1 General Information -...

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1 General Information

1.1 Intended Use of Bilirubin Direct Gen. 2

This is an in-vitro test for the quantitative determination of direct bilirubin in serum and plasma on Roche/Hitachi cobas c 111/311/501/502/701/702 , Roche/Hitachi 902, Modular P and COBAS INTEGRA® 400/800 systems.

1.2 Clinical Significance / Clinical Utility of Direct Bilirubin Testing

Bilirubin is a breakdown product of heme which is the prosthetic group of hemoglobin. Bilirubin is

excreted in bile and to a tiny extent in urine. Clearance of bilirubin is facilitated by conjugation with

glucuronic acid , making the bilirubin water-soluble. The conjugated bilirubin (direct bilirubin) is

secreted into the bile, which is excreted into the intestine.

Increased total bilirubin causes jaundice of different origin:

Prehepatic Increased bilirubin production. This can be due to a number of causes, including hemolytic anemias and internal hemorrhage.

Hepatic Deficiencies in bilirubin metabolism (e.g. reduced hepatocyte uptake, impaired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin), as with liver cirrhosis or viral hepatitis.

Posthepatic Obstruction of the bile ducts, reflected as deficiencies in bilirubin excretion.

The diagnosis is narrowed down further by looking at the levels of direct bilirubin:

If direct (i.e. conjugated plus δ-) bilirubin is normal, then the problem is an excess of unconjugated bilirubin, and the location of the problem is upstream of bilirubin excretion. Hemolysis, viral hepatitis, or liver cirrhosis can be suspected.

If direct bilirubin is elevated, then the liver is conjugating bilirubin normally, but is not able to excrete it. Bile duct obstruction by gallstones or cancer can be suspected.

Test priniple

This new direct bilirubin reagent is based on the diazo method. Conjugated and δ-bilirubin (direct bilirubin) react directly with 3.5 dichlorophenyl diazonium salt in acid buffer to form the red-colored azobilirubin.

Standardization

This new method has been standardized against the manual test performance using the Jendrassik-Grof method.

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1.3 Discont. Direct Bilirubin Kits

The following products will be replaced by Bilirubin Direct Gen. 2:

Product Cat. No. System

Bilirubin Direct SYS 1 11 109 774 216 Roche/Hitachi

902/904/911/912 Bilirubin Direct HIT 917/MODULAR

P 11 555 413 216

Roche/Hitachi 917/MODULAR P

cobas c pack Bil-D 20 737 496 322 cobas c 311/501/502,

COBAS INTEGRA

cobas c pack large Bil-D 05 172 063 190 cobas c 701/702

cobas c 111 Bilirubin direct 05 401 747 190 cobas c 111

For the USA only, please note:

Bilrubin, Direct (RDC), cat. nos. 12 217 716 001 (Roche/Hitachi Modular P),

11 986 660 018 11 (Modular P/D, R1) and 11 986 651 018 (Roche/Hitachi Modular P/D, R2) will

stay in the market. 1.4 Bilirubin Direct Gen. 2 Portfolio

Product Name Cat. No. System

Bilirubin Direct Gen. 2 (HIT 902)

05 588 987 190

Roche/Hitachi 902

Bilirubin Direct Gen. 2 (MODULAR P)

05 589 037 190

Roche/Hitachi Modular P

Bilirubin Direct Gen. 2 (cobas c, C. INTEGRA)

05 589 061 190

cobas c 501/502/311, Integra 400/800

Bilirubin Direct Gen. 2 (cobas c 701/702)

05 168 384 190

cobas c 701/702

Bilirubin Direct Gen. 2 (cobas c 111)

05 589 134 190

cobas c 111

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1.5 Bilirubin Direct Gen. 2 Kit Concept

Kit Cat. No. Volume on

Label Bottle

Format

Tests per Kit

SYS 1 05 588 987 190 R1: 50 mL 50 mL

4 x 173 R2: 12 mL 22 mL

MOD P 05 589 037 190 R1: 66 mL 70 mL

4 x 304 R2: 16 mL 20 mL

cobas c 311/501/502,

C. INTEGRA 400/800

05 589 061 190/529

Not required

60 mL/20 mL

350

cobas c 701/702 05 168 384 190 Not

required 90 mL/30

mL 500

cobas c 111 05 589 134 190 R1: 8.3 mL 22 mL

2 x 50 R2: 3.4 mL 22 mL

1.6 Bilirubin Direct Gen. 2, Cassette Short Name / Cassette ID / ACN

Instrument c 311/501 c 502 C 701/702

Application Code Number (ACN) 734 8734 8734

Cassette Short Name BILD2 BILD2 BILD2

Cassette ID 07 7479 0 07 7479 0 03 7479 0

1.7 Summary of Improvements attained by Bilrubin Direct Gen. 2

Overall improved on-board stability No falsely low test results due to heparin interference (as with current BIL-D reagent) Suited for analysis of neonatal samples Improvement of accuracy to distinctly minimize BILD > BILT problem Ready-to-use reagents, manual preparation not required No recovery problems in Precinorm U, Precinorm U plus, PreciControl Clin. Chem. Multi 1 Homogeneous calibration on all platforms (2-point each lot) instead of using different

calibration factors (c 501/502, c 311, and c701/702) or blanking at frequent intervals (Hitachi, blanking every 24 h/after reagent bottle change)

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1.8 Bilirubin Direct Gen.2 Supplementary Facts

1. Availability ex Mannheim June 01, 2011

2. Kit shelf life 12 months for the introduction lot; scheduled for next lots: 18 months

3. Storage conditions 2 - 8 °C

4. Shipping conditions 2 – 8 °C

5. Reagent composition See package inserts (annex)

6. Reagent stability on-board 6 weeks

7. System software version Current versions

8. Pipetting volumes

Sample Hit. 902: 14 µL Mod. P: 10 µL INTEGRA: 7 µL c 311/501/502/701/702: 6.7 µL c 111: 7 µL

Reagent (R1/R2) Hit. 902: 250/50 µL Mod. P: 180/36 µL INTEGRA: 120/24 µL C 311/501/502/701/702: 120/24 µL C 111: 120/24 µL

9. Sample material Serum, Li-heparin, K2-, K3-EDTA plasma

10. Sample stability 2 days at 15-25 °C 7 days at 2-8 °C

6 months at (-15)-(-25) °C

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1.8 Bilirubin Direct Gen.2 Supplementary Facts, cont.

11. Calibration mode 2-point calibration

12. Calibration frequency 2-pointcalibration after reagent lot change or as required following quality control procedures

13. Calibrator C.f.a.s., 10 759 350 190

14. Controls

Precinorm U (10 171 743 122) Precipath U (10 171 778 122) Precinorm U plus (12 149 435 122) Precipath U plus (12 149 443 122)

Precibil (10 158 046 122) PreciControl ClinChem Multi 1 (05 117 003 190) PreciControl ClinChem Multi 2 (05 117 216 190)

15. External quality assessment Reference method for direct bilirubin not available. Recovery with BILD2 will be higher compared to current direct bilirubin tests (separate peer group required).

16. Expected values ≤ 5 μmol/L (≤ 0.30 mg/dL)

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2 Performance Data

2.1 cobas c 501

2.1.1 c 501, Overview of Specified vs. Measured Data

Feature Specification Measured Data Spec. met

Low end of measuring range

1.5 µmol/L 0.9 µmol/L YES

High end of measuring range 291 µmol/L

400 µmol/L YES

Lower detection limit 1 µmol/L 0.2 µmol/L YES Limit of blank 1 µmol/L 0.1 µmol/L YES Limit of detection 1.5 µmol/L ≤ 0.9 µmol/L YES Limit of quantification 3 µmol/L ≤ 2.8 µmol/L YES Within-run imprecision

≤ 10 µmol/L: SD ≤ 0.30 > 10 µmol/L: CV ≤ 3 %

Mean SD CV (%) Precinorm U 16.2 0.09 0.6 YES Precipath U 42.0 0.14 0.3 YES

Human serum 1 2.5 0.07 2.9 YES Human serum 2 174 0.55 0.3 YES Human serum 3 280 0.84 0.3 YES

Recovery in controls Median 100 % ± 10 100.5 % YES

<17 µmol/L: < + 1.7 Repeatability acc. to CLSI

< 10 µmol/L: SD < 0.30 > 10 µmol/L: CV < 3 %



Total imprecision acc. to CLSI

< 10 µmol/L: SD <0.50 > 10 µmol/L: CV < 5 %



2.1.1 c 501, Overview of Specified vs. Measured Data, cont.


Method comparison Analyzer / Method DBIL/MOD P vs. BILD2/c

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501 Slope (Passing/Bablok) Slope and intercept not

specified 1.010 YES

Intercept 1.17 µmol/L YES Coefficient of Correlation r ≥ 0.975 0.997 YES

Analyzer / Method BILDF vs. BILD2/c 501 Slope (Passing/Bablok) 0.988

Intercept not specified 6.24 µmol/L Coefficient of correlation 0.995

Analyzer / Method c 501 vs. I. 800/ BILD2 Slope (Passing/Bablok) 1.00 ± 0.05 0.995 YES

Intercept < 5.1 µmol/L 0.42 µmol/L YES Coefficients of correlation r ≥ 0.975 0.999 YES

Analyzer / Method c 501 vs. MODP/BILD2 Slope (Passing/Bablok) 1.00 ± 0.05 1.013 YES

Intercept < 5.1 µmol/L -0.54 µmol/L YES Coefficient of correlation r ≥ 0.975 1.000 YES

Analyzer / Method BILD2 MP lot vs. BILD2

P2 lot /c 501 Slope (Passing/Bablok) 1.00 ± 0.05 0.991 YES

Intercept < 5.1 µmol/L 0.25 µmol/L YES Coefficient of correlation r ≥ 0.975 0.999 YES

Anticoagulants

Median recovery

(%)

Median deviation (µmol/L)

Li-Heparin

≤ 34.2 µmol/L: < 3.4 > 34.2 µmol/L: < ± 10 %

102 0.31 YES Li-Heparin, half-filled

tubes -0.01 YES

K2-EDTA 100 -0.07 YES

K2-EDTA, half-filled tubes - -0.49 YES

K3-EDTA 100 -0.24 YES

K3-EDTA, half-filled tubes - -0.49 YES Gel separation tube 104 0.43 YES

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2.1.1 c 501, Overview of Specified vs. Measured Data, cont.


On board stability 6 weeks 6 weeks YES Calibration frequency

during 6 weeks on-board no no YES after cassette change no no YES

after lot change yes yes YES

Interference, endogeneous

No interf. up to index of

DBIL concn.

(µmol/L) Lipemia, DBIL low ≤ 750 L-Index 879 34.0 YES

Lipemia, DBIL high ≤ 750 L-Index 1100 80.0 YES Hemoglobin, DBIL low ≤10 H-Index 14 34.0 YES

Hemoglobin, DBIL high ≤10 H-Index 25 110 YES

Therap. drug interference recovery ≤ ± 10 %

Phenylbutazone interferes

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2.1.2 c 501, Method Comparison

BILD2, c 501 vs. Mod P

c 501, BILDF vs. BILD2

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BILD2/ c 501 vs. DBIL/Mod P

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BILD2, I. 800 vs. c 501

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BILD2, c 501, MP lot vs. P2 lot

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7.1.3 c 501, Linearity

c 501 (diluent: serum)

Target Direct Bil. [µmol/L]

Measured

Direct Bil.

[µmol/]

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2.1.4 c 501, Endogeneous Interferences

c 501, Hb Interference at approx. 34 µmol/L of Direct Bilirubin

H Serum Index

Dotted lines represent acceptance limits.

c 501, Hb Interference at approx. 110 µmol/L of Direct Bilirubin

H Serum Index


Deviation

[µmol/L]

Recovery

[%]

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c 501, Lipemia Interference at approx. 34 µmol/L of Direct Bilirubin

L Serum Index


c 501, Lipemia Interference at approx. 80 µmol/L of Direct Bilirubin

L Serum Index


Deviation

[µmol/L]

Recovery

[%]

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2.1.5 c 501, Suitability of Plasma as Sample Material

c 501, Li-Heparin Plasma vs. Serum

c 501, K2-EDTA Plasma vs. Serum

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c 501, K3-EDTA Plasma vs. Serum

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2.2 cobas c 311



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Low end of measuring range 1.5 µmol/L

0.2 µmol/L YES

Lower detection limit 1 µmol/L 0.3 µmol/L YES Limit of blank 1 µmol/L 0.1 µmol/L YES Limit of detection 1.5 µmol/L 0.8 µmol/L YES Limit of quantification 3 µmol/L 2.3 µmol/L YES Within-run precision

≤ 10 µmol/L: SD ≤ 0.30 > 10 µmol/L: CV ≤ 3 %

Mean SD CV ( % ) Precinorm U 15.8 0.09 0.6 YES Precipath U 39.8 0.10 0.3 YES


Recovery in controls Median 100 % ± 10

102.4 % YES < 17 µmol/L: < + 1.7

Method comparison Analyzer / Method c 311 vs. c 502/BILD2 YES

Slope (Passing/Bablok) 1.00 ± 0.05 1.020 YES Intercept < 5.1 µmol/L 0.354 µmol/L YES

Coefficient of correlation r ≥ 0.975 1.000 YES

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BILD2, c 501 vs. c 311

2.3 cobas c 701


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1 µmol/L YES

Lower detection limit 1 µmol/L 0.3 µmol/L YES Limit of blank 1 µmol/L 0.3 µmol/L YES Limit of detection 1.5 µmol/L 1.0 µmol/L YES Limit of quantification 3 µmol/L 2.3 µmol/L YES Within-run precision Mean SD CV ( % )

Precinorm U 15.7 0.13 0.9 YES Precipath U ≤ 10 µmol/L: SD ≤ 0.30 42.3 0.13 0.3 YES

Human serum 1 > 10 µmol/L: CV ≤ 3 % 4.9 0.09 1.9 YES Human serum 2 188 0.38 0.2 YES Human serum 3 285 0.75 0.3 YES


101.3 % YES <17 µmol/L: < + 1.7

Method comparison Analyzer / Method c 501 vs. c 701/BILD2 YES



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BILD2, c 501 vs. c 701

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2.4 cobas c 111



Low end measuring range 1.5 µmol/L 1.1 µmol/L YES Lower detection limit 1 µmol/L 0.4 µmol/L YES High end measuring range 291 µmol/L 362 µmol/L YES Limit of blank 1 µmol/L 0.4 µmol/L YES Limit of detection 1.5 µmol/L 1.1 µmol/L YES Limit of quantitation 3 µmol/L 1.9 µmol/L YES Within-run precision Mean SD CV (%)




97.5 % YES < 17 µmol/L: < + 1.7

Method comparison Analyzer / Method I. 800 vs. c 111/BILD2 YES



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I. 800 vs. c 111, BILD2

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2.5 Roche/Hitachi Modular P

2.5.1 Mod P, Overview of Specified vs. Measured Data


Low end measuring range 1.5 µmol/L 0.5 µmol/L YES High end measuring range 291 µmol/L 361 µmol/L YES Lower detection limit 1 µmol/L 0.2 µmol/L YES Limit of blank 1 µmol/L 0.1 µmol/L YES Limit of detection 1.5 µmol/L 0.5 µmol/L YES Limit of quantification 3 µmol/L 0.7 µmol/L YES Within-run precision Mean SD CV ( % )

Precinorm U 16.6 0.12 0.70 YES PrecipathU ≤ 10 µmol/L: SD ≤ 0.30 40.6 0.25 0.60 YES

Human serum 1 > 10 µmol/L: CV ≤ 3 % 2.2 0.04 1.90 YES Human serum 2 82.6 0.34 0.40 YES Human serum 3 266.0 1.44 0.50 YES

Recovery in controls 99.1 % YES Median 100 % ± 10

< 17 µmol/L: < + 1.7 Method comparison

Analyzer / Method Mod P/current DBIL vs. BILD2 YES Slope (Passing/Bablok) slope and intercept not

specified 1.013 YES

Intercept 0.54 µmol/L YES Coefficient of correlation r ≥ 0.975 0.996 YES

Analyzer / Method Mod P vs. c 501/BILD2 YES



Analyzer / Method Mod P vs. I. 800/BILD2 YES Slope (Passing/Bablok) 1.00 ± 0.05 1.011 YES


On board stability 6 weeks 6 weeks YES Calibration

during 6 weeks on board no no YES bottle change no no YES

lot change 2 point yes YES 2.5.2 Modular P, Method Comparison

Mod P, DBIL vs. BILD2

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BILD2, Mod P vs. c 501

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BILD2, Mod P vs. I. 800

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2.6 Roche/Hitachi 902

2.6.1 Hit 902, Overview of Specified vs. Measured Data



1.2 µmol/L YES

High end measuring range 291 µmol/L 347 µmol/L YES Lower detection limit 1 µmol/L 0.7 µmol/L YES Limit of blank 1 µmol/L 0.4 µmol/L YES Limit of detection 1.5 µmol/L 1.2 µmol/L YES Limit of quantification 3 µmol/L 2.4 µmol/L YES Within-run precision Mean SD CV ( % )





Analyzer / Method Hit 902 vs. Mod P/BILD2 YES Slope (Passing/Bablok) 1.00 ± 0.05 0.997 YES


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2.6.2 Roche/Hitachi 902, Method Comparison

BILD2, Hit 902 vs. Mod P

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2.7 COBAS INTEGRA 800

2.7.1 I. 800, Overview of Specified vs. Measured Data


Low end measuring range 1.5 µmol/L 0.6 µmol/L YES High end measuring range 291 µmol/L 359 µmol/L YES Lower detection limit 1 µmol/L 0.3 µmol/L YES MP Lot P2 Lot P3 Lot Limit of blank 1 µmol/L 0.3 0.2 0.1 YES Limit of detection 1.5 µmol/L 0.7 0.6 0.5 YES Limit of quantification 3 µmol/L 2.3 1.8 0.9 YES Within-run imprecision Mean SD CV (%)

Precinorm U < 10 µmol/L: SD < 0.30 16.2 0.14 0.9 YES Precipath U > 10 µmol/L: CV < 3 % 41.5 0.22 0.5 YES

Human serum 1 2.6 0.17 6.4 YES Human serum 2 80.5 0.19 0.2 YES Human serum 3 282 0.63 0.2 YES

Repeatability acc. to CLSI Mean SD CV (%) Precinorm U < 10 µmol/L: SD < 0.30 15.9 0.19 1.2 YES Precipath U > 10 µmol/L: CV < 3 % 40.5 0.24 0.6 YES


Total imprecision acc. to CLSI Mean SD CV (%)

Precinorm U

< 10 µmol/L: SD < 0.50 > 10 µmol/L: CV < 5 %

15.9 0.25 1.6 YES Precipath U 40.5 0.40 1.0 YES


Recovery in controls 100 % + 10 99.6 % YES

Median < 17 µmol/L < + 1.7

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2.7.1 I. 800, Overview of Specified vs. Measured Data, cont.


Method comparison Analyzer / Method BIL-D vs. BILD2/Integra 800

Slope (Passing/Bablok) slope and intercept not specified

1.012 YES Intercept 2.62 YES


Analyzer / Method I. 800 vs. c501/BILD2 Slope (Passing/Bablok) 1.00 ± 0.05 0.994 YES

Intercept < 5.1 µmol/L 0.86 YES Coefficient of correlation r ≥ 0.975 1.000 YES

Analyzer / Method I. 800 vs. Mod P/BILD2

Slope (Passing/Bablok) 1.00 ± 0.05 0.989 YES Intercept < 5.1 µmol/L -0.15 YES


Analyzer / Method Lot vs. Lot/BILD2 MP vs. P2 Lot/I. 800/BILD2 Slope (Passing/Bablok) 1.00 ± 0.05 0.980 YES

Intercept < 5.1 µmol/L 0.372 YES Coefficient of correlation r ≥ 0.975 1.000 YES

Anticoagulants

Median recovery (%)

Median dev. (µmol/L)

Li-Heparin

< 34.2 µmol/L: < 3.4 > 34.2 µmol/L:< ± 10 %

102 0.45 YES Li-Heparin dc - -0.23 YES

K2-EDTA 101 0.03 YES

K2-EDTA dc - -0.39 YES

K3-EDTA 100 -0.06 YES

K3-EDTA dc - -0.63 YES Gel separation tube 104 0.50 YES

On board stability 6 weeks 6 weeks YES

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2.7.1 I. 800, Overview of Specified vs. Measured Data, cont.


Calibration during 6 weeks on-board no no YES

after cassette change no no YES after lot change yes yes YES

Interference, endogeneous Lipemia low 750 1098 YES

Lipemia high 750 1100 YES Hemolysis low (as Hb) 10 14 YES

Hemolysis high (as Hb) 10 25 YES Therap. drug interference < +/-10 % Phenylbutazone interferes

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2.7.2 I. 800, Method Comparison

I. 800, BILD vs. BILD2

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BILD2, I. 800 vs. c 501

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BILD2, I. 800 vs. Mod P

BILD2, I. 800, MP lot vs. P2 lot

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2.7.3 I. 800, Linearity

I. 800 (diluent: serum)

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Target Direct Bil. [µmol/L]

Measured Direct Bil. [µmol/L]

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2.7.4 I. 800, Endogeneous Interference

I. 800, Hb Interference at approx. 34 µmol/L of Direct Bilirubin

H Serum Index


I. 800, Hb Interference at approx. 115 µmol/L of Direct Bilirubin

H Serum Index


Deviation [µmol/L]

Recovery [%]

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I. 800, Lipemia Interference at approx. 37 µmol/L of Direct Bilirubin

L Serum Index


I. 800, Lipemia Interference at approx. 80 µmol/L of Direct Bilirubin

L Serum Index


Deviation [µmol/L]

Recovery [%]

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2.7.5 I. 800, Suitability of Plasma as Sample Material

I. 800, Li Heparin Plasma vs. Serum

I. 800, K2-EDTA Plasma vs. Serum

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I. 800, K3-EDTA Plasma vs. Serum

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2.8 COBAS INTEGRA 400

2.8.1 I. 400, Overview of Specified vs. Measured Data



0.5 µmol/L YES

High end measuring range 291 µmol/L 351 µmol/L YES Lower detection limit 1 µmol/L 0.4 µmol/L YES Limit of blank 1 µmol/L 0.2 µmol/L YES Limit of detection 1.5 µmol/L 0.5 µmol/L YES Limit of quantification 3 µmol/L 1.3 µmol/L YES Within-run precision Mean SD CV ( % )

Control level 1

< 10 µmol/L: SD < 0.30 > 10 µmol/L: CV < 3 %

15.2 0.15 1.0 YES Control level 2 41.1 0.57 1.4 YES

Human serum A 2.9 0.14 4.7 YES Human serum B 78.2 0.57 0.7 YES Human serum C 285 2.07 0.7 YES



Analyzer / Method I. 400 vs. I. 800/BILD2 YES Slope (Passing/Bablok) 1.00 ± 0.05 1.005 YES

Intercept ≤ ± 5.1 µmol/L -0.213 µmol/L YES Coefficient of correlation r ≥ 0.975 1.000 YES

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2.7.2 I. 400, Method Comparison

BILD2, I. 400 vs. I. 800

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