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    Cholesterol & HDL Cholesterol

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    Dinesh Sreedharan
    The document describes a method for determining cholesterol and HDL cholesterol levels in serum or plasma samples. It involves using enzymatic reactions to measure total cholesterol and HDL cholesterol concentrations, which are important biomarkers for cardiovascular health. The method details the reagents, assay procedure, calculations, reference values and notes on sample stability and interference.

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    Cholesterol & HDL Cholesterol

    Uploaded by

    Dinesh Sreedharan
    9 views1 page
    The document describes a method for determining cholesterol and HDL cholesterol levels in serum or plasma samples. It involves using enzymatic reactions to measure total cholesterol and HDL cholesterol concentrations, which are important biomarkers for cardiovascular health. The method details the reagents, assay procedure, calculations, reference values and notes on sample stability and interference.

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    Chol

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    The document describes a method for determining cholesterol and HDL cholesterol levels in serum or plasma samples. It involves using enzymatic reactions to measure total cholesterol and HDL cholesterol concentrations, which are important biomarkers for cardiovascular health. The method details the reagents, assay procedure, calculations, reference values and notes on sample stability and interference.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    9 views1 page

    Cholesterol & HDL Cholesterol

    Uploaded by

    Dinesh Sreedharan
    The document describes a method for determining cholesterol and HDL cholesterol levels in serum or plasma samples. It involves using enzymatic reactions to measure total cholesterol and HDL cholesterol concentrations, which are important biomarkers for cardiovascular health. The method details the reagents, assay procedure, calculations, reference values and notes on sample stability and interference.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 1

    CHOLESTEROL & HDL CHOLESTEROL

    (CHOD- PAP Method) Step 1 - For HDL Cholesterol:


    For the determination of Cholesterol and Pipette into a centrifuge tube :
    HDL Cholesterol in serum. Serum / Plasma 200 µl
    (For In vitro Diagnostic Use Only) HDL Reagent (A2) 300 µl
    SUMMARY: Mix well allow to stand at R.T. for 5 minutes. Centrifuge at 3000 rpm for 10 minutes to get a
    Cholesterol is the main lipid found In blood, bile and brain tissues. It Is the main lipid associated with clear supernatant.
    arteriosclerotic vascular diseases. It Is required for the formation of steroids and cellular If the supernatant is not clear (high TGL Level) dilute the sample 1:1 with normal Saline, (result
    membranes. x 2)
    The liver metabolises the cholesterol and It is transported In the blood stream by lipoproteins. Step 2 - For Total and HDL Cholesterol:
    Pipette into 4 test tubes labeled Blank (B), Standard (S), Total Cholesterol (Tc) and HDL
    PRINCIPLE: Cholesterol (TH) as shown below :
    Cholesterol Esterase
    Cholesterol esters + H2O Cholesterol + Fatty acids Addition Sequence B(µl) S(µl) TC(µl) TH(µl)
    Cholesterol Oxidase Cholesterol Reagent (A1) 1000 1000 1000 1000
    Cholesterol + O2 Cholestenone+ H2O2
    Standard (S) - 10 - -
    Peroxidase
    H202 + 4 Aminoantipyrine+ Phenol Red Quinoneimine dye + H2O Specimen - - 10 -
    Supernatant (from Step 1) - - - 100
    Intensity of the colour formed is directly proportional to the amount of cholesterol present in the Mix well and Incubate for 5 minutes at 37°C (or) 10 minutes at R.T. Read the absorbances of
    sample. Standard (S), Total Cholesterol (Tc) and HDL Cholesterol (TH ) against Blank (B) at 505 nm or
    with green filter (500 - 540 nm).
    CONTENTS:
    Pack size Cholesterol Reagent(A1) HDL Reagent(A2) Standard (s) CALCULATIONS:
    Abs.Tc
    2x25ml 2x25ml 5ml 1ml 1. Cholesterol in mg/dl= -------------------------- × 200
    Abs.S
    1x60ml 1x60ml 5ml 1ml
    Abs.TH
    2x50ml 2x50ml 10ml 2ml 2. HDL Cholesterol= -------------------- × 50
    Abs.S
    STORAGE/STABILITY:
    Contents are stable at 2 - 8°C till the expiry mentioned on the labels. NORMAL REFERENCE VALUES:
    Total Cholesterol : 130 - 250 mg/dl
    REAGENT PREPARATION:
    Reagents are ready to use for the given procedure. HDL Cholesterol (Male) : 30 - 70 mg/dl
    (Female) : 35 - 90 mg/dl
    SAMPLE : It is recommended that laboratories establish their own normal range representing its patient
    Serum, EDTA Plasma. Cholesterol is reported to be stable in the sample for 7 days when stored population
    at 2 - 8°C. The sample should preferably of 12 to 14 hours fasting.
    LINEARITY:
    GENERAL SYSTEM PARAMETER: This procedure is linear upto 1000 mg/dl. If values exceed this limit, dilute the serum with
    Reaction End Point Sample Vol. 10µl normal saline (NaCI 0.9%) and repeat the assay. Calculate the value using the proper dilution
    Wavelength 505nm (500-540nm) Reagent Vol. 1000µl factor.
    The linearity of HDL Cholesterol is 150 mg/dl.
    Blank Reagent blank Standard 200 mg/dl
    NOTE:
    Incubation 37°C 5 min / R.T.10 min React. Slope Increasing
    Anticoagulants such as fluorides and oxalates result in false low values. The test is not
    Delay Time 5 Sec Linearity 1000 mg/dl influenced by Hb values upto 20 mg/ dl and bilirubin upto 10 mg/dl.
    Read Time 5 Sec Units mg/dl REFERENCES:
    Trinder, P., (1969) Ann. Clin. Biochem, 6:24
    ASSAY PROCEDURE:
    Allain, C.C., etal, (1974) Clin. Chem, 20:470
    Wavelength / filter : 505 nm (Hg 546 nm) / Green Flegg, H.M., (1972) Ann. Clin. Biochem. 10:79
    Temperature : 37°C / R.T.
    Light Path : 1 cm

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