Rx Only

cobas® EBV

Quantitative nucleic acid test

for use on the cobas® 5800/6800/8800 Systems
For in vitro diagnostic use

cobas® EBV P/N: 09040943190

For use on the cobas® 5800 System

cobas® EBV/BKV Control Kit P/N: 09040951190

cobas® Buffer Negative Control Kit P/N: 09051953190

For use on the cobas® 6800/8800 Systems

cobas® EBV/BKV Control Kit P/N: 08688214190 or

P/N: 09040951190
cobas® Buffer Negative Control Kit P/N: 07002238190 or
P/N: 09051953190
 cobas® EBV

Table of contents
Intended use ............................................................................................................................ 4

Summary and explanation of the test................................................................................. 4

Reagents and materials ......................................................................................................... 7

cobas® EBV reagents and controls ............................................................................................................ 7

cobas® omni reagents for sample preparation ....................................................................................... 10
Reagent storage requirements ................................................................................................................. 11
Reagent handling requirements for the cobas® 5800 System .............................................................. 12
Reagent handling requirements for the cobas® 6800/8800 Systems................................................... 13
Additional materials required for the cobas® 5800 System .................................................................. 14
Additional materials required for the cobas® 6800/8800 Systems ...................................................... 15
Instrumentation and software required ................................................................................................. 15
Precautions and handling requirements ......................................................................... 16

Warnings and precautions ...................................................................................................................... 16

Reagent handling ...................................................................................................................................... 16
Good laboratory practice ......................................................................................................................... 17
Sample collection, transport, and storage........................................................................ 17

Samples ...................................................................................................................................................... 17
Instructions for use............................................................................................................... 18

Procedural notes ....................................................................................................................................... 18

Running cobas® EBV on the cobas® 5800 System ................................................................................. 19
Running cobas® EBV on the cobas® 6800/8800 Systems ...................................................................... 20
Results ..................................................................................................................................... 21

Quality control and validity of results on the cobas® 5800 System .................................................... 21
Control results on the cobas® 5800 System ................................................................................... 21
Quality control and validity of results on the cobas® 6800/8800 Systems ......................................... 21
Control flags on the cobas® 6800/8800 Systems ........................................................................... 22

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Interpretation of results ........................................................................................................................... 22

Interpretation of results on the cobas® 5800 System ................................................................... 23
Interpretation of results on the cobas® 6800/8800 Systems ........................................................ 23
Procedural limitations.............................................................................................................................. 23
Non-clinical performance evaluation ............................................................................... 24

Key performance characteristics performed on the cobas® 6800/8800 Systems ............................... 24

Limit of Detection (LoD) ................................................................................................................ 24
Linear range ...................................................................................................................................... 25
Precision – within laboratory ......................................................................................................... 26
Genotype verification ...................................................................................................................... 26
Specificity .......................................................................................................................................... 27
Analytical specificity ........................................................................................................................ 27
Analytical specificity – interfering substances .............................................................................. 27
Method correlation .......................................................................................................................... 28
Whole system failure ....................................................................................................................... 29
Cross contamination ....................................................................................................................... 29
Clinical performance evaluation performed on the cobas® 6800/8800 Systems .. 30

Reproducibility of cobas® EBV................................................................................................................ 30

Performance of cobas® EBV .................................................................................................................... 31
System equivalency / system comparison ............................................................................................. 33
Additional information ......................................................................................................... 34

Key test features ........................................................................................................................................ 34

Symbols ...................................................................................................................................................... 35
Technical support ..................................................................................................................................... 36
Manufacturer and importer .................................................................................................................... 36
Trademarks and patents .......................................................................................................................... 36
Copyright................................................................................................................................................... 36
References .................................................................................................................................................. 37
Document revision ................................................................................................................................... 39

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Intended use
cobas® EBV is an in vitro nucleic acid amplification test for the quantitation of Epstein-Barr virus (EBV) DNA in human
EDTA plasma on the cobas® 5800/6800/8800 Systems.
cobas® EBV is intended for use as an aid in the diagnosis and management of EBV in transplant patients. In patients
undergoing monitoring of EBV, serial DNA measurements can be used to indicate the need for potential treatment
changes and to assess viral response to treatment.
The results from cobas® EBV must be interpreted within the context of all relevant clinical and laboratory findings.

Summary and explanation of the test

Background
Transplant recipients are at increased risk for many viral and bacterial infections that are more likely to cause severe
adverse health outcomes in the transplant patient population compared to the general healthy population. This increased
risk is partly attributable to diminished immune system function conferred by the immunosuppressive medications that
transplant patients receive in order to reduce their likelihood of graft rejection.1,2
EBV is a member of the herpes virus family. It is a double stranded, enveloped deoxyribonucleic acid (DNA) virus
(~172kb). Two main EBV genotypes, type 1 and type 2, have been defined by the differences in the EBNA-2 gene. EBV
infections in humans are quite common; more than 90% of adults are infected, and latent infection persists over the
lifetime. EBV causes infectious mononucleosis in a subset of newly infected adolescents and adults, and is associated with
several types of cancer, including nasopharyngeal carcinoma, Burkitt lymphoma, and Hodgkin lymphoma. EBV can be
the cause of lymphoproliferative disorders in persons with congenital or acquired immunodeficiency, including transplant
recipients and patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS).3
In transplant recipients, EBV can cause disease either through reactivation of latent virus from memory B cells, or through
a new primary infection, especially in EBV-negative transplant recipients who receive grafts from EBV-positive donors.3
For these patients, the most severe form of EBV-related disease is post-transplant lymphoproliferative disorder (PTLD),
which results from uncontrolled proliferation of lymphocytes, typically B cells.4 Overall, > 70% of PTLD cases among
transplant recipients are linked to EBV infection. The highest risk for PTLD occurs during the first year after transplant,
and >90% of PTLD cases that occur during this period are linked to EBV. Up to 20% of PTLD cases that occur after the
first year post-transplant are EBV-negative.4,5
Risk factors for early-onset PTLD include EBV-negative recipient serostatus at transplantation, younger age, exposure to
lymphocyte-depleting antibodies, and type of organ transplanted.5,6
Early identification of primary EBV infections and DNA level monitoring can support prompt therapeutic intervention to
prevent progression to EBV-related disease. Guidelines recommend regular EBV monitoring using quantitative nucleic
acid tests (NATs), especially in transplant recipients who are EBV-negative in high risk transplant patients.4,5 While the
exact medically relevant viral threshold is still a subject of debate due to inter-assay variability, the critical threshold
concept appears valid and has been reported in natural history studies showing that higher EBV DNA levels correlate with
increased risk for the development of EBV disease and PTLD.4,5,7 Both plasma and whole blood sample types have been
used for EBV testing, but evidence suggests that plasma is more specific for detection of PTLD.4,5,7,8

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Common therapeutic interventions to reduce EBV DNA levels and prevent onset of PTLD include reduction of
immunosuppression medication doses and treatment with B cell depleting antibodies.7 Pre-emptive therapy to reduce
EBV DNA levels is successful in most patients, although up to 20% of patients may still develop PTLD, especially those
who are more than one year post-transplant.7
Most laboratory tests for EBV quantitation are not standardized, which has resulted in inter-laboratory and inter-assay
variability in DNA level results and precludes comparison of DNA levels generated from different laboratories and tests.7
To address this problem, the WHO has created an International Standard for EBV quantitation, allowing standardized
tests to report in IU/mL.9 Formal assessment of the reproducibility and validity of EBV DNA levels is critical to ensure
consistent results across laboratories in order to improve the clinical management of patients at increased risk for
developing EBV-related diseases and PTLD.

Rationale for NAT testing

EBV serologies of donor and recipient are determined before transplantation to help determine a transplant patient’s risk
of EBV-related complications, but serology is not sufficiently sensitive or precise for monitoring patients after
transplantation. EBV culture methods are slow and have poor predictive value in this setting. Direct detection of EBV
DNA by real-time PCR potentially offers a wide dynamic range, precision, and high sensitivity and specificity.

Explanation of the test

cobas® EBV is a quantitative test that is run on the cobas® 5800/6800/8800 Systems. cobas® EBV enables the detection and
quantitation of EBV DNA in EDTA plasma of infected patients. The viral load is quantified against a non-EBV DNA
quantitation standard (DNA-QS), which is introduced into each specimen during sample processing. The DNA-QS also
functions to monitor for the entire sample preparation and PCR amplification process. In addition, the test utilizes three
external controls: a high titer positive, a low titer positive, and a negative control. The high positive and low positive external
controls are manufactured by dilution from stock material with a titer traceable to EBV WHO International Standard. Each
Amplification/Detection kit lot is calibrated traceable to EBV WHO International Standard.

Principles of the procedure

cobas® EBV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by
PCR amplification and detection. The cobas® 5800 System is designed as one integrated instrument. The cobas®
6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the
analytic module. Automated data management is performed by the cobas® 5800 or cobas® 6800/8800 System software
which assigns test results for all tests as either target not detected, EBV DNA detected < LLoQ (lower limit of
quantitation), EBV DNA detected > ULoQ (upper limit of quantitation), or a value in the linear range LLoQ < x <
ULoQ. Results can be reviewed directly on the system screen, exported, or printed as a report.
Nucleic acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, viral
nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica
surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris
and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the
glass particles with elution buffer at elevated temperature.

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Selective amplification of target nucleic acid from the sample is achieved by the use of a dual target virus specific approach
from highly-conserved regions of the EBV located in the EBV EBNA-1 gene and the EBV BMRF gene. Selective
amplification of DNA-QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have
no homology with the EBV genome. A thermostable DNA polymerase enzyme is used for amplification. The target and
DNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined
temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of
deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon).10-12 Any
contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme, which is included in the PCR mix,
when heated in the first thermal cycling step. However, newly formed amplicon are not eliminated since the AmpErase
enzyme is inactivated once exposed to temperatures above 55°C.
The cobas® EBV master mix contains two detection probes specific for EBV target sequences and one for the DNA-QS. The
probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of EBV target and
DNA-QS in two different target channels.13,14 The fluorescent signal of the intact probes is suppressed by the quencher dye.
During the PCR amplification step, hybridization of the probe to the specific single-stranded DNA templates results in
cleavage by the 5'-to-3' nuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes
and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the
cumulative signal of the reporter dye is concomitantly increased. Real-time detection and discrimination of PCR products is
accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and DNA-QS.

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Reagents and materials

cobas® EBV reagents and controls
The materials provided for cobas® EBV can be found in Table 1. Materials required, but not provided can be found in
Table 2 through Table 4, and Table 8 through Table 9.
Refer to the Reagents and materials section and Precautions and handling requirements section for the hazard
information for the product.

Table 1 cobas® EBV

cobas® EBV
Store at 2-8°C
192 test cassette (P/N 09040943190)
Kit components Reagent ingredients Quantity per kit
192 tests

Proteinase Solution Tris buffer, < 0.05% EDTA, calcium chloride, calcium acetate, 22.3 mL
(PASE) 8% proteinase, glycerol
EUH210: Safety data sheets available on request.
EUH208: Contains subtilisin from Bacillus subtilis. May
produce an allergic reaction.
DNA Quantitation Tris buffer, < 0.05% EDTA, < 0.001% non-EBV DNA 21.2 mL
Standard construct containing non-EBV primer binding and a unique
(DNA QS) probe region (non-infectious DNA), < 0.002% Poly rA RNA
(synthetic), < 0.1% sodium azide
Elution Buffer Tris buffer, 0.2% methyl-4 hydroxybenzoate 21.2 mL
(EB)
Master Mix Reagent 1 Manganese acetate, potassium hydroxide, < 0.1% sodium 7.5 mL
(MMX-R1) azide

EBV Master Mix Tricine buffer, potassium acetate, < 18% dimethyl sulfoxide, 9.7 mL
Reagent 2 glycerol, < 0.1% Tween 20, EDTA, < 0.12% dATP, dCTP, dGTP,
(EBV MMX-R2) dUTPs, < 0.01% upstream and downstream EBV primers,
< 0.01% Quantitation Standard forward and reverse primers,
< 0.01% fluorescent-labeled oligonucleotide probes specific
for EBV and the EBV Quantitation Standard, < 0.01%
oligonucleotide aptamer, < 0.01% Z05D DNA polymerase,
< 0.10% AmpErase (uracil-N- glycosylase) enzyme (microbial),
< 0.1% sodium azide

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Table 2 cobas® EBV/BKV Control Kit

cobas® EBV/BKV Control Kit
Store at 2–8°C
For use on the cobas® 5800 System (P/N 09040951190)
For use on the cobas® 6800/8800 Systems (P/N 08688214190 or P/N 09040951190)
Kit components Reagent ingredients Quantity per kit Safety symbol and warning*
EBV/BKV < 0.001% synthetic (plasmid) 4 mL
Low Positive EBV DNA encapsulated in (8 x 0.5 mL)
Control Lambda bacteriophage coat
(EBV/BKV L(+)C) protein, normal human plasma,
EBV DNA not detectable by PCR
methods. WARNING
H317: May cause an allergic skin reaction.
0.1% ProClin® 300 preservative**
H412: Harmful to aquatic life with long lasting
effects.
P261: Avoid breathing dust/fume/gas/
mist/vapours/spray.
P273: Avoid release to the environment.
P280: Wear protective gloves.
P333 + P313: If skin irritation or rash occurs:
Get medical advice/ attention.
P362 + P364: Take off contaminated clothing
and wash it before reuse.
P501: Dispose of contents/ container to an
approved waste disposal plant.
55965-84-9 Reaction mass of: 5-chloro-2-
methyl-4-isothiazolin-3-one and 2-methyl-2H-
isothiazol-3-one (3:1)
EBV/BKV < 0.001% synthetic (plasmid) 4 mL
High Positive EBV DNA encapsulated in (8 x 0.5 mL)
Control Lambda bacteriophage coat
(EBV/BKV H(+)C) protein, normal human plasma,
EBV DNA not detectable by PCR
methods. WARNING
H317: May cause an allergic skin reaction.
0.1% ProClin® 300 preservative**
H412: Harmful to aquatic life with long lasting
effects.
P261: Avoid breathing dust/fume/gas/
mist/vapours/spray.
P273: Avoid release to the environment.
P280: Wear protective gloves.
P333 + P313: If skin irritation or rash occurs:
Get medical advice/ attention.
P362 + P364: Take off contaminated clothing
and wash it before reuse.
P501: Dispose of contents/ container to an
approved waste disposal plant.
55965-84-9 Reaction mass of: 5-chloro-2-
methyl-4-isothiazolin-3-one and 2-methyl-2H-
isothiazol-3-one (3:1)
* Product safety labeling primarily follows EU GHS guidance
**Hazardous substance or mixture
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Table 3 cobas® Buffer Negative Control Kit

cobas® Buffer Negative Control Kit
Store at 2-8°C
For use on the cobas® 5800 System (P/N 09051953190)
For use on the cobas® 6800/8800 Systems (P/N 07002238190 or P/N 09051953190)
Kit components Reagent ingredients Quantity per kit

cobas® Buffer Negative Tris buffer, < 0.1% sodium azide, EDTA, 0.002% Poly rA RNA 16 mL
Control (synthetic) (16 x 1 mL)
(BUF (-) C)

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cobas® omni reagents for sample preparation

Table 4 cobas® omni reagents for sample preparation

Reagents Reagent ingredients Quantity Safety symbol and warning*

per kit
cobas® omni Magnetic glass particles, Tris buffer, 480 tests Not applicable
MGP Reagent 0.1% methyl-4 hydroxybenzoate,
(MGP) < 0.1% sodium azide
Store at 2–8°C
(P/N 06997546190)
cobas® omni Tris buffer, 0.1% methyl-4 4 x 875 mL Not applicable
Specimen Diluent hydroxybenzoate, < 0.1% sodium azide
(SPEC DIL)
Store at 2–8°C
(P/N 06997511190)
cobas® omni 43% (w/w) guanidine thiocyanate**, 4 x 875 mL
Lysis Reagent 5% (w/v) polydocanol**, 2% (w/v)
(LYS) dithiothreitol**, dihydro sodium citrate
Store at 2–8°C
(P/N 06997538190) DANGER
H302 + H332: Harmful if swallowed or if inhaled.
H314: Causes severe skin burns and eye damage.
H411 Toxic to aquatic life with long lasting effects.
EUH032: Contact with acids liberates very toxic gas.
P273: Avoid release to the environment.
P280: Wear protective gloves/ protective clothing/ eye
protection/ face protection/ hearing protection.
P303 + P361 + P353: IF ON SKIN (or hair): Take off
immediately all contaminated clothing. Rinse skin with
water.
P304 + P340 + P310: IF INHALED: Remove person to
fresh air and keep comfortable for breathing.
Immediately call a POISON CENTER/doctor.
P305 + P351 + P338 + P310: IF IN EYES: Rinse
cautiously with water for several minutes. Remove
contact lenses, if present and easy to do. Continue
rinsing. Immediately call a POISON CENTER/ doctor.
P391: Collect spillage.
593-84-0 Guanidinium thiocyanate
9002-92-0 Polidocanol
3483-12-3 (R*,R*)-1,4-dimercaptobutane-2,3-diol
cobas® omni Sodium citrate dihydrate, 0.1% methyl-4 4.2 L Not applicable
Wash Reagent hydroxybenzoate
(WASH)
Store at 15–30°C
(P/N 06997503190)
* Product safety labeling primarily follows EU GHS guidance
**Hazardous substance or mixture

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Reagent storage requirements

Reagents shall be stored and will be handled as specified in Table 5, Table 6 and Table 7.
When reagents are not loaded on the cobas® 5800 or cobas® 6800/8800 Systems, store them at the corresponding
temperature specified in Table 5.
Table 5 Reagent storage (when reagent is not on the system)
Reagent Storage temperature

cobas® EBV 2–8°C

cobas® EBV/BKV Control Kit 2–8°C

cobas® Buffer Negative Control Kit 2–8°C

cobas® omni Lysis Reagent 2–8°C

cobas® omni MGP Reagent 2–8°C

cobas® omni Specimen Diluent 2–8°C

cobas® omni Wash Reagent 15–30°C

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Reagent handling requirements for the cobas® 5800 System

Reagents loaded onto the cobas® 5800 System are stored at appropriate temperatures and their expiration is monitored by
the system. The system allows reagents to be used only if all of the conditions shown in Table 6 are met. The system
automatically prevents use of expired reagents. Table 6 allows the user to understand the reagent handling conditions
enforced by the cobas® 5800 System.

Table 6 Reagent expiry conditions enforced by the cobas® 5800 System

Number of runs for which On-board
Reagent Kit expiration date Open-kit stability
this kit can be used stability
cobas® EBV Date not passed 90 days from first usage Max 40 runs Max 36 days**
cobas® EBV/BKV Control Kit Date not passed Not applicable* Not applicable Max 36 days**

cobas® Buffer Negative Control Kit Date not passed Not applicable* Not applicable Max 36 days**

cobas® omni Lysis Reagent Date not passed 30 days from loading** Not applicable Not applicable

cobas® omni MGP Reagent Date not passed 30 days from loading** Not applicable Not applicable
cobas® omni Specimen Diluent Date not passed 30 days from loading** Not applicable Not applicable

cobas® omni Wash Reagent Date not passed 30 days from loading** Not applicable Not applicable

* Single use reagents.

**Time is measured from the first time that reagent is loaded onto the cobas® 5800 System.

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Reagent handling requirements for the cobas® 6800/8800 Systems

Reagents loaded onto the cobas® 6800/8800 Systems are stored at appropriate temperatures and their expiration is
monitored by the system. The cobas® 6800/8800 Systems allow reagents to be used only if all of the conditions shown in
Table 7 are met. The system automatically prevents use of expired reagents. Table 7 allows the user to understand the
reagent handling conditions enforced by the cobas® 6800/8800 Systems.
Table 7 Reagent expiry conditions enforced by the cobas® 6800/8800 Systems

Reagent Kit expiration Open-kit stability Number of runs On-board stability

date for which this kit (cumulative time on board
can be used outside refrigerator)

cobas® EBV Date not passed 90 days from first usage Max 40 runs Max 40 hours

cobas® EBV/BKV Control Kit Date not passed Not applicable* Not applicable Max 8 hours

cobas® Buffer Negative Control Kit Date not passed Not applicable* Not applicable Max 10 hours
®
cobas omni Lysis Reagent Date not passed 30 days from loading** Not applicable Not applicable

cobas® omni MGP Reagent Date not passed 30 days from loading** Not applicable Not applicable
®
cobas omni Specimen Diluent Date not passed 30 days from loading** Not applicable Not applicable

cobas® omni Wash Reagent Date not passed 30 days from loading** Not applicable Not applicable

* Single use reagents

**Time is measured from the first time that reagent is loaded onto the cobas® 6800/8800 Systems.

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Additional materials required for the cobas® 5800 System

Table 8 Materials and consumables for use on the cobas® 5800 System

Material P/N

cobas® omni Processing Plate 24 08413975001

cobas® omni Amplification Plate 24 08499853001

cobas® omni Liquid Waste Plate 24 08413983001

Tip CORE TIPS with Filter, 1mL 04639642001

Tip CORE TIPS with Filter, 300µL 07345607001

cobas® omni Liquid Waste Container 07094388001

cobas® omni Lysis Reagent 06997538190

cobas® omni MGP Reagent 06997546190

cobas® omni Specimen Diluent 06997511190

cobas® omni Wash Reagent 06997503190

Solid Waste Bag 07435967001

or
or
08030073001
Solid Waste Bag With Insert

cobas® omni Secondary Tubes 13x75 (optional)* 06438776001

*Contact your local Roche representative for a detailed order list for sample racks.

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Additional materials required for the cobas® 6800/8800 Systems

Table 9 Materials and consumables for use on the cobas® 6800/8800 Systems

Material P/N
cobas® omni Processing Plate 05534917001

cobas® omni Amplification Plate 05534941001

cobas® omni Pipette Tips 05534925001

cobas® omni Liquid Waste Container 07094388001

cobas® omni Lysis Reagent 06997538190

cobas® omni MGP Reagent 06997546190

cobas® omni Specimen Diluent 06997511190

cobas® omni Wash Reagent 06997503190

Solid Waste Bag and Solid Waste Container 07435967001 and 07094361001
or or
Solid Waste Bag With Insert and Kit Drawer Solid Waste Update 08030073001 and 08387281001

Instrumentation and software required

The cobas® 5800 software and cobas® EBV analysis package for the cobas® 5800 System must be installed on the cobas®
5800 instrument. The Data Manager software and PC for the cobas® 5800 System will be provided with the system.
The cobas® 6800/8800 software and cobas® EBV analysis package must be installed on the instrument(s). The Instrument
Gateway (IG) server will be provided with the system.

Table 10 Instrumentation

Equipment P/N

cobas® 5800 System 08707464001

cobas® 6800 System (Option Moveable) 06379672001

cobas® 6800 System (Fix) 05524245001

cobas® 8800 System 05412722001

Sample Supply Module 06301037001

Refer to the cobas® 5800 System or cobas® 6800/8800 Systems – User Assistance and/or User Guides for additional information.
Note: Contact your local Roche representative for a detailed order list for primary and secondary sample tubes, for sample racks, racks for clotted
tips and rack trays accepted on the instruments.

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Precautions and handling requirements

Warnings and precautions
As with any test procedure, good laboratory practice is essential to the proper performance of this assay. Due to the high
sensitivity of this test, care should be taken to keep reagents and amplification mixtures free of contamination.
 For in vitro diagnostic use only.
 cobas® EBV has not been evaluated for use as a screening test for the presence of EBV in blood or blood products.
 All patient samples should be handled as if infectious, using good laboratory procedures as outlined in Biosafety
in Microbiological and Biomedical Laboratories and in the CLSI Document M29-A4.15,16 Only personnel
proficient in handling infectious materials and the use of cobas® EBV and cobas® 5800/6800/8800 Systems should
perform this procedure.
 All human-sourced materials should be considered potentially infectious and should be handled with universal
precautions. If spillage occurs, immediately disinfect with a freshly prepared solution of 0.6% sodium or potassium
hypochlorite in distilled or deionized water or follow appropriate site procedures.
 cobas® EBV/BKV Control Kit contains plasma derived from human blood. The source material has been tested by
PCR methods and showed acceptable traces of low levels of EBV DNA. No known test method can offer complete
assurance that products derived from human blood will not transmit infectious agents.
 Do not freeze whole blood or any samples stored in primary tubes.
 Use only supplied or specified required consumables to ensure optimal test performance.
 Safety Data Sheets (SDS) are available on request from your local Roche representative.
 Closely follow procedures and guidelines provided to ensure that the test is performed correctly. Any deviation
from the procedures and guidelines may affect optimal test performance.
 False positive results may occur if carryover of samples is not adequately controlled during sample handling
and processing.
 Since > 90% of adults are chronic EBV carriers who may shed up to 10 8 EBV copies/mL in their saliva, and
given the high sensitivity of the assay, it is important to implement adequate contamination control measures
in laboratories.17
 Inform your local competent authority and manufacturer about any serious incidents which may occur when
using this assay.

Reagent handling
 Handle all reagents, controls, and samples according to good laboratory practice in order to prevent carryover of
samples or controls.
 Before use, visually inspect each reagent cassette, diluent, lysis reagent, and wash reagent to ensure that there are
no signs of leakage. If there is any evidence of leakage, do not use that material for testing.
 cobas® omni Lysis Reagent contains guanidine thiocyanate, a potentially hazardous chemical. Avoid contact of
reagents with the skin, eyes, or mucous membranes. If contact does occur, immediately wash with generous
amounts of water; otherwise, burns can occur.
 cobas® EBV test kits, cobas® omni MGP Reagent, and cobas® omni Specimen Diluent contain sodium azide as a
preservative. Avoid contact of reagents with the skin, eyes, or mucous membranes. If contact does occur,
immediately wash with generous amounts of water; otherwise, burns can occur. If these reagents are spilled, dilute
with water before wiping dry.
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 Do not allow cobas® omni Lysis Reagent, which contains guanidine thiocyanate, to contact sodium hypochlorite
(bleach) solution. This mixture can produce a highly toxic gas.
 Dispose of all materials that have come in contact with samples and reagents in accordance with country, state,
and local regulations.

Good laboratory practice

 Do not pipette by mouth.
 Do not eat, drink, or smoke in designated work areas.
 Wear laboratory gloves, laboratory coats, and eye protection when handling samples and reagents. Gloves must
be changed between handling samples and cobas® EBV kits, EBV/BKV Low Positive Control (EBV/BKV L(+)C),
EBV/BKV High Positive Control (EBV/BKV H(+)C), cobas® Buffer Negative Control (BUF (-) C) and cobas®
omni reagents to prevent contamination. Avoid contaminating gloves when handling samples and controls.
 Wash hands thoroughly after handling samples and kit reagents, and after removing the gloves.
 Thoroughly clean and disinfect all laboratory work surfaces with a freshly prepared solution of 0.6% sodium or
potassium hypochlorite in distilled or deionized water. Follow by wiping the surface with 70% ethanol.
 If spills occur on the cobas® 5800 or cobas® 6800/8800 instrument, follow the instructions in the cobas® 5800 or
cobas® 6800/8800 Systems – User Assistance and/or User Guide to properly clean and decontaminate the surface
of instrument(s).

Sample collection, transport, and storage

Note: Handle all samples and controls as if they are capable of transmitting infectious agents.
Store all samples at specified temperatures.
Sample stability is affected by elevated temperatures.
If using frozen samples in secondary tubes, place the samples at room temperature (15-30ºC) until
completely thawed and then briefly mix (e.g. vortex for 3-5 seconds) and centrifuge to collect all sample
volume at the bottom of the tube.
After centrifugation, if there is potential that cells have re-suspended into the plasma consider re-centrifugation
before processing on the instrument.

Samples
 Whole blood should be collected in BD Vacutainer® PPT™ Plasma Preparation Tubes for Molecular Diagnostic
Test Methods or in sterile tubes using EDTA as the anticoagulant. Follow the sample collection tube
manufacturer instructions. Refer to Figure 1.
 Whole blood collected in BD Vacutainer® PPT™ Plasma Preparation Tubes for Molecular Diagnostic Test Methods
or in sterile tubes using EDTA as the anticoagulant may be stored and/or transported for up to 24 hours at 2-25°C
prior to plasma preparation. Centrifugation should be performed according to manufacturer instructions.
 Upon separation plasma samples may be stored for 24 hours at 2-30°C in primary or secondary tubes, followed by:
o Storage in primary or secondary tubes for up to 6 days at 2-8 °C.
o Storage in secondary tubes for up to 6 months at ≤ -18°C.
 Plasma samples are stable for up to four freeze/thaw cycles when frozen at ≤ -18°C.

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Figure 1 Sample storage conditions

 If samples are to be shipped, they should be packaged and labeled in compliance with applicable country and/or
international regulations covering the transport of samples and etiologic agents.

Instructions for use

Procedural notes
 Do not use cobas® EBV reagents, cobas® EBV/BKV Control Kit, cobas® Buffer Negative Control Kit, or cobas® omni
reagents after their expiry dates.
 Do not reuse consumables. They are for one-time use only.
 Refer to the cobas® 5800 System or cobas® 6800/8800 Systems – User Assistance and/or User Guides for proper
maintenance of instruments.

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Running cobas® EBV on the cobas® 5800 System

cobas® EBV can be run with a minimum sample volume of 350 µL of which 200 µL is processed. The test procedure is
described in detail in the cobas® 5800 Systems User Assistance and/or User Guide. Figure 2 below summarizes the
procedure.

Figure 2 cobas® EBV test procedure on the cobas® 5800 System

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Running cobas® EBV on the cobas® 6800/8800 Systems

cobas® EBV can be run with a minimum sample volume of 350 µL of which is processed 200 µL. The test procedure is
described in detail in the cobas® 6800/8800 Systems – User Assistance and/or User Guide. Figure 3 below summarizes the
procedure.

Figure 3 cobas® EBV procedure on the cobas® 6800/8800 Systems

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Results
The cobas® 5800/6800/8800 Systems automatically determine the EBV DNA concentration for the samples and controls.
The EBV DNA concentration is expressed in International Units per milliliter (IU/mL).

Quality control and validity of results on the cobas® 5800 System

 One negative control [(-) Ctrl] and two positive controls, a low positive control [EBV/BKV L (+) C] and a high
positive control [EBV/BKV H (+) C] are processed at least every 72 hours and with every new kit lot. Positive
and/or negative controls can be scheduled more frequently based on laboratory procedures and/or local
regulations.
 In the cobas® 5800 software and/or report, check for flags and their associated results to ensure the result
validity.
Invalidation of results is performed automatically by the cobas® 5800 software based on negative or positive control failures.
NOTE: The cobas® 5800 System will be delivered with the standard setting of running a set of controls (positive and
negative) with every run, but can be configured to a less frequent scheduling up to every 72 hours based on laboratory
procedures and/or local regulations. Please contact your Roche service engineer and/or Roche customer technical support
for more information.

Control results on the cobas® 5800 System

The results of the controls are shown in the cobas® 5800 software in the “Controls” app.
 Controls are marked with “Valid” in the column “Control result” if all Targets of the control are reported valid.
Controls are marked with “Invalid” in the column “Control result” if all or one Target of the control are
reported invalid.
 Controls marked with “Invalid” show a flag in the “Flags” column. More information on why the control is
reported invalid including flag information is shown in the detail view.
 If one of the controls is invalid, repeat testing of all controls and all associated samples is required.

Quality control and validity of results on the cobas® 6800/8800 Systems

 One negative control [(–) Ctrl] and two positive controls, a low positive control [EBV/BKV L (+) C ] and a high
positive control [EBV/BKV H (+) C] is processed with each batch.
 In the cobas® 6800/8800 software and/or report, check for flags and their associated results to ensure the batch validity.
 The batch is valid if no flags appear for all three controls, which includes one negative control and two positive
controls: EBV/BKV L (+) C, EBV/BKV H (+) C. The negative control result is displayed as (–) Ctrl and the low
and high positive controls are displayed as EBV/BKV L (+) C and EBV/BKV H (+) C.
Invalidation of results is performed automatically by the cobas® 6800/8800 software based on negative and positive
control failures.

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Control flags on the cobas® 6800/8800 Systems

Table 11 Control flags for negative and positive controls

Negative Control Flag Result Interpretation

(–) Ctrl Q02 Invalid An invalid result or the calculated titer result for the negative
(Control batch failed) control is not negative.

Positive Control Flag Result Interpretation

EBV/BKV L (+) C Q02 Invalid An invalid result or the calculated titer result for the low positive
(Control batch failed) control is not within the assigned range.

EBV/BKV H (+) C Q02 Invalid An invalid result or the calculated titer result for the high positive
(Control batch failed) control is not within the assigned range.

If the control batch is invalid, repeat testing of all samples of affected batch.

Interpretation of results
For a valid batch, check each individual sample for flags in the cobas® 5800 and cobas® 6800/8800 Systems software and/or
reports. The result interpretation should be as follows:
 A valid batch may include both valid and invalid sample results.

Table 12 Target results for individual target result interpretation

Results Interpretation
Target Not Detected EBV DNA not detected.
Report results as “EBV not detected”.
< Titer Mina Calculated titer is below the Lower Limit of Quantitation (LLoQ) of the assay. Report results as
“EBV detected, less than (Titer Min)”.
Titer min = 35.0 IU/mL
Titer Calculated titer is within the Linear Range of the assay – greater than or equal to Titer Min and less than or
equal to Titer Max.
Report results as “(Titer) of EBV detected”.
> Titer Maxb Calculated titer is above the Upper Limit of Quantitation (ULoQ) of the assay. Report results
as “EBV detected, greater than (Titer Max)”.
Titer max = 1.0E+08 IU/mL
a
Sample results < Titer min (Target Detected < LLoQ) should be interpreted with the context of other clinical data and should not be the sole basis
for treatment decisions.
b
Sample result > Titer Max refers to EBV positive samples detected with titers above the upper limit of quantitation (ULoQ). If a quantitative result is
desired, the original sample should be diluted with EBV-negative human EDTA plasma and the test should be repeated. Multiply the reported result
by the dilution factor.

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Interpretation of results on the cobas® 5800 System

The results of the samples are shown in the cobas® 5800 software in the “Results” app.
For a valid control batch, check each individual sample for flags in the cobas® 5800 software and/or report. The result
interpretation should be as follows:
 Samples associated with a valid control batch are shown as ‘Valid’ in the “Control result” column if all Control Target
Results reported valid. Samples associated with a failed control batch are shown as ‘Invalid’ in the “Control result”
column if all Control Target Results reported invalid.
 If the associated controls of a sample result are invalid, a specific flag will be added to the sample result as follows:
o Q05D : Result validation failure because of an invalid positive control
o Q06D :Result validation failure because of an invalid negative control
 The values in “Results” column for individual sample target result should be interpreted as shown in Table 11 above.
If one or more sample targets are marked with “Invalid” the cobas® 5800 software shows a flag in the “Flags”column. More
information on why the sample target(s) is reported invalid including flag information is shown in the detail view.

Interpretation of results on the cobas® 6800/8800 Systems

For a valid batch, check each individual sample for flags in the cobas® 6800/8800 Systems software and/or report. The
result interpretation should be as follows:
 Samples are marked with “Yes” in the column ‘Valid’ if all requested Target Results reported valid results. Samples
marked with “No” in the column ‘Valid’ may require additional interpretation and action.
 The values for individual sample target result should be interpreted as shown in Table 12 above.

Procedural limitations
 cobas® EBV has been evaluated only for use in combination with the cobas® EBV/BKV Control Kit, cobas® Buffer
Negative Control Kit, cobas® omni MGP Reagent, cobas® omni Lysis Reagent, cobas® omni Specimen Diluent,
and cobas® omni Wash Reagent for use on the cobas® 5800/6800/8800 Systems.
 Reliable results depend on proper sample collection, storage and handling procedures.
 This test has been validated only for use with EDTA plasma. Testing of other sample types with cobas® EBV may result
in inaccurate results. Plasma viral load measurements are not directly comparable to those of other sample types.
 Quantitation of EBV DNA may be affected by sample collection methods, patient factors (i.e., age, presence of
symptoms), and/or stage of infection.
 As with any molecular test, mutations within the target regions of cobas® EBV could affect primer and/or probe
binding resulting in the under-quantitation of virus or failure to detect the presence of virus.
 Due to inherent differences between technologies, it is recommended that, prior to switching from one
technology to the next, users perform method correlation studies in their laboratory to qualify technology
differences. Users should follow their own specific policies/procedures.
 cobas® EBV is not intended for use as a screening test for the presence of EBV in blood or blood products.

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Non-clinical performance evaluation

Key performance characteristics performed on the cobas® 6800/8800 Systems

Limit of Detection (LoD)

WHO International Standard
The limit of detection of cobas® EBV was determined by analysis of serial dilutions of the 1st WHO International Standard
for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (1st EBV WHO International Standard) obtained from
NIBSC (NIBSC 09/260), in EBV-negative human EDTA plasma. Panels of six concentration levels plus a blank were tested
over three lots of cobas® EBV reagents, multiple runs, days, operators, and instruments.
The results for EDTA plasma are shown in Table 13 through Table 15. The study demonstrates that with the least sensitive
lot, the concentration for which 95% hit rate is expected by PROBIT is 18.8 IU/mL with a 95% confidence range of 14.5 to
27.5 IU/mL in EDTA plasma. The lowest concentration with a hit rate 95% is 20.0 IU/mL in EDTA plasma.

Table 13 Limit of detection in EDTA plasma, Lot 1

Input titer concentration Number of valid replicates Number of positives Hit rate in %
(EBV DNA IU/mL)
50.0 63 63 100.0
35.0 63 62 98.4
20.0 63 61 96.8
10.0 63 53 84.1
5.0 63 37 58.7
2.5 63 26 41.3
0.0 63 0 0.0

LoD by PROBIT at 95% hit rate 18.8 IU/mL

95% confidence range: 14.5 – 27.5 IU/mL

Table 14 Limit of detection in EDTA plasma, Lot 2

Input titer concentration Number of valid replicates Number of positives Hit rate in %
(EBV DNA IU/mL)
50.0 63 63 100.0
35.0 63 63 100.0
20.0 63 63 100.0
10.0 63 58 92.1
5.0 63 35 55.6
2.5 63 20 31.8
0.0 63 0 0.0

LoD by PROBIT at 95% hit rate 12.4 IU/mL

95% confidence range: 10.0 – 17.0 IU/mL

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Table 15 Limit of detection in EDTA plasma, Lot 3

Input titer concentration

(EBV DNA IU/mL) Number of valid replicates Number of positives Hit rate in %

50.0 63 63 100.0

35.0 63 63 100.0

20.0 63 62 98.4

10.0 63 48 76.2

5.0 63 38 60.3

2.5 63 26 41.3

0.0 63 0 0.0

LoD by PROBIT at 95% hit rate 18.6 IU/mL

95% confidence range: 14.4 – 27.1 IU/mL

Linear range
Linearity of cobas® EBV was evaluated using a dilution series consisting of 17 panel members with EBV genotype 1 DNA
spanning the assay linear range. A high titer lambda DNA stock was used to prepare 11 panel members spanning the
entire linear range. A clinical specimen was used to prepare 6 panel members covering the intermediate and lower levels of
the linear range.
Each panel member was tested in 36 replicates across three lots of cobas® EBV reagents and the results of the study are
presented in Figure 4.
cobas® EBV was demonstrated to be linear from 1.40E+01 IU/mL to 2.30E+08 IU/mL and shows an absolute deviation
from the better fitting non-linear regression of less or equal than ± 0.1 log10 in human EDTA plasma (see Figure 4). Across
the linear range, the accuracy of the test was within ± 0.2 log10.

Figure 4 Linear range determination in EDTA plasma

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Precision – within laboratory

Precision of cobas® EBV was determined by analysis of serial dilutions of high titer EBV DNA (genotype 1) in EBV-negative
EDTA plasma. Six dilution levels were tested in 72 replicates for each level across three lots of cobas® EBV reagents using
three instruments and three operators over 12 days. Each sample was carried through the entire cobas® EBV procedure on
fully automated cobas® 6800/8800 Systems. Therefore, the precision reported here represents all aspects of the test
procedure. The results are shown in Table 16.
cobas® EBV showed high precision for three lots of reagents tested across a concentration range of 1.08E+02 IU/mL to
5.40+07 IU/mL.

Table 16 Within-laboratory precision of cobas® EBV

EDTA plasma
Nominal Assigned
concentration concentration Lot 1 Lot 2 Lot 3 All lots
[IU/mL] [IU/mL]
SD SD SD Pooled SD
5.00E+07 5.40E+07 0.03 0.04 0.04 0.04
1.00E+06 1.08E+06 0.02 0.03 0.02 0.02
1.00E+05 1.08E+05 0.02 0.02 0.03 0.02
1.00E+04 1.08E+04 0.04 0.02 0.03 0.03
1.00E+03 1.08E+03 0.05 0.05 0.05 0.05
1.00E+02 1.08E+02 0.17 0.18 0.15 0.17

Genotype verification
The performance of cobas® EBV on EBV genotype 2 was evaluated by:
 Verification of the limit of detection
 Verification of the linear range

Verification of limit of detection for genotype 2

EBV DNA for genotype 2 was diluted to three different concentration levels in EBV-negative EDTA plasma. The hit
rate determination was performed with 63 replicates for each level. Testing was conducted with three lots of cobas® EBV
reagents, multiple runs, days, operators, and instruments. The results verify that cobas® EBV detected EBV DNA for
genotype 2 at a concentration of 18.8 IU/mL with a hit rate of 95%.

Verification of linear range for genotype 2

The dilution series used in the verification of genotypes linearity study of cobas® EBV consisted of eight panel members
spanning the linear range of the assay. Testing was conducted with three lots of cobas® EBV reagent, 12 replicates per level
were tested in EDTA plasma.
The linear range of cobas® EBV was verified for genotype 2.

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Specificity
The specificity of cobas® EBV was determined by analyzing EBV-negative EDTA plasma samples from individual
donors. One hundred and one individual EDTA plasma samples were tested with three lots of cobas® EBV reagents. All
samples were tested negative for EBV DNA. In the test panel the specificity of cobas® EBV was 100% (lower one-sided
95% confidence interval 97.08%).

Analytical specificity
The analytical specificity of cobas® EBV was evaluated by diluting a panel of microorganisms to a concentration of
1.00E+06 units/mL (cells/mL, CFU/mL, IFU/mL, CCU/mL ) for bacteria and yeast and between 3.00E+05 and 1.00E+06 units/mL
(IU/mL, copies/mL, cells/mL, TCID50/mL) for viruses with EBV DNA positive and EBV DNA negative EDTA plasma. The specific
organisms tested are listed in Table 17. Each panel member was evaluated with cobas® EBV. None of the non-EBV pathogens were
shown to interfere with test performance.
Table 17 Microorganisms tested for cross-reactivity

Viruses Bacteria Yeast

Adenovirus Type 5 Propionibacterium acnes Aspergillus niger
BK Polyomavirus Staphylococcus aureus Candida albicans
Cytomegalovirus Chlamydia trachomatis Cryptococcus neoformans
Hepatitis B Virus Clostridium perfringens -
Hepatitis C Virus Enterococcus faecalis -
Herpes Simplex VirusType 1 Escherichia coli -
Herpes Simplex VirusType 2 Klebsiella pneumoniae -
Human Herpes Virus Type-6 Listeria monocytogenes -
Human Herpes Virus Type-7 Mycobacterium avium -
Human Herpes Virus Type-8 Neisseria gonorrhoeae -
Human Immunodeficiency Virus-1 Staphylococcus epidermidis -
Human Immunodeficiency Virus-2 Streptococcus pyogenes -
Human Papillomavirus Mycoplasma pneumoniae -
JC Virus Salmonella enterica -
Parvovirus B19 Streptococcus pneumoniae -
Simian Virus 40
Varicella-Zoster Virus - -

Analytical specificity – interfering substances

Elevated levels of triglycerides (33.0 g/L), conjugated bilirubin (0.2 g/L), unconjugated bilirubin (0.2 g/L), albumin (60.0 g/L),
hemoglobin (2.0 g/L) and human DNA (2 mg/L) in samples were tested in the presence and absence of EBV DNA. The
tested endogenous interferences were shown not to interfere with the test performance of cobas® EBV.
In addition, drug compounds listed in Table 18 were tested at three times the Cmax in presence and absence of EBV DNA.
All potentially interfering substances have been shown to not interfere with the test performance.

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Table 18 Drug compounds tested for interference with the quantitation of EBV DNA by cobas® EBV

Class of drug Generic drug name

Antimicrobial Cefotetan Sulfamethoxazole
Clavulanate potassium Ticarcillin disodium
Fluconazole Trimethoprim
Piperacillin Vancomycin
Tazobactam sodium Micafungin
Compounds for Treatment of Herpes Ganciclovir Cidofovir
Viruses Valganciclovir Foscarnet
Acyclovir Letermovir
Immune suppressant Azathioprine Prednisone
Cyclosporine Sirolimus
Everolimus Tacrolimus
Mycophenolate mofetil Mycophenolic acid

Method correlation
The performance of cobas® EBV was assessed against a comparator assay by analyzing EDTA plasma specimens from
EBV-infected patients. EDTA plasma specimens, within the quantitation range of both tests, were tested as single
replicates. Deming regression analysis was performed.
The Deming regression results are shown in Figure 5.

Figure 5 Regression analysis of cobas® EBV versus comparator assay

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Whole system failure

The whole system failure rate for cobas® EBV was determined by testing 100 replicates of EDTA plasma spiked with an
EBV positive clinical specimen. These samples were tested at a concentration of 3 x LoD.
The results of this study determined that all replicates were valid and positive for the EBV target, resulting in a whole system
failure rate of 0% (upper one-sided 95% confidence interval 2.95%).

Cross contamination
The cross-contamination rate for cobas® EBV was determined by testing 240 replicates of an EBV-negative matrix
sample and 225 replicates of a high titer EBV sample at approximately 2.00E+07 IU/mL. In total, five runs were performed
with positive and negative samples in a checkerboard configuration.
All 240 replicates of the negative sample were negative, resulting in a cross-contamination rate of 0% (upper one-sided
95% confidence interval 1.24%).

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Clinical performance evaluation performed on the cobas®

6800/8800 Systems
Reproducibility of cobas® EBV
The reproducibility of cobas® EBV was evaluated across factors (reagent lot, test site, batch and testing days) that could
affect reported results in routine clinical testing. The evaluation was conducted at 3 testing sites, using 3 reagent lots, of a
positive and a negative sample panel with a total number 270 tests (not including controls). The panels were made from
EDTA plasma that was EBV VCA IgG negative and were tested for EBV with a plasma NAT release protocol, and spiked
with an EBV WHO international standard, EBV cell culture supernatant or lambda phagemid with EBV DNA. Two
operators at each site tested each reagent lot for 5 days. Two runs (1 run = 1 batch; 1 batch = 1 panel + 3 controls) per
operator were performed each day and 3 replicates of each panel member were performed for each run. The evaluation
results are summarized in Table 19.
Table 19 Attributable percentage of total variance (%TV), total precision Standard Deviation (SD), and lognormal CV(%) of EBV DNA
concentration (log10 IU/mL) by positive panel member
Expected Observed
EBV DNA Meana Day/ Within -
Lot Site Batch Total Total
Concentra EBV DNA Number Operator Batch
%TVc %TVc %TVc Precision Precision
tion (log10 Concentra of Testsb %TVc %TVc
(CV%)d (CV%)d (CV%)d SDe CV(%)d
IU/mL) tion (log10 (CV%)d (CV%)d
IU/mL)
11% 2% 3% 84%
2.02 2.09 270 0% (0.00) 0.158 37.56
(11.97) (5.30) (6.34) (34.25)
43% 15% 16% 26%
3.70 3.68 270 0% (0.00) 0.067 15.43
(10.07) (5.92) (6.23) (7.81)
39% 10% 24% 28%
4.70 4.68 270 0% (0.00) 0.059 13.70
(8.54) (4.24) (6.63) (7.18)
7% 58% 21% 15%
5.70 5.50 268 0% (0.00) 0.191 46.16
(11.39) (34.36) (20.18) (17.08)
27% 15% 13% 45%
7.70 7.76 270 0% (0.88) 0.073 16.83
(8.63) (6.52) (6.01) (11.26)
a
Calculated using SAS MIXED procedure.
b
Number of valid tests with detectable DNA level.
c
%TV = Percent contribution to Total Variance.
d
CV% = Lognormal percent coefficient of variation = sqrt(10^[SD^2 * ln(10)] - 1) * 100.
e
Calculated using the total variability from the SAS MIXED procedure.
Note: The table only includes results with detectable DNA level. SD = standard deviation. CV = coefficient of variation; EBV = Epstein Barr Virus.

cobas® EBV showed acceptable clinical reproducibility on the respective comparative concentration. In addition, the
system detected 100% of the 3 x LLoQ samples. The cobas® 6800 and cobas® 8800 Systems share a modular design and
they showed equivalency when using the cobas® EBV. All of the estimated 95% confidence limits (CLs) for the difference
between 2 measurements from the same subject were within ± 0.53 log10 IU/mL, indicating that the assay can assess
changes in EBV DNA levels that are thought to be clinically significant.
Of the 270 valid tests for the negative panel members performed on the cobas® 6800/8800 Systems, 14 samples (5.19%)
showed detection of < LLoQ positivity. The results were not associated with a particular instrument/site or reagent lot.
Heminested PCR and DNA sequencing confirmed the presence of EBV DNA in these samples.

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Performance of cobas® EBV

The clinical performance of cobas® EBV was further evaluated at three testing sites by measuring EBV DNA levels in
clinical samples (neat and diluted) of EBV infected and non-infected patients and contrived EDTA plasma samples spiked
with cultured EBV virus, compared with a well-established laboratory developed nucleic acid test (LDT)(comparator EBV
LDT). From all samples tested with cobas® EBV and the comparator EBV test, there were a total of 464 samples (439 neat
or diluted clinical samples of 72 transplant subjects and 25 contrived samples) that were valid on both assays and evaluable
for the clinical concordance analysis.
Table 20 Concordance analysis between cobas® EBV and the comparator LDT on EBV DNA level results for all samples
cobas® EBV (log10 Comparator Comparator Comparator Comparator Comparator Comparator Total
IU/mL) EBV LDT EBV LDT EBV LDT EBV LDT EBV LDT EBV LDT
(log10 IU/mL) (log10 IU/mL) (log10 IU/mL) (log10 IU/mL) (log10 IU/mL) (log10 IU/mL)
Target Not < LLoQ (< 2) 2 to < 2.6 2.6 to < 3.2 3.2 to 3.8 > 3.8
Detected

Target Not Detected 95 17 17 0 0 0 129

< LLoQ (< 2) 39 46 75 11 0 0 171

2 to < 2.6 1 2 16 37 6 0 62

2.6 to < 3.2 1 0 5 15 30 1 52

3.2 to 3.8 0 0 0 0 9 11 20

> 3.8 0 0 0 0 1 29 30

Total 136 65 113 63 46 41 464

Column Agreement (134/136)
(65/65) 100% (96/113) 85.0% (52/63) 82.5% (40/46) 87.0% (40/41) 97.6% -
(%) 98.5%
(95% Score CI)a (94.8%, 99.6%) (94.4%, 100%) (77.2%, 90.4%) (71.4%, 90.0%) (74.3%, 93.9%) (87.4%, 99.6%) -

Note: LLoQ = lower limit of quantitation of comparator EBV LDT (100 IU/mL).
Standard Deviation of comparator EBV LDT estimated at 0.3 log10 IU/mL (EBV LDT analytical precision study).
Paired samples evaluable for clinical concordance analysis were included in this table.
a
Assumed independence between all samples.
CI = Confidence Interval.

DNA sequencing on representative samples from subjects with results consistently offset by more than 1 log10 IU/ml DNA
level did not reveal any sequence mismatches for any primer or probe targets for the cobas® EBV assay.
Discordant results were defined as those that are more than one box away from the diagonal (indicated by shading). For
Target Not Detected by LDT Column Agreement the cobas® EBV Target Not Detected and < LLoQ (< 2) cells were
combined. The rationale for adding the adjacent < LLoQ and TND cells for the TND column is that the difference
between a TND and < LLoQ is not clinically meaningful and that these are analytically at the lower end of the measuring
range, which may be impacted by random error.

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Out of the 43 comparator EBV LDT negative samples collected for the estimation of the Negative Percent Agreement
(NPA) with the cobas® EBV, 41 samples were negative by cobas® EBV therefore the NPA was 95.4% with the 95% Exact CI
of 84.2% to 99.4%. The two comparator EBV LDT negative samples were positive (< LLoQ) by cobas® EBV and were
seropositive for EBV VCA IgG and EBNA-1 IgG by supplemental serology testing.
Concordance between cobas® EBV and the comparator EBV LDT was also evaluated using different clinical thresholds.
Table 21 Summary of concordance of cobas® EBV and comparator EBV LDT using different thresholds for all samples
Percent Agreement Percent Agreement
< Threshold  Threshold
95% CIb 95% CIb
(n/N) (n/N)
Target Not Detected 98.5% (134/136) 89.6% (294/328)
(94.8%, 99.6%) (85.9%, 92.5%)
LLoQa (2.0 Log10 IU/mL) 98.0% (197/201) 60.8% (160/263)
(95.0%, 99.2%) (54.8%, 66.5%)
3.0 Log10 IU/mL 100.0% (363/363) 64.4% (65/101)
(99.0%, 100.0%) (54.6%, 73.0%)
4.0 Log10 IU/mL 100.0% (431/431) 84.8% (28/33)
(99.1%, 100.0%) (69.1%, 93.3%)
a
LLoQ = lower limit of quantitation of comparator EBV LDT (100 IU/mL)
b
CI = Confidence Interval

From all samples tested with cobas® EBV that were EBV positive with the comparator EBV test, there were a total of 158
(139 neat or diluted clinical samples of 28 transplant subjects and 19 contrived samples), which were evaluable for the
correlation analysis at the three testing sites.

Figure 6 Correlation between cobas® EBV and comparator EBV LDT for all samples: Deming linear regression plot of DNA levels (log10 IU/mL)

5
cobas EBV(log10 IU/mL)

Deming Regression (N=158): Y = -0.45 + 1.025 X

R = 0.95
1 95% CIs: Intercept=(-0.637, -0.263); Slope=( 0.974, 1.076)

1 2 3 4 5 6 7

Comparator EBV LDT (log10 IU/mL)

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Additional bias plot analysis of DNA level differences indicated a systematic difference between both assays that is
constant across the overlapping linear range. The 95% CI of the intercept of the fitted line in the bias plots was -0.456 to
0.104, which is within ±0.6 log10 IU/mL (± 2 times analytical precision standard deviation of comparator EBV LDT).
Furthermore, the mean bias was estimated at -0.364 log10 IU/mL and the systematic difference between both assays was
-0.352 log10 IU/mL and -0.376 log10 IU/mL for samples with DNA levels at 3 and 4 log10 IU/mL, respectively.

System equivalency / system comparison

System equivalency of the cobas® 5800, cobas® 6800 and cobas® 8800 Systems was demonstrated via performance studies.
The results presented in the Instructions for Use support equivalent performance for all systems.

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Additional information
Key test features
Sample type EDTA plasma

Minimum amount of sample required 350 µL*

Sample processing volume 200 µL

Analytical sensitivity 18.8 IU/mL

Linear range 35.0 IU/mL to 1E+08 IU/mL

Specificity 100%

Genotypes detected EBV Genotypes 1 and 2

*Dead volume of 150 µL identified for the cobas® omni Secondary tubes. Other tubes used for testing may have different dead volume and require
more or less minimum volume. Contact your local Roche service representative for further information.

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Symbols
The following symbols are used in labeling for Roche PCR diagnostic products.
Table 22 Symbols used in labeling for Roche PCR diagnostics products

Device not for

Age or Date of Birth QS IU per PCR reaction,
near-patient testing
use the QS International
Units (IU) per PCR reaction
Device not for in calculation of
Ancillary Software self-testing the results.

Distributor
Assigned Range (Note: The applicable
Serial number
(copies/mL) country/region may be
designated beneath the symbol)

Assigned Range (IU/mL) Do not re-use Site

Authorized representative in
Female Standard Procedure
the European Community

Barcode Data Sheet For IVD performance Sterilized using

evaluation only ethylene oxide

Batch code Global Trade Item Number Store in dark

Biological risks Importer Temperature limit

In vitro diagnostic
Catalogue number Test Definition File
medical device

CE marking of conformity; Lower Limit of

this device is in conformity This way up
Assigned Range
with the applicable
requirements for CE marking
of an in vitro diagnostic Male Ultrasensitive Procedure
medical device

Collect date Manufacturer Unique Device Identifier

Consult instructions Upper Limit of

Negative control
for use Assigned Range

Contains sufficient
Non-sterile Urine Fill Line
for <n> tests
US Only: Federal law
restricts this device to sale
Content of kit Patient Name
by or on the order of a
physician.

Control Patient number Use-by date

Date of manufacture Peel here

Device for
Positive control
near-patient testing

QS copies per PCR

reaction, use the QS copies
Device for self-testing
per PCR reaction in
calculation of the results.

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Technical support
For technical support (assistance) please reach out to your local affiliate:
https://www.roche.com/about/business/roche_worldwide.htm

Manufacturer and importer

Table 23 Manufacturer and importer

Roche Molecular Systems, Inc.

1080 US Highway 202 South
Branchburg, NJ 08876 USA
www.roche.com

Made in USA

Roche Diagnostics GmbH

Sandhofer Strasse 116
68305 Mannheim, Germany

Trademarks and patents

See https://diagnostics.roche.com/us/en/about-us/patents

Copyright
©2023 Roche Molecular Systems, Inc.

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References
1. Tomblyn M, Chiller T, Einsele H, et al. Guidelines for preventing infectious complications among hematopoietic
cell transplant recipients: a global perspective. Preface. Bone Marrow Transplant. 2009;44:453-5. PMID: 19861977.

2. Green M. Introduction: Infections in solid organ transplantation. Am J Transplant. 2013;13 Suppl 4:3-8. PMID:
23464993.

3. Cohen JI. Epstein-Barr virus infection. N Engl J Med. 2000;343:481-92. PMID: 10944566.

4. Styczynski J, van der Velden W, Fox CP, et al. Management of Epstein-Barr Virus infections and post-transplant
lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European
Conference on Infections in Leukemia (ECIL-6) guidelines. Haematologica. 2016;101:803-11. PMID: 27365460.

5. Allen UD, Preiksaitis JK. Epstein-Barr virus and posttransplant lymphoproliferative disorder in solid organ
transplantation. Am J Transplant. 2013;13 Suppl 4:107-20. PMID: 23465004.

6. San-Juan R, Comoli P, Caillard S, et al. Epstein-Barr virus-related post-transplant lymphoproliferative disorder in

solid organ transplant recipients. Clin Microbiol Infect. 2014;20 Suppl 7:109-18. PMID: 24475976.

7. Nijland ML, Kersten MJ, Pals ST, Bemelman FJ, Ten Berge IJ. Epstein-Barr virus-positive posttransplant
lymphoproliferative disease after solid organ transplantation: Pathogenesis, clinical manifestations, diagnosis, and
management. Transplant Direct. 2016;2:e48. PMID: 27500242.

8. Tsai DE, Douglas L, Andreadis C, et al. EBV PCR in the diagnosis and monitoring of posttransplant
lymphoproliferative disorder: Results of a two-arm prospective trial. Am J Transplant. 2008;8:1016-24. PMID:
18312608.

9. Fryer JF, Heath AB, Wilkinson DE, Minor PD. A collaborative study to establish the 1st WHO International
Standard for Epstein-Barr virus for nucleic acid amplification techniques. Biologicals. 2016;44:423-33. PMID:
27461128.

10. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in
polymerase chain reactions. Gene. 1990;93:125-8. PMID: 2227421.

11. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA
glycosylase. Nature. 1995;373:487-93. PMID: 7845459.

12. Mol CD, Arvai AS, Slupphaug G, et al. Crystal structure and mutational analysis of human uracil-DNA
glycosylase: Structural basis for specificity and catalysis. Cell. 1995;80:869-78. PMID: 7697717.

13. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA
sequences. Biotechnology (N Y). 1992;10:413-7. PMID: 1368485.

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14. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6:986-94. PMID:
8908518.

15. Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical Laboratories, 5th ed.
U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and
Prevention, National Institutes of Health HHS Publication No. (CDC) 21-1112, revised December 2009.

16. Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory Workers From Occupationally
Acquired Infections; Approved Guideline, 4th ed. CLSI Document M29-A4. Wayne, PA: CLSI, 2014.

17. Hadinoto V, Shapiro M, Sun CC, Thorley-Lawson DA. The dynamics of EBV shedding implicate a central role for
epithelial cells in amplifying viral output. PLoS Pathog. 2009;5:e1000496. PMID: 19578433.

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Document revision
Document Revision Information
Doc Rev. 2.0 Updated front page and table 2 and 3 with additional P/N for the control kits.
09/2022 Updated Trademarks and patents section, including the link.
Please contact your local Roche Representative if you have any questions.
Doc Rev. 3.0 Return minimum sample volume to original level in Instructions for use and Key test features section.
05/2023 Updated competent authority statement.
Updated cobas® branding.
Minor wording corrections.
Please contact your local Roche Representative if you have any questions.

The summary of safety and performance report can be found using the following link: https://ec.europa.eu/tools/eudamed.

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