Standard Operating Procedure: Fasting Lipid Profile

1. Purpose and Scope

To measure total cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol (calculated or direct) for cardiovascular risk assessment, dyslipidemia diagnosis, and monitoring of lipid-lowering therapy. Calculated VLDL and non-HDL cholesterol are reported as derived parameters.

2. Specimen Requirements

• Type: Serum (or EDTA plasma where validated). Fasting of 9-12 hours is preferred, particularly when triglycerides and calculated LDL are required.
• Volume: Minimum 1.0 mL.
• Fasting guidance: For screening of total cholesterol, HDL, and non-HDL, a non-fasting sample is acceptable in adults; if triglycerides are elevated (> 400 mg/dL) or LDL is to be calculated, a fasting sample is required.
• Lipemia: Grossly lipemic samples must be ultracentrifuged or flagged; extreme lipemia can interfere with turbidimetric and enzymatic reactions.
• Stability: Separate serum within 2 hours. Cholesterol and HDL stable 2-3 days at 2-8°C; triglycerides stable 24-48 h refrigerated if cells are removed.

3. Safety and Precautions

• Standard PPE (gloves, lab coat, eye protection). All blood samples are potentially infectious.
• Dispose of sharps and biohazard waste per laboratory policy.
• Do not pipette by mouth. Use mechanical pipetting devices.

4. Equipment and Reagents

• Automated chemistry analyzer (spectrophotometric).
• Total Cholesterol: Enzymatic CHOD-PAP method (cholesterol esterase + cholesterol oxidase + peroxidase), measured at 500 nm.
• Triglycerides: Enzymatic GPO-PAP method (lipase + glycerol kinase + glycerol phosphate oxidase + peroxidase), measured at 500 nm.
• HDL: Direct homogeneous assay using selective polymers/detergents to mask non-HDL lipoproteins, measured enzymatically.
• LDL: Calculated by the Friedewald equation (valid for triglycerides < 400 mg/dL) or measured directly by a homogeneous (beta-quantification-validated) assay.
• Two levels of quality control material for each assay.

5. Per-Analyte Procedures

5.1 Total Cholesterol Procedure

1. Load the CHOD-PAP reagent (cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, phenol) at 37°C.
2. Piper 200 µL of reagent and 10 µL of serum; mix.
3. Cholesterol esterase frees esterified cholesterol; cholesterol oxidase oxidizes it to cholest-4-en-3-one with hydrogen peroxide.
4. Peroxidase couples H2O2 with aminoantipyrine and phenol to form a red quinoneimine.
5. Measure absorbance at 500 nm after incubation and read total cholesterol in mg/dL from the calibration curve.

5.2 Triglycerides Procedure

1. Load the GPO-PAP reagent (lipase, glycerol kinase, glycerol phosphate oxidase, peroxidase) at 37°C.
2. Piper 200 µL of reagent and 10 µL of serum; mix.
3. Lipase hydrolyzes triglycerides to glycerol and fatty acids; glycerol kinase converts glycerol to glycerol-3-phosphate.
4. Glycerol phosphate oxidase generates H2O2, coupled by peroxidase to a colored chromogen.
5. Measure absorbance at 500 nm and read triglycerides in mg/dL; flag grossly lipemic samples for ultracentrifugation.

5.3 HDL Cholesterol Procedure (Direct Homogeneous)

1. Load the direct HDL reagent containing polymers/detergents that selectively mask chylomicrons, VLDL, and LDL.
2. Piper 200 µL of reagent and 10 µL of serum; mix.
3. Non-HDL lipoproteins are masked; HDL cholesterol remains available to react through the CHOD-PAP enzymatic endpoints.
4. Measure the resulting color at 500 nm.
5. Read HDL in mg/dL; no precipitation/centrifugation step is required with the homogeneous assay.

5.4 LDL Cholesterol Procedure (Calculated and Direct)

1. Confirm triglycerides are < 400 mg/dL before using calculation.
2. Calculate LDL by the Friedewald equation: LDL = Total cholesterol - HDL - (Triglycerides / 5).
3. For triglycerides >= 400 mg/dL, chylomicronemia, or type III dyslipidemia, measure LDL directly.
4. For direct LDL, load the homogeneous direct-LDL reagent (detergents unmask LDL while leaving other lipoproteins masked), add 10 µL serum to 200 µL reagent, mix, and measure at 500 nm.
5. Read LDL in mg/dL and report derived VLDL = Triglycerides / 5 and Non-HDL = Total cholesterol - HDL.

6. Common Calibration and Quality Control

• Run two levels (normal and abnormal) of control for each assay daily, with every calibration, and with each new reagent lot.
• Enforce Levey-Jennings rules; investigate trends or shifts before reporting patient results.
• Participate in an external quality assessment (EQA) program for all lipid analytes, including standardization against CDC (CRMLN) reference methods where available.
• Verify control values fall within acceptable target ranges before patient reporting.

7. Decision limits

The cardiovascular-risk categories below are interpretive decision thresholds stated as prose; the canonical adult reference intervals are displayed in the Reference Ranges panel.

• Total cholesterol (mg/dL): < 200 desirable; 200-239 borderline high; >= 240 high.
• LDL cholesterol (mg/dL): < 100 optimal; 100-129 near optimal; 130-159 borderline high; 160-189 high; >= 190 very high.
• HDL cholesterol (mg/dL): < 40 in men and < 50 in women is considered low; >= 60 is protective.
• Triglycerides (mg/dL): < 150 normal; 150-199 borderline high; 200-499 high; >= 500 very high (risk of pancreatitis).
• Non-HDL cholesterol (mg/dL): target is LDL target + 30; generally < 130 desirable for most adults.
• Friedewald limitation: calculated LDL is unreliable when triglycerides >= 400 mg/dL, in chylomicronemia, and in type III dyslipidemia; use direct LDL or an alternate equation in these settings.
• Fasting note: elevated triglycerides are common causes of false high results; confirm borderline elevations on a repeat fasting specimen before change of management.

Note on units: conventional units are mg/dL; SI conversion for cholesterol fractions is mg/dL x 0.02586 = mmol/L and for triglycerides mg/dL x 0.01129 = mmol/L.