Bilirubin Auto Direct FS*

Diagnostic reagent for quantitative in vitro determination of direct bilirubin in serum or plasma on
photometric systems

Order Information Waste Management

Cat. No. Kit size Please refer to local legal requirements.
1 0821 99 10 021 R1 5x 20 mL + R2 1x 25 mL
Reagent Preparation
1 0821 99 10 026 R1 5x 80 mL + R2 1x 100 mL
1 0821 99 10 023 R1 1x 800 mL + R2 1x 200 mL The reagents are ready to use.
1 0821 99 10 704 R1 8x 50 mL + R2 8x 12.5 mL
Materials required but not provided
1 0821 99 10 930 R1 4x 20 mL + R2 2x 10 mL
NaCl solution 9 g/L
Summary [1,2] General laboratory equipment
Bilirubin is a breakdown product of hemoglobin. Free,
unconjugated bilirubin is extremely apolar and nearly insoluble in
Specimen
water, thus forming a complex with albumin for the transport in the Serum or heparin plasma
blood from the spleen to the liver. In the liver, bilirubin is It is very important to store the sample protected from light!
conjugated with glucoronic acid and the resulting water soluble Stability [3]: 2 days at 20 – 25°C
bilirubin glucoronic acid is excreted via the bile ducts. 7 days at 4 – 8°C
Hyperbilirubinemia can be caused by increased bilirubin 6 months at –20°C
production due to hemolysis (pre-hepatic jaundice), by in case of immediate freezing.
parenchymal damages of the liver (intra-hepatic jaundice) or by Freeze only once!
occlusion of bile ducts (post-hepatic jaundice). A chronic Discard contaminated specimens!
congenital (predominantly unconjugated) hyperbilirubinemia called
Gilbert’s syndrome is quite frequent in the population. High levels Assay Procedure
of total bilirubin are observed in 60 – 70% of neonates due to an Application sheets for automated systems are available on
increased postpartal breakdown of erythrocytes and because of request.
delayed function of enzymes for bilirubin degradation. Common
bilirubin methods detect either total bilirubin or direct bilirubin. Wavelength 546 nm (540 – 560 nm)
Determinations of direct bilirubin measure mainly conjugated, Optical path 1 cm
water soluble bilirubin. Therefore, the value of unconjugated Temperature 20 – 25°C/37°C
bilirubin may be estimated from the difference between total Measurement Against reagent blank
bilirubin and direct bilirubin.
Blank Sample or calibrator
Method Sample or calibrator - 50 µL
Photometric test using 2,4-dichloroaniline (DCA) Dist. Water 50 µL -
Reagent 1 1000 µL 1000 µL
Principle Mix, incubate for 3 – 5 min. at 20 – 25°C/37°C, read absorbance
A1, then add:
Direct bilirubin in presence of diazotized 2,4-dichloroaniline forms
Reagent 2 250 µL 250 µL
a red colored azocompound in acidic solution.
Mix, incubate for exactly 5 min. at 37°C or 10 min. at 20 – 25°C,
Reagents then read absorbance A2.

Components and Concentrations A = (A2 – A1) Sample or calibrator

R1: EDTA-Na2 0.1 mmol/L Calculation
NaCl 150 mmol/L
Sulfamic acid 100 mmol/L With calibrator
R2: 2,4-Dichlorophenyl-diazonium salt 0.5 mmol/L ΔA Sample
Bilirubin [mg/dL] = × Conc. Cal. [mg/dL]
HCl 900 mmol/L ΔA Cal.
EDTA-Na2 0.13 mmol/L
Conversion factor
Storage Instructions and Reagent Stability Bilirubin [mg/dL] x 17.1 = Bilirubin [µmol/L]
The reagents are stable up to the end of the indicated month of
expiry, if stored at 2 – 8°C and contamination is avoided. Do not Calibrators and Controls
freeze the reagents! For the calibration of automated photometric systems, DiaSys
Reagent 2 must be protected from light. TruCal U calibrator is recommended. This method has been
Warning and Precautions standardized against the manual Jendrassik-Gróf test. DiaSys
TruLab N and P controls should be assayed for internal quality
1. Reagents: Warning. H290 May be corrosive to metals. P234
control. Each laboratory should establish corrective action in case
Keep only in original container. P390 Absorb spillage to
of deviations in control recovery.
prevent material damage.
2. In very rare cases, samples of patients with gammopathy Cat. No. Kit size
TruCal U 5 9100 99 10 063 20 x 3 mL
might give falsified results [6].
5 9100 99 10 064 6 x 3 mL
3. Please refer to the safety data sheets and take the necessary TruLab N 5 9000 99 10 062 20 X 5 mL
precautions for the use of laboratory reagents. For diagnostic 5 9000 99 10 061 6 X 5 mL
purposes, the results should always be assessed with the TruLab P 5 9050 99 10 062 20 X 5 mL
patient’s medical history, clinical examinations and other 5 9050 99 10 061 6 X 5 mL
findings.
4. For professional use only!

Bilirubin Auto Direct FS – Page 1 * fluid stable

Performance Characteristics Reference Range [1]
Measuring range Adults and children  0.2 mg/dL ( 3.4 µmol/L)
The test has been developed to determine bilirubin concentrations Each laboratory should check if the reference ranges are
within a measuring range from 0.1 - 10 mg/dL. When values transferable to its own patient population and determine own
exceed this range, samples should be diluted 1 + 1 with reference ranges if necessary.
NaCl solution (9 g/L) and the result multiplied by 2.
Specificity/Interferences Literature
No interference was observed by ascorbic acid up to 30 mg/dL, 1. Thomas L ed. Clinical Laboratory Diagnostics. 1st ed.
naproxen up to 1 mmol/L and lipemia up to 1000 mg/dL Frankfurt: TH-Books Verlagsgesellschaft, 1998: p. 192–202.
triglycerides. Interference by hemoglobin occurs starting at 2. Tolman KG, Rej R. Liver function. In: Burtis CA, Ashwood
hemoglobin concentrations of 50 mg/dL. ER, editors. Tietz Textbook of Clinical Chemistry. 3rd ed.
For further information on interfering substances refer to Young DS Philadelphia: W.B Saunders Company; 1999. p. 1125-77.
[5]. 3. Guder WG, Zawta B et al. The Quality of Diagnostic Samples.
1st ed. Darmstadt: GIT Verlag; 2001; p. 18-9.
Sensitivity/Limit of Detection
4. Rand RN, di Pasqua A. A new diazo method for the
The lower limit of detection is 0.1 mg/dL. determination of bilirubin. Clin Chem 1962;6:570-8.
Precision (at 37°C) 5. Young DS. Effects of Drugs on Clinical Laboratory Tests. 5th
ed. Volume 1 and 2. Washington, DC: The American
Intra-assay Mean SD CV
Association for Clinical Chemistry Press 2000.
n = 20 [mg/dL] [mg/dL] [%]
6. Bakker AJ, Mücke M. Gammopathy interference in clinical
Sample 1 0.36 0.01 3.12
chemistry assays: mechanisms, detection and prevention.
Sample 2 0.76 0.01 1.46
ClinChemLabMed 2007;45(9):1240–1243.
Sample 3 2.07 0.03 1.30
Manufacturer
Inter-assay Mean SD CV
DiaSys Diagnostic Systems GmbH
n = 20 [mg/dL] [mg/dL] [%] IVD Alte Strasse 9 65558 Holzheim Germany
Sample 1 0.35 0.01 3.34
Sample 2 0.75 0.01 1.00
Sample 3 2.13 0.02 0.71
Method Comparison
A comparison of DiaSys Bilirubin Auto Direct FS (y) with a
commercially available test (x) using 85 samples gave following
results: y = 0.95 x + 0.04 mg/dL; r = 0.995

Bilirubin Auto Direct FS – Page 2 844 0821 10 02 00 January 2016/15