This document provides instructions for using a reagent kit to quantify cholesterol and HDL cholesterol levels in serum or plasma samples. It describes the intended use, clinical significance, principles, procedures, calculations, quality controls, limitations and references for both tests. The cholesterol assay uses enzymatic reactions to produce a red quinoneimine dye complex, while the HDL cholesterol assay uses precipitation to separate HDL from other lipoproteins before quantification. Both methods are end-point assays measuring absorbance at specific wavelengths to determine concentrations.
This document provides instructions for using a reagent kit to quantify cholesterol and HDL cholesterol levels in serum or plasma samples. It describes the intended use, clinical significance, principles, procedures, calculations, quality controls, limitations and references for both tests. The cholesterol assay uses enzymatic reactions to produce a red quinoneimine dye complex, while the HDL cholesterol assay uses precipitation to separate HDL from other lipoproteins before quantification. Both methods are end-point assays measuring absorbance at specific wavelengths to determine concentrations.
This document provides instructions for using a reagent kit to quantify cholesterol and HDL cholesterol levels in serum or plasma samples. It describes the intended use, clinical significance, principles, procedures, calculations, quality controls, limitations and references for both tests. The cholesterol assay uses enzymatic reactions to produce a red quinoneimine dye complex, while the HDL cholesterol assay uses precipitation to separate HDL from other lipoproteins before quantification. Both methods are end-point assays measuring absorbance at specific wavelengths to determine concentrations.
This document provides instructions for using a reagent kit to quantify cholesterol and HDL cholesterol levels in serum or plasma samples. It describes the intended use, clinical significance, principles, procedures, calculations, quality controls, limitations and references for both tests. The cholesterol assay uses enzymatic reactions to produce a red quinoneimine dye complex, while the HDL cholesterol assay uses precipitation to separate HDL from other lipoproteins before quantification. Both methods are end-point assays measuring absorbance at specific wavelengths to determine concentrations.
INTENDED USE: The reagent kit is intended for the “in vitro” quantitative determination of Cholesterol in serum/plasma. PROCEDURE: Pipette into clean dry test tubes labeled as Blank (B), Standard (S) CLINICAL SIGNIFICANCE: and Test (T) : Cholesterol is the main lipid found in blood, bile and brain tissue. It is the main lipid associated with arteriosclerotic vascular Addition Sequence B. S. T. diseases. It is required for the formation of steroids and cellular membranes. The liver metabolizes the cholesterol and it is Enzyme reagent 1 ml 1 ml 1 ml transported in the blood stream by lipoproteins. Increased levels Standard - 10 µl - are found in hypercholesterolaemia , hyperlipidaemia Sample - - 10 µl hypothyroidism, uncontrolled diabetes, nephrotic syndrom and cirrhosis. Decreased levels are found in malabsorption, malnutrition, hyperthyroidism anemias and liver diseases. Mix well and incubate at 37° C for 5 mins. Measure absorbance of the Standard (Abs.S) and Test (Abs.T) against Reagent Blank at 505 nm. PRINCIPLE: Cholesterol esterase hydrolyses esterified cholesterols to free cholesterol. The free cholesterol is oxidised to form hydrogen CALCULATION : Abs. T peroxide which further reacts with phenol and 4-aminoantipyrine Cholesterol mg/dl = X 200 by the catalytic action of peroxidase to form a red coloured Abs.S quinoneimine dye complex. Intensity of the colour formed is directly propotional to the amount of cholesterol present in the NORMAL VALUE : sample. Serum : 130 - 250 mg/dl Each Laboratory should establish it's own normal range REACTION: representing its patient population. Cholesterol Esterase Cholesterol esters + H2O Cholesterol + Fatty LINEARITY : Acids The procedure is linear upto 1000 mg/dl. If the value exceeds this Cholesterol Oxidase limit, dilute the serum with normal saline (NaCl 0.9%) and repeat Cholesterol + O2 Cholestenone + H2O2 the assay. Multiply result by dilution factor.
Peroxidase QUALITY CONTROL :
H2O2 + Phenol + 4 amino antipyrine Red quinonimine For accuracy it is necessary to run known controls with every dye + H2O assay. CONTENTS: Reagent 1 : Cholesterol Enzyme Reagent LIMITATION & PRECAUTIONS : Reagent 2 : Cholesterol Standard 200 mg/dl. 1. Storage condition as mentioned on the kit to be adhered. Reagent 3 : Cholesterol Precipitating Reagent (Free) 2.Do not freeze or expose reagents to high temperature. Protect from direct sun light as it may affect the performance of MATERIALS REQUIRED BUT NOT PROVIDED: the Kit. - Clean & Dry Glassware. 3. The Enzyme reagent contains Sodium Azide. - Laboratory Glass Pipettes or Micropipettes & Tips. 4. Before the assay bring all the reagents to room temperature . - Colorimeter or Bio-Chemistry Analyzer. 5. Avoid contamination of the reagents during the assay process. 6. Use clean glassware free from dust or debris. SAMPLES: Serum, heparinized/ EDTA plasma. Cholesterol is reported to be BIBLIOGRAPHY : stable for 7 days when stored at 2 - 8°C. 1. Richmond, N.Clin. Chem. 1973: 19 : 1350 - 1356 2. Overview and Recommendations : Current Status of Blood PREPARATION OF REAGENT & STABILITY : Cholesterol Measurement clin.chem. 34/1, 193-201 (1988) All Reagents are ready to use and are Stable till the expiry date 3. Allain C.C and al. Clin. Chem., 20 (1974), 470. mentioned on the label, when stored at 2 - 8 °C. CODE NO. PACK SIZE Reagent 1 Reagent 2 Reagent 3 GENERAL SYSTEM PARAMETERS: S06 2 x 30 ml 2 x 30 ml 3.0 ml 3.0 ml Reaction type : End point S06D 2 x 50 ml 2 x 50 ml 3.0 ml 3.0 ml Wave Iength : 505 nm. (490-530nm) S06E 6 x 50 ml 6 x 50 ml 3.0 ml 3.0 ml Temperature : 37°C Incubation : 5 minutes IVD Reagent volume : 1.0 ml. UNIONPROJET S.R.L.S. EC REP Via Aliprandi 41-20851 Lissone (MB) www.jas-anz.com.au/register
Sample volume : 10µl. ISO 9001:2015 ISO 13485:2003
Standard concentration : 200 mg/dl
Zero setting : Reagent blank Light path : 1 cm HDL CHOLESTEROL INTENDED USE: The reagent kit is intended for the in-vitro quantitative determination of HDL Cholesterol in serum/plasma.
MATERIALS REQUIRED BUT NOT PROVIDED:-
- Clean & Dry Glassware. - Laboratory Glass Pipettes or Micropipettes & Tips. - Colorimeter or Bio-Chemistry Analyzer.
SAMPLES: Unhemolysed Serum, heparinized plasma or EDTA plasma.
GENERAL SYSTEM PARAMETERS:
Reaction type : End point Wave Iength : 510 nm. (490-530nm) Temperature : 37°C Incubation : 5 minutes Reagent volume : 1.0 ml Sample volume : 10µl Standard concentration : 200 mg/dl Zero setting : Reagent blank Light path : 1 cm
PREPARATION: Take 0.5 ml of serum / plasma in to glass tube. Add 50 µl precipitating reagent. Mix well, leave it at R.T. for 10 minutes. Centrifuge at 3000 r.p.m. for 10 minutes.Take the clear supernatant for HDL cholesterol estimation. PROCEDURE: 1. Bring all the reagents of assay to room temperature. 2. Working assay table B. S. T.
Enyzyme reagent 1 ml 1 ml 1 ml
Standard - 10µ l -
Supernatant sample - - 10 µl 0 Mix well and incubate for 5 minutes at 37 c Measure the absorbance of test & Std at 510 nm.
CALCULATION: Absorbance of HDL Test HDL Cholesterol mg/dl = x 200 Absorbance of std
NORMAL VALUE: HDL cholesterol : Men :30-60 mg% , Women :40-70 mg%
NOTE: If Triglycerides level is more then 300 mg/dl in sample please dilute the same test sample tube 1:1 with normal saline, add 50 µl precipitating reagent & use the diluted sample for HDL cholesterol estimation. Final results should be multiplied by the dilution factor (Two).
