0105795397190c501V7.

BILT3
Bilirubin Total Gen.3
Order information
Analyzer(s) on which cobas c pack(s) can be used
05795397 190 Bilirubin Total Gen.3 (250 tests) System‑ID 07 7483 9 Roche/Hitachi cobas c 311, cobas c 501/502
10759350 190 Calibrator f.a.s. (12 × 3 mL) Code 401
12149435 122 Precinorm U plus (10 × 3 mL) Code 300
12149443 122 Precipath U plus (10 × 3 mL) Code 301
10171743 122 Precinorm U (20 × 5 mL) Code 300
10171735 122 Precinorm U (4 × 5 mL) Code 300
10171778 122 Precipath U (20 × 5 mL) Code 301
10171760 122 Precipath U (4 × 5 mL) Code 301
10158046 122 Precibil (4 × 2 mL) Code 306
05117003 190 PreciControl ClinChem Multi 1 (20 × 5 mL) Code 391
05947626 190 PreciControl ClinChem Multi 1 (4 × 5 mL) Code 391
05117216 190 PreciControl ClinChem Multi 2 (20 × 5 mL) Code 392
05947774 190 PreciControl ClinChem Multi 2 (4 × 5 mL) Code 392
04489357 190 Diluent NaCl 9 % (50 mL) System‑ID 07 6869 3

English Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.
System information
For cobas c 311/501 analyzers: This kit contains components classified as follows in accordance with the
Regulation (EC) No. 1272/2008:
BILT3: ACN 712
SBIL3: ACN 711 (STAT, reaction time: 4)
For cobas c 502 analyzer:
BILT3: ACN 8712
SBIL3: ACN 8711 (STAT, reaction time: 4)
Danger
Intended use
In vitro test for the quantitative determination of total bilirubin in serum and H290 May be corrosive to metals.
plasma of adults and neonates on Roche/Hitachi cobas c systems.
H314 Causes severe skin burns and eye damage.
Summary1
Bilirubin is formed in the reticuloendothelial system during the degradation H360FD May damage fertility. May damage the unborn child.
of aged erythrocytes. The heme portion from hemoglobin and from other
heme‑containing proteins is removed, metabolized to bilirubin, and Prevention:
transported as a complex with serum albumin to the liver. In the liver,
bilirubin is conjugated with glucuronic acid for solubilization and subsequent P201 Obtain special instructions before use.
transport through the bile duct and elimination via the digestive tract.
Diseases or conditions which, through hemolytic processes, produce P280 Wear protective gloves/ protective clothing/ eye protection/
bilirubin faster than the liver can metabolize it, cause the levels of face protection.
unconjugated (indirect) bilirubin to increase in the circulation. Liver Response:
immaturity and several other diseases in which the bilirubin conjugation
mechanism is impaired cause similar elevations of circulating unconjugated P303 + P361 IF ON SKIN (or hair): Take off immediately all contaminated
bilirubin. Bile duct obstruction or damage to hepatocellular structure causes + P353 clothing. Rinse skin with water/shower.
increases in the levels of both conjugated (direct) and unconjugated
(indirect) bilirubin in the circulation. P304 + P340 IF INHALED: Remove person to fresh air and keep
Test principle2 + P310 comfortable for breathing.
Colorimetric diazomethod Immediately call a POISON CENTER/ doctor.
Total bilirubin, in the presence of a suitable solubilizing agent, is coupled
with 3,5‑dichlorophenyl diazonium in a strongly acidic medium. P305 + P351 IF IN EYES: Rinse cautiously with water for several
+ P338 minutes. Remove contact lenses, if present and easy to do.
acid
+ P310 Continue rinsing. Immediately call a POISON CENTER/
Bilirubin + 3,5‑DPD azobilirubin doctor.
The color intensity of the red azo dye formed is directly proportional to the
total bilirubin and can be determined photometrically. P308 + P313 IF exposed or concerned: Get medical advice/attention.
Reagents - working solutions Product safety labeling follows EU GHS guidance.
Contact phone: all countries: +49-621-7590
R1 Phosphate: 25 mmol/L; detergents; stabilizers, pH 1.0
Reagent handling
R2 3,5‑dichlorophenyl diazonium salt: ≥ 1.35 mmol/L
Ready for use
R1 is in position B and R2 is in position C.
Storage and stability
Precautions and warnings
For in vitro diagnostic use. BILT3
Exercise the normal precautions required for handling all laboratory
reagents.

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BILT3
Bilirubin Total Gen.3

Shelf life at 2‑8 °C: See expiration date cobas c 501 test definition
on cobas c pack Assay type 2‑Point End
label.
Reaction time / Assay points 10 / 10‑25 (STAT 4 / 10‑25)
On‑board in use and refrigerated on the analyzer: 6 weeks
Wavelength (sub/main) 600/546 nm
Diluent NaCl 9 %
Reaction direction Increase
Shelf life at 2‑8 °C: See expiration date
on cobas c pack Units µmol/L (mg/dL, mg/L)
label. Reagent pipetting Diluent (H2O)
On‑board in use and refrigerated on the analyzer: 12 weeks R1 120 µL –
Specimen collection and preparation R2 24 µL –
For specimen collection and preparation only use suitable tubes or Sample volumes Sample Sample dilution
collection containers.
Sample Diluent (NaCl)
Only the specimens listed below were tested and found acceptable.
Serum Normal 2 µL – –
Plasma: Li‑heparin, K2‑, K3‑EDTA plasma Decreased 8 µL 15 µL 105 µL
(The use of EDTA‑plasma with elevated hematocrit may lead to slightly
lower values.) Increased 2 µL – –
The sample types listed were tested with a selection of sample collection
tubes that were commercially available at the time of testing, i.e. not all cobas c 502 test definition
available tubes of all manufacturers were tested. Sample collection systems Assay type 2‑Point End
from various manufacturers may contain differing materials which could
affect the test results in some cases. When processing samples in primary Reaction time / Assay points 10 / 10‑25 (STAT 4 / 10‑25)
tubes (sample collection systems), follow the instructions of the tube Wavelength (sub/main) 600/546 nm
manufacturer.
Reaction direction Increase
Centrifuge samples containing precipitates before performing the assay.
Units µmol/L (mg/dL, mg/L)
Stability:a,3 1 day at 15‑25 °C
Reagent pipetting Diluent (H2O)
7 days at 2‑8 °C
R1 120 µL –
6 months at (-15)‑(-25) °C
R2 24 µL –
a) If care is taken to prevent exposure to light
Sample volumes Sample Sample dilution
Materials provided
Sample Diluent (NaCl)
See “Reagents – working solutions” section for reagents.
Normal 2 µL – –
Materials required (but not provided)
Decreased 8 µL 15 µL 105 µL
▪ See “Order information” section
▪ General laboratory equipment Increased 4 µL – –
Assay Calibration
For optimum performance of the assay follow the directions given in this Calibrators S1: H2O
document for the analyzer concerned. Refer to the appropriate operator’s
manual for analyzer‑specific assay instructions. S2: C.f.a.s.
The performance of applications not validated by Roche is not warranted Calibration mode Linear
and must be defined by the user.
Calibration frequency 2‑point calibration
Application for serum and plasma
• after reagent lot change
cobas c 311 test definition • as required following quality control
Assay type 2‑Point End procedures
Reaction time / Assay points 10 / 6‑17 (STAT 4 / 6‑17) Calibration interval may be extended based on acceptable verification of
calibration by the laboratory.
Wavelength (sub/main) 600/546 nm Traceability: The method was standardized against the Doumas method.4
Reaction direction Increase Quality control
Units µmol/L (mg/dL, mg/L) For quality control, use control materials as listed in the "Order information"
Reagent pipetting Diluent (H2O) section.
In addition, other suitable control material can be used.
R1 120 µL –
The control intervals and limits should be adapted to each laboratory’s
R2 24 µL – individual requirements. Values obtained should fall within the defined
Sample volumes Sample Sample dilution limits. Each laboratory should establish corrective measures to be taken if
values fall outside the defined limits.
Sample Diluent (NaCl) Follow the applicable government regulations and local guidelines for
Normal 2 µL – – quality control.
Decreased 8 µL 15 µL 105 µL Calculation
Roche/Hitachi cobas c systems automatically calculate the analyte
Increased 2 µL – – concentration of each sample.

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BILT3
Bilirubin Total Gen.3

Conversion factors: µmol/L × 0.0585 = mg/dL The Limit of Detection is determined based on the Limit of Blank and the
standard deviation of low concentration samples.
mg/dL × 10 = mg/L
The Limit of Detection corresponds to the lowest analyte concentration
mg/dL × 17.1 = µmol/L which can be detected (value above the Limit of Blank with a probability of
95 %).
Limitations - interference
Criterion: Recovery within ± 3.4 µmol/L (0.199 mg/dL) of initial values of The Limit of Quantitation is the lowest analyte concentration that can be
samples ≤ 34 µmol/L (1.99 mg/dL) and ± 10 % of samples > 34 µmol/L. reproducibly measured with a total error of 30 %. It has been determined
using low concentration bilirubin samples.
Hemolysis:5 No significant interference up to an H index of 800
(approximate hemoglobin concentration: 497 µmol/L or 800 mg/dL). Expected values
Criterion: Recovery within ± 1.7 µmol/L (0.099 mg/dL) of initial values of Adults9 up to 21 µmol/L (up to 1.2 mg/dL)
samples ≤ 17 µmol/L (0.995 mg/dL) and ± 10 % of samples > 17 µmol/L.
Children with age up to 17 µmol/L (up to 1.0 mg/dL)
Hemolysis in neonates:5 No significant interference up to an H index of
1000 (approximate hemoglobin concentration: 621 µmol/L or 1000 mg/dL). ≥ 1 month
Lipemia (Intralipid):5 No significant interference up to an L index of 1000. Reference range study with 500 well‑characterized human serum
There is poor correlation between the L index (corresponds to turbidity) and samples:10
triglycerides concentration.
Males up to 24 µmol/L (up to 1.4 mg/dL)
Drugs: No interference was found at therapeutic concentrations using
common drug panels.6,7 Females up to 15 µmol/L (up to 0.9 mg/dL)
Indican: No significant interference from indican up to levels of 0.12 mmol/L High risk for developing clinically significant hyperbilirubinemia:
or 3 mg/dL.
Newborns: Term and near-term11
Cyanokit (Hydroxocobalamin) may cause falsely low results.
Samples containing indocyanine green must not be measured. Age of newborn:
Results from certain multiple myeloma patients may show a positive bias in 24 hours ≥ 137 µmol/Lb) (≥ 8.0 mg/dLb))
recovery. Not all multiple myeloma patients show the bias and the severity 48 hours ≥ 222 µmol/Lb) (≥ 13.0 mg/dLb))
of the bias may vary between patients.
84 hours ≥ 290 µmol/Lb) (≥ 17.0 mg/dLb))
In very rare cases, gammopathy, in particular type IgM (Waldenström’s
macroglobulinemia), may cause unreliable results.8 b) 95th percentile

For diagnostic purposes, the results should always be assessed in Levels > 95th percentile: Such levels of hyperbilirubinemia have been
conjunction with the patient’s medical history, clinical examination and other deemed significant and are generally considered to require close
findings. supervision, possible further evaluation, and sometimes intervention.
In certain cases specimens may give a direct bilirubin result slightly greater Nomogramm for designation of risk in 2840 well newborns11
than the total bilirubin result. This is observed in patient samples when Serum Bilirubin
nearly all the reacting bilirubin is in the direct form. In such cases the result
for the total bilirubin should be reported for both D‑bilirubin and total
bilirubin values.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory
when certain test combinations are run together on Roche/Hitachi
cobas c systems. The latest version of the carry‑over evasion list can be
found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets. For further
instructions refer to the operator’s manual. cobas c 502 analyzer: All
special wash programming necessary for avoiding carry‑over is available
via the cobas link, manual input is not required.
Where required, special wash/carry‑over evasion programming must
be implemented prior to reporting results with this test.
Limits and ranges
Measuring range
2.5‑650 µmol/L (0.146‑38.0 mg/dL)
Determine samples having higher concentrations via the rerun function.
Dilution of samples via the rerun function is a 1:2 dilution. Results from Postnatal Age (hours)
samples diluted using the rerun function are automatically multiplied by a
factor of 2. * 95th percentile
Lower limits of measurement A High risk zone C Low intermediate risk zone
Limit of Blank, Limit of Detection and Limit of Quantitation B High intermediate risk zone D Low risk zone
Limit of Blank = 1.7 µmol/L (0.099 mg/dL) Each laboratory should investigate the transferability of the expected values
Limit of Detection = 2.5 µmol/L (0.146 mg/dL) to its own patient population and if necessary determine its own reference
ranges.
Limit of Quantitation = 2.5 µmol/L (0.146 mg/dL)
Specific performance data
The Limit of Blank, Limit of Detection and Limit of Quantitation were Representative performance data on the analyzers are given below.
determined in accordance with the CLSI (Clinical and Laboratory Standards Results obtained in individual laboratories may differ.
Institute) EP17‑A2 requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of Precision
analyte‑free samples over several independent series. The Limit of Blank Repeatability and intermediate precision were determined using human
corresponds to the concentration below which analyte‑free samples are samples and controls in accordance with the CLSI (Clinical and Laboratory
found with a probability of 95 %. Standards Institute) EP5 requirements (2 aliquots per run, 2 runs per day,
21 days). The following results were obtained:

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BILT3
Bilirubin Total Gen.3

Repeatability Mean SD CV 8 Bakker AJ, Mücke M. Gammopathy interference in clinical chemistry

assays: mechanisms, detection and prevention.
µmol/L (mg/dL) µmol/L (mg/dL) % Clin Chem Lab Med 2007;45(9):1240-1243.
Control level 1 15.4 (0.901) 0.3 (0.018) 2.1 9 Thomas L, ed. Labor und Diagnose. Indikation und Bewertung von
Control level 2 52.8 (3.09) 0.3 (0.02) 0.6 Laborbefunden für die Medizinische Diagnostik, 7th ed.: TH-Books
Verlagsgesellschaft 2007:259-273.
Human serum A 8.69 (0.508) 0.25 (0.015) 2.9
10 Löhr B, El-Samalouti V, Junge W, et al. Reference Range Study for
Human serum B 302 (17.7) 2 (0.1) 0.6 Various Parameters on Roche Clinical Chemistry Analyzers. Clin Lab
2009;55:465-471.
Human serum C 544 (31.8) 2 (0.1) 0.4
11 Subcommittee on Hyperbilirubinemia. Management of
Intermediate precision Mean SD CV Hyperbilirubinemia in the Newborn Infant 35 or More Weeks of
Gestation. Pediatrics 2004;114:297-316.
µmol/L (mg/dL) µmol/L (mg/dL) %
12 Bablok W, Passing H, Bender R, et al. A general regression procedure
Control level 1 15.4 (0.901) 0.3 (0.018) 2.1 for method transformation. Application of linear regression procedures
for method comparison studies in clinical chemistry, Part III. J Clin
Control level 2 52.8 (3.09) 0.4 (0.02) 0.8 Chem Clin Biochem 1988 Nov;26(11):783-790.
Human serum A 8.69 (0.508) 0.29 (0.017) 3.3 A point (period/stop) is always used in this Method Sheet as the decimal
Human serum B 302 (17.7) 2 (0.1) 0.8 separator to mark the border between the integral and the fractional parts of
a decimal numeral. Separators for thousands are not used.
Human serum C 544 (31.8) 3 (0.2) 0.6
Symbols
Method comparison Roche Diagnostics uses the following symbols and signs in addition to
Total bilirubin values for human serum and plasma samples obtained on a those listed in the ISO 15223‑1 standard (for USA: see
Roche/Hitachi cobas c 501 analyzer (y) using the Roche Bilirubin Total https://usdiagnostics.roche.com for definition of symbols used):
Gen.3 reagent were compared with those determined on a
COBAS INTEGRA 800 analyzer using the corresponding reagent (x). Contents of kit
Sample size (n) = 64 Volume after reconstitution or mixing
Passing/Bablok12 Linear regression GTIN Global Trade Item Number
y = 0.995x + 0.734 µmol/L y = 0.993x + 1.20 µmol/L COBAS, COBAS C, COBAS INTEGRA, PRECIBIL, PRECICONTROL, PRECINORM and PRECIPATH are
trademarks of Roche.
τ = 0.990 r = 1.00
All other product names and trademarks are the property of their respective owners.
The sample concentrations were between 3.6 and 618 µmol/L (0.211 and Additions, deletions or changes are indicated by a change bar in the margin.
36.2 mg/dL). © 2017, Roche Diagnostics

Total bilirubin values for human serum and plasma samples obtained on a
Roche/Hitachi cobas c 501 analyzer (y) using the Roche Bilirubin Total
Gen.3 reagent were compared with those determined using the Roche
Total Bilirubin Special reagent on the same analyzer (x). Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim
www.roche.com
Sample size (n) = 152
Passing/Bablok12 Linear regression
y = 0.962x + 1.55 µmol/L y = 0.936x + 3.01 µmol/L
τ = 0.981 r = 1.00
The sample concentrations were between 2.4 and 561 µmol/L (0.140 and
32.8 mg/dL).
References
1 Balistreri WF, Shaw LM. Liver function. In: Tietz NW, ed. Fundamentals
of Clinical Chemistry. 3rd ed. Philadelphia: WB Saunders
1987;729-761.
2 Wahlefeld AW, Herz G, Bernt E. Modification of the Malloy-Evelyn
method for a simple, reliable determination of total bilirubin in serum.
Scand J Clin Lab Invest 1972;29 Supplement 126:Abstract 11.12.
3 Quality of Diagnostic Samples, Recommendations of the Working
Group on Preanalytical Quality of the German Society for Clinical
Chemistry and Laboratory Medicine, 3rd completely revised ed. 2010.
4 Doumas BT, Kwok-Cheung PP, Perry BW, et al. Candidate Reference
Method for Determination of Total Bilirubin in Serum: Development and
Validation. Clin Chem 1985;31:1779-1789.
5 Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of
Interferences in Clinical Chemistry Instrumentation. Clin Chem
1986;32:470-475.
6 Breuer J. Report on the Symposium “Drug effects in Clinical Chemistry
Methods”. Eur J Clin Chem Clin Biochem 1996;34:385-386.
7 Sonntag O, Scholer A. Drug interference in clinical chemistry:
recommendation of drugs and their concentrations to be used in drug
interference studies. Ann Clin Biochem 2001;38:376-385.

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