was found

to give a linear response from 20 (Log10 = 1.30) HBV DNA IU/mL to 1.7E+08 (Log10 = 8.23) HBV DNA

IU/mL in both EDTA plasma and serum. The evaluation was performed according to CLSI Guideline EP6-A

using two lots of COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 reagents and serial dilutions of a

high titer HBV DNA (+) EDTA plasma or serum specimen. Thirteen concentration levels for EDTA plasma

and twelve levels for serum were tested, with 10 replicates per level.

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Figure 10

Linearity for the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

in EDTA Plasma Specimens

y = 1.0183x - 0.0182

R2 = 0.9987

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8 9 10

Nominal Concentration

Log10 (HBV DNA IU/mL)

C O B A S ® A m p l i P r e p / C O B A S ® T a q M

a n ®

H B V T e s t , v 2 . 0 R e s u l t s

L o g 1 0 ( H B V D N A I U / m L )

Mean

Figure 11

Linearity for the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

in Serum Specimens

y = 1.0202x - 0.1142

R2 = 0.9987

0

12

3

4

5

6

7

8

9

0 1 2 3 4 5 6 7 8 9



C O B A S ® A m p l i P r e p / C O B A S ® T a q M a n ®

H B V T e s t , v 2 . 0 R e s u l t s

L

o g 1 0 ( H B V D N A I U / m L )

Mean

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E. Genotype Titer Quantitation

To date, eight genotype categories have been proposed for HBV based on nucleotide divergence within

the genome of greater than 8%38, 39, 40

. These genotypes are designated with capital alphabetical letters

from A through H. The HBV genotypes have characteristic geographic distributions41

.

The performance of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 on HBV genotypes was

evaluated by analysis of 25 purified, linearized and quantitated plasmid DNAs containing representative

sequence inserts from HBV genotypes A through H and the most common pre-core mutant (see Table 7).The assignment of nominal concentrations to the plasmid stock solutions was performed by the PicoGreen

method. Each plasmid DNA was diluted to nominal concentrations of approximately 3.00E+03, 3.00E+05

and 3.00E+07 PicoGreen copies/mL in both EDTA plasma and serum. The concentrations (IU/mL) were

then determined by the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 using two reagent lots and

the results of all tested plasmids were compared.

The evaluation of all 25 plasmids analyzed by the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

demonstrates equivalent quantification of all plasmids and genotypes for EDTA plasma and serum.

Table 7COBAS

® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

Inclusivity Testing – Typed Plasmid DNAs Tested

Plasmid Designation Genotype Parent Specimen Origin

p8423-c1

A

India

p1115-c1 Burundi

p3952-c1 Cameroon

p4199-c2 Norway

p1764-c1

B

China

p1767-c1 China

p3958-c1 East Asia

p830-c1 Society Island

p3982-c1 Vietnam

p1786-c1C

China

p11549-1 Bangladesh

p3872-c1

D

Iran

p1103-c1 Tunisia

p3953-c2 North Africa

p18-c1 Sweden

p30893-5 Sweden

p4244-c1 Denmarkp3217-c1

ESenegal

p3963-c2 Nigeria

p9203-c1

F

Colombia

p479-c1 Venezuela

p1009-c1 Spain

p00042975-4 G United States

pEFHBV20 H Nicaragua

pITmut1896 Pre-core mutant Italy

Also, for eight of these plasmids, representing all genotypes, inclusivity was shown over the linear range(Figure 12 and 13). For each plasmid, three concentration levels for EDTA plasma and serum were tested,

with 10 replicates per level.

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Figure 12

Performance of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

on HBV Genotypes A through H in EDTA Plasma

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

2.5 3.5 4.5 5.5 6.5 7.5



D e v i a t i o n

L o g 1 0 ( H B V D N A I U / m L ) Genotype A

Genotype B

Genotype C

Genotype D

Genotype E

Genotype F

Genotype G

Genotype H

+/- 0.3 log

Figure 13

Performance of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

on HBV Genotypes A through H in Serum

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

2.5 3.5 4.5 5.5 6.5 7.5


Log10

(HBV DNA IU/mL)

e v a

o n

L o g 1 0 ( H B V D N A I U / m L ) Genotype A

Genotype B

Genotype C

Genotype D

Genotype E

Genotype F

Genotype G

Genotype H

+/- 0.3 log

F. Clinical Specificity

The specificity of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was determined by analysis of

HBV-negative EDTA plasma and serum from blood donors. A total of 647 individual EDTA plasma and 649serum specimens were tested. The results of all specimens were negative for HBV DNA. Based on these

results, the specificity of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 is 100% with a

confidence limit of 99.54%.

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G. Cross Reactivity

The analytical specificity of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was evaluated by

adding cultured organisms (viruses, bacteria, yeast) or DNA (HTLV-2, 2.5E+07 cp/PCR) into HBV-negative

human EDTA plasma (see Table 8).

None of the non-HBV organisms tested was positive for HBV DNA.

Table 8

Analytical Specificity Specimens

Virus

Adenovirus type 5

Cytomegalovirus

Epstein-Barr virus

Human Herpes Virus type 6

Herpes simplex virus type 1

Herpes simplex virus type 2

Human T-Cell Lymphotropic virus type 1

Human T-Cell Lymphotropic virus type 2

Influenza A

Hepatitis A virus

Hepatitis C virus

HIV-1

Bacteria

Staphylococcus aureus

Propionibacterium acnes

Yeast

Candida albicans

H. Correlation of EDTA Plasma and Serum

The correlation of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 was tested by analysis of HBV

positive matched clinical EDTA plasma and serum specimens. 279 matched sets of specimens from HBV

infected patients were analyzed. The COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 results showed

high correlation between EDTA plasma and serum specimens (Figure 14).

Figure 14

Correlation of the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0

on EDTA Plasma and Serum Specimens from HBV Infected Patients (n=279)

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I. Performance of COBAS® AmpliPrep/COBAS

® TaqMan

® HBV Test, v2.0 compared to the


® TaqMan

® HBV Test


® TaqMan

® HBV Test, v2.0 was compared to the


® TaqMan

® HBV Test

30 by analysis of HBV positive (' 54 IU/mL) clinical

specimens. Left over EDTA plasma specimens from routine diagnosis were analyzed at two external sites.

The specimen titers spread over the dynamic range of the COBAS® AmpliPrep/COBAS

® TaqMan

® HBV

Test, v2.0. Linear regression analysis was performed on 275 valid samples (143 tested at one site and 132

at the other) (Figure 15).

Figure 15


® TaqMan

® HBV Test, v2.0

and the COBAS ® AmpliPrep/COBAS

® TaqMan

® HBV Test

on EDTA Plasma Specimens from HBV Infected Patients (n=275)

y = 0.9958x + 0.0443

R2 = 0.9743

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

COBAS ® AmpliPrep/COBAS ® TaqMan ® HBV Test

Log10

HBV DNA Titer

C O B A S ® A m p l i P r e p / C O B A S ® T a q M a n

® H B

V T e s t , v 2 . 0

L o g 1 0 H

B V D N A T i t e r

J. Method correlation


® TaqMan

® HBV Test, v2.0 was compared to the

COBAS® TaqMan

® HBV Test For Use with the High Pure System (HPS HBV, Figures 16 and 17), the

COBAS

®

AMPLICOR

®

HBV MONITOR Test (COBAS

®

AMPLICOR

®

HBV, Figures 18 and 19) and theVERSANT HBV DNA 3.0 Assay (Figures 20 and 21) by regression analysis of undiluted clinically

characterized specimens from HBV infected patients. Specimens were obtained from Teragenix (Fort

Lauderdale, FL, USA). Regression analysis was performed for serum and EDTA-plasma specimens that

yielded results within the linear range.

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05382874001-10EN 34 Doc Rev. 10.0

Figure 16


® TaqMan

® HBV Test, v2.0

and the COBAS ® TaqMan

® HBV Test For Use with the High Pure System


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0.0 2.0 4.0 6.0 8.0 10.0

High Pure System HBVPlasma Specimen (log

10 Titer )

C O B A S ® A m p l i P r e p / C O B

A S ® T a q M a n ® H B V T e s t , v 2

. 0

P l a s m a S p e c i m e n ( L o g 1 0 T i t e r )

Y = 0.974 * X + 0.107

R2 = 0.964

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Figure 17


® TaqMan

® HBV Test, v2.0

and the COBAS ® TaqMan

® HBV Test For Use with the High Pure System

on Serum Specimens from HBV Infected Patients (n=142)

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Figure 18


® TaqMan

® HBV Test, v2.0

and the COBAS ® AMPLICOR

® HBV MONITOR Test


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0.0 1.0 2.0 3.0 4.0 5.0

COBAS ® AMPLICOR ® HBVPlasma Specimen (log10 Titer)

C O B A S ® A m p l i P r e p / C O

B A S ® T a q M a n ® H B V T e s t ,

v 2 . 0

P l a s m a S p

e c i m e n ( L o g 1 0 T i t e r )

Y = 0.899 * X + 0.546

R2 = 0.872

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Figure 19


® TaqMan

® HBV Test, v2.0

and the COBAS ® AMPLICOR

® HBV MONITOR Test


0

1

2

3

4

5

6

0 1 2 3 4 5 6

COBAS ® AMPLICOR ® HBVSerum Specimen (log10Titer)


A S ® T a q M a n

® H B V T e s t , v 2 . 0

S e r u m S

p e c i m e n ( L o g 1 0 T i t e r )

Y = 0.911 * X + 0.461

R2 = 0.821

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Figure 20


® TaqMan

® HBV Test, v2.0

and the VERSANT HBV DNA 3.0 Assay


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

VERSANT HBV DNA 3.0 AssayPlasma Specimen (log10 Titer)


A S ® T a q M a n ® H B V T e s t , v 2

. 0

P l a s m a S p e c i m e n ( L o g 1 0 T i t e r )

Y = 0.892 * X + 0.260

R2 = 0.810

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05382874001-10EN 39 Doc Rev. 10.0

Figure 21


® TaqMan

® HBV Test, v2.0

and the VERSANT HBV DNA 3.0 Assay


0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8

VERSANT HBV DNA 3.0 Assay

Serum Specimen (log10

Titer)

C O B A S ® A m p l i P r e p / C O B A S ® T a q M a n ® H B V T e s t , v 2 . 0

S e r u m S p e c i m

e n ( L o g 1 0 T i t e r )

Y = 0.913 * X + 0.125

R2 = 0.837

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