CHOLESTEROL HDL

COD 11648 50 mL
PRECIPITATING REAGENT
STORE AT 2-8ºC
Reagents for measurement of HDL cholesterol concentration CHOLESTEROL HDL
Only for in vitro use in the clinical laboratory
PRECIPITATING REAGENT

PRINCIPLE OF THE METHOD REFERENCE VALUES

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in the sample precipitate HDL cholesterol concentrations vary considerably with age and sex. The following cut-off point
with phosphotungstate and magnesium ions. The supernatant contains high density lipoproteins has been recommended for identifiying individuals at high risk of coronary artery disease3.
(HDL). The HDL cholesterol is then spectrophotometrically measured by means of the coupled
reactions described below1,2. Up to 35 mg/dL = 0.91 mmol/L High
chol. esterase > 60 mg/dL = > 1.56 mmol/L Low
Cholesterol ester + H2O Cholesterol + Fatty acid
chol. oxidase QUALITY CONTROL
Cholesterol + ½ O2 + H2O Cholestenone + H2O2
peroxidase It is recommended to use the Lipid Control Serum level I (cod. 18040) and II (cod. 18041) to
2 H2O2 + 4 – Aminoantipyrine + Phenol Quinoneimine + 4 H2O verify the performance of the measurement procedure.

CONTENTS AND COMPOSITION Each laboratory should establish its own internal Quality Control scheme and procedures for
corrective action if controls do not recover within the acceptable tolerances.
A. Reagent: 1 x 50 mL. Phosphotungstate 0.4 mmol/L, magnesium chloride 20 mmol/L.
S. HDL Cholesterol Standard: 1 x 5 mL. Cholesterol 15 mg/dL. Aqueous primary standard. METROLOGICAL CHARACTERISTICS
− Detection limit: 0.3 mg/dL = 0.008 mmol/L
STORAGE
− Linearity limit: 150 mg/dL = 3.9 mmol/L.
Store at 2-8ºC. − Repeatibility (within run):
Reagent and Standard are stable until the expiry date shown on the label when stored tightly
Mean Concentration CV n
closed and if contaminations are prevented during their use.
30 mg/dL = 0.78 mmol/L 3.3 % 20
Indications of deterioration:
55 mg/dL = 1.42 mmol/L 2.0 % 20
− Reagent: Presence of particulate material, turbidity.
− Standard: Presence of particulate material, turbidity. − Reproducibility (run to run):
ADDITIONAL REAGENTS Mean Concentration CV n
These auxiliary reagents are to be used together with the Cholesterol Reagent contained in any 30 mg/dL = 0.78 mmol/L 4.2 % 20
of the BioSystems Cholesterol kits (cod. 11805, 11505, 11506, 11539). 55 mg/dL = 1.42 mmol/L 3.2 % 20

REAGENT PREPARATION − Sensitivity: 1.75 mA⋅dL/mg = 67.6 mA⋅L/mmol

Reagent and Standard are provided ready to use. − Trueness: Results obtained with this reagent did not show systematic differences when
compared with reference reagents (Note 4). Details of the comparison experiments are
ADDITIONAL EQUIPMENT available on request.
− Interferences: Lipemia (triglycerides 10 g/L) does not interfere. Bilirubin (10 mg/dL) and
− Desktop centrifuge
hemoglobin (5 g/L) may intefere. Other drugs and substances may interfere4.
− Thermostatic water bath at 37ºC
These metrological characteristics have been obtained using an analyzer. Results may vary if a
− Analyzer, spectrophotometer or photometer able to read at 500 ± 20 nm
different instrument or a manual procedure are used.
SAMPLES DIAGNOSTIC CHARACTERISTICS
Serum or plasma collected by standard procedures.
HDL play an important part in the removal of cholesterol from tissues and its transportation to
HDL cholesterol in serum or plasma is stable for 7 days at 2-8ºC. Heparin, EDTA, oxalate and the liver for removal as bile acids.
fluoride may be used as anticoagulants.
Decreased plasma HDL-cholesterol concentrations are positively correlated with the incidence
PROCEDURE of atherosclerosic diseases, basis of myocardial infarction and cerebrovascular accidents5,6.
There are several disease states or environmental influences associated with reduced levels of
Precipitation HDL: acute or chronic hepatocellular diseases, intravenous hyperalimentation, severe
1. Pipette into labelled centrifuge tubes (Note 1): malnutrition, diabetes, chronic anemia, myeloproliferative disorders, Tangier disease,
analphalipopro-teinemia, acute stress, some drugs and smoking5,6.
Sample 0.2 mL Clinical diagnosis should not be made on the findings of a single test result, but should integrate
Reagent (A) (Cholesterol HDL kit) 0.5 mL
both clinical and laboratory data.
2. Mix thoroughly and let stand for 10 minutes at room temperature.
NOTES
3. Centrifuge at a minimum of 4000 r.p.m. for 10 minutes.
4. Carefully collect the supernatant (Note 2). 1. Sample and Reagent A volumes may be varied as long as the same ratio is maintained.
2. Supernatant must be clear. When supernatant is turbid or the pellet floats, add again 0.5 mL
Colorimetry of Reagent A, mix thoroughly and centrifuge. Multiply the obtained concentration by 1.7
5. Bring the Reagent (Cholesterol kit) to room temperature. (dilution).
6. Pipette into labelled test tubes: (Note 3) 3. These reagents may be used in several automatic analysers. Instructions for many of them
are available on request.
Blank Standard Sample
4. Calibration with the provided aqueous standard may cause a matrix related bias, specially in
Distilled water 100 µL   some analyzers. In these cases, it is recommended to calibrate using a serum based
HDL Cholesterol Standard (S)  100 µL  standard (Biochemistry Calibrator, cod. 18011 and 18044).
Sample supernatant   100 µL
Reagent (A) (Cholesterol kit) 1.0 mL 1.0 mL 1.0 mL BIBLIOGRAPHY
7. Mix thoroughly and incubate the tubes for 30 minutes at room temperature (16-25ºC) or for 10 1. Grove TH. Effect of reagent pH on determination of high-density lipoprotein cholesterol by
minutes at 37ºC. precipitation with sodium phosphotungstate-magnesium. Clin Chem 1979; 25: 560-564.
8. Measure the absorbance (A) of the Standard and Sample at 500 nm against the Blank. The 2. Burstein M, Scholnick HR and Morfin R. Rapid method for the isolation of lipoproteins from
colour is stable for at least 30 minutes. human serum by precipitation with polyanions. Scand J Clin Lab Invest 1980; 40: 583-595.
CALCULATIONS 3. National Cholesterol Educarion Program Expert Panel. Third report of the National
Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and
The HDL cholesterol concentration in the sample is calculated using the following general Treatment of High Blood Cholesterol in Adults (ATP III). NIH Publication. Bethesda: National
formula: Heart, Lung, and Blood Institute; 2001.
A Sample
x C Standard x Sample dilution factor = C Sample 4. Young DS. Effects of drugs on clinical laboratory tests, 4th ed. AACC Press, 1995.
A Standard
5. Tietz NW. Clinical guide to laboratory tests, 2nd ed. Saunders Co, 1991.
6. Friedman and Young. Effects of disease on clinical laboratory tests, 3th ed. AACC Press,
If the HDL Cholesterol Standard provided has been used to calibrate (Note 4): 1997.

A Sample x 52.5 = mg/dL HDL cholesterol

A Standard x 1.36 = mmol/L HDL cholesterol

M11648i-0515 BioSystems S.A. Costa Brava 30, Barcelona (Spain)

Quality System certified according to
EN ISO 13485 and EN ISO 9001 standards