Sect 7.2 - Testing Methods

Coronary Heart Disease (CHD)

Lipids and Lipoproteins are important indicators of

> 60 mg/dl

optimal levels of HDL set by NCEP

< 100 mg/dl

optimal levels of LDL set by NCEP

< 150 mg/dl

optimal levels of Triglycerides set by NCEP

< 200 mg/dl

optimal levels of Total Cholesterol

< 3.3

optimal TC:HDL ratio

< 1.6

optimal LDL:HDL ratio

total cholesterol

HDL

LDL

triglycerides

What is included in a lipid panel?

every 5 years

NCEP recommends that all adults (20+) have a fasting lipid panel performed how often?

10-12hr fast

A lipid panel requires

**hint: testing triglycerides in VLDL form

Risk factors of CHD

Smoking

Hypertension (Systolic >140 mmHg)

Age (Men: 45+ / Women: 55+)

Abdominal obesity

Family hx of premature CHD in first degree relative

Dx of Diabetes or Metabolic Syndrome

Low risk

0-1 risk factors

LDL outside optimal range (>100 mg/dl)

Intermediate/Moderate risk

2+ risk factors

LDL (>100) and HDL (

Moderately High risk

2+ risk factors

LDL (>100), HDL (

High risk

presence of Clinical Atherosclerotic Disease (CHD, Carotid Artery Disease, Peripheral arterial disease or abdominal aortic aneurysm)

Cholesterol esterase

Cholesterol oxidase

Peroxidase

3 enzymes involved in Total Cholesterol measurement

GC-MS

Gold Standard method for Total Cholesterol measurment

interferences in Cholesterol testing

false pos: bilirubin, hemoglobin

false neg: uric acid, ascorbic acid

Friedwald formula

most commonly used method to calculate LDL Cholesterol

TC - (HDL + (Triglycerides / 5))

Friedewald Formula

1. Cannot be used when triglycerides >400 mg/dL

2. Chylomicrons not accounted for (cannot be used with non-fasting specimen)

3. Modify formula when triglycerides are normal to low

Limitations of Friedewald formula

Modified Friedewald formula

When triglycerides are lower to normal, what formula should be used?

TC - (HDL - Triglycerides / 5))

Modified Friedewald formula

Direct Measurement of LDL Cholesterol

LDL is measured using the cholesterol rxn, but with the addition of reagents that block the contribution of HDL and VLDL

allows for only LDL Cholesterol to form chromogen

2 methods available

Lipase (bacterial)

What enzyme is used in triglyceride measurements to cleave the fatty acids from glycerol?

NAD+

end product that is measured in the first triglyceride measurement method

enzymes: Lipase (bacterial) → Glycerol kinase → Pyruvate kinase → Lactate dehydrogenase

proportional to the amount of triglycerides present

colored product

end product that is measured in the second, most common triglyceride measurement method

enzymes: Lipase (bacterial) → Glycerol kinase → Glycerophosphate oxidase → Peroxidase

proportional to the amount of triglycerides present

Endogenous free glycerol

the most common interference with triglycerides measurement

leads to false increase (overestimation of triglycerides)

chylomicrons in non-fasting state will falsely elevate results

Why is fasting important for triglyceride measurement?

Precipitation Reaction

Homogenous Reaction

2 methods of HDL-Cholesterol measurment

Precipitation Reaction

HDL-Cholesterol method that uses

dextran sulfate or phosphotungstic acid with MgCl2 to precipitate LDL and VLDL from fasting serum sample

the supernatant is assayed for cholesterol (using cholesterol rxn) and represents amount of HDL in the serum sample

dextran sulfate

phosphotungstic acid with MgCl2

enzymes used in the precipitation reaction for HDL-Cholesterol

Chylomicrons from non-fasting specimens

High triglyceride levels

What are some potential interferences of HDL measurement?

Immunoassay

Chemical reagent

2 methods of Homogenous Reaction to measure HDL-Cholesterol

Apo-B100

type of antibody used to bind LDL and VLDL so HDL can be measured in the homogenous reaction of HDL measurement

synthetic polyanion reagent

type of chemical used as a blocking agent (blocks VLDL and LDL) to enable only HDL to react and form colored product in the homogenous reaction of HDL measurement

Nephelometry

Immunoassay

2 methods used to measure Apoprotein levels

Nephelometry

most common method to measure apoprotein levels

Ag-Ab complex produces light scatter pattern and amount of scatter is measured; proportional to concentration of apoprotein present

Immunoassay

method to measure apoprotein levels

presence of specific patient antigen to a specific apoprotein is used to combine with apoprotein antibody in reagent

label used to detect presence of combined Ab-Ag complex and measures concentration of the complex

Lipoprotein (a)

unusual type of LDL particle with an attached Apo (a)

plasminogen

Lp(a) is structurally similar to

inactive form of plasmin

plasmin

aids in the degradation of incidental clots that are formed periodically in blood vessels

needed to breakdown little clots

attracting the plasminogen activators and blocks their action

High Lp(a) levels inhibit plasmin formation by

clot formation increases, clot sizes grow and occlusions can occur

result of inhibited plasmin formation due to high Lp(a) levels

Myocardial infarction (heart attack)

Pts with elevated levels of Lp(a) in their blood are at risk for circulatory blocks, resulting in

increased

Women with high levels of Lp(a) & homocysteine levels have (increased / decreased) risk of atherosclerosis

CAD

Women with high levels of Lp(a) and homocysteine is a strong predictor of what medical condition?

Enzyme Linked Immunosorbent Assay (ELISA)

method utilized for the measurement of Lp(a)

ELISA

polyclonal Ab specific for Lpa(a) and a second monoclonal Ab specific for apoprotein-B is added to pts sample

label specific for Apoprotein-B Ab is added, catalyzing the formation of a colored product