PROCEDURE

REAGENTS 1. Assay conditions:

Sulfanilic acid 30 mmol/L Wavelength: ................... ………………… 555 nm (530-580)
Cuvette: ................................................. 1 cm light path
R1 Hydrochloric acid (HCl) 50 mmol/L
Temperature ...................................................... 15-25°C
Dimethylsulphoxide (DMSO) 7 mol/L
2. Adjust the instrument to zero with distilled water.
Total Bilirubin DMSO Colorimetric R2 Sodium nitrite 29 mmol/L 3. Pipette into a cuvette:
Blank B. Total
Optional BILIRUBIN CAL R 1 (mL) 1.5 1.5
For in-vitro diagnostic use only
R 2 (µL) -- 50
Store at 2-8  C ADDITIONAL EQUIPMENT Sample /Calibrator (µL) 100 100
 Spectrophotometer or colorimeter measuring at
INTENDED USE 4. Mix and incubate for exactly 5 minutes at room
555 nm.
For the quantitative determination of total bilirubin temperature.
 Matched cuvettes 1.0 cm light path.
concentrationin human serum or plasma. 5. Read the absorbance (A).
 General laboratory equipment
CLINICAL SIGNIFICANCE CALCULATIONS
PRECAUTIONS
Bilirubin is a breakdown product of hemoglobin,
 Dimethylsulphoxide (DMSO): Irritant to eyes and With Calibrator:
insoluble in water. It is transported from the spleen to (A)Sample-(A) Sample Blank x Conc. Calibrator = mg/dL
skin. In case of contact with eyes, rinse
the liver and excreted into bile. Hyperbilirubinemia
immediately with plenty of water and seek (A) Calibrator-(A)Calibrator Blank bilirubin
results from the increase of bilirubin concentrations in
medical advice.
plasma.
Causes of hyperbilirubinemia:  Hydrochloric acid (HCL): Irritant to eyes, With Factor:
respiratory system and skin. In case of contact (A)Sample-(A) Sample Blank x 19.1 = mg/dL bilirubin
-Total bilirubin: Increase hemolysis, genetic errors,
with eyes, rinse immediately with plenty of in the sample
neonatal jaundice, ineffective erythrpoiesis, and drugs.
water and seek medical advice. Conversion factor: mg/dL x 17.1 = µmol/L.
-Direct bilirubin: Hepatic cholestasis, genetic errors,
hepatocellular damage. PREPARATION QUALITY CONTROL
Clinical diagnosis should not be made on a single test All the reagents are ready to use.
 Control sera are recommended to monitor the
result; it should integrate clinical and other laboratory performance of assay procedure.
data. STORAGE AND STABILITY
 Control values are found outside the defined
 All the components of the kit are stable until
range, check the instrument, reagents and
PRINCIPLE OF THE METHOD the expiration date on the label when stored
0 calibrator for problems.
Bilirubin is converted to colored azobilirubin by diazotized tightly closed at 2-8 C, protected from light and
 Each laboratory should establish its own Quality
sulfanilic acid and measured photometrically. Of the two contaminations prevented during their use.
Control scheme and corrective actions if controls
fractions presents in serum, bilirubin-glucuromide and  Do not use reagents over the expiration date.
do not meet the acceptable tolerances.
free bilirubin loosely bound to albumin, only the former  Signs of reagent deterioration:
reacts directly in aqueous solution (bilirubin direct),  Presence of particles and turbidity. REFERENCE VALUES
while free bilirubin requires solubilization with  Color development in R 2. Bilirubin Total Up to 1.10 mg/dL = Up to 18.81 µmol/L
dimethylsulphoxide (DMSO) to react (bilirubin indirect).
SAMPLES These values are for orientation purpose; each laboratory
In the determination of indirect bilirubin the direct is also
Serum or plasma: free of hemolysis. Protect samples should establish its own reference range.
determined, the results correspond to total bilirubin.
The intensity of the color formed is proportional to the from direct light. Stability: Bilirubin is stable at 2-8°C for 4
bilirrubin concentration in the sample. days and 2 months at –20°C.
PERFORMANCE CHARACTERISTICS
1.Measuring range:
From detection limit of 0.00526 mg/L to linearity limit of 4. Young DS. Effects of drugs on Clinical Lab. Tests,
18 mg/dL. 4th ed AACC Press, 1995.
If the results obtained were greater than linearity limit, 5. Young DS. Effects of disease on Clinical Lab.
dilute the sample 1/2 with NaCl 9 g/L and multiply the Tests, 4th ed AACC 2001.
result by 2. 6. Burtis A et al. Tietz Textbook of Clinical
Chemistry, 3rd ed AACC 1999.
2.Precision: 7. Tietz N W et al. Clinical Guide to Laboratory
Intra-assay Inter-assay Tests, 3rd ed AACC 1995.
(n=20) (n=20)
Mean (mg/dL) 1.53 5.06 1.53 5.02 ATLAS MEDICAL
SD 0.03 0.05 0.03 0.11 Ludwig-Erhard Ring 3
CV % 1.73 1.01 1.92 2.18 15827 Blankenfelde-Mahlow
Germany
3.Sensitivity: Tel: +49 - 33708 – 3550 30
1 mg/dL = 0.05074 A. Email: Info@atlas-medical.com

4.Accuracy: PPI1443A01
Results obtained using reagents (y) did not show Rev A (02.09.2019)
systematic differences when compared with other Temperature
commercial reagents (x). The results obtained using 50 Catalogue Number
limit
samples were the following: In Vitro diagnostic
2 Caution
Correlation coefficient (r) : 0.991. medical device
Regression equation: y= 0.82743x-0.0382. Contains sufficient Consult
The results of the performance characteristics depend on for <n> tests and instructions for
the analyzer used. Relative size use (IFU)
Batch code Manufacturer
INTERFERENCES
Fragile,
Hemolysis causes decreased bilirubin values. handle with care
Use-by date
A list of drugs and other interfering substances with Do not use if
bilirubin has been reported. Manufacturer fax
package is
number
damaged
Note
Manufacturer Date of
 For bilirubin determination in newborns, pipette telephone number Manufacture
50 µL of sample. Multiply the result by 2.
Keep away from
Keep dry
REFERENCES sunlight
1. Kaplan A et al. Bilirubin. Clin Chem The C.V.
Mosby Co. St Louis. Toronto. Princeton 1984;
1238-1241. 436 and 650.
2. Malloy H T. et al. The determination of bilirubin
with the photoelectric colorimeter. J. Biol Chem
1937; 112, 2; 481-491.
3. Martinek R. Improved micro-method for
determination of serum bilirubin. Clin Chim
1966: Acta 13: 61-170.