HIGH-DENSITY LIPOPROTEIN - (HDL) CHOLESTEROL. Information about test and diagnosis of diseases

HIGH-DENSITY LIPOPROTEIN — (HDL) CHOLESTEROL

Quantitative estimation of HDL cholesterol in human serum by magnesium-phosphotungstate precipitation followed by enzymatic cholesterol assay.

1. Purpose

Quantitative estimation of HDL cholesterol in human serum by precipitation method — precipitation of VLDL and LDL (using magnesium ions and phosphotungstic acid), followed by estimation of HDL cholesterol using the cholesterol esterase–cholesterol oxidase method.

Measurement of serum HDL cholesterol is useful to screen lipid status, detect atherosclerotic risk, monitor response to lipid-lowering measures, and classify hyperlipidemias. An inverse relationship exists between serum HDL cholesterol and coronary heart disease risk. HDL < 30 mg/dL is considered a risk factor for coronary and cerebrovascular disease.

2. Principle

Phosphotungstate / Mg2+ precipitate VLDL, LDL and chylomicrons; HDL remains in the supernatant. After centrifugation the supernatant (HDL fraction) is assayed for cholesterol using the cholesterol esterase–cholesterol oxidase enzymatic method.

Schematic:
Serum/Plasma —(Phosphotungstate / Mg²⁺)→ HDL (supernatant) + (LDL + VLDL + CM in precipitate)

3. Performance specifications

Characteristic	Value
Linearity	Up to 400 mg/dL of serum
Measurement range	1 – 400 mg/dL
Sensitivity	Minimum detection limit: 1 mg/dL
Specificity	Cholesterol oxidase oxidizes some cholesterol analogs (e.g., dihydrocholesterol, 7-dehydrocholesterol) — these analogs are not normally present in appreciable amounts in serum.

4. Primary sample

• Use only fasting serum as specimen.
• Collect 4 mL venous blood in a heparin vacutainer.
• Allow to stand 30 minutes; separate serum by centrifugation at 2500–3000 rpm for 5–10 minutes.
• Avoid icteric/hemolyzed or turbid/contaminated samples — they may give falsely high results.
• Process sample the same day within 3 hours of collection. If delayed, separate serum and store at 2–8 °C for up to 7 days.

5. Container & additive

Heparin vacutainer. No preservatives required.

6. Reagents / Consumables

• Precipitating reagent: phosphotungstic acid (2.4 mM) + magnesium chloride (40 mM)
• Cholesterol reagent components: 4-aminophenazone, phenol
• Enzymes: cholesterol esterase, cholesterol oxidase, horseradish peroxidase
• Buffer (pH 6.8), stabilizers, and fillers
• HDL-cholesterol standard: 50 mg/dL

7. Instrument

Semi-automatic analyzer (follow instrument specific warm-up and maintenance procedures).

8. Procedure

1. Bring reagents to room temperature.
2. Add 500 µL serum + 500 µL HDL precipitating reagent. Mix and centrifuge at 4000 rpm for 10 min to obtain a clear supernatant.
3. Assay the supernatant for HDL cholesterol using the cholesterol reagent (same steps as total cholesterol assay).
4. Instrument prep: flush tubing with 2% detergent for 2 minutes, then distilled water for 2 minutes.
5. Select Absorbance mode, choose the test, note reagent and sample volumes.
6. Run a reagent blank with each batch; blank absorbance must be < 0.100. Discard reagent if blank >= 0.100.
7. Feed patient samples and record values.
8. Check samples for hemolysis or icterus. If hemolyzed — recollect. If highly icteric or lipemic, dilute 1:10 with distilled water and multiply result by 10.

Assay details (end point):
Reagent volume: 1000 µL  •  Sample volume: 50 µL  •  Wavelength: 510 nm  •  Temperature: 37 °C
Zero: reagent blank  •  Incubation: 10 minutes  •  Standard concentration: 50 mg/dL

9. Interferences & precautions

• Avoid blood collection in fed state — use fasting samples.
• Do not use samples kept above 2–8 °C for ≥1 day.
• Hemolyzed samples may give falsely elevated values; turbid reagent or high reagent blank indicates contamination — do not use.

10. Calculations

Calculate HDL cholesterol from absorbances using the standard:

Sample concentration = (Sample absorbance / Standard absorbance) × Concentration of standard

11. Biological reference range

Normal HDL cholesterol: 30 – 60 mg/dL

12. Alert / Critical values

HDL cholesterol < 30 mg/dL — considered a risk factor for coronary and cerebral vascular disease.

13. Laboratory interpretation

• HDL/Total cholesterol ratio < 0.2 indicates increased coronary heart disease risk.
• Equivalently, a Total cholesterol / HDL cholesterol ratio ≈ 5 is considered unfavorable.

14. Potential sources of variability

• Hemolysis → falsely elevated values.
• Turbid reagents or high reagent blank (≥ 0.100) → reagent contamination; do not use.

Bibliography

1. Castelli WP. Circulation 1977;55:767.
2. Castelli WP. Metabolic Therapy 1977;6:1.
3. Gordon T, et al. Am J Med 1977;62:707.

Prepared as a laboratory test protocol for educational and clinical reference. Always correlate results clinically and follow local laboratory regulations and instrument manufacturer instructions.