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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

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