Bilirubin Total and Direct
DMSO. Colorimetric
BSIS36-I 28/06/11 SPINREACT,S.A./S.A.U. Ctra.Santa Coloma, 7 E-17176 SANT ESTEVE DE BAS (GI) SPAIN
Tel. +34 972 69 08 00 Fax +34 972 69 00 99. e-mail: [email protected]
BILIRUBIN T&D
Quantitative determination of bilirubin
IVD
Store at 2-8ºC
PRINCIPLE OF THE METHOD
Bilirubin is converted to colored azobilirubin by diazotized sulfanilic acid and
measured photometrically. Of the two fractions presents in serum, bilirubin-
glucuromide and free bilirubin loosel y bound t o albumin, only t he former reacts
directly in aqueous solution (bilirubin direct), while free bilirubin requires
solubilization with dimethylsulfoxide (DMSO) to react (bi lirubin indirect). In the
determination of indirec t bilirubin the direct is als o determined, the results
correspond to total bilirubin.
The intensity of the color formed is proportional to the bilirrubin concentration in
the sample1,2.
CLINICAL SIGNIFICANCE
Bilirubin is a breakdown product of hemoglobin.
It is transported from the spleen to the liver and excreted into bil e.
Hyperbilirubinemia results from the i ncrease of bilirubin concentrations in
plasma. Causes of hyperbilirubinemia:
Total bilirubin (T): Increase hemolysis, genetic errors , neonatal jaundice,
ineffective erythrpoiesis, and drugs.
Direct bilirubin (D): Hepatic cholestasis, genetic errors, hepatocellular
damage1,5,6.
Clinical diagnosis should not be made on a s ingle test result; it should i ntegrate
clinical and other laboratory data.
REAGENTS
R 1 (D)
Sulfanilic acid
Hydrochloric acid (HCl)
30 mmol/L
150 mmol/L
R 2 (T)
Sulfanilic acid
Hydrochloric acid (HCl)
Dimethylsulfoxide (DMSO)
30 mmol/L
50 mmol/L
7 mol/L
R 3
Sodium nitrite
29 mmol/L
Optional
BILIRRUBIN CAL Ref: 1002250
PRECAUTIONS
R1/R2/RT: Corrosive (C):R35:Causes severe burns.
S26: In case of contact with eyes, rinse immedi ately with plenty of water and
seek medical advice.
PREPARATION
All the reagents are ready to use.
STORAGE AND STABILITY
All the components of the kit are stable until the expiration date on the label
when stored ti ghtly closed at 2-8ºC, protected from light and contaminations
prevented during their use. Do not use reagents over the expirati on date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
- Color development in R 2.
ADDITIONAL EQUIPMENT
- Spectrophotometer or colorimeter measuring at 555 nm.
- Matched cuvettes 1.0 cm light path.
- General laboratory equipment.
SAMPLES
Serum or plasma, free of haemolysis (s eparated from red blood cells as s oon as
possible). Protect samples from direct light.
Sample Stabili ty (without red blood cells): 2-8ºC for 4 days and 2 months at –
20ºC.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . … . . . . 555 nm (530-580)
Cuvette: . . . . . . . . . . . . . . . ... . . … … . . .1 cm light path
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-25ºC
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
Blank
Total B.
Blank
Direct B.
R 1 (D) (mL)
--
--
1.5
1.5
R 2 (T) (mL)
1.5
1.5
--
--
R 3 ( L)
--
50
--
50
Sample(Note 1) / Calibrator
(L)
100
100
100
100
4. Mix and incubate exactly for 5 minutes at 15-25ºC.
5. Read the absorbance (A).
CALCULATIONS
With Calibrator:
BlankCalibrator)A(Calibrator)A(
BlankSample)A(Sample)A(
x Conc. Calibrator = mg/dL bilirubi n
With Factor:
((A) Sample - (A) Sample Blank) x Factor* = mg/dL bilirubin in the sam ple
*Factor:
BlankCalibrator)A(Calibrator)A(
CalibratorofionConcentrat
Theoretical factor: Bilirubin (T) = 19,1 ; Bilir ubin (D) = 14
Conversion factor: mg/dL x 17.1 = mol/L.
QUALITY CONTROL
Control sera are recommended to monitor the performance of ass ay procedures:
SPINTROL H Normal and Pathologic (Ref. 1002120 and 1002210).
If control values are found outside the defined range, check the instrument, reagents
and calibrator for problems.
Each laboratory should establish its own Qual ity Control scheme and correcti ve actions
if controls do not meet the acceptable tolerances.
REFERENCE VALUES1
Bilirubin Total
Up to 1.10 mg/dL 18.81 mol/L
Bilirubin Direct
Up to 0.25 mg/dL 4.27 mol/L
These values are for orientation purpose; each laboratory sho uld establis h its own
reference range.
PERFORMANCE CHARACTERISTICS
Measuring range: From detection limit of (T) 0,099 mg/dL (D) 0.04 mg/dL to linearity
limit of 18 mg/dL.
If the results obtained were greater than linearity limi t, dilute the sample 1/2 with NaCl
9 g/L and multiply the result by 2.
Precision:
Bilirubin T
Intra-assay (n=20)
Inter-assay (n=20)
Mean (mg/dL)
1.12
5.36
1.01
5.28
SD
0.02
0.12
0.03
0.12
CV (%)
2.33
2.27
2.70
2.32
Bilirubin D
Intra-assay (n=20)
Inter-assay (n=20)
Mean (mg/dL)
0.64
2.28
0.68
2.53
SD
0.01
0.02
0.02
0.05
CV (%)
1.91
1.10
2.51
1.95
Sensitivity: 1 mg/dL = 1 mg/dL = 0.015 A (T ).
1 mg/dL = 0.073 A (D).
Accuracy: Results obtained using SPINREACT reagents did not show systematic
differences when compared with other commercial reagents.
The results obtained using 50 samples for Bilirubin D were the following:
Correlation coefficient (r): 0.99
Regression equation: y=0.9933x + 0.0039
The results obtained using 50 samples for Bilirubin T were the following:
Correlation coefficient (r): 0.996
Regression equation: y=0.0884x + 0.0208
The results of the performance characteris tics depend on the analyzer used.
INTERFERENCES
Hemolysis causes decreased bilirubin values1,2.
A li st of drugs and other interfering substances with bilirubin determination has been
reported by Young et. al3,4.
NOTES
1. For bilirubin determination i n newborns, pipette 50 L of sample. Multiply the
result by 2.
2. SPINREACT has instruction sheets for severa l au tomatic analyzers.
Instructions for many of them are available on request.
BIBLIOGRAPHY
1. Kaplan A et al . Bili rubin. Cli n Chem The C.V. Mosby Co. St Louis. Toronto.
Princeton 1984; 1238-1241. 436 and 650.
2. Malloy H T. et al. The determi nation of bilirubin with the photoelectric colorimeter.
J. Biol Chem 1937; 112, 2; 481-491.
3. Young DS. Effects of drugs on Clinical Lab. Tes ts, 4th ed AACC Press, 1995.
4. Young DS. Effects of disease on Clinical Lab. T ests, 4th ed AACC 2001.
5. Burtis A et al. Tietz Textbook of Clinical Chemistr y, 3rd ed AACC 1999.
6. Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed A ACC 1995.
PACKAGING
Ref: 1001044
R 1 (D):
R 2 (T):
R 3:
1 x 150 mL
1 x 150 mL
1 x 10 mL
Cont.
