Lipid Profile Test

Components of a Standard Lipid Profile

LDL-C = Total Cholesterol - HDL-C - (Triglycerides ÷ 5)

• Textbook of Family Medicine 9e, p. 234

Reference Ranges (mg/dL)

• Biochemistry, 8th ed - Lippincott Illustrated Reviews, p. 1419
• UW Lab Medicine lipid panel interpretation (updated March 2026)

Key Facts About Each Component

LDL-C

• Carries ~60-70% of plasma cholesterol
• Direct association with coronary heart disease (CHD) risk
• Primary therapeutic target in dyslipidemia management
• For high-risk patients (prior ASCVD), target is typically < 70 mg/dL; statin therapy is recommended when LDL-C ≥ 190 mg/dL

HDL-C

• Carries ~20-30% of total cholesterol
• Functions in reverse cholesterol transport (removes cholesterol from tissues back to the liver)
• Every 1 mg/dL decrease in HDL raises CAD risk by 2-3%
• Low HDL can be caused by: elevated triglycerides, liver disease, amyloidosis, inborn errors of metabolism

Triglycerides

• Transported primarily in VLDL and chylomicrons
• Marker of TG-rich lipoproteins; causally related to ASCVD
• TG > 500 mg/dL carries very high risk of acute pancreatitis
• Measured in fasting state (postprandial rise is normal and expected)

When and How to Test

• Fasting: Standard test requires a 9-12 hour fast. Failure to fast elevates triglycerides and underestimates LDL-C.
• Non-fasting: Total cholesterol and HDL-C are reliable without fasting. If non-fasting non-HDL-C ≥ 220 mg/dL or TG ≥ 500 mg/dL, a fasting panel is required.
• Timing: Morning specimens are preferred since triglycerides have diurnal variation (lowest in the morning).
• Stable diet required: Dietary changes appear in lipid values within 1-2 weeks, so patients should maintain a stable diet for 3 weeks before testing.
• Avoid testing after: Recent illness, surgery, MI, or stroke - cholesterol drops within 24 hours of MI and stays depressed up to 12 weeks. Wait 2-3 months after major illness.
• Screening interval: Every 4-6 years for adults aged 40-75 without indications for therapy. Hospitalized patients with ACS should have a lipid panel within 24 hours.
• Washington Manual of Medical Therapeutics, p. 97

Limitations of the Friedewald Formula

1. Chylomicrons present (non-fasting sample)
2. Triglycerides > 400 mg/dL
3. Dysbetalipoproteinemia (Type III hyperlipidemia)

Common Causes of Secondary Dyslipidemia

• Hypothyroidism
• Diabetes mellitus
• Nephrotic syndrome
• Obstructive liver disease
• Medications: estrogens, progestins, anabolic steroids, corticosteroids, cyclosporine, retinoids, atypical antipsychotics, antiretrovirals (protease inhibitors)

Clinical Significance

• Risk-based LDL-C targets under the 2018 ACC/AHA guidelines are set per ASCVD risk category, not single cutoffs
• The greater the LDL-C reduction, the greater the cardiovascular risk reduction
• Non-HDL-C is a useful secondary target since it includes VLDL, IDL, and Lp(a) - all atherogenic particles
• Lipoprotein subfractions and particle size testing is not recommended for routine cardiovascular risk assessment in asymptomatic adults (AHA 2010)I'll read the PDF skill first, then extract the content from your uploaded file.

Experiment No. 20 - Lipid Profile Tests

AIM

• Total Cholesterol (TC)
• Triglycerides (TG)
• High-Density Lipoprotein Cholesterol (HDL-C)
• Low-Density Lipoprotein Cholesterol (LDL-C) - calculated
• Very Low-Density Lipoprotein Cholesterol (VLDL-C) - calculated

Chemicals and Reagents

• Standard Total Cholesterol calibrator
• Standard Triglyceride calibrator
• Standard HDL calibrator
• Enzymatic reagent kits:
  • CHOD-PAP reagent (Cholesterol Oxidase - Peroxidase) for Total Cholesterol
  • GPO-PAP reagent (Glycerol-3-Phosphate Oxidase - Peroxidase) for Triglycerides
  • HDL reagent (precipitating agent for manual method, or direct HDL reagent for automated assays)
  • Lipase (for some TG methods)
• Distilled water
• Serum sample (fasted sample, preferably 8-11 hours fasting)
• Standards, controls, and blanks as provided by kit manufacturer

Principle

1. Total Cholesterol (TC) - CHOD-PAP Method

1. Cholesterol esters are hydrolyzed by cholesterol esterase to free cholesterol and fatty acids.
2. Cholesterol oxidase then oxidizes cholesterol to cholest-4-en-3-one with production of hydrogen peroxide.
3. In the presence of peroxidase, H₂O₂ reacts with a chromogen to form a colored quinoneimine dye.
4. Absorbance (measured at ~500 nm) is proportional to cholesterol concentration.

2. Triglycerides (TG) - Enzymatic GPO-PAP Method

1. Triglycerides are hydrolyzed by lipase to glycerol and free fatty acids.
2. Glycerol is phosphorylated and subsequently oxidized, producing hydrogen peroxide.
3. H₂O₂ reacts with a chromogen (peroxidase-coupled) to give a colored product.
4. Measured spectrophotometrically; intensity is proportional to TG concentration.

3. HDL-C

• Can be estimated by precipitation method: polyanions + divalent cations precipitate LDL, VLDL, and chylomicrons; cholesterol is measured in the supernatant = HDL-C.
• OR by direct homogeneous assays that selectively block non-HDL particles.

4. LDL-C & VLDL-C - Calculated (Friedewald Formula)

Valid only when TG < 400 mg/dL and patient is fasting.

Procedure (General Laboratory Steps)

1. Prepare reagents, standards, and controls as per manufacturer instructions. Bring to room temperature if required.
2. Calibrate spectrophotometer/autoanalyzer with blank and calibrator(s) at the appropriate wavelength (~500 nm) following CHOD-PAP and GPO-PAP kit instructions.
3. Total Cholesterol:
   • a. Pipette appropriate volume of reagent and sample/standard into cuvette or well.
   • b. Incubate at specified temperature (often 37°C or room temperature) for the stated time.
   • c. Read absorbance against blank and calculate concentration using standard or calibration curve.
4. Triglycerides:
   • a. Mix sample/standards with GPO-PAP reagent as per kit.
   • b. Incubate and read absorbance at specified temperature (often 37°C or room temperature) for stated time.
   • c. Read absorbance against blank and calculate concentration.
5. HDL-C (Precipitation Method):
   • a. To 0.5 mL serum add precipitating reagent (e.g., phosphotungstic acid + magnesium chloride) as per kit.
   • b. Vortex and centrifuge at 3000 rpm for 10 min.
   • c. Carefully collect supernatant (contains HDL) and perform cholesterol assay on supernatant using CHOD-PAP reagent.
   • d. For direct HDL assay, follow the kit protocol for reagent/sample volumes and incubation.
   • e. Run quality control samples with each batch and record results. Repeat any out-of-range control or sample after troubleshooting.
6. Calculate LDL-C using Friedewald formula (when TG < 400 mg/dL).
7. Record results in report format and interpret according to reference ranges.

Calculations (Example)

Note: Friedewald is valid only when TG < 400 mg/dL and patient is fasting. For TG ≥ 400 mg/dL, use direct LDL measurement.

Reference Ranges and Interpretation

Report Format (Sample)

Precautions

• Use non-hemolyzed serum - haemolysis can interfere with some assays.
• Ensure fasting status for triglyceride accuracy (8-11 hrs).
• Follow kit instructions for reagent preparation, incubation times, and wavelength.
• Use proper calibration and run controls with each batch.
• Avoid lipemic samples for some direct assays, or note limitations.
• Always correlate laboratory results with clinical findings and patient history.