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CLINCHEM LIPIDS LAB


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Ellyn Mae Armada


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Cholesterol Assay Based on Agappe INTENDED USE
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This reagent is intended for in vitro quantitative determination of Cholesterol in serum or plasma

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Cholesterol Assay Based on Agappe INTENDED USE
This reagent is intended for in vitro quantitative determination of Cholesterol in serum or plasma
What is in vitro testing
- testing within the tube
Importance of LCF
If the patient has an increase lipid contents the serum/plasma will be blurry and the clarity will be decrease because of the increase lipids. For the machine to read the linearity we need a clearing factor.
Clinical Significance Cholesterol Assay
• It is the main lipid found in the blood, bile & brain tissues . It is also one of the most important steroids of the body & is a precursor of many steroid hormones. • Two thirds of cholesterol present in the blood is esterified. The liver metabolizes the cholesterol & it is transported in the blood stream by lipoproteins • Increased levels are found in hypercholesterolaemia , hyperlipidaemia , hypothyroidism, uncontrolled diabetes, nephritic syndrome & cirrhosis • Decreased levels are found in malabsorption , malnutrition , hyperthyroidism, anemia , & liver diseases.
Triglycerides Assay Based on Agappe INTENDED USE
This reagent is intended for in vitro quantitative determination of triglycerides in serum or plasma.
TAG METHODOLOGY
GPO TOPS methodology
Linearity
1000 mg/ dL
Clinical Significance Triglycerides Assay
• Triglycerides are simple lipids, formed in the liver by glycerol & fatty acids • They are transported by VLDL, LDL & constitute about 95% of fat, stored as source of energy in the tissue & plasma. • Increased levels are found in hyperlipidemias , diabetes , nephrotic syndrome & hypothyroidism • Increased levels are risk factor for arteriosclerotic coronary disease , peripheral vascular disease, acute pancreatitis & hyperlipoproteinemia • Decreased levels are found in malnutrition & hyperthyroidism.
HDL Assay Based on Agappe INTENDED USE
This reagent is intended for in vitro quantitative determination of HDL cholesterol in serum • Direct determination of HDL Cholesterol
HDL assay method
Selective method
HDL linearity
Up to 150 mg/ dL
Clinical Significance HDL Assay
Blood total cholesterol levels have long been known to be related to coronary heart disease ( • In recent years, in addition to total cholesterol, high density lipoprotein cholesterol (HDL C ) has become an important tool used to assess an individual risk of developing CHD since a strong negative relationship between HDL C concentration and the incidence of CHD was reported
Principle of HDL Assay
The reaction between cholesterol other than HDL and the enzyme for cholesterol assay is suppressed by the electrostatic interaction between polyanions & cationic substances. • Hydrogen peroxide is formed by the free cholesterol in HDL by cholesterol oxidase. Oxidative condensation of EMSE and 4 AA is caused by hydrogen peroxide in the presence of peroxidase , and the absorbance of the resulting red purple quinone is measured to obtain the cholesterol value in HDL.
METHODOLOGIES General Sample and Patient Consideration
Cholesterol values rise with age • 12 hrs fasting ; CMs almost completely cleared within 6 9hrs after food ingestion • Total cholesterol and HDL C generally don’t need for fasting • Plasma or serum can be used for only cholesterol , TAG , and HDL C are measured; LDL C is calculated Friedewald equation • Plasma should be separated from cells within 3 hrs • Plasma (EDTA) is preferred for lipoprotein measurement
Cholesterol Assay considerations
-px should be in his/her usual diet 2 weeks prior to testing -total cholesterol is measured rather than its forms -Serum is the preferred specimen AC cause water shifting from RC dilution -For Colorimetric methods , sample for cholesterol assay is incubated 20 mins and 10min at 37 degrees Celsius.
Cholesterol Assay CHEMICAL METHODS Definition of terms:
• Saponification hydrolysis/splitting up cholesterol ester to free cholesterol and FAs • Extraction separation of cholesterol from protein carriers • Precipitation further isolation of FAs • Colorimetry addition of color reagent as an indicato
Triglyceride Assay Patient Preparation and Specimen Considerations
12 14 hrs fasting fresh serum best sample • Plasma has slightly higher TAG level • Heparinized or EDTA treated plasma can be used • Glucose and phospholipids cause interference in non enzymatic mtd addition of zeolite (magnesium aluminum silicate) removes interference
WHAT ARE THE QUANTITATIVE ANALYSIS OF LIPOPROTEINS
ELECTROPHORESIS AND ULTRACENTRIFUGATION
Quantitative Analysis of Lipoprotein
• Polyanion Precipitation Method and standing plasma test
Polyanion Precipitation Method
- lipoproteins are precipitated with polyanion heparin sulfate , dextran sulphate , phosphotungstate ) in the presence of calcium , magnesium , or manganese. most commonly used for HDL assay
Lipoprotein Disorders
Familial Hypercholesterolemia(Type 2A) defective or deficient LDL receptors • Familial Dystbetalipoproteinemia ( Type 3 accumulation of plasma VLDL rich in cholesterol VLDL fraction migrates abnormally in beta region Beta VLDL Abetalipoproteinemia Bassen Kornzweig Syndrome) defective apo B synthesis VLDL, LDL, CM = absent in plasma deficient Vitamins A,E,K; Vit D is not affected • Tangier’s Disease complete absence of HDL due to mutation in the ABCA1 gene on chromosome 9 • Tay Sachs Disease neurodegenerative disorder of lipid metabolism characterized by a deficiency of the enzyme Hexosaminidase A = accumulation of sphingolipids in brain