2 x 40 mL / 2 x 60 mL / 4 x 60 mL

HDL CHOLESTEROL (D) WITH CALIBRATOR 11414003, 11414005, 11414004

INTENDED USE Wavelength II 630 nm (630-700)
This reagent is intended for in vitro quantitative determination of HDL Cholesterol in Temperature 370C
serum Calibrator Con As on the vial
- Direct determination of HDL Cholesterol Linearity 150 mg/dL
- Selective Inhibition Method Incubation time 5 min+5 min
- Linear up to 150 mg/dL Blank Reagent
- Ready to use liquid stable reagents Sample volume 5 μL
CLINICAL SIGNIFICANCE Reagent volume 450 μL+150 μL
Blood total cholesterol levels have long been known to be related to coronary heart Cuvette 1 cm light path
disease (CHD). In recent years, in addition to total cholesterol, high density LABORATORY PROCEDURE
lipoprotein cholesterol (HDL-C) has become an important tool used to assess an
individual risk of developing CHD since a strong negative relationship between Blank Calibrator Sample
HDL - C concentration and the incidence of CHD was reported. Reagent1 450 μL 450 μL 450 μL
Calibrator - 5 μL -
PRINCIPLE
Sample - - 5 μL
The reaction between cholesterol other than HDL and the enzyme for cholesterol
assay is suppressed by the electrostatic interaction between polyanions & cationic Mix and incubate for 5 minutes at 370C.
substances. Hydrogen peroxide is formed by the free cholesterol in HDL by cholesterol Reagent 2 150 μL 150 μL 150 μL
oxidase. Oxidative condensation of EMSE and 4-AA is caused by hydrogen peroxide Mix and incubate for 5 minutes at 370C. Measure the absorbance of calibrator &
in the presence of peroxidase, and the absorbance of the resulting red-purple quinone sample against reagent blank at 578nm/ 630 nm.
is measured to obtain the cholesterol value in HDL
CALCULATION
Polyanions
Absorbance of Sample
Other lipoproteins than HDL --------------> Suppress reaction with enzyme
HDL-C Concentration (mg/dL) = ------------------------------ x Calibrator Concentration
Cationic substances
Absorbance of Calibrator
cholesterol esterase
HDL (cholesterol esters) + H2O --------------------------> HDL (free cholesterol) + ALTERNATIVE PROCEDURE
Free fatty acids Mode of Reaction Differential
cholesterol oxidase Slope of reaction Increasing
HDL (free cholesterol) + O2 + H+ -------------------------> Cholestenone + H2O2
Wavelength I 600 nm
Peroxidase
2H2O2 + 4-AA + EMSE + H3 + O ---------------> Violet quinone + 5H2O Wavelength II 700 nm
Temperature 370C
REAGENT COMPOSITION
HDL–C DIRECT R1 2 x 30 mL / 2 x 45 mL / 4 x 45 mL Calibrator Concentration As on the vial label
N—ethyl-N-(3-methylphenyl)-N’succinylethyenediame(EMSE) Linearity 150 mg/dL
HDL–C DIRECT R2 2 x 10 mL / 1 x 30 mL / 2 x 30 mL Blank Reagent
Cholesterol Oxidase Incubation time 5 minutes+5 minutes
4-Aminoantipyrin(4-AA) Sample volume 3 μL
HDL–C DIRECT CALIBRATOR 1 x 3 mL
Reagent volume 270 μL + 90 μL
STORAGE AND STABILITY Cuvette 1 cm light path
The sealed reagents are stable upto the expiry date stated on the label, when stored LABORATORY PROCEDURE
at 2 - 80C, protected from light. Do not freeze.
Blank Calibrator Sample
LINEARITY Reagent1 270 μL 270 μL 270 μL
This reagent is linear upto 150 mg/dL Calibrator - 3 μL -
If the concentration is greater than linearity (150 mg/dL), dilute the sample with normal Sample - - 3 μL
saline and repeat the assay. Multiply the result with dilution factor Mix and incubate for 5 minutes at 370C. Measure the absorbance (OD1) at 600 nm/
NORMAL RANGE 700 nm.
It is recommended that each laboratory establish its own reference values. Reagent 2 90 μL 90 μL 90 μL
The following value may be used as guide line. Mix and incubate for 5 minutes at 370C. Measure the absorbance (OD2) at 600 nm/
Male : 35 - 80 mg/dL 700 nm.
Female : 42 - 88 mg/dL CALCULATION
PREPARATION AND STABILITY OF WORKING REAGENT (OD2-OD1) Sample
The Reagent1 & Reagent 2 are ready to use. HDL-C Concentration (mg/dL) = ---------------------------- x Calibrator Conc.
Calibrator : Reconstitute with 3 mL of distilled water. Let it stand for 30 minutes at (OD2-OD1) Calibrator
room temperature. Dissolve the content of the vial by swirling gently to avoid the INTERFERING SUBSTANCES
formation of foam. Test will not be affected by:
Stability : Reconstituted calibrator is stable only for 7 days at 2- 80C. Bilirubin up to 40 mg/dL
PRECAUTION Ascorbic acid up to 50 mg/dL
To avoid contamination, use clean laboratory wares. Use clean, dry disposable Hemoglobin up to 500 mg/dL
pipette tips for dispensing. Close reagent bottles immediately after use. Avoid direct Triglyceride up to 1000 mg/dL
exposure of reagent to light. Do not blow into the reagent bottles. *(when triglyceride in a sample exceeds 1000 mg/dL, dilute the sample 1+9 with
SAMPLE saline, repeat the assay and multiply result by 10)
Fresh serum (free of haemolysis) BIBLIOGRAPHY
GENERAL SYSTEM PARAMETER 1. Williams, P., et al.; High density lipoprotein and coronary risk factor, Lancet. 1:72
(1979)
Mode of Reaction End Point 2. Gordon,T., Castelli, W.P., Hjortland, M.C. et al. Am. J.Med. 62, 707-714 (1977)
Slope of reaction Increasing 3. Rifai,N.and Warnick, G.R.,Ed. Laboratory Measurement of Lipids, Lipoproteins
Wavelength I 578 nm(578-610) and Apolipoproteins AACC Press. Washington, DC,USA,1994

ADL/V.03/180114