SAMPLES

R2 GOOD PH 7.0 Serum or heparinazed plasma: Free of Hemolysis.

Cholesterol esterase <1500 U/L Removed from the blood clot as soon as possible.

4-Aminoantipyrine(4-AP) <1mM Stability : HDL Cholesterol is stable for 7 days at 2-8C .

HDL cholesterol Detergent < 2%
Direct Enzymatic colorimetric PROCEDURE
Ascorbic oxidase <3000 U/L 1. Assay conditions:
Quantitative determination Wavelength: . . . . . . . . 600 -700 nm
Peroxidase <1300 U/L
For In-Vitro and professional use only Cuvette: ... . . . . 1 cm light path
HDLc CAL Calibrator .Lyophilized human serum. Temperature . . . .. . 37C
Store at 2 to 8C 2. Adjust the instrument to zero with distilled
INTENDED USE water.
For direct determination of human serum HDL cholesterol PREPARATION
3. Pipette into a cuvette:
concentration The reagent is ready to use.
Dissolve HDL/LDL Calibrator with 1ml of Distilled
INTRODUCTION Blank Calibrator Sample
water.
HDL particles carry cholesterol from the cells back to the R1(L) 300 300 300
Mix thoroughly, avoiding foam forming.
liver. Calibrator -- 3 --
Bring to room temperature for about 30 min
HDL is known as good cholesterol because high levels Sample (L) -- -- 3
.before use.
are thought to lower the risk of heart disease. 4. Mix well and incubate for 5 min at 37 C.
Improper handing and/ or storage can affect
A low HDL cholesterol levels, is considered a greater heart 5. Read the absorbance (A1) of the sample and
results.
disease risk. Calibrator
Inaccurate reconstitution and errors in assay
Clinical diagnosis should not be made on a single test 6. Add
technique can cause erroneous results.
result; it should integrate clinical and other laboratory Blank Calibrator Sample
data. R2(L) 100 100 100
STORAGE AND STABILITY
PRINCIPLE OF THE METHOD 7. Mix well and incubate for 5 min at 37 C.
All the components of the kit are stable until the
Directly determination of serum HDLc and( high-density 8. Read the absorbance (A2) of the sample and
expiration date on the label when stored tightly
lipoproteins cholesterol ) levels without the need for any Calibrator, against the Blank.
closed at 2-8C, and contaminations prevented
pre-treatment or centrifugation of the sample. 9. Calculate the increase of the absorbance
during their use.
The method depends on the properties of a detergent A=A2-A1 .
Do not freeze the reagent
which solubizies only the HDL so that the HDL-c is Do not use reagents over the expiration date.
released to react with the cholesterol esterase, After reconstitution, HDL/LDL calibrator is stable CALCULATIONS
cholesterol oxidase and Chromogens to give color .The for: (A) Sample x Calibrator (conc.) = mg/dL HDLc (in the
non HDL lipoproteins LDL,VLDL and chylomicrons are o R1/R2: Once opened is stable 8weeks at
inhibited from reacting with the enzymes due to (A)Calibrator sample).
2-8c
absorption of the detergents on their surfaces . o HDLc/LDLc CAL : Once reconstitute CONVERSION FACTOR: mg/dL x0.0259 =mmol/L
The intensity of the color formed is proportional to the 2weeks at 2-8c or 3 montha at -20c
HDL-c concentration in the sample . Signs of reagent deterioration: QUALITY CONTROL
REAGENTS Presence of particles and turbidity. Control sera are recommended to monitor the
GOOD Ph 7.0 EQUIPMENTS NEEDED BUT NOT PROVIDED performance of assay procedures. If control values are
R1 Spectrophotometer or colorimeter measuring at found outside the defined range, check the instrument,
Cholesterol oxidase <1000U/L
DSBmT <1mM 600 nm. reagents and calibrator for problems.
Matched cuvettes 1.0 cm light path. Each laboratory should establish its own Quality Control
General laboratory equipment. scheme and corrective actions if controls do not meet
the acceptable tolerances. following:
HDL/LDL cholesterol calibrator values are: 45mg/dl Correlation coefficient (r): 0.996.
HDL Cholesterol Calibrator: 1.03mmol/l Regression equation: y= 0,98 x + 3.42
The results of the performance characteristics depend on
REFERENCE VALUES the analyzer used.
Men Women
INTERFERENCES
No interferences were observed to bilirubin T.and D up
Lower risk > 50 mg/dl > 60 mg/dl to 60 mg/dL
Hemoglobin up to 1000mg/dL.or lipemia up to 1800
mg/dL
A list of drugs and other interfering substances with HDL
Standard risk 35-50 mg/dl 45-60 mg/dl cholesterol determination has been reported by Young
et. Al.
Notes
Increased risk < 35 mg/dl < 45 mg/dl
The reagent 2 presents yellowish coloration due to the
peroxidaes ,but it dose not affect its functionality.

These values are for orientation purpose; each laboratory

REFERENCES
should establish its own reference range.
1. Naito H K. High-density lipoprotein (HDL) cholesterol.
PERFORMANCE CHARACTERISTICS Kaplan A et al. Clin Chem The C.V. Mosby Co. St
Measuring range: Louis. Toronto. Princeton 1984; 1207-1213 and 437.
From detection limit of 2.5 mg/dL to linearity limit of 2. Grove T H. Effect of reagent pH on Determination of
200 mg/dL. HDL Cholesterol by precipitation with Sodium
If the results obtained were greater than linearity limit, Phosphotungstate-magnesium Clin Chem 25:560,
dilute the sample 1/2 with NaCl 9 g/L and multiply the 1979.
result by 2. 3. US National Cholesterol Education Program of the
Precision: National Institutesof Health.
Intra-assay Inter-assay 4. Young DS. Effects of drugs on Clinical Lab. Tests, 4th
(n=20) (n=20) ed AACC Press, 1995.
Mean 32.9 50.6 32.8 50.0 5. Young DS. Effects of disease on Clinical Lab. Tests,
(mg/dL) 4th ed. AACC 2001.
6. Burtis A. et al. Tietz Textbook of Clinical Chemistry,
SD 0.3 0.2 0.4 0.7
3rd ed. AACC 1999.
CV (%) 0.8 0.5 1.3 1.5 7. Tietz N W et al. Clinical Guide to Laboratory Tests,
3rd ed. AACC 1995.
Sensitivity:
ATLAS Medical
1 mg/dL = 0.0016 A.
William James House,
Accuracy:
Cowley Road, Cambridge, CB4 4WX, UK
Results obtained using ATLAS reagents (y) did not show
Tel: ++44 (0) 1223 858 910
systematic differences when compared with other
Fax: ++44 (0) 1223 858 524
commercial reagents (x).
PPI690A01
The results obtained using 50 samples were the
Rev B (03.01.2010)