N BIO - Direct HDL
(Direct clearance method)
KIT NAME KIT SIZE CAT. NO
N BIO - Direct HDL 1 x 40 ml DHDL01040M GENUINE BIOSYSTEM
N BIO - Direct HDL 2 x 40 ml DHDL01080M
INTRODUCTION CONCENTRATIONS IN THE TEST
Plasma lipoproteins are spherical particles containing varying
Cholesterol Esterase > 1000 U/L
amounts of cholesterol, triglycerides, phospholipids and proteins.
Cholesterol Oxidase > 800 U/L
The relative protein and lipid determine the density of these
Catalase > 900 U/L
lipoproteins and provide the basis on which to begin their
TOOS
classification. The classes are: chylomicron, very-low-density
4 – aminoantipyrine > 4000 U/L
lipoprotein (VLDL), low-density-lipoprotein (LDL) and high-density
peroxidase ≥3.5 KU/L
lipoprotein (HDL). The principle role of HDL in lipid metabolism is
sodium azide 0.05 %
the uptake and transport of cholesterol from peripheral tissues to
surfactants 1.4 %
the liver. Low HDL cholesterol (HDL-C) levels are strongly
associated with an increased risk of coronary artery disease. WARNINGS AND NOTES
METHOD PRINCIPLE - Product for in vitro diagnostic use only.
The assay consists of 2 distinct reaction steps: - The reagents are ready to use.
1. Elimination of chylomicron, VLDL and LDL by cholesterol - Do not pipette by mouth.
esterase, cholesterol oxidase and subsequently catalase. - The reagents contain 0.05% sodium azide as a preservative.
- Avoid contact with skin and mucous membranes.
cholesterol esterase
SPECIMEN
cholesterol ester -----------------------> cholesterol + fatty acid
Serum, heparinized or EDTA plasma.
cholesterol oxidase Blood should be collected only if the patient has been fasting for 12
cholesterol + O2 -----------------------> cholestenone + H2O2 -14 hours.
Serum and plasma can be stored up to 6 days at 2 - 8°C. Sample are
Catalase stable for 1 year when stored at for -70°C. Samples may be frozen
2 H2O2 -----------> H2O + O2 once. If any samples show precipitates, centrifuge before using
2. Specific measurement of HDL-Cholesterol after release of HDL .Nevertheless it is recommended to perform the assay with freshly
Cholesterol by detergents in R2 Reagent. In the second reaction collected samples.
catalase is inhibited by sodium azide in R2-Reagent. PROCEDURE
cholesterol esterase These reagents may be used both for manual assay and in several
cholesterol ester -----------------------> cholesterol + fatty acid automatic analysers. Applications for them are available on
request.
cholesterol oxidase
cholesterol + O2 -----------------------> cholestenone + H2O2 Wavelength 578 nm
Temperature 37°C
peroxidase Cuvette 1 cm
2 H2O2 + 4-AA + HDAOS -----------> quinone pigment + 4 H2O
Pipette into the cuvette:
The colour intensity is proportional to the HDL-cholesterol
Reagent Blank (B) Calibrator (C) Test (T)
concentration when measured at 600 nm.
R1 HDL Reagent 450 µl 450 µl 450 µl
KIT CONTENTS
Distilled Water 5 µl - -
Reagent Name DHDL01040M DHDL01080M
R3 - Calibrator - 5 µl -
R1 HDL Reagent 1 x 30 ml 2 x 30 ml
Sample - - 5 µl
R2 HDL Reagent 1 x 10 ml 2 x 10 ml
Mix well and incubate for 5 mins at 37° C, than add
R3 - Calibrator 1 vial 1 vial R2 HDL Reagent 150 µl 150 µl 150 µl
R3-Calibrator will be provided, the concentration will be printed Mix well & incubate for 5 min. at 37 C. Measure the absorbance of
o
on the label. calibrator & sample against reagent blank.
WORKING REAGENT PREPARATION AND CALCULATION
STABILITY Concentration in mg/dl = [Link] X Calibrator Concentration
The reagents R1 & R2 are stable up to the kit expiry date printed on [Link]
the package when stored at 2-8°C. Protect from light. REFERENCE VALUE
Calibrator: Reconstitute with required distilled water mentioned Male 35 to 80 mg / dl
on the bottle label. Let it stand for 30 minutes at room Female 42 to 88 mg / dl
temperature. Dissolve the content of the vial by swirling gently to
avoid the formation of foam. The reconstituted calibrator is stable It is recommended for each laboratory to establish its own
only for 7 days at 2-8°C. reference ranges for local population.
QUALITY CONTROL SYSTEM PARAMETERS
To ensure adequate quality control, each run should include
Method End Point
assayed normal and abnormal controls. If commercial controls are
not available it is recommended that known value samples be Wavelength 578 nm
aliquoted, frozen and used as controls. Zero Setting Reagent Blank
PERFORMANCE CHARACTERISTICS Temperature Setting 37° C
Linearity: The procedure is linear upto 180 mg/dl. Patient Incubation Temperature 37° C
samples with HDL cholesterol levels exceeding 100 mg/dl should Incubation Time 5 min + 5 min
be diluted with physiological saline before assaying. Multiply the
result obtained from the manual dilution by the appropriate Delay Time ----
dilution factor. Read Time ----
Sensitivity / Limit of Quantitation: 0.55 mg/dl No. of Reading ----
Specificity / Interferences Interval Time ----
Bilirubin up to 20 mg/dl, , haemoglobin up to 20 g/dl, ascorbate up Sample Volume 0.005 ml (5 ul)
to 62 mg/l and triglycerides up to 500 mg/dl do not interfere with
Reagent Volume 0.6 ml (600 ul)
the test.
Standard Concentration Refer Calibrator vial
WASTE MANAGEMENT
Units mg / dl
Please refer to local legal requirements.
Factor ----
LITERATURE
1. National Institutes of Health Consensus Development Reaction Slope Increasing
Conference Statement: Triglyceride, High Density Lipoprotein Linearity 180 mg / dl
and Coronary Heart Disease. Washington D.C. Feb 26-28, 1992.
2. Izawa S., Okada M., Matusui H., and Horita Y. J. Medicine and
Pharmaceutical Sci., 1385-1388, 37 (1997).
3. Shih WJ, Bachorik PS, Haga JA, Myers GL, Stein EA; Clinical
Chemistry, 2000; 46:3:351 – 364.
4. Third Report of the National Cholesterol Education Programme
(NCEP) Expert Panel on Detection, Evaluation and treatment of
High Blood Cholesterol in Adults (Adult Treatment Panel III)
JAMA Publication, Vol 285, No. 19, P2486 – 2497; 2001.
5. Jacobs D., et al. In Laboratory and Test Handbook; Jacobs D.S;
Kasten B.L., De Mott W.R., Wolfson W.L., Eds; Lexi-Comp Inc:
Hudson (Cleveland), 1990; P.219.
6. Gordon T. et al.: Am. J. Med., 62; 707 (1977).
7. Wiliams P., Robinson D., Baily A.: Lancet, 1/72 (1979).
8. Goto A.M.: Hospital Practice, 23; Suppl., 1, 4 (1988).
9. Crouse J.R. et al.: J. Lipid Res., 26; 566 (1985).
10. Badmion J.J., Badmion L., Fuester V.: Journal of Clinical
Investigation, 85:1234-41 (1990).
11. Castelli W.P. et all.: Circulation, 55; 767 (1977).
12. Barr D.P., Russ E.M., Eder H.A.: Am. J. Med., 11; 480 (1951).
13. Kannel W.B., Castelli W.P., Gordon T.: Ann. Intern. Med., 90:85
(1979).
Genuine Biosystem Private Limited
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Parvathy Nagar Extension,
Old Perungalathur, Chennai - 600063, India.
Ph: +91-44-48681845
GBPL/DHDL/04 12.20 Email: genuinebiosystem@[Link]
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