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www.biomaghreb.com
IVD
CHOLESTEROL
Enzymatic Colorimetric test
(CHOD- PAP)
Reagent for the quantitative determination
of Total Cholesterol in human plasma
FT En 22 Date of updating: 01/ 2020 Version B
IN VITRO DIAGNOSTIC USE
CLINICAL SIGNIFICANCE
Cholesterol is a molecule essential to the proper functioning of the body, which comes from food
where is synthesized in the liver. It is an insoluble molecule in the blood, transported by proteins
called «lipoproteins»: LDL, HDL and VLDL. Cholesterol levels are measured in order to detect hyper-
cholesterolemia, which could lead to the development of atheromatous plaques (atherosclerosis),
and can also be used to diagnose liver and thyroid diseases. In some cases, cholesterol levels are
monitored before initiating drug therapy.
PRINCIPLE
The cholesterol level is measured after enzymatic hydrolysis and then oxidation. The quinoneimine
indicator is formed from hydrogen peroxide and amino 4 antipyrine in the presence of phenol and
peroxidase according to the following reactions:
Cholesterol esterase
Cholesterol esters + H
2
O Cholesterol + Fatty Acid
Cholesterol oxidase
Cholesterol + O
2
Cholestene-4-one-3 + H
2
O
2
Peroxidase
H
2
O
2
+ Phenol +Amino-4–antipyrine Quinoneimine rose
The quantity of quinoneimine formed is proportional to the concentration of cholesterol.
REAGENT COMPOSITION
Reagent 1
Buffer
solution
Pipes pH 6.9
Phenol
90 mmol/l
26 mmol/l
Reagent 2
Enzymes
Cholesterol oxidase
Peroxidase
Cholesterol esterase
Amino-4-antipyrine
300 U/l
1250 U/l
300 U/l
0.4 mmol/l
Reagent 3
Standard
Standard Cholesterol
200 mg/dl
2 g/l
5.17 mmol/l
SAFETY CAUTIONS
Biomaghreb reagents are intended for use by qualified personnel for in vitro use (do not pipette by
mouth).
• Refer to the current SDS available on request or at www.biomaghreb.com;
• Verify the integrity of the reagents before use; and
• Disposal of waste: comply with the legislation in force.
For safety reasons, treat any specimen or reagent of biological origin as potentially infectious.
Respect the legislation in force.
REAGENT PREPARATION
Working solution: Dissolve the lyophilisate R2 with the contents of a Buffer R1 vial.
SAMPLE PREPARATION
Serum, plasma heparinized non-hemolyzed.
PRESERVATION AND STABILITY
• Before opening: Until the expiry date indicated on the label of the box at 2-8°C;
• After opening (Working Solution) :
1 month at 20-25°C;
4 months at 2-8°C.
ADDITIONAL EQUIPMENT
• Basic equipment of the medical analysis laboratory ;
• Spectrophotometer or Clinical Biochemistry Analyzer.
QUALITY CONTROL
External quality control program.
It is recommended to control in the following cases:
• At least one test per series.
• Change of reagent bottle.
• After maintenance work on the analyzer.
If a control value is outside the confidence limits, repeat the procedure using the same control.
Use normal and pathological control sera.
CALIBRATION
The standard of the kit (Reagent 3) or any calibrator connected to a method or reference material.
The frequency of calibration depends on analyzer performance and reagent storage conditions.
Recalibration is recommended in the following cases:
1. Changing the reagent lot;
2. After maintenance work on the analyzer; and
3. Control values are outside the confidence limits.
LINEARITY
The method is linear up to 6 g/l (600 mg/dl - 15.4 mmol/l). If the cholesterol concentration is higher
than 6 g/l, dilute the sample 1/2 with 9 g/l NaCl solution and repeat the test. Multiply the result by 2.
PROCEDURE
Wavelength: 505 nm (500-550);
Temperature: 37°C ;
Tank: 1 cm thick;
Adjust the spectrophotometer zero on the reagent blank.
Blank Standard Sample
Standard
- - 10 µl - -
Sample
- - - - 10 µl
Working solution
1 ml 1 ml 1 ml
Mix, read absorbances after incubation for 5 minutes at 37°C or 10
minutes at 20 - 25°C. Staining is stable 30 minutes.
CALCUL
Cholesterol =
OD. Sample
OD. Standard
x n
n = Standard value
n = 200 mg/dl;
n = 2 g/l;
n = 5, 17 mmol/l.
REFERENCE VALUES
Serum or
plasma
3,6 at 5,7 mmol/l
1,4 at 2,2 g/l
140 at 220 mg/dl
REFERENCES
Trinder P., Ann. Clin. Biochem. 6, 24 (1969) ;
Richmond, Clin. Chem. 19, 1350 (1973) ;
Fasce C.F., Clin. Chem. 18901 (1982).
Manufacturer Use by In Vitro Diagnostic Temperature
Limitation
Catalogue number See insert Store away from light Sufcient
for < n > essais
Batch number
