| Cholesterol Assay Based on Agappe
INTENDED USE | This reagent is intended for in vitro quantitative
determination of Cholesterol in serum or plasma |
| Cholesterol assay Methodology | • CHOD PAP |
| What is in vitro testing | - testing within the tube |
| Cholesterol assay linearity | 600 mg/ dL |
| 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. |
| Principle: Cholesterol Assay | Principle |
| Reagent composition, Linearity, Storage and stability of cholesterol assay | Composition |
| Normal range, sample used, preparation | Normal range |
| Cholesterol assay general system procedure | GSP |
| Laboratory procedure | Lab procedure |
| Cholesterol assay calculation | Calc |
| 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. |
| Principle of TAG | TAG |
| Reagent composition, linearity, standard, storage and stability | TAG |
| Normal range, sample, preparation and stability of reagent | TAG |
| General System Parameter | TAG |
| TAG Lab Procedure | TAG |
| TAG Calculations | tag |
| 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. |
| HDL reagent composition, linearity, storage | HDL |
| HDL normal range, sample, preparation and stability, precaution | HDL |
| HDL general system procedure | hdl |
| HDL laboratory procedure | HDL |
| HDL Calculation | HDL |
| HDL interfering substance | 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 |
| EXPLAIN CHOLESTEROL ASSAY CHEMICAL METHODS | Cholesterol assay |
| Explain cholesterol assay enzymatic method | Cholesterol assay |
| 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 |
| Triglyceride assay van handel and hantzch | TAG |
| TAG enzymatic methods | TAG |
| WHAT ARE THE QUANTITATIVE ANALYSIS OF LIPOPROTEINS | ELECTROPHORESIS AND ULTRACENTRIFUGATION |
| EXPLAIN ELECTROPHOREISIS | ELECTRO |
| Explain centrifugation | ultra |
| 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 |
| Standing plasma test | Quali |
| Explain Friedewal calculation | Calc |
| Risk for coronary heart disease | Risk |
| Classification of lipoprotein diseases | Diseases |
| 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 |
