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Photometric Test for Direct (D) Bilirubin
Calculation
Package Size A546 = A S/[CAL] - A RB
[REF] 10741 375 ml Reagent Kit
A sample A sample
[IVD] C = C [CAL] x [mg/dl] or C = C [CAL] x [mmol/l]
1 A [CAL] A [CAL]
Principle
The stabilised diazonium salt 3,5-dichlorophenyl-diazonium-tetrafluoro- For calibration use AUTOCAL ([REF] 13160). Recalibrate, if control sera are
borate (DPD) couples directly with direct (conjugated) bilirubin in an acid found outside the permitted ranges.
medium to yield the corresponding azobilirubin. The absorbance of this
Performance Characteristics
dye at 546 nm is directly proportional to the direct bilirubin concentration
Linearity: The assay is linear up to 9 mg/dl. For bilirubin concentrations
in the sample.
exceeding 9 mg/dl dilute the sample 1+4 with physiological saline (0.9%)
Reaction Principle and repeat the assay. Multiply the result by 5.
Bilirubin + DPD DIRECT Azobilirubin Typical performance date can be found in the Verification Report,
accessible via
Contents
www.human.de/data/gb/vr/su-abild.pdf or
[RGT1] 3 x 100 ml Hydrochloric Acid (red cap)
Hydrochloric acid (pH < 1.0) 170 mmol/l www.human-de.com/data/gb/vr/su-abild.pdf
[RGT2] 1 x 75 ml Colour reagent (white cap) Normal Values 2
Hydrochloric acid (pH < 1.0) 170 mmol/l Adults and children: up to 0.2 mg/dl or 3.42 µmol/l

3,5-dichlorophenyl-diazonium-tetrafluoroborate 0.24 mmol/l Quality Control

All control sera with bilirubin values determined by this method may be
Additional Material not Supplied with the Kit employed.
[REF] 13160
We recommend to use our HumaTrol quality control sera based on animal
[CAL] 4 x for 5 ml AUTOCAL serum or our SERODOS based on human serum.
lyophilised Calibrator for HUMAN Clinical Chemistry Systems
Notes
Reagent Preparation and Stability 1. Bilirubin is light sensitive, samples should be kept in the dark.
[RGT1] and [RGT2] are ready for use.
2. Bilirubin levels may be reduced if the samples are exposed to light.
They are stable up to the given expiry date if unopened and stored at Hemolysis will also lower the bilirubin value due to the inhibitory effect
2...8°C. Once opened, reagents stored on board the instrument at 2...8°C of hemoglobin on the diazo reaction.
are stable for 30 days. Protect [RGT2] from light.
References
Specimen 1. Tietz N.W., Clinical guide to laboratory tests, Saunders Co.
Serum, Heparinplasma
2. Thomas L., Clinical Laboratory Diagnostics, TH-Books
Avoid hemolytic and lipemic samples! Samples must be protected from (1998)
light.
Stability: Bilirubin is stable for 3 days when stored light-protected at
2...8°C or for up to 3 months at -20°C.
SU-ABILD INF 1074101 GB 03-2013-09M
Assay
Wavelength: 546 nm (520 - 560 nm)
Optical path: 1 cm
Temperature: 25 or 37°C
Measurement: Against reagent blank

Pipetting Scheme
Analyzer settings (2-shot version)
Proposals to apply the reagents on analyzers are available on request.
Each laboratory has to validate the application in its own responsibility.

Manual Application
Pipette into cuvettes Reagent Blank (RB) Sample (S) or [CAL]
Sample or [CAL] -- 100 µl
dist. Water 100 µl --
[RGT1] 1000 µl 1000 µl
Mix thoroughly, incubate for 2-5 min. at 25°C or 37°C
Read absorbance A1.
[RGT2] 250 µl 250 µl
Mix thoroughly, incubate for exactly 5 min. at 37°C or for exactly
10 min at 25°C, read absorbance A2. A = A2-A1.

Human Gesellschaft für Biochemica und Diagnostica mbH

Max-Planck-Ring 21 · 65205 Wiesbaden · Germany
Telefon +49 6122-9988-0 · Telefax +49 6122-9988-100 · e-Mail human@human.de