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    LIPID PROFILES

    (block 12)

    Clinical Pathology Department

    2008

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    Measurement of lipids and

    lipoproteins

    Many analytical techniques have been

    developed : chemical, enzymaticand

    immunochemical methods as well as

    physical methods, such asultracentrifugation, electrophoresis, column

    chromatography, precipitation method.

    Enzymat ic method :

    accurate, precise, simple to use

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    TOTAL CHOLESTEROL

    (CHOD-PAP method)

    Principle: hydrolisis & oxydation

    Serum + reagents

    Incubation measure the

    absorbance

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    The reaction is as follow:

    Cholesterol

    ester + H2O

    Chol ester

    hydrolase

    cholesterol + fatty

    acid

    Cholesterol + O2Chol oxydase Cholest-4-en-3-

    one + H2O2

    H2O2 + phenol +

    4 aminoantipyrine

    Quinoneimine dye

    + 2H2O

    peroxidase

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    Reagents:

    Goods buffer pH 6.7 50 mmol/l

    Phenol 5 mmol/l

    4-Aminoantipyrine 0.3 mmol/l

    Cholesterol esterase (CHE) 200 U/l

    Cholesterol oxidase (CHO) 50 U/l

    Peroxidase (POD) 3 kU/l

    Standard : 200 mg/dl (5.2 mmol/l)

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    Specimen

    Serum, heparin plasma or EDTA plasma.Stability : 7 days at 20-250C

    3 months at - 200C

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    Assay Procedure

    Blank Sample or standard

    Sample or standard - 10 l

    Dist. Water 10 l -Reagent 1000 l 1000 l

    Mix, incubate for 20 min. at 20 250C or for 10 min. at370C . Read absorbance within 60 min against reagentblank.

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    Calculation

    With standard or calibrator

    Cholesterol [mg/dl] = x Conc.Std / Cal

    [mg/dl]

    A Sample

    A Std /Cal

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    Measuring range3 750 mg/dl (0.08 19.4 mmol/l).

    Specificity / InterferencesNo interference was observed by:

    - ascorbic acid up to 5 mg/dl,

    - bilirubin up to 20 mg/dl,

    - hemoglobin up to 20 g/dl and

    - lipemia up to 2,000 mg/dl triglycerides.

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    Sensitivity / Limit of Detection

    The lower limit of detection is 3 mg/dl (0.08 mmol/l).

    Reference Range

    Desirable 200 mg/dl (5.2 mmol/l)

    Borderline high risk 200 240 mg/dl(5.2 6.2 mmol/l)

    High risk > 240 mg/dl (> 6.2 mmol/l)

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    HDL-CHOLESTEROL

    (CHOD-PAP method)

    principleSample + precipitation reagen

    measureclear

    supernatant

    centrifuge

    Precipi tate VLDL,

    IDL,Lp (a), LDL

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    Reagents

    Precipitating reagent (1.4 mmol/l

    phosphotungstic acid, 8.6 mmol/l magnesium

    chloride), 250 ml.

    Storage

    150C - 250C until expiry date.

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    Sample Material

    Serum , heparin-or EDTA plasma

    The HDL-Cholesterol in serum is stable- 7 days (150C - 250C)

    -14 days ( 20C - 80C)

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    Procedure

    Precipitation :

    Pipette into centrifuge tubes:

    Sample 200 lPrecipitating reagent 1 500 l

    Mix well and incubatefor 10 min at RT, then centrifuge

    for 5 min at 5000g.

    0.1 ml clear supernatant cholesterol determination

    (CHOD-PAP method)

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    Photometric measurementWavelength:500nm,546nm

    Sample reagent blank

    Pippete :

    Supernatant 0.1 ml -

    Water - 0.1 ml

    Reaction solution 1.0 ml 1.0 ml

    Mix well and incubatefor 10 min (RT) or 5 min ( 370C)

    Then measure the absorbance(A) against the reagent

    blank value.

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    HDL-Cholesterol concentration = A x F

    F = Factor (obtained from reagent kit)

    e.g. F (mg/dl) = 222

    [CHO-PAP Prod.No.1.14366.1.14165-67]

    Calculation:

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    Notes

    The supernatant after the centrifugation should

    be a clear solution.

    Sera with a triglyceride content over 1000 mg/dl

    tend to turbid supernatants or flotatingprecipitates.

    In this case a predilution of sample with the

    same volume of physiologic sodium chloride

    solution ( 9 g/l =^ 154 mmol/l) followed by theprecipitation procedure is recommended.

    Multiply the result by 2.

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    Principle: determination of triglycerides

    after enzymatic splitting with lipoprotein

    lipaseSerum + reagents

    Incubation measure theabsorbance

    TRIGLYCERIDES

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    Method

    Colorimetric enzymatic test using glycerol-3-

    phosphate-oxidase (GPO)

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    Reagents :

    Goods buffer pH 7.2 50 mmol/l

    4-Chlorophenol 4 mmol/l

    ATP 2 mmol/l

    Mg2+

    15 mmol/lGlycerokinase (GK) 0.4 kU/l

    Peroxidase (POD) 2 kU/l

    Lipoprotein lipase (LPL) 2 kU/l

    4-Aminoantipyrine 0.5 mmol/lGlycerol-3-phosphate-oxidase (GPO) 2 kU/l

    Standard : 200 mg/dl (2.3 mmol/l)

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    Specimen

    Serum, heparin plasma or EDTA plasma

    Stability: 2 days at 20-250C7 days at 4-80C

    at least one year at -200C

    Discard contaminated specimens.

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    Assay Procedure

    Blank Sample or standard

    Sample or standard - 10 l

    Dist. Water 10 l -Reagent 1000 l 1000 l

    Mix, incubate20 min (20 250C) or 10 min (370c).

    Read absorbanceagainst reagent blank within 60 min.

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    Calculation

    With standard or calibrator

    Triglycerides [mg/dl] = x Conc.Std / Cal

    [mg/dl]

    A Sample

    A Std /

    Cal

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    Measuring range

    1 1000 mg/dl (0.01 11.3 mmol/l).

    Specificity / Interferences

    No interference was observed by

    -bilirubin up to 40 mg/dl.

    -Ascorbic < 6 mg/dl,

    -Haemoglobin < 25 g/dl.

    Sensitivity / Limit of detectionThe lower limit of detection is 1 mg/dl

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    Reference Range

    Desirable : < 200 mg/dl (fasting)

    (2.3 mmol/l)

    Borderline high : 200-400 mg/dl(2.3 4.5mmol/l)

    Elevated : > 400 mg/dl

    (4.5 mmol/l)

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    LDL-Cholesterols

    measurement :

    - assume that total cholesterols is

    composed primarily of cholesterolsin VLDL, LDL and HDL

    - method : indirect or direct

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    Indirect method

    Friedewald equation

    LDL = Total chol. HDL ( TG/5)

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    Direct Method LDL

    Methods:

    1. Immunoseparation (IS)

    - Anti-human lipoprotein antibody binds &

    inactivates chylomicrons, HDL, and VLDL- LDL then measured using enzymatic method

    2. Zwitterionic detergents (ZI)

    - Zwitterionic detergents mask the VLDL

    - After addition of reagen, a LDL polyanion

    complex is formed which can be measured

    turbidimetrically

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    Direct Method LDL

    Methods:

    3. Clearance Method

    - selective detergents release cholesterol from

    chylomicrons, HDL, and VLDL- this cholesterol is then removed by the

    action of chol-esterase and oxidase

    - LDL- chol is then released in the 2nd step,

    and measured

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    erima kasih