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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