Document Information Repository: Document Number: Document Version: Document Title: Document State: Effective Date:

EMEA_PROD_DDM C_c501_GENT-CH_AR_004771 1.0 GENT2_system Effective 24.05.2006 14:28:52

Details: Business Area: Site: Language:

Centralized Diagnostics Penzberg English

Type of record: Project:

Application Report C_Gentamycin_CobHit

Print Type:

C_proj_Print_pub

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

Coverpage 1 of 1

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

Page 1 of 58

Table of Contents Page Summary Information Test Principle Critical Issues Conclusion/Recommendation References Specifications/Actual Data Calibration Curve Materials and Methods

3 5 5 5 6 13 14

Results Within - Run Imprecision Imprecision acc. to NCCLS Daily QC Lower Detection Limit (LDL) Method Comparison Linearity Recovery in Controls Interferences (Glick) Drug Interferences Calibration Log

16 17 20 21 22 24 25 26 30 31

Attachments (see GENT2_c501_Version 1.0.pdf) Instrument Settings c501 Appendix: Roche/Hitachi 917 Data Anticoagulants Sample Handling Cross Reactivity Additional Interferences Cal-OB Stability

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

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Test Principle

Studies

See ONLINE TDM Gentamicin Package Insert, Mat # 03800504190, for Test Principle to be used in the cobas Method Sheet.

The following studies were performed as required for a System level application, according to the Application Policy NewGen Systems V1.0, 01.04.04, and Global Application Policy, C_RP0008 Version 7.0 and associated SOPs: Study

SOP

Within Run Imprecision

RS03-03e

NCCLS Imprecision

RS06-04e

Sensitivity (LDL)

RS23-04e

Method Comparison

RS13-04e

Linearity

RS21-02e

Control Recovery

RS10-02e

Calibration Stability

NT

On-board Stability Interferences:

NT

- Bilirubin, Hemolysis, Lipemia - Anticoagulants

RS15-03e NT

- Drugs

RS02-05e

- Cross Reactivity

NT

Additional Interferences: - HAMA

NT

- Rhumatoid Factor

NT

System Specific: - Carryover Sample

NT

Reagent (Hitachi)

NT

NT = Not Tested for this application level

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Reportable Range The reportable range for this application should be stated on the application sheet as follows: Reportable Range:

0.3

to

10.0 µg/mL

Sample Storage Recommendation Recommended Sample Storage

Room Temperature (25°C)

Sample Storage Comments

No recommendation

Refrigerator (2 - 8°C)

7 days capped at 2 - 8°C

Freezer (- 20°C)

28 days capped at -20°C

See ONLINE TDM Gentamicin Package Insert, Mat # 03800504190, for reference and statements regarding specimen collection and preparation. See Appendix: Roche/Hitachi 917 Data.

Calibration Frequency • after bottle/cassette change • after reagent lot change • as required following quality control procedures

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Critical Issues

The pertinent observations made during these studies are: •

No critical issues to report from application studies on the cobas c501 system.

• •

Conclusions

Application studies on the cobas c501 met all criteria at a System level. The present application report will be used as the basis for the specifications/claims in the global method sheet. Per Global Regulatory requirements, specifications must be presented in the Limitations - interference and Measuring range sections of method sheets. In accordance with the Application Policy NewGen Systems V1.0 01.04.04, select performance data are transferred from the Hitachi 917 in support of the cobas c501 application. These data are included in the appendix. Calibration SDs are not available on cobas systems. Therefore #DIV/0! appears for calibration SD calculations and calibration SD input cells are blank in this report.

Recommendations

References

The application for the cobas c501 can be released.

The following laboratory notebooks and data binders contain supporting information and data: Laboratory notebook no:

NB 37855

Data binder no.:

37855-A / 37855-B

Electronic data caputure system version and study not applicable name:

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Performance Parameter Within Run imprecision N=21

Specification

Observed Performance

SD ≤ 0.09 up to 1.7 µg/mL CV ≤ 5% conc > 1.7 µg/mL

TDM I Mean SD CV

Pass/Fail

1.88 0.04 2.3%

Pass

TDM II Mean SD CV

4.16 0.06 1.4%

Pass

TDM III Mean SD CV

6.17 0.11 1.8%

Pass

HSP 1 Mean SD CV

1.96 0.05 2.6%

Pass

HSP 2 Mean SD CV

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6.06 0.07 1.2%

Pass

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Performance Parameter NCCLS Within-run Imprecision N=63 (master) N=30 (A I)

Specification

Observed Performance

SD ≤ 0.09 up to 1.7 µg/mL CV ≤ 5% conc > 1.7 µg/mL

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Total Mean Within-run SD Within-run CV

TDM I 1.87 0.08 4.2%

Total Mean Within-run SD Within-run CV

TDM II 4.37 0.08 1.8%

Total Mean Within-run SD Within-run CV

TDM III 6.48 0.12 1.8%

Total Mean Within-run SD Within-run CV

HSP 1 1.90 0.07 3.6%

Total Mean Within-run SD Within-run CV

HSP 2 6.04 0.13 2.2%

CONFIDENTIAL

Pass/Fail

Pass

Pass

Pass

Pass

Pass

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Performance Parameter NCCLS Between Day Imprecision

Specification

Observed Performance

SD ≤ 0.12 up to 1.7 µg/mL CV ≤ 7% conc > 1.7µg/mL

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Total Mean Between-day SD Between-day CV

TDM I 1.87 0.05 2.8%

Total Mean Between-day SD Between-day CV

TDM II 4.37 0.04 0.9%

Total Mean Between-day SD Between-day CV

TDM III 6.48 0.09 1.3%

Total Mean Between-day SD Between-day CV

HSP 1 1.90 0.03 1.8%

Total Mean Between-day SD Between-day CV

HSP 2 6.04 0.11 1.8%

CONFIDENTIAL

Pass/Fail

Pass

Pass

Pass

Pass

Pass

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Performance Parameter NCCLS Total Imprecision

Specification

Observed Performance

SD ≤ 0.12 up to 1.7 µg/mL CV ≤ 7% conc > 1.7µg/mL

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Total Mean Total SD Total CV

TDM I 1.87 0.09 5.1%

Total Mean Total SD Total CV

TDM II 4.37 0.09 2.0%

Total Mean Total SD Total CV

TDM III 6.48 0.15 2.3%

Total Mean Total SD Total CV

HSP 1 1.90 0.08 4.1%

Total Mean Total SD Total CV

HSP 2 6.04 0.17 2.8%

CONFIDENTIAL

Pass/Fail

Pass

Pass

Pass

Pass

Pass

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Performance Parameter

Specification

Observed Performance

Pass/Fail

Linearity Actual vs Theoretical ± 10% recovery Fresh Reagent observed: up to 12.1 µg/mL claim: up to 10.0 µg/mL Aged on-board Reagent observed: NT for System level application claim:

Target: Linear to 10 µg/mL

Minimum Accepted: No minimum spec.

Recovery of Controls Recovery to sheet values

Number of RD controls checked Number of competitor controls checked Recovery 110%

Sensitivity (LDL)

n (RD controls) 3 5 7

Pass

15 3 n (competitors)

Pass

2 1

Target: ≤ 0.30 µg/mL Minimum:

0.11

@ 2SD

Pass

≤ No minimum spec.

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Performance Parameter

Specification

Observed Performance

Pass/Fail

Method Comparison vs. H 917 0.976 0.999 -0.014

Slope Correlation (r) Intercept (b)

0.90-1.10 ≥ 0.95 ± 0.3

Slope Correlation (r) Intercept (b)

No specification c501 vs. Integra

Pass

vs. I800 0.983 0.997 -0.214

FIO

Interference Studies (Glick) No Interference up to: Lipemia ≤ 10% up to 150 mg/dL Intralipid (L Index)

208 mg/dL

Pass

1062 mg/dL

Pass

79 mg/dL

Pass

73 mg/dL

Pass

Hemolysis ≤ 10% up to 1000 mg/dL Hemoglobin (H Index)

Conjugated Bilirubin ≤ 10% up to 50 mg/dL (I Index)

Unconjugated Bilirubin ≤ 10% up to 50 mg/dL (I Index)

See interferences section for additional interferences tested

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Performance Parameter

Specification

Observed Performance

Pass/Fail

Drug Cross Reactivity See Appendix: Roche/Hitachi 917 Data.

Drug Interferences Target: ≤ ± 10% in recovery

16 common theraputic drugs were tested, no significant interference found - see drug interference section for individual results

Pass

Anticoagulants Acceptable anticoagulants include: sodium, lithium and ammonium heparin, K2 and K3 EDTA, sodium citrate, fluoride oxalate. See Appendix: Roche/Hitachi 917 Data.

Calibration Stability No claim in cobas method sheet. See calibration frequency recommendations.

On Board Stability Minimum claim transferred from Hitachi systems: 30 days opened and refrigerated on analyzer. See Appendix: Roche/Hitachi 917 Data.

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Analyzer (primary): Serial number: Software version:

cobas c501 s/n 0403-03 B1 - 14

Additional Analyzers used in studies: Analyzer: cobas c501 cobas c501 Serial number: s/n 0403-05 s/n 0403-04 Software version: B1 - 14 B1 - 14

H917 s/n 0600-7 02 - 48

General running parameters: sample volume (µL) R1 volume (µL) R2 volume (µL) wavelength (nm) read window calibration type assay type

2 100 95 600/800 18 -49 RCM 2-point End

Average Calibration: Calibrator Concentration:

S1 0.00 4811

S2 0.50 4404

SD #DIV/0!

Total S1 22073

S1 abs 4779

K 2.654

S3 1.50 3399

S4 4.00 1486

S5 7.00 676

A 1.607

B -184.600

C

S6 10.00 355

Typical Calibration Curve Gentamicin 6000

∆ ABS x 10,000

5000 4000 3000 2000 1000 0 0

2

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4

6 Concentration (µg/mL)

CONFIDENTIAL

8

10

12

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Materials used to generate this application report are listed below Calibrator Information Calibrator Catalog number

Lot number

Expiration date

03375790

15586201

Aug-06

Section

Intentionally

Left

Blank

Control Information Control

Preciset TDM 1

Catalog number

Lot number

Expiration date

Cobas FP TDM Cobas FP TDM BioRad Liquicheck Cliniqa TDM Control

20716626322 20716626322 360 04521536190

657364 659272 40660 0773/0774/0775

Jun-07 Jun-06 Jun-07 Mfr. 9/20/04

Cliniqa TDM Control Cliniqa TDM Control High Serum Pool I Low Serum Pool I

04521536190 04521536190 n/a n/a

502071/502072/502073 674373 37855-1A 37855-1B

Mfr. 3/1/05 Jun-07 Jan-07 Jan-07

Section

Intentionally

Left

Blank

Reagent Information Reagent

Catalog number

Lot number

Expiration date

Online Gentamicin

03500241190

15757800

Jan-06

Section

Intentionally

Left

Blank

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

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Glick and Endogenous Interference Materials Manufacturer

Catalog Number

Lot Number

Frontier JT Baker Frontier Fresenius Kabi R/D Internal Prep

B584-9 5635-02 B85 68100-278 NB 37855

050703 367101 JL04-14 1034873 37855-42

Intentionally

Left

Blank

Drug

Manufacturer

Catalog Number

Lot Number

Acetyl cysteine Ampicillin-Na Ascorbic acid Ca-Dobesilate Cyclosporine Cefoxitin Heparin Intralipid Levodopa Methyldopa + 1,5 H2O Metronidazole Phenylbutazone Doxycycline

USP Sigma In House Aldrich Novartis Sigma NT NT Sigma USP Sigma Sigma Sigma

900 A9518 11582909001 H18402 SD2027-400 C4786 NT NT D9628 1426002 M1547 P8386 T9823

1HB169 085K0485 52115231 09417PR 97800010 095K0605 NT NT 110K1340 11E059 29H05151 065K1492 092K16072

Acetylsalycilic acid Rifampicin Acetaminophen Ibuprofen Theophylline

Sigma Sigma Sigma Sigma Sigma

A5376 R3501 A5000 I4883 T1633

045K0101 074K1601 124K0165 026H1368 125K0661

Anticoagulant

Manufacturer

Catalog Number

Lot Number

Section

Intentionally

Left

Blank

Drug Bilirubin Sodium Hydroxide (1N) Conjugated Bilirubin Intralipid (20%) Fresh Hemolysate

Section Drug Interference Materials

Anticoagulant Materials

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

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Precision Notebook: 37855

Material µg/mL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Mean SD CV% min max N

Page: 4 - 11

TDM I

TDM II

TDM III

HSP 1

HSP 2

1.83 1.84 1.92 1.84 1.74 1.90 1.89 1.90 1.91 1.84 1.94 1.87 1.84 1.89 1.88 1.92 1.86 1.92 1.88 1.88 1.89

4.16 4.16 4.17 4.17 4.15 3.95 4.14 4.16 4.17 4.15 4.15 4.23 4.14 4.24 4.16 4.23 4.15 4.20 4.16 4.15 4.13

6.22 6.22 6.22 5.89 5.91 6.25 6.14 6.16 6.18 6.13 6.22 6.19 6.24 6.31 6.22 6.33 6.15 6.19 6.05 6.15 6.24

1.89 1.93 1.94 1.98 1.94 1.99 1.97 1.86 1.93 1.89 1.96 2.01 1.95 1.93 1.98 1.91 2.03 1.95 2.01 2.03 2.05

6.14 5.97 5.99 6.05 5.97 6.12 6.10 6.01 6.02 6.05 6.04 6.05 6.00 5.98 6.11 6.11 6.24 6.17 6.05 6.04 6.12

1.88

4.16

6.17

1.96

6.06

0.04 2.3%

0.06 1.4%

0.11 1.8%

0.05 2.6%

0.07 1.2%

1.74 1.94 21

3.95 4.24 21

5.89 6.33 21

1.86 2.05 21

5.97 6.24 21

Comments: All results meet specifications.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

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NCCLS Precision Notebook: 37855

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Specimen

TDM I

TDM II

TDM III

HSP 1

HSP 2

Total Mean Within Run Imprecision SD Within Run Imprecision CV% Total Imprecision SD Total Imprecision CV% Between-day Imprecision SD Between-day Imprecision CV%

1.87 0.079 4.2% 0.095 5.1% 0.052 2.8%

4.37 0.078 1.8% 0.088 2.0% 0.041 0.9%

6.48 0.119 1.8% 0.147 2.3% 0.087 1.3%

1.90 0.069 3.6% 0.078 4.1% 0.035 1.8%

6.04 0.134 2.2% 0.172 2.8% 0.107 1.8%

Date

Result 1

Result 2

Result 3

Mean

1/1

2/21/2006

1.90

1.89

1.87

1.89

2/1

2/22/2006

1.58

1.80

1.82

1.73

3/1

2/23/2006

1.83

1.94

2.00

1.92

4/1

2/24/2006

1.89

1.97

1.91

1.92

5/1

2/26/2006

1.96

1.62

1.90

1.83

6/1

2/27/2006

1.96

1.93

1.92

1.94

7/1

2/28/2006

1.94

1.96

1.96

1.95

8/1 9/1

3/1/2006 3/2/2006

1.89 1.93

1.97 1.95

1.93 1.83

1.93 1.90

10/1

3/3/2006

1.79

1.94

1.88

1.87

11/1

3/4/2006

1.94

2.00

1.97

1.97

12/1 13/1

3/5/2006 3/6/2006

1.79 1.85

1.98 1.91

1.95 1.82

1.91 1.86

14/1

3/7/2006

1.85

1.92

1.88

1.88

15/1

3/8/2006

1.92

1.89

1.83

1.88

16/1

3/9/2006

1.86

1.90

1.92

1.89

17/1

3/10/2006

1.71

1.85

1.70

1.75

18/1

3/11/2006

1.92

1.81

1.80

1.84

19/1

3/12/2006

1.84

1.74

1.58

1.72

20/1

3/13/2006

1.78

1.93

1.87

1.86

21/1

3/14/2006

1.86

1.71

1.85

1.81

21

21

21

Comments: All results meet specifications.

Material: Day/Run

TDM I

N MEAN

1.86

1.89

1.87

SD

0.091

0.099

0.095

CV

4.9%

5.2%

5.1%

MIN

1.58

1.62

1.58

MAX

1.96

2.00

2.00

Total Mean:

1.87

Within-run Imprecision:

SD =

0.079

Total Imprecision:

SD =

0.095

CV = 5.1%

Between-day Imprecision:

SD =

0.052

CV = 2.8%

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CONFIDENTIAL

CV = 4.2%

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NCCLS Precision Material: Day/Run

TDM II Date

Result 1

Result 2

Result 3

Mean

1/1

2/21/2006

4.51

4.28

4.36

4.38

2/1

2/22/2006

4.37

4.26

4.08

4.24

3/1

2/23/2006

4.24

4.38

4.20

4.27

4/1

2/24/2006

4.45

4.35

4.42

4.41

5/1

2/26/2006

4.40

4.36

4.27

4.34

6/1

2/27/2006

4.37

4.33

4.28

4.33

7/1

2/28/2006

4.36

4.38

4.39

4.38

8/1 9/1

3/1/2006 3/2/2006

4.50 4.46

4.38 4.53

4.46 4.43

4.45 4.47

10/1

3/3/2006

4.46

4.34

4.42

4.41

11/1

3/4/2006

4.25

4.23

4.47

4.32

12/1 13/1

3/5/2006 3/6/2006

4.30 4.34

4.35 4.39

4.39 4.13

4.35 4.29

14/1

3/7/2006

4.41

4.32

4.36

4.36

15/1

3/8/2006

4.37

4.42

4.38

4.39

16/1

3/9/2006

4.39

4.48

4.47

4.45

17/1

3/10/2006

4.37

4.34

4.33

4.35

18/1

3/11/2006

4.38

4.46

4.30

4.38

19/1

3/12/2006

4.38

4.44

4.38

4.40

20/1

3/13/2006

4.45

4.39

4.40

4.41

21/1

3/14/2006

4.40

4.31

4.20

4.30

21

21

21

N MEAN

4.39

4.37

4.34

SD

0.071

0.072

0.110 2.5%

CV

1.6%

1.7%

MIN

4.24

4.23

4.08

MAX

4.51

4.53

4.47

Total Mean:

4.37

Within-run Imprecision:

SD =

0.078

Total Imprecision:

SD =

0.088

CV = 2.0%

Between-day Imprecision:

SD =

0.041

CV = 0.9%

Material: Day/Run

CV = 1.8%

TDM III Date

Result 1

Result 2

Result 3

Mean

1/1

2/21/2006

6.44

6.21

6.29

6.31

2/1

2/22/2006

6.32

6.47

6.30

6.36

3/1

2/23/2006

6.51

6.57

6.46

6.51

4/1

2/24/2006

6.58

6.60

6.51

6.56

5/1

2/26/2006

6.54

6.67

6.69

6.63

6/1

2/27/2006

6.48

6.42

6.63

6.51

7/1

2/28/2006

6.45

6.50

6.14

6.36

8/1 9/1

3/1/2006 3/2/2006

6.58 6.28

6.63 6.36

6.75 6.64

6.65 6.43

10/1

3/3/2006

6.59

6.40

6.47

6.49

11/1

3/4/2006

6.54

6.39

6.59

6.51

12/1 13/1

3/5/2006 3/6/2006

6.58 6.63

6.47 6.52

6.53 6.45

6.53 6.53

14/1

3/7/2006

6.53

6.43

6.53

6.50

15/1

3/8/2006

6.55

6.69

6.68

6.64

16/1

3/9/2006

6.46

6.59

6.25

6.43

17/1

3/10/2006

6.38

6.57

6.40

6.45

18/1

3/11/2006

6.63

6.58

6.28

6.50

19/1

3/12/2006

6.40

6.65

6.52

6.52

20/1

3/13/2006

6.52

6.44

6.19

6.38

21/1

3/14/2006

6.12

6.29

6.19

6.20

21

21

21

N MEAN

6.48

6.50

6.45

SD

0.126

0.128

0.182

CV

1.9%

2.0%

2.8%

MIN

6.12

6.21

6.14

MAX

6.63

6.69

6.75

Total Mean:

6.48

Within-run Imprecision:

SD =

0.119

Total Imprecision:

SD =

0.147

CV = 2.3%

Between-day Imprecision:

SD =

0.087

CV = 1.3%

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

CV = 1.8%

Approved CN: cdman6835 18 of 32

Page 18 of 58

NCCLS Precision Material: Day/Run

HSP 1 Date

Result 1

Result 2

Result 3

Mean

1/1

2/21/2006

1.95

1.95

1.94

1.95

2/1

2/22/2006

1.92

1.94

1.82

1.89

3/1

2/23/2006

1.93

1.89

1.93

1.92

4/1

2/24/2006

2.00

1.95

1.93

1.96

5/1

2/26/2006

1.96

1.94

1.94

1.95

6/1

2/27/2006

1.96

1.86

1.90

1.91

7/1

2/28/2006

1.91

1.88

1.87

1.89

8/1 9/1

3/1/2006 3/2/2006

1.99 1.82

1.99 1.97

1.96 1.94

1.98 1.91

10/1

3/3/2006

1.79

1.95

1.93

1.89

11/1

3/4/2006

1.78

1.92

1.94

1.88

12/1 13/1

3/5/2006 3/6/2006

1.91 1.88

1.89 1.94

1.92 1.86

1.91 1.89

14/1

3/7/2006

1.90

1.92

1.94

1.92

15/1

3/8/2006

1.81

1.94

1.93

1.89

16/1

3/9/2006

1.97

1.98

1.96

1.97

17/1

3/10/2006

1.74

1.91

1.91

1.85

18/1

3/11/2006

1.90

1.88

1.87

1.88

19/1

3/12/2006

1.88

1.85

1.95

1.89

20/1

3/13/2006

1.93

1.61

1.84

1.79

21/1

3/14/2006

1.84

1.58

1.84

1.75

21

21

21

N MEAN

1.89

1.89

1.91

SD

0.073

0.106

0.043 2.2%

CV

3.8%

5.6%

MIN

1.74

1.58

1.82

MAX

2.00

1.99

1.96

Total Mean:

1.90

Within-run Imprecision:

SD =

0.069

Total Imprecision:

SD =

0.078

CV = 4.1%

Between-day Imprecision:

SD =

0.035

CV = 1.8%

Material: Day/Run

CV = 3.6%

HSP 2 Date

Result 1

Result 2

Result 3

Mean

1/1

2/21/2006

6.14

6.16

5.81

6.04

2/1

2/22/2006

5.39

6.08

5.73

5.73

3/1

2/23/2006

6.24

6.09

6.10

6.14

4/1

2/24/2006

6.10

6.19

5.98

6.09

5/1

2/26/2006

6.10

6.11

6.17

6.13

6/1

2/27/2006

6.14

6.04

6.15

6.11

7/1

2/28/2006

6.14

6.10

6.10

6.11

8/1 9/1

3/1/2006 3/2/2006

6.10 6.13

6.30 6.11

6.21 6.26

6.20 6.17

10/1

3/3/2006

6.02

6.07

5.96

6.02

11/1

3/4/2006

5.87

5.80

5.83

5.83

12/1 13/1

3/5/2006 3/6/2006

6.13 5.75

6.03 5.97

6.15 6.04

6.10 5.92

14/1

3/7/2006

5.98

6.03

6.05

6.02

15/1

3/8/2006

6.26

6.09

6.18

6.18

16/1

3/9/2006

6.19

6.10

6.23

6.17

17/1

3/10/2006

5.91

6.20

5.74

5.95

18/1

3/11/2006

5.90

6.12

6.24

6.09

19/1

3/12/2006

6.04

5.95

5.95

5.98

20/1

3/13/2006

6.04

6.05

5.86

5.98

21/1

3/14/2006

5.86

5.92

5.56

5.78

21

21

21

N MEAN

6.02

6.07

6.01

SD

0.196

0.106

0.196

CV

3.3%

1.7%

3.3%

MIN

5.39

5.80

5.56

MAX

6.26

6.30

6.26

Total Mean:

6.04

Within-run Imprecision:

SD =

0.134

Total Imprecision:

SD =

0.172

CV = 2.8%

Between-day Imprecision:

SD =

0.107

CV = 1.8%

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

CV = 2.2%

Approved CN: cdman6835 19 of 32

Page 19 of 58

Daily QC Results QC FLUID results (µg/mL) Date 1/27/2006 1/27/2006 1/31/2006 2/9/2006 2/14/2006 2/14/2006 2/20/2006 2/21/2006 2/22/2006 2/22/2006 3/15/2006 3/15/2006 3/15/2006 3/20/2006

Run precision ldl linearity control recovery glick NCCLS glick glick glick glick method comp run 1 method comp run 2 method comp run 3 drug interference

Count Mean SD CV (%) Mean-2SD Mean+2SD

TDM I

TDM II

TDM III

1.85 1.87 1.90 1.83 1.76 1.67 1.77 1.71 1.95 1.97 1.85 1.88 1.77 1.86

4.13 4.11 4.10 3.99 3.88 4.01 4.17 4.10 4.12 4.20 4.07 4.19 4.15 4.01

5.84 6.10 6.20 6.08 6.01 6.04 6.25 6.23 6.26 6.29 6.10 5.91 6.20 6.16

14 1.83 0.1 4.7% 1.66 2.00

14 4.09 0.1 2.2% 3.91 4.27

14 6.12 0.1 2.2% 5.85 6.39

Comments nominal value of control N=3 ran median value recorded N=3 ran median value recorded

N=3 ran median value recorded

QC Gentamicin Concentration (µg/mL)

7.00 6.00

TDM I TDM II TDM III

5.00 4.00 3.00 2.00 1.00 0.00 0

2

4

6

8

10

12

14

Data Point

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 20 of 32

Page 20 of 58

Sensitivity (LDL) Notebook: 37855

Rep

mAbs

Cal A

1

4627

2 3 4 5 6 7 8

4702 4615 4582 4613 4586 4607 4614

9 10 11

4570 4666 4625

12 13 14 15 16 17 18 19 20 21

4589 4610 4611 4667 4579 4678 4564 4597 4611 4507

1 2 3 4 5

4213 4225 4218 4180 4209

Cal B

Comments:

Page: 12 - 13, 162 - 166

Sample

Calculations Conc Mean SD 2SD 3SD Slope Intercept 2SD LDL=

Cal A 0

Cal B 0.50

4610.5 43.2 86.4 129.6 -0.0012 5.7419

4209

0.11

µg/mL

Lower detection limit meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 21 of 32 Page 21 of 58

METHOD COMPARISON SUMMARY Notebook: 37855 Reference Method (x): Reference Instrument (x): Comparative Method (y): Comparative Instrument (y): Passing Bablok Regression equation: y = 0.976x - 0.0143 Slope: 0.976 Intercept: -0.014 md95: 0.158 t: 0.962 n: 64 Bias at Low MDL (conc.L): -0.050 Bias at High MDL (conc.H): -0.158

Page: 88 - 95, 127 - 134 OnLine Gentamicin H 917 Gentamicin cobas c501 Linear Regression equation: y = 0.976x - 0.0161 Slope: 0.976 Intercept: -0.016 Sy.x: 0.2195 r: 0.999 n: 64

12

OnLine Gentamicin c501

10

8

6

4

2

0 0

2

4

6

8

10

12

OnLine Gentamicin H917 Comments: Results meet specifications for slope, intercept and correlation. The sample concentrations were between 0.5 and 8.7 µg/mL. Bias calculated at 1.5 and 6 µg/mL.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 22 of 31 Page 22 of 58

METHOD COMPARISON SUMMARY Reference Method (x): Reference Instrument (x): Comparative Method (y): Comparative Instrument (y): Regression equation: Slope: Intercept: md95: t: n: Bias at Low MDL (conc.L): Bias at High MDL (conc.H):

FP Gentamicin I800 Gentamicin cobas c501 Passing Bablok y = 0.983x - 0.2144 0.983 -0.214 0.229 0.965 63 -0.240 -0.316

Regression equation: Slope: Intercept: Sy.x: r: n:

Linear y = 0.988x - 0.2194 0.988 -0.219 0.3103 0.997 63

10 9

OnLine Gentamicin c501

8 7 6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

9

10

FP Gentamicin I800 Comments: Bias calculated at 1.5 and 6 µg/mL. The sample concentrations were between 0.5 and 8.8 µg/mL. I800 s/n 38-5211 sw 9864 A1 used for reference testing.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 23 of 31 Page 23 of 58

Linearity FRESH REAGENT Notebook: 37855 Page: 14 - 19 dilution level n measured theoretical low high value value 1.00 0.00 3 0.0 0.0000 0.90 0.10 * 3 1.4 1.2917 0.80 0.20 * 3 2.7 2.5833 0.70 0.30 * 3 4.0 3.8750 0.60 0.40 * 3 5.2 5.1667 0.50 0.50 * 3 6.4 6.4583 0.40 0.60 * 3 7.7 7.7500 0.30 0.70 * 3 8.9 9.0417 0.20 0.80 * 3 10.0 10.3333 0.10 0.90 3 10.9 11.6250 0.00 1.00 3 12.1 12.9167 Standard regression procedure (Passing/Bablok): y = 12.9167x + 0

recovery % 0.0 108.4 104.5 103.2 100.7 99.1 99.4 98.4 96.8 93.8 93.7

Recovery [%]

120 110 100 90 80 0

2

4

6

8

10

12

14

Measured Concentration µg/mL

Measured Concentration µg/mL

14 12 10 8 6 4 2 0 0

5

10

15

Theoretical Concentration µg/mL

Comments:

Linearity meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 24 of 32 Page 24 of 58

Control Recovery Roche Controls and Calibrators Notebook: 37855 Control Material Cal B Cal C Cal D Cal E Cal F TDM I TDM II TDM III TDM I TDM II TDM III TDM I TDM II TDM III

Method

Lot Number

Target

cobas c501 cobas c501 cobas c501 cobas c501 cobas c501 OnLine OnLine OnLine OnLine OnLine OnLine OnLine OnLine OnLine

15586201 15586201 15586201 15586201 15586201 Cliniqa P3 Lot Cliniqa P3 Lot Cliniqa P3 Lot Cliniqa Master Lot Cliniqa Master Lot Cliniqa Master Lot Cliniqa P2 Lot Cliniqa P2 Lot Cliniqa P2 Lot

0.50 1.50 4.00 7.00 10.00 2.1 5.4 8.3 2.2 5.9 8.6 2.1 5.2 8.0

Page: 31 - 38 Range Low High

1.7 4.4 6.7 1.7 4.5 6.3 1.6 4.0 5.9

Obs. Median c501

Ref. Median % Recovery % Recovery (inst. used) to Sheet to Ref.

0.5 1.3 3.9 6.8 9.4 2.0 5.3 7.9 2.1 5.7 8.2 1.9 5.0 7.7

100.0 89.3 97.3 97.1 94.2 94.7 97.2 94.8 94.1 96.6 94.3 89.7 96.2 96.5

Obs. Median c501

Ref. Median % Recovery % Recovery (inst. used) to Sheet to Ref.

2.7 5.7 7.0 1.8 4.0 6.1 1.8 3.8 5.8

95.7 101.3 95.3 98.4 98.8 98.5 87.5 86.6 85.5

2.5 6.5 10.0 2.7 7.3 11.0 2.7 6.5 10.1

#DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0!

Other Controls (if tested) Control Material

Method

Lot Number

Target

BioRad Liquichek BioRad Liquichek BioRad Liquichek TDM I TDM II TDM III TDM I TDM II TDM III

Roche Hitachi Roche Hitachi Roche Hitachi OnLine OnLine OnLine OnLine OnLine OnLine

40661 40662 40663 657364 657364 657364 659272 659272 659272

2.8 5.6 7.3 1.9 4.0 6.2 2.0 4.3 6.8

Range Low High 2.3 4.5 5.8 1.4 3.1 4.5 1.5 3.3 5.0

3.4 6.7 8.8 2.3 5.0 7.9 2.5 5.4 8.6

#DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0!

Comments: Control recovery meets specification. All controls recovered within the target range.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 25 of 32

Page 25 of 58

Interferences LIPEMIA High

Notebook: 37855 Page: 61 - 65 Percent * Denotes Observed Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 >10% Result 9 5.68 ----41 5.80 102.1 0.12 71 5.76 101.4 0.08 101 5.63 99.1 -0.05 133 5.64 99.3 -0.04 160 5.57 98.1 -0.11 195 5.40 95.1 -0.28 215 5.43 95.6 -0.25 253 5.38 94.7 -0.30 285 5.16 90.8 -0.52 313 5.14 90.5 -0.54 ** The Index value corresponds approximately to mg/dL Intralipid LIPEMIA Low Percent * Denotes Observed Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 >10% Result 10 1.80 ----40 1.74 96.7 -0.06 69 1.75 97.2 -0.05 101 1.71 95.0 -0.09 128 1.74 96.7 -0.06 151 1.62 90.0 -0.18 181 1.62 90.0 -0.18 208 1.63 90.6 -0.17 239 1.57 87.2 * -0.23 268 1.52 84.4 * -0.28 297 1.46 81.1 * -0.34

Lipemia Interference Lipemia High

% Recovery (vs. level 0)

120.0

Lipemia Low

110.0 100.0 90.0 80.0 0

100

200

300

L Index

Comments: Lipemia interference meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 26 of 32

Page 26 of 58

Interferences HEMOGLOBIN High

Notebook: 37855 Page: 42 - 46 Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 2 5.23 ----104 5.05 96.6 -0.18 209 5.14 98.3 -0.09 315 5.21 99.6 -0.02 433 5.23 100.0 0.00 543 5.19 99.2 -0.04 654 5.16 98.7 -0.07 766 5.21 99.6 -0.02 861 5.23 100.0 0.00 960 5.18 99.0 -0.05 1062 5.23 100.0 0.00 ** The Index value corresponds approximately to mg/dL Hemoglobin HEMOGLOBIN Low Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 2 1.70 ----97 1.67 98.2 -0.03 198 1.67 98.2 -0.03 291 1.72 101.2 0.02 405 1.73 101.8 0.03 508 1.73 101.8 0.03 598 1.66 97.6 -0.04 692 1.73 101.8 0.03 817 1.69 99.4 -0.01 902 1.76 103.5 0.06 998 1.76 103.5 0.06

Hemoglobin Interference Hemoglobin High

% Recovery (vs. level 0)

120.0

Hemoglobin Low

110.0 100.0 90.0 80.0 0

400

800

1200

H Index

Comments: Hemoglobin interference meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

Approved CN: cdman6835 27 of 32

Page 27 of 58

Interferences CONJUGATED BILIRUBIN High Notebook: 37855 Page: 57 - 60 Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 1 5.91 ----11 5.88 99.5 -0.03 20 5.89 99.7 -0.02 31 5.83 98.6 -0.08 42 5.87 99.3 -0.04 46 6.06 102.5 0.15 55 5.88 99.5 -0.03 62 5.91 100.0 0.00 71 5.95 100.7 0.04 79 6.03 102.0 0.12 88 6.05 102.4 0.14 ** The Index value corresponds approximately to mg/dL Bilirubin CONJUGATED BILIRUBIN Low Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 1 1.68 ----10 1.69 100.6 0.01 19 1.68 100.0 0.00 31 1.77 105.4 0.09 39 1.72 102.4 0.04 40 1.65 98.2 -0.03 49 1.70 101.2 0.02 55 1.72 102.4 0.04 61 1.70 101.2 0.02 70 1.76 104.8 0.08 79 1.79 106.5 0.11

Conjugated Bilirubin Interference Conjugated Bilirubin High

% Recovery (vs. level 0)

120.0

Conjugated Bilirubin Low

110.0 100.0 90.0 80.0 0

30

60

90

I Index

Comments: Conjugated bilirubin interference meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

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CONFIDENTIAL

Approved CN: cdman6835 28 of 32

Page 28 of 58

Interferences UNCONJUGATED BILIRUBIN High Notebook: 37855 Page: 53 - 56 Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 1 5.65 ----9 5.67 100.4 0.02 17 5.54 98.1 -0.11 24 5.62 99.5 -0.03 32 5.65 100.0 0.00 38 5.58 98.8 -0.07 48 5.62 99.5 -0.03 55 5.59 98.9 -0.06 63 5.64 99.8 -0.01 70 5.63 99.6 -0.02 77 5.64 99.8 -0.01 ** The Index value corresponds approximately to mg/dL Bilirubin UNCONJUGATED BILIRUBIN Low Percent Observed * Denotes Bias vs Index Value** Recovery deviation Analyte Level 0 vs Level 0 Result >10% 1 1.65 ----8 1.68 101.8 0.03 15 1.64 99.4 -0.01 22 1.66 100.6 0.01 30 1.66 100.6 0.01 39 1.63 98.8 -0.02 45 1.65 100.0 0.00 53 1.60 97.0 -0.05 60 1.61 97.6 -0.04 66 1.62 98.2 -0.03 73 1.63 98.8 -0.02

% Recovery (vs. level 0)

Unconjugated Bilirubin Interference 120.0

Unconjugated Bilirubin High

110.0

Unconjugated Bilirubin Low

100.0 90.0 80.0 0

30

60

90

I Index

Comments: Unconjugated bilirubin interference meets specification.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

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Approved CN: cdman6835 29 of 32

Page 29 of 58

Drug Interferences Notebook: 37855 LDL

Page: 79, 151 - 160

0.30 µg/mL

C1

Median

C1 %

C2

Median

C2 %

µg/mL

µg/mL

Recovery

µg/mL

µg/mL

Recovery

1.82

---

1.82

---

Acetyl cysteine

150

1.81

99.5%

30

1.89

103.8%

Ampicillin-Na

1000

1.76

96.7%

200

1.91

104.9%

Ascorbic acid

300

1.82

100.0%

30

1.89

103.8%

Ca-Dobesilate

200

1.80

98.9%

20

1.80

98.9%

Theophylline

100

1.84

101.1%

10

1.90

104.4%

Cefoxitin

2500

1.82

100.0%

250

1.90

104.4%

Heparin

5000 U

NT

10 U

NT

Intralipid

Drug Control 1

10000

NT

2000

NT

Levodopa

20

1.78

97.8%

4

1.93

Methyldopa + 1,5 H2O

20

1.81

99.5%

2

1.88

103.3%

Metronidazole

200

1.83

100.5%

10

1.90

104.4%

Phenylbutazone

400

1.81

99.5%

100

1.76

96.7%

Doxycycline

50

1.81

99.5%

10

1.84

101.1%

1000

1.80

98.9%

300

1.92

105.5%

10000

NT

2000

NT

1.89

---

1.89

---

Acetaminophen

200

1.89

100.0%

20

1.93

102.1%

Ibuprofen

500

1.86

98.4%

50

1.93

102.1%

Rifampicin

60

1.73

91.5%

20

1.90

100.5%

Cyclosporine

5

1.87

98.9%

1

1.92

101.6%

Acetylsalycilic acid Control 2

106.0%

---

Intralipid Control 3

---

Comments: No significant interference at C1 and C2 drug levels.

GENT2_c501_Version 1.0 TDM - Hitachi Application Report Template Roche Diagnostics FORM-01162

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CONFIDENTIAL

Approved CN: cdman6835 30 of 32

Page 30 of 58

For INTERNAL USE Only

SUCCESSFUL CALIBRATIONS HISTORY LOG date 1/27/2006

run 1

1/27/2006

2

1/31/2006

3

2/9/2006

4

2/14/2006

5

2/14/2006

6

2/20/2006

7

2/21/2006

8

2/22/2006

9

2/22/2006

10

3/15/2006

11

3/15/2006

12

3/15/2006

13

3/20/2006

14

15

ng/mL: 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff

S1 0.0 4779 4938 159 3.3% 4641 4719 78 1.7% 4790 4762 -28 -0.6% 4851 4808 -43 -0.9% 4895 4923 28 0.6% 4956 4800 -156 -3.2% 4922 4924 2 0.0% 4857 4912 55 1.1% 4694

S1Abs Total 22183 22664 481 2.1% 21452 21731 279 1.3% 22006 21926 -80 -0.4% 22049 21837 -212 -1.0% 22223 22307 84 0.4% 22354 21847 -507 -2.3% 22375 22393 18 0.1% 22207 22363 156 0.7% 21614

S2 0.5 4502 4500 -2 0.0% 4195 4314 119 2.8% 4449 4547 98 2.2% 4378 4370 -8 -0.2% 4441 4451 10 0.2% 4421 4579 158 3.5% 4437 4525 88 2.0% 4422 4441 19 0.4% 4359

S3 1.5 3469 3348 -121 -3.5% 3179 3125 -54 -1.7% 3418 3596 178 5.1% 3374 3427 53 1.6% 3479 3473 -6 -0.2% 3569 3473 -96 -2.7% 3414 3452 38 1.1% 3528 3427 -101 -2.9% 3482

S4 4.0 1508 1512 4 0.3% 1375 1343 -32 -2.4% 1538 1519 -19 -1.2% 1496 1508 12 0.8% 1534 1551 17 1.1% 1527 1552 25 1.6% 1520 1506 -14 -0.9% 1464 1533 69 4.6% 1483

S5 7.0 666 678 12 1.8% 599 592 -7 -1.2% 700 705 5 0.7% 665 666 1 0.2% 714 693 -21 -3.0% 725 713 -12 -1.7% 659 677 18 2.7% 707 684 -23 -3.3% 680

S6 10.0 351 332 -19 -5.6% 286 294 8 2.8% 365 371 6 1.6% 352 371 19 5.3% 369 371 2 0.5% 391 386 -5 -1.3% 358 361 3 0.8% 351 367 16 4.5% 353

4805 111 2.3% 4797 4905 108 2.2% 4787 4779 -8 -0.2% 4751 4776 25 0.5% 4723 4683 -40 -0.9% 4769 4761 -8 -0.2%

22064 450 2.1% 21914 22269 355 1.6% 22179 22040 -139 -0.6% 22022 22146 124 0.6% 21929 21784 -145 -0.7% 22099 22053 -46 -0.2%

4361 2 0.0% 4407 4386 -21 -0.5% 4329 4343 14 0.3% 4335 4389 54 1.2% 4328 4297 -31 -0.7% 4356 4437 81 1.8%

3419 -63 -1.8% 3376 3489 113 3.3% 3338 3369 31 0.9% 3292 3272 -20 -0.6% 3331 3293 -38 -1.1% 3393 3376 -17 -0.5%

1517 34 2.3% 1462 1495 33 2.2% 1472 1437 -35 -2.4% 1443 1430 -13 -0.9% 1468 1453 -15 -1.0% 1475 1488 13 0.9%

672 -8 -1.2% 677 676 -1 -0.1% 669 657 -12 -1.8% 682 656 -26 -3.9% 663 668 5 0.8% 690 681 -9 -1.3%

366 13 3.6% 360 361 1 0.3% 356 352 -4 -1.1% 345 350 5 1.4% 352 342 -10 -2.9% 361 367 6 1.6%

GENT2_c501_Version 1.0 DAT - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

CONFIDENTIAL

S1Abs. 4848

K 2.64

A 1.602

B -199.7

4666

2.516

1.528

-251.0

4761

2.698

1.757

-68.8

4788

2.698

1.558

-241.6

4866

2.736

1.551

-253.4

4842

2.68

1.674

-110.3

4892

2.644

1.561

-232.6

4835

2.681

1.609

-193.1

4703

2.73

1.679

-147.1

4803

2.651

1.604

-186.9

4743

2.634

1.579

-200.0

4747

2.553

1.569

-173.3

4678

2.659

1.59

-191.3

4740

2.638

1.639

-135.3

C

SD

Approved CN: cdman6835 31 of 32

Page 31 of 58

For INTERNAL USE Only

FAILED CALIBRATIONS HISTORY LOG date

run 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

ng/mL: 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff 1 2 Diff (2-1) % Diff

S1 0

S1Abs Total

S2 S3 S4 0.5 1.5 4 no failed calibrations during this study

GENT2_c501_Version 1.0 DAT - Hitachi Application Report Template Roche Diagnostics FORM-01162

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

S5 7

S6 10 Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

Alarm:

CONFIDENTIAL

Approved CN: cdman6835 32 of 32

Page 32 of 58

cobas c501 Instrument Settings

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GENT2_c501_Instrument Settings Cover Parameters Page 33 of 58

cobas c501

C

ANALYZE

GENT2

Serum

2 POINT END

10

Assay/Time/Point

Code (ACN) 416

Wave (2nd/Primary)

800

600

Sample Volume Normal Decrease Increase

2.0 2.0 2.0

0 0 0

Diluent Water Diluent

Code

0

0

Reagent Vol. R1 100 R2 95 R3 0

1

Test 100 Dummy Inactive

30

Bottle A R3 B R1 C R2

0 0 0

Tests

Vol.

Cancel 100 100

** ** **

Dilution

0

Prozone Limit

0

1 0

0 0

0

32000

Cell Detergent

49

Code 0769223 Expiration Days

0 0 0

951

Linearity Limit Abs. Limit

18

0 0

0

0

Inside

0

0

INCREASE

DETERGENT1

3

Stirring Level

** Refer to applicable reagent carrier fill tool

CALIBRATION Auto Calibration Calibration Type Point Span Weight Update Type

RCM 6 6 0 DIFFERENCE

Lot Calibration to Lot Time Out

1

Full Cancel -

6 Cassette Calibration to Cassette Time out

SD Limit Duplicate Limit Sensitivity Limit S1-Abs Limit Auto Masking

999 10 -99999 -32000 NO

%

80 Abs 999999 32000

QC Failed CANCEL

2 of 3

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Full Cancel -

Filename: GENT2_c501_Instrument Settings Tab: Setting GENT2AnalyzeCalibration

Page 34 of 58

cobas c501

Code (ACN)

C

416

RANGE App Code Unit

416 ug/ml

Report Name Automatic Rerun NO Data Mode Technical Limit Repeat Limit Control Interval Time On Board Stability Time

-

Expected Ranges Male 100 Years 100 Years

10 999999

Female 100 Years 100 Years

Factor

Unit 2 Unit 3

umol/L -

2.09

-99999 -99999 -99999

999999 999999 999999

-99999 -99999 -99999

999999 999999 999999

Gentamicin ACTIVE 0.3 -99999

Qualitative (1) (2) (3) (4) (5) (6)

-+/++ +++ ++++

L H I

150 1000 50

Default

(1) 691 0.0

(2) 692 0.5

(3) 693 1.5

(4) 694 4.0

(5) 695 7.0

(6) 696 10.0

2.0 0 0

2.0 0 0

2.0 0 0

2.0 0 0

2.0 0 0

2.0 0 0

Sex Male

Female

Range Range 1

Range 2

Range 3

OTHERS Std Calib. Code Concentration Position Sample Volume Diluent S. Volume Diluent Volume

3 of 3

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Page 35 of 58

Appendix of Application Report cobas c501 Gentamicin Data in this appendix were measured on a Roche/Hitachi 917 and 911 System References: R&D Memorandum No. 2070, R&D Report No. 2062

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GENT2_c501_Appendix Page 36 of 58

Summary References Hitachi 917

The following laboratory notebooks and data binders contain supporting information and data: Laboratory notebook no:

# 33486

Data binder no.:

# 01D73 (Book 1 & Book 2 & Book 3)

CAEv version and study name:

Version 2.51h GENT THEO MASTER LOT PEP

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GENT2_C501_Appendix Filename: Gent-ML-PEP Tab: Summary Page 37 of 58

Appendix 7. Anticoagulant Purpose

To test the suitability of various plasma types and sample primary tubes.

Protocol

Following Global SOP RS17-03e, test serum/plasma paired donations for each plasma type. The median value of 3 reps was used. Passing-Bablok regression is performed on the results.

Acceptance Criteria

The criteria for the Passing-Bablok regression statistics are slope of 0.90-1.10 intercept ± 2 X LDL correlation coefficient, r ≥ (0.95)

Results

All of the criteria for the regression statistics were met for heparin (Lithium, Sodium and Ammonia) plasma, EDTA K3 and K2 plasma, sodium citrate plasma, potassium oxalate plasma, and serum gel separator tubes. Sera (Red Top) is x scale reference

P/B Slope P/B Intercept r Correlation LSR slope n

Gel Sera 1.002 0.014 1.000 1.004 15

K3EDTA 0.995 -0.008 0.999 0.993 30

P/B Slope P/B Intercept r Correlation LSR slope n

Oxalate Fluorate 1.002 -0.015 0.999 0.996 10

0.5x Ox Fluor 0.992 -0.002 1.000 0.985 5

K2EDTA 0.994 -0.035 0.999 0.992 20

Li Heparin Na Heparin 0.998 1.007 -0.028 -0.115 0.999 1.000 1.002 1.008 15 10

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All EDTA 0.5x EDTA Na Citrate 0.5x Na Citr 0.994 0.982 1.000 0.997 -0.033 -0.054 -0.030 -0.026 0.999 0.999 0.999 1.000 0.992 0.991 1.002 0.998 50 10 30 5 NH4 Heparin 1.000 -0.030 0.999 1.004 10

All Heparin 1.000 -0.040 0.999 1.004 30

0.5x Heparin 1.004 -0.094 0.999 1.001 15

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 7 Page 38 of 58

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Page 39 of 58

Results

Acceptance Criteria

Following Global SOP RS55-01e, test serum and plasma samples were spiked to cover measuring range of Gentamicin. Samples were tested under varying storage temperatures over time. Samples were aliquotted at day 0

Protocol

4 of 23

Serum and plasma samples can be frozen and thawed up to six iterations. Heparin samples showed clot formation by second freeze/thaw cycle but recovery of drug was unaffected.

Serum and plasma samples can be stored at room temperature for 3 days (longer is probably acceptable but not encouraged because of bacterial growth), refrigerated for 7 days (data indicates 21 days or longer) and frozen at -20C for 4 weeks (longer periods possible but not verified.) Gentamicin samples were able to be stored under several conditions and recovery of drug was unaffected.

The criteria for freeze/thaw is number of cycles where all samples are within +/-10% of day 0 values.

The criteria for storage conditions (time and temperature) is time point (for each storage temperature) where all samples are within +/-10% of day 0 values.

Following Global SOP RS10-02e, test serum and plasma samples were spiked to cover measuring range of Gentamicin. Samples were aliquotted at day 0 and frozen. Samples were frozen and thawed up to 6 times; all samples were run at same time. The median value of 3 reps was used. Values were compared to day 0.

To determine analyte stability in human samples under defined storage conditions. To review various sample pretreatment (freezing only) procedures.

Purpose

Appendix 7b. Sample Handling

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 7b Sample Handling

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Page 40 of 58

Data 1.03 2.90 4.79 6.71 8.13 1.98 5.78 3.87 7.42 1.44 4.94

1- Sera 2- Sera Pool 3- Sera Pool 4- Sera Pool 5- Sera Pool 6- K2EDTA 7- K2 EDTA 8- NH4 Heparin 9- Na Heparin 10- Heparin Pool 11- NH4 Heparin

1.03 2.90 4.79 6.71 8.13 1.98 5.78 3.87 7.42 1.44 4.94

Stored at 25C Sample Day 0

1- Sera 2- Sera Pool 3- Sera Pool 4- Sera Pool 5- Sera Pool 6- K2EDTA 7- K2 EDTA 8- NH4 Heparin 9- Na Heparin 10- Heparin Pool 11- NH4 Heparin

Stored at 2-8C Sample Day 0

1.12 3.09 5.01 6.82 8.39 1.97 5.83 3.94 7.55 1.52 5.10

Day 1

1.12 2.99 4.93 6.73 8.24 2.00 5.81 3.87 7.50 1.53 5.06

Day 1

Appendix 7b. Sample Handling

108.7 106.6 104.6 101.6 103.2 99.5 100.9 101.8 101.8 105.6 103.2

108.7 103.1 102.9 100.3 101.4 101.0 100.5 100.0 101.1 106.3 102.4

1.02 2.98 4.99 6.79 8.25 1.93 5.79 3.95 7.49 1.51 5.08

Day 2

1.08 2.95 4.78 6.63 8.17 1.98 5.75 3.83 7.39 1.47 5.00

Day 2

99.0 102.8 104.2 101.2 101.5 97.5 100.2 102.1 100.9 104.9 102.8

104.9 101.7 99.8 98.8 100.5 100.0 99.5 99.0 99.6 102.1 101.2

5 of 23

1.13 3.03 5.09 6.87 8.36 1.99 5.88 4.02 7.58 1.55 5.18

Day 3

1.11 2.99 4.83 6.77 8.18 1.99 5.84 3.92 7.46 1.51 5.01

Day 3

109.7 104.5 106.3 102.4 102.8 100.5 101.7 103.9 102.2 107.6 104.9

107.8 103.1 100.8 100.9 100.6 100.5 101.0 101.3 100.5 104.9 101.4

1.06 3.02 4.83 6.81 8.30 1.94 5.87 4.01 7.46 1.55 5.10

Day 6 102.9 104.1 100.8 101.5 102.1 98.0 101.6 103.6 100.5 107.6 103.2

1.03 3.01 4.86 6.86 8.30 2.01 5.80 4.00 7.50 1.54 5.09

Day 7 100.0 103.8 101.5 102.2 102.1 101.5 100.3 103.4 101.1 106.9 103.0

1.22 3.12 5.05 6.93 8.48 2.08 6.09 4.05 7.64 1.62 5.18

Day 14

0.97 2.85 4.84 6.72 8.12 1.92 5.70 3.98 7.44 1.49

Day 21 94.2 98.3 101.0 100.1 99.9 97.0 98.6 102.8 100.3 103.5 0.0

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 7b Sample Handling

118.4 107.6 105.4 103.3 104.3 105.1 105.4 104.7 103.0 112.5 104.9

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Page 41 of 58

1.03 2.90 4.79 6.71 8.13 1.98 5.78 3.87 7.42 1.44 4.94

1- Sera 2- Sera Pool 3- Sera Pool 4- Sera Pool 5- Sera Pool 6- K2EDTA 7- K2 EDTA 8- NH4 Heparin 9- Na Heparin 10- Heparin Pool 11- NH4 Heparin

1X

1.06 2.97 4.95 6.91 8.34 1.97 5.78 3.92 7.48 1.62 5.06

Day 3 102.9 102.4 103.3 103.0 102.6 99.5 100.0 101.3 100.8 112.5 102.4

2X

1.10 3.06 5.00 6.85 8.37 1.95 5.89 3.95 7.57 1.54 5.15

Day 7 106.8 105.5 104.4 102.1 103.0 98.5 101.9 102.1 102.0 106.9 104.3

3X

1.16 3.11 4.99 6.88 8.40 1.99 5.83 4.04 7.58 1.48 5.05

Day 14 112.6 107.2 104.2 102.5 103.3 100.5 100.9 104.4 102.2 102.8 102.2

4X 1.08 3.08 4.98 6.95 8.48 2.06 6.02 4.00 7.72 1.54 5.20

1.01 2.94 4.83 6.82 8.40 1.89 5.75 3.91 7.52 1.53 4.99

Day 21

104.9 106.2 104.0 103.6 104.3 104.0 104.2 103.4 104.0 106.9 105.3

98.1 101.4 100.8 101.6 103.3 95.5 99.5 101.0 101.3 106.3 101.0

5X 1.10 3.10 5.06 6.95 8.40 2.00 5.94 3.96 7.60 1.61 5.23

1.04 2.93 4.83 6.73 8.34 1.97 5.78 3.95 7.47 1.52 4.96

Day 28

106.8 106.9 105.6 103.6 103.3 101.0 102.8 102.3 102.4 111.8 105.9

101.0 101.0 100.8 100.3 102.6 99.5 100.0 102.1 100.7 105.6 100.4

6 of 23

**Note: Sample 10 was problematic in recovery but can be disregarded because it was a pool of 3 different donors and heparin types mixed.

1.03 1.10 106.8 1.13 109.7 1.00 97.1 2.90 2.93 101.0 3.07 105.9 3.05 105.2 4.79 4.99 104.2 5.01 104.6 5.07 105.8 6.71 6.87 102.4 6.92 103.1 7.00 104.3 8.13 8.33 102.5 8.43 103.7 8.50 104.6 1.98 2.03 102.5 2.03 102.5 2.02 102.0 5.78 5.88 101.7 5.99 103.6 5.99 103.6 3.87 4.00 103.4 4.08 105.4 4.02 103.9 7.42 7.56 101.9 7.72 104.0 7.54 101.6 1.44 1.52 105.6 1.62 112.5 1.61 111.8 4.94 5.15 104.3 5.18 104.9 5.14 104.0 *Note: Samples 8, 9, 10, 11 showed clotting after 1 freeze/thaw cycle

Freeze/Thaw Cycles Sample Day 0

1- Sera 2- Sera Pool 3- Sera Pool 4- Sera Pool 5- Sera Pool 6- K2EDTA 7- K2 EDTA 8- NH4 Heparin 9- Na Heparin 10- Heparin Pool 11- NH4 Heparin

Stored at -20C Sample Day 0

Appendix 7b. Sample Handling

6X 1.09 3.06 4.94 6.99 8.51 2.00 5.92 3.96 7.51 1.60 5.16

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 7b Sample Handling

105.8 105.5 103.1 104.2 104.7 101.0 102.4 102.3 101.2 111.1 104.5

Appendix 10. Cross Reactivity and Drug Interference Purpose

To determine significant cross reactivity within the test. Validation of in vitro interferences by drugs.

Protocol

Global SOP no. RS35-01e, using a 4 level dilution series of human sample or pool. Global SOP no. RS02-02e Initially one level screening at the highest drug concentration was done. Drugs with cross-reactivity were rerun using the 4 level dilution series..

Materials, crossreactivity

The materials used for cross reactivity testing include the following:

Level Tested

Product

Lot #

ug/mL

Manufacturer

Code

90

Sigma

A-1593

20K0844

500

Sigma

C-3270

19H0576

Netilmicin

70

SUSP

46050

G

Oxytetracycline

40

Sigma

O-5750

112H0123

Sisomicin

200

Sigma

S-7796

58H0573

Amikacin

250

Sigma

A-2324

54H0144

Amphotericin

50

Sigma

A-4888

11K4004

Carbenicillin

500

Sigma

C-1387

60K0662

Cephalexin

500

Sigma

C-4895

99H0521

Cephalothin

63

Sigma

C-4520

57H0423

Chloramphenical

300

Sigma

C-0378

113H0860

Clindamycin

500

Sigma

C-5269

118H4099

Erythromycin

200

Sigma

E-6376

87F-0539

5-Flourorcytosine

700

Sigma

F-7129

110K4012

Furosemide

100

Sigma

F-4381

53F-0099

Kanamycin

250

Sigma

K-4000

73H0811

23

USP

Methylprednisolone

500

Sigma

M-0639

80K1859

Neomycin

100

Sigma

N-1876

66F-0151

Prednisolone

500

USP

Spectinomycin

200

Sigma

Cross Reactant Ampicillin Cephalosporin C

Methotrexate

H

L S-9007

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109H1046 GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 42 of 58

Appendix 10. Cross Reactivity and Drug Interference Materials, continued Level Tested

Product

Lot #

ug/mL

Manufacturer

Code

Streptomycin

200

Sigma

S-9137

105H07665

Tetracycline

40

Sigma

T-3383

103F-0350

Tobramycin

100

Sigma

T-1783

64H0221

Vancomycin

400

Sigma

V-2002

129H0713

Cross Reacta

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Appendix 10. Cross Reactivity and Drug Interference Materials, drug interference

The materials used for drug interference testing include the following:

Manufacturer Drug Interference

Product

Lot #

Code

Acetyl cysteine

Fluka

1039

385631/1

Ampicillin-Na

Fluka

10044

385561/1

Ascorbic acid

Aldrich

124200S

25,556-4

K-Dobesilate

Aldrich

09417PR

H1,840-2

Cyclosporine

Sigma

C-3662

107H4019

Cefoxitin

Sigma

C-4786

10K0786

Levodopa

Sigma

D-9628

39H0745

Methyldopa + 1,5

RBI

M-129

TK-590B

Metronidazole

Sigma

M-1547

29H05151

Aldrich

21,186-9

13019PR

Fluka

44577

367296/1

Acetylsalycilic acid

ALdrich

23,963-1

02928T5

Rifampicin

Fluka

83907

304324/1

Acetaminophen

Sigma

!-7085

107H0332

Sigma Fluka

I-4883

117F0797

88308

371622/1

Heparin

Phenylbutazone Doxycycline (Tetracycline)

Ibuprofen Theophylline

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 44 of 58

Appendix 10. Cross Reactivity and Drug Interference

Results

Cross Reactivity LDD= Drug

0

* Set LDD to 0 ug/mL so that all data would calculate

Drug

XXX (ug/mL)

%Cross-

XXX (ug/mL)

%Cross-

Level

Median

Reactivity

Median

Reactivity

Hitachi 917

Tested Control 1

Integra 700

5.37

5.67

Ampicillin

78

5.17

-0.26

5.28

-0.50

Ampicillin

39

5.20

-0.44

5.46

-0.54

Ampicillin

20

5.31

-0.31

5.37

-1.54

Ampicillin

10

5.29

-0.82

5.50

-1.74

Netilmicin

70

10.28

7.01

>20

Netilmicin

35

8.46

8.83

13.40

22.09

Netilmicin

18

7.09

9.83

9.02

19.14

Netilmicin

9

6.32

10.86

7.26

18.17

Oxytetracycline

37

5.25

-0.32

5.48

-0.51

Oxytetracycline

19

5.28

-0.49

5.36

-1.68

Oxytetracycline

9

5.37

0.00

5.56

-1.19

Oxytetracycline

5

5.32

-1.08

5.26

-8.86

Sisomicin

131

13.01

5.83

>20

Sisomicin

66

10.52

7.86

>20

Sisomicin

33

8.38

9.19

13.38

23.54

Sisomicin

16

6.97

9.77

8.80

19.11

Cephalosporin C

432

5.12

-0.06

5.28

-0.09

Cephalosporin C

216

5.27

-0.05

5.19

-0.22

Cephalosporin C

108 54

5.27 5.22

-0.09 -0.28

5.30 5.40

-0.34 -0.50

Cephalosporin C

10 of 23 Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

I700 H917 Average Average -0.46 -1.08

9.13

19.80

-0.47

-3.06

8.16

21.33

-0.12

-0.29

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 45 of 58

Appendix 10. Cross Reactivity and Drug Interference Results

Cross Reactivity, continued ≈ 2 µg/mL Gentamicin Spike LDD= Drug

0

* Set LDD to 0 ug/mL so that all data would calculate

Drug

XXX (ug/mL)

%Cross-

XXX (ug/mL)

%Cross-

Level

Median

Reactivity

Median

Reactivity

Hitachi 917

Tested

Integra 700

1.70

Control 2 (H2O)

1.79

Amikacin

250

1.71

0.00

1.86

0.03

Carbenicillin

500

1.55

-0.03

1.50

-0.06

Cephalexin

500

1.73

0.01

1.84

0.01

Cephalothin

63

1.70

0.00

1.76

-0.05

Clindamycin

500

1.74

0.01

1.85

0.01

5-Fluorocytosine

700

1.69

0.00

1.68

-0.02

Kanamycin

250

1.75

0.02

1.81

0.01

23

1.74

0.17

1.73

-0.26

Neomycin

100

1.73

0.03

1.80

0.01

Spectinomycin

200

1.71

0.01

1.78

-0.01

Streptomycin

200

1.70

0.00

1.86

0.04

Tetracycline

40

1.72

0.05

1.76

-0.08

Tobramycin

100

1.72

0.02

1.84

0.05

Vancomycin

400

1.76

0.02

1.78

0.00

Chloramphical

300

1.71

0.01

1.81

0.02

Erythromycin

200

1.69

0.00

1.76

0.01

Methylprednisolon

500

1.71

0.00

1.80

0.01

Prednisolone

500

1.70

0.00

1.73

0.00

Amphotericin

50

1.74

-0.02

1.84

0.16

Furosemide

100

1.75

0.00

1.76

0.00

Methotrexate

1.69

Control 3 (EtOH)

1.75

1.75

Control 4 (DMSO)

1.76

Samples were run concurrently on the Integra 700 as a reference

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 46 of 58

Appendix 10. Cross Reactivity and Drug Interference ≈ 6 µg/mL Gentamicin Spike LDD= Drug

0

* Set LDD to 0 ug/mL so that all data would calculate

Drug

XXX (ug/mL)

%Cross-

XXX (ug/mL)

%Cross-

Level

Median

Reactivity

Median

Reactivity

Hitachi 917

Tested

Integra 700

5.39

Control 2 (H2O)

5.96

Amikacin

250

5.44

0.02

5.74

-0.09

Carbenicillin

500

4.92

-0.09

5.33

-0.13

Cephalexin

500

5.41

0.00

5.69

-0.05

Cephalothin

63

5.38

-0.02

5.83

-0.21

Clindamycin

500

5.40

0.00

5.70

-0.05

5-Fluorocytosine

700

5.31

-0.01

5.71

-0.04

Kanamycin

250

5.39

0.00

5.78

-0.07

23

5.44

0.22

5.87

-0.39

Neomycin

100

5.39

0.00

5.75

-0.21

Spectinomycin

200

5.45

0.03

5.82

-0.07

Streptomycin

200

5.40

0.01

5.67

-0.15

Methotrexate

Tetracycline

40

5.43

0.10

5.84

-0.30

Tobramycin

100

5.38

-0.01

5.69

-0.27

Vancomycin

400

5.34

-0.01

5.86

-0.02

Chloramphical

300

5.35

-0.01

5.70

-0.06

Erythromycin

200

5.39

0.00

5.72

-0.08

Methylprednisolon

500

5.39

0.00

5.85

-0.01

Prednisolone

500

5.37

0.00

5.93

0.01

5.38

Control 3 (EtOH)

5.88

5.40

Control 4 (DMSO)

5.84

Amphotericin

50

5.39

-0.02

5.61

-0.46

Furosemide

100

5.36

-0.04

5.94

0.10

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 47 of 58

Appendix 10. Cross Reactivity and Drug Interference Drug Interference: ≈ 2 µg/mL Gentamicin Spike Hitachi 917 Drug

C1

Aceta (C1)

C1 %

C2

ug/mL

Median (ug/mL)

Interference

ug/mL

150

1.68

0.6

30

1.68

0.6

Ampicillin-Na

1000

1.64

-1.8

200

1.64

-1.8

Ascorbic Acid

300

1.67

0.0

30

1.64

-1.8

Cyclosporine

5

1.69

1.2

1

1.68

0.6

Cefoxitin

2500

1.68

0.6

250

1.69

1.2

Levodopa

20

1.73

3.6

4

1.74

4.2

Methyldopa + 1,5

20

1.69

1.2

2

1.76

5.4

Rifampicin

60

1.67

0.0

20

1.65

-1.2

1.67

Control 1 Acetyl cysteine

C2 %

Median (ug/mL) Interference 1.67

1.75

Control 2

Aceta (C2)

1.75

Acetaminophen

200

1.67

-4.6

20

1.65

-5.7

Ibuprofen

500

1.63

-6.9

50

1.66

-5.1

Theophylline

100

1.68

-4.0

10

1.66

-5.1

1.75

1.75

Control 3 K-Dobesilate

200

1.79

2.3

20

1.75

0.0

Metronidazole

200

1.75

0.0

10

1.72

-1.7

Phenylbutazone

400

1.72

-1.7

100

1.76

0.6

Doxycycline (Tetra

50

1.67

-4.6

10

1.77

1.1

Acetylsalicylic Acid

1000

1.71

-2.3

300

1.73

-1.1

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 48 of 58

Appendix 10. Cross Reactivity and Drug Interference Drug Interference: ≈ 6 µg/mL Gentamicin Spike

Hitachi 917 Drug

C1

Aceta (C1)

C1 %

C2

ug/mL

Median (ug/mL)

Interference

ug/mL

5.07

Control 1

Aceta (C2)

C2 %

Median (ug/mL) Interference 5.07

150

5.00

-1.4

30

4.91

-3.2

Ampicillin-Na

1000

4.98

-1.8

200

5.06

-0.2

Ascorbic Acid

300

5.13

1.2

30

5.09

0.4

Cyclosporine

5

4.98

-1.8

1

5.08

0.2

Cefoxitin

2500

5.04

-0.6

250

5.08

0.2

Levodopa

20

5.08

0.2

4

5.11

0.8

Methyldopa + 1,5

20

5.03

-0.8

2

5.22

3.0

Rifampicin

60

5.00

-1.4

20

4.91

-3.2

Acetaminophen

200

5.19

1.2

20

5.01

-2.3

Ibuprofen

500

5.08

-1.0

50

5.04

-1.8

Theophylline

100

5.05

-1.6

10

4.94

-3.7

Acetyl cysteine

5.13

Control 2

5.13

5.04

5.04

Control 3 K-Dobesilate

200

5.18

2.8

20

5.09

1.0

Metronidazole

200

5.23

3.8

10

5.07

0.6

Phenylbutazone

400

5.11

1.4

100

5.09

1.0

Doxycycline (Tetra

50

5.08

0.8

10

5.15

2.2

Acetylsalicylic Acid

1000

5.12

1.6

300

5.24

4.0

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App 10 Xreactiv Page 49 of 58

Appendix 12. Additional Interference (Endogenous) Purposes

To assess the interference of total protein concentration, endogenous cholesterol, triglycerides and rheumatoid factor on the Gentamicin assay.

Protocol

The recovery of analyte spiked at the MDL(s) is assessed for low and high protein, cholesterol, triglyceride and RF samples vs. a reference normal human sample or pool spiked with analyte.

Materials

Normal human pool for total protein Cholesterol samples ranging from 300 mg/dL: ID. Nos. 1605108, 1605258, 1604514, 1605086, 1604518, 1604526 Triglyceride samples ranging from 1000 mg/dL: ID. Nos. 1604085, 1605558, 1605559, Follas 4, 1604571, Follas 3, 1605624 RF samples ranging from ~20 to 200 IU/mL: ID. Nos. 160536, 1604478, 1605671, 1605336, 1605334, 1605672

Acceptance Criteria

Interference is considered to be insignificant if the positive or negative bias observed on an analyte at the 2 and 6 ug/mL decision level is less than or equal to 10 % at the following level of interferents: •

total protein level of 2 - 12 g/dL

•

cholesterol level of 300 mg/dL

•

triglycerides at a level of 1000 mg/dL

•

Rheumatoid Factor (RF) level of 100 IU/mL

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GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App12 AdditionInterfer Page 50 of 58

Appendix 12. Additional Interference (Endogenous)

Total Protein Interference Result

Gentamicin shows no significant interference up to a level of 12.2 g/dL total protein. The results (10%

Bias vs Control

GENT2_c501_Appendix Filename: Gent-ML-PEP Tab: App12 AdditionInterfer Page 51 of 58

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Page 52 of 58

273 383 333 130 279 308 259 386 318 362

1604514

1605086

1604518

Ctl High

1605108

1605258

1604514

1605086

1604518

1604526

305

593

244

78

575

260

289

588

246

620 6.59

6.84

6.81

6.62

6.75

6.75

6.85

1.78

1.84

1.72

1.82

1.76

1.81

Observed Analyte Result

96.2

99.9

99.4

96.6

98.5

98.5

100.0

98.3

101.7

95.0

100.6

97.2

100.0

Percent * Denotes Recovery deviation >10% vs. Control

-0.26

-0.01

-0.04

-0.23

-0.10

-0.10

0.00

-0.03

0.03

-0.09

0.01

-0.05

0.00

Bias vs Control

17 of 23

Gentamaicin shows no significant interference up to a level of 360 mg/dL endogenous cholesterol. The results are within specifications for cholesterol. No endogeneous samples were available to test higher values to set higher claim.

324

1605258

300

Endogenous Cholesterol Interference Results

294

1605108

609

Interferent Conc. (mg/dL) 138

Ctl Low

Sample ID. No.

The following table shows the effect of endogenous cholesterol on percent recovery of Gentamicin at values of ~2 ug/mL and ~6 ug/mL.

94

Lipemia Index

Endogenous Cholesterol Interference Data

Appendix 12. Additional Interference (Endogenous)

6.22

6.21

6.27

6.38

6.44

6.23

6.43

1.57

1.57

1.68

1.6

1.52

1.68

CEDIA Ref Value

6.45

6.33

6.97

6.16

6.55

6.43

7.03

1.82

1.76

1.82

1.74

1.84

1.95

FP Ref Value

GENT2_c501_Appendix Gent-ML-PEP Tab: App12 AdditionInterfer (2)

Chol > 300, LDL > 200

Chol > 300, LDL > 200

Chol > 300, Trig > 900

LDL > 200

Chol > 300, LDL > 200

Trig > 300

Chol > 300, LDL > 200

Chol > 300, Trig > 900

LDL > 200

Chol > 300, LDL > 200

Trig > 300

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Page 53 of 58

812 1060 864 131 130 586 771 1060 864 1466 2940

1605559

Follas 4

1604571

Ctl High

1604085

1605558

1605559

Follas 4

1604571

Follas 3

1605624

732

16

792

85

160

656

704

24

770

48

2756 4.42

6.79

6.72

6.6

6.81

6.8

6.78

6.82

1.8

1.71

1.76

1.78

1.85

1.79

Observed Analyte Result

64.8

99.6

98.5

96.8

99.9

99.7

99.4

100.0

100.6

95.5

98.3

99.4

103.4

100.0

*

Percent * Denotes Recovery deviation >10% vs. Control

-2.40

-0.03

-0.10

-0.22

-0.01

-0.02

-0.04

0.00

0.01

-0.08

-0.03

-0.01

0.06

0.00

Bias vs Control

18 of 23

Gentamicin shows no significant interference up to a level of 1466 mg/dL triglycerides. The results of 300, Chol > 300

Trig > 300

Trig > 300

Trig > 300, Chol > 300, LDL > 200

Trig > 300

Trig > 300

Trig > 300

Trig > 300, Chol > 300, LDL > 200

Trig > 300

Trig > 300

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Page 54 of 58

Endogenous RF Interference Results

L > 300

L > 300

L > 300

Endogenous RF Interference Data

47 131 199

1604478

1605671

1605672 6.32

20 47 131 142 196 199

1605367

1604478

1605671

1605336

1605334

1605672

97.2

-0.18

-0.52

-0.34

-0.11

-0.12

-0.11

0.00

-0.08

-0.10

-0.10

-0.08

0.00

Bias vs Control

5.88

6.09

6.02

5.93

5.86

5.96

6.3

1.63

1.59

1.52

1.56

1.67

CEDIA Ref Value

19 of 23

Gentamicin shows no significant interference up to a level of 199 IU/mL Rheumatoid Factor. The results are within specifications for RF interference.

5.97

6.21

6.21

6.41

5.88

6.06

6.35

1.85

1.71

1.75

1.76

2.01

FP Ref Value

Note: Samples were double spiked with Theophylline because of sample volume constraints

6.25

91.9

94.7

5.91

98.3

6.09

98.1

98.3

100.0

95.6

94.5

94.5

95.6

100.0

Percent * Denotes Recovery deviation >10% vs. Control

6.32

6.31

6.43

Ctl Hi

1.75

1.73

1.73

1.75

20

1605367

Observed Analyte Result

1.83

Interferent Conc. (mg/dL)

Ctl Lo

Sample ID. No.

The following table shows the effect of endogenous Rheumatoid Factor on the percent recovery of Gentamicin at ~ 2 ug/mL and ~ 6 ug/mL.

Appendix 12. Additional Interference (Endogenous)

GENT2_c501_Appendix Gent-ML-PEP Tab: App12 AdditionInterfer (2)

Trig > 300

Trig > 300

Summary References Hitachi 911

The following laboratory notebooks and data binders contain supporting information and data: Laboratory notebook no: Data binder no.:

33727 01D76

CAEv version:

2.51h

CAEv study name:

GENT THEO MASTER LOT PEP

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GENT2_c501_Appendix Filename: H 911 Gent A1 App Report Approved 3-11 Tab: Summary Page 55 of 58

Appendix 8. Standard Curve and On-Board Stability (SCS / OBS) Purpose

To assess SCS/OBS vs. the product specification requirements using fresh and aged reagen

Protocol

Global SOP numbers: RS25-02e and RS42-01e

Materials

Refer to Materials Section Between time points, reagent bottles remained on board the analyzer, uncapped.

Acceptance Criteria

Minimum SCS of 7 days based on recovery of controls within ± 20% of mean established at T=0. Minimum OBS of 30 days based on recovery of controls within ± 20% of mean established at T=0.

Results

The observed SCS and OBS met the minimum product specifications of 7 days and 30 days, respectively.

21 of 23

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Page 56 of 58

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Page 57 of 58

CONC ug/mL

STANDARDS

6.20 6.11 6.06 6.12

8.05 8.13 8.16 8.11

Rep 1 Rep 2 Rep 3 Mean % Deviation from Day 0 % Deviation from Target % Recovery Day 0

Rep 1 Rep 2 Rep 3 Mean % Deviation from Day 0 % Deviation from Target % Recovery Day 0

Level III

Level II

3.55 3.44 3.68 3.56

Rep 1 Rep 2 Rep 3 Mean % Deviation from Day 0 % Deviation from Target % Recovery Day 0

Level I

Control Lot 62019501

11/30/01

A

DATE

Stability Days

0

B

0

8.29 8.24 8.11 8.21

6.40 6.40 6.49 6.43

3.76 3.76 3.56 3.69

CONC ug/mL

11/30/01

K

12416

-610

UNCAPPED on 911

S1Abs

C

0

8.23 8.23 8.31 8.26

6.51 6.47 6.49 6.49

3.80 3.87 3.72 3.80

CONC ug/mL

11/30/01

2135.70

A

Day 0 (Bottle Set RED A) Cal Monitor:

8.22 8.21 8.22 8.22

7.98

Op Target

6.32

Op Target

8.32 8.25 8.29 8.29

6.41 6.36 6.38 6.38

6.53 6.45 6.49 6.49

3.40

Op Target

Means

CONC ug/mL

T=0

Totals

3.69 3.70 3.65 3.68

CONC ug/mL

11/30/01

0

3.64 3.72 3.65 3.67

D

1.5625

B

A

3

3.71

2.88 99.9

-0.09

0.35 3.34 100.4

8.32 8.31 8.00 8.21

8.31 8.38 8.05 8.25

4.75 103.7

2.09 3.11 102.1

6.59 6.66 6.61 6.62

6.51 6.46 6.58 6.52

7.35 99.2

-0.79

7.35 99.2

-0.79

CONC ug/mL % dev Day 5

12/5/01

3.66 3.64 3.65 3.65

B

5

3.54 3.73 3.68 3.65

CONC ug/mL % dev Day 3

12/3/01

1. Standard Curve Stability / Cassette On-Board Stability Roche-TDM OnLine Gentamicin Assay / Hitachi 911 S/N 9204-19

Standard Curve and On-Board Stabilities: With Means and Target

C

3.22 100.2

0.23

8.40 8.24 8.07 8.24

2.58 101.6

1.57

6.46 6.53 6.46 6.48

-0.10 92.3

-7.68

3.29 3.43 3.47 3.40

CONC ug/mL % dev Day 7

12/7/01

7 D

-2.38 94.8

-5.20

7.78 7.88 7.71 7.79

-3.85 95.2

-4.80

5.98 6.14 6.11 6.08

-6.57 86.3

-13.66

3.06 3.19 3.28 3.18

CONC ug/mL % dev Day 10

12/10/01

10

14

A

17

19

3/2002

12/31/02

21

26

New Cal.

28

31

-1.17 96.0

-4.03

7.88 7.98 7.80 7.89

-2.37 96.7

-3.34

6.10 6.15 6.26 6.17

-6.18 86.7

-13.30

3.16 3.15 3.26 3.19

CONC ug/mL % dev Day 12

22/23 of 23

-5.35 91.9

-8.08

7.63 7.63 7.40 7.55

-8.12 91.0

-9.03

5.66 5.87 5.89 5.81

-12.84 80.5

-19.46

2.95 2.97 2.97 2.96

CONC ug/mL % dev Day 14

2.46 99.5

-0.50

8.32 8.16 8.05 8.18

0.32 99.3

-0.68

6.34 6.34 6.34 6.34

-2.25 90.3

-9.67

3.26 3.33 3.38 3.32

CONC ug/mL % dev Day 17

1.55 98.6

-1.39

8.17 8.20 7.94 8.10

1.37 100.4

0.37

6.42 6.38 6.42 6.41

4.61 96.7

-3.33

3.70 3.52 3.45 3.56

CONC ug/mL % dev Day 19

-1.09 96.1

-3.94

7.88 7.91 7.89 7.89

3.64 102.6

2.61

6.53 6.56 6.56 6.55

5.69 97.7

-2.33

3.58 3.62 3.58 3.59

CONC ug/mL % dev Day 21

-10.61 86.8

-13.19

7.17 7.21 7.02 7.13

-12.24 86.9

-13.11

5.54 5.61 5.49 5.55

-15.98 77.6

-22.36

2.86 2.89 2.82 2.86

CONC ug/mL % dev Day 26

-0.71 96.4

-3.58

7.89 8.11 7.77 7.92

-3.85 95.2

-4.80

6.17 6.02 6.04 6.08

-2.75 89.9

-10.12

3.31 3.26 3.35 3.31

CONC ug/mL % dev Day 28

0.50 97.6

-2.40

8.12 8.08 7.86 8.02

-2.90 96.1

-3.86

6.14 6.18 6.09 6.14

-0.98 91.5

-8.49

3.17 3.43 3.50 3.37

CONC ug/mL % dev Day 31

B C D A C D A B 12/12/01 12/014/01 12/17/01 12/19/01 12/21/01 12/26/01 12/28/01 12/31/01

New Cal.

Exp:

12

Exp:

Calibrator Lot:

61740801

Reagent Master Lot:

-5.10 92.2

-7.84

7.63 7.51 7.58 7.57

-7.81 91.3

-8.72

5.78 5.82 5.88 5.83

-6.27 86.6

-13.39

3.11 3.22 3.23 3.19

CONC ug/mL % dev Day 33

1/2/02

33 C

-12.16 85.3

-14.69

7.18 7.04 6.81 7.01

-14.87 84.3

-15.72

5.46 5.43 5.25 5.38

-18.14 75.7

-24.35

2.90 2.67 2.78 2.78

CONC ug/mL % dev Day 35

1/4/02

35 C

35

1.46 98.5

-1.47

7.98 8.09 8.22 8.10

2.85 101.8

1.83

6.56 6.53 6.41 6.50

4.31 96.4

-3.60

3.66 3.55 3.43 3.55

CONC ug/mL % dev Day 35

1/4/02

New Cal.

D

A

-12.87 84.6

-15.38

7.00 7.12 6.74 6.95

-16.30 82.9

-17.13

5.25 5.26 5.36 5.29

-21.08 72.9

-27.07

2.69 2.69 2.67 2.68

CONC ug/mL % dev Day 40

1/9/02

40

A

40

0.84 97.9

-2.08

7.94 8.14 8.06 8.05

-1.79 97.2

-2.77

6.21 6.25 6.16 6.21

2.16 94.4

-5.59

3.64 3.42 3.36 3.47

CONC ug/mL % dev Day 40

1/9/02

New Cal

B

42

6.47 103.4

3.40

8.61 8.54 8.34 8.50

7.33 106.3

6.27

6.82 6.78 6.75 6.78

12.65 104.1

4.10

3.90 3.79 3.80 3.83

CONC ug/mL % dev Day 42

1/11/02

GENT2_c501_Appendix Filename: H 911 Gent A1 App Report Approved 3-11 Tab: App 8 OBS SCS Chart

-5.76 91.5

-8.49

7.62 7.51 7.43 7.52

-7.86 91.2

-8.77

5.85 5.77 5.85 5.82

-10.00 83.2

-16.83

3.07 3.06 3.05 3.06

CONC ug/mL % dev Day 38

1/7/02

38

Day 40

Day 35

Day 28

Day 17

Day 0

27293 27805 27549 -1.8%

28501 29254 28878 -2.6%

30208 30809 30509 -2.0%

14822 15236 15029 -2.7%

15786 16374 16080 -3.6%

16894 17292 17093 -2.3%

Avg. %rep. dev

Avg. %rep. dev

Avg. %rep. dev

Avg. %rep. dev

0

2000

4000

6000

8000

10000

12000

14000

16000

0

24373 25635 25004 -4.9%

12690 13704 13197 -7.4%

Avg. %rep. dev

18000

23569 24448 24009 -3.6%

11996 12704 12350 -5.6%

s1

Calibration Monitor Data

Day 0

2

16582 16736 16659 -0.9%

15029 15769 15399 -4.7%

14086 14447 14267 -2.5%

12026 12643 12335 -4.9%

11012 11609 11311 -5.1%

s2

14680 15034 14857 -2.4%

13255 13507 13381 -1.9%

11466 11936 11701 -3.9%

9470 10261 9866 -7.7%

8307 9025 8666 -8.0%

s3

27579 27972 27776 -1.4%

25631 26018 25825 -1.5%

23569 24204 23887 -2.6%

20939 21960 21450 -4.6%

19601 20411 20006 -4.0%

8732 9302 9017 -6.1%

6919 7373 7146 -6.2%

5523 5686 5605 -2.9%

4357 4683 4520 -7.0%

3603 3941 3772 -8.6%

s4

Day 17

6

Day 35

CALIBRATOR CONCENTRATION

4

Day 28

8

21052 21724 21388 -3.1%

18735 19351 19043 -3.2%

17108 17322 17215 -1.2%

15359 15760 15560 -2.5%

14373 14858 14616 -3.3%

DOSE-RESPONSE CURVES

29788 30014 29901 -0.8%

27512 28562 28037 -3.7%

26453 26956 26705 -1.9%

23646 24440 24043 -3.2%

22462 23256 22859 -3.4%

s5

Day 40

3939 4331 4135 -9.1%

2975 3170 3073 -6.2%

2308 2408 2358 -4.2%

1796 1927 1862 -6.8%

1559 1652 1606 -5.6%

22/23 of 23

10

15654 16227 15941 -3.5%

14366 14651 14509 -1.9%

13449 13639 13544 -1.4%

12639 12663 12651 -0.2%

12203 12241 12222 -0.3%

2011 2102 2057 -4.3%

1566 1671 1619 -6.3%

1232 1279 1256 -3.7%

981 1032 1007 -4.9%

845 894 870 -5.5%

s6

12

13431 13553 13492 -0.9%

12605 12985 12795 -2.9%

12245 12361 12303 -0.9%

11468 11586 11527 -1.0%

11207 11358 11283 -1.3%

Calibration Monitor The calibration monitor data and standard curve graphs from the Gentamicin On-board and Standard curve stability study using OnLine Gentamicin reagent on and Graphics for the Hitachi 911: OBS/SCS

ABSORBANCE SIGNAL

Doc No: C_c501_GENT-CH_AR_004771 Version: 1.0, Effective, Date: 24.05.2006 14:28:52

Page 58 of 58 GENT2_c501_Appendix Filename: H 911 Gent A1 App Report Approved 3-11 Tab: App 8 OBS SCS Chart