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www.biomaghreb.com
IVD
FT En 47 Date of updating : 10/ 2020 Version B
IN VITRO DIAGNOSTIC USE
Manufacturer Use by In Vitro Diagnostic Temperature
Limitation
Catalogue number See insert Store away from light Sufcient
for < n > essais
Batch number
HDL-CHOLESTEROL DIRECT
CLINICAL SIGNIFICANCE
The principal role of high density lipoprotein (HDL) in lipid metabolism is the uptake and transport
of cholesterol from peripherical tissue to the liver through a process known as reverse cholesterol
transport. Low HDL cholesterol levels are strongly associated with an increased risk of coronary
heart disease and coronary artery disease. Hence, the determination of serum HDL-Cholesterol is
a useful tool in identifying high-risk patients. Increased Total Cholesterol/HDL-Cholesterol ratio is
significant of an increased risk of atheroslerosis.
PRINCIPLE
“Selective detergent and accelerator» methodology
Direct method, without pre-treatment of the specimen.
During the first phase, LDL, VLDL, and chylomicron particles release free cholesterol which under-
goes an enzymatic reaction, producing hydrogen peroxide, which is degraded by the reaction with
POD and DSSmT. No coloured derivatives are formed.
During the second phase, a specific detergent solubilises the HDL cholesterol. Under the combined
action of CO and CE, the POD + 4- AAP couple develops a coloured reaction proportional to the HDL
cholesterol concentration. The reading is taken at 600 nm.
LDL= Low Density Lipoproteins
HDL = High Density Lipoprotein
VLDL= Very Low Density Lipoproteins - POD = Peroxidase
CO = Cholesterol Oxidase - CE = Cholesterol Esterase – 4
AAP = 4-Aminoantipyrine - AAO = Ascorbate Oxidase
DSSmT = N, N-bis(sulphobutyl)-m-toluldine-disodium
REAGENT COMPOSITION
Reagent 1
GOOD
Cholesterol oxidase
Peroxidase
DSBmT
pH = 7
< 1000 U/I
< 1300 U/ l
< 1mM
Reagent 2
GOOD
Cholestérol oxydase
4 Amino Antipyrine
Detergent
Ascorbate oxidase
pH = 7
< 1500 U/l
< 1mM
< 2%
< 3000 U/ l
Reagent 3 HDLc/LDLc calibrator: freeze-dried human serum
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
Reagents R1 and R2 are ready for use.
- Reconstitute the vial of the HDLc/LDLc calibrator with 1 ml of distilled water, then homogenize the
contents of the bottle gently.
- Wait 30 minutes before use.
For safety reasons, treat the calibrator as potentially infectious.
SAMPLE PREPARATION
The patient must be taken after at least 12h -14h fasting
-Plasma : collected on EDTA or sodium heparinate or lithium ; citrate must not be used.
Separate plasma from blood cells by centrifugation within 3 hours after collection.
-Serum: Centrifugally separate the Serum from the blood cells within 3 hours after collection.
Sera and plasma should not be left at room temperature for more than 14 hours.
HDL-cholesterol is stable in the specimen:
- 7 days at 2-8°C ;
- 1 month at -20°C.
PRESERVATION AND STABILITY
Store at 2-8°C, in the original bottle, tightly stoppered and protected from light.
• Before opening : if stored under the recommended conditions, the reagents are stable until
the expiry date indicated on the label.
• After opening and in the absence of contamination: Reagents R1 and R2 are stable for 8
weeks at 2-8°C.
• After reconstitution: The calibrator is stable 2 weeks at 2-8°C and 3 months at -20°C.
LIMITS
Do not use the reagents if they are cloudy or after the expiry date.
ADDITIONAL EQUIPMENT
• Basic equipment of the medical analysis laboratory ;
• Spectrophotometer or Clinical Biochemistry Analyzer.
LINEARITY
The reaction is linear from 25mg/dl up to 200mg/dl.
Above this concentration, dilute the sample 1+1 with a
9 g/l NaCl solution and repeat the determination.
Multiply the result by 2.
PROCEDURE
Wavelength…………………………..............600-700 nm
Tank: ……………………………………………1 cm thick
Temperature...............................................…………37°C
Adjusting the spectrophotometer zero with distilled water
Blank Calibrator Dosage
Reagent R1 300 µl 300 µl 300 µl
Calibrator - - 3 µl - -
Sample - - - - 3 µl
Stir well, leave to stand for 5 minutes at 37°C.
Record absorbances A1 at 600 nm against the reagent blank.
Add Blank Calibrator Dosage
Reagen R2 100 µl 100 µl 100 µl
Mix well, let stand 5 minutes at 37°C.
Record absorbances A2 against the reagent blank
CALCULATION
Calculating the increase in absorbance A = A2-A1
A sample
A calibrator
x Calibrator Concentration
= mg/dl of HDL direct
with mg/dl x 0,0259 = mmol/l
REFERENCE VALUES
Men Women
Low risk > 50mg/dl > 60mg/dl
Moderate risk 35-50mg/dl 45-60mg/dl
High risk <35mg/dl <45mg/dl
-Concentrations tested (mg/dl) without significant interference (+10%):
Conjugated bilirubin: 60 mg/dl
Total Bilirubin: 60 mg/dl
Hemoglobin : 1000 mg/dl
Ascorbic Acid: 100 mg/dl
Fat (intralipid) 1800 mg/dl
-The reagent may interfere with the magnesium determination.
REFERENCES
- bNaito N K HDL.Cholesterol, Kaplon A et al.Clin Chem the C.V.Mosby Co.St Louis. Toronto. Prince-
ton 1984 : 1207 - 1213 and 437.
- Us Naffonal Cholesterol Education Program of the National instites of Health.
- Young DS. Effects of drugs on Clinical Lab. tests, 4th ed AACC Press.1995.
- Young DS. Effects ofdisease on Clinical Lab. Tests, 4th ed AACC 2001.
- Burits A et al. Tietz Texibook of Clinical Chemistry, 3rd ed AACC 1999.
- Tietz N W et al. Clinical Guide to laboratory tests, 3rd ed AACC 1995. PCT/JP97/04442.
