CHOLESTEROL TEST. Information about test and diagnosis of diseases

 

CHOLESTEROL TEST (CHOD-PAP METHOD)

1. Purpose

Quantitative estimation of total cholesterol in human serum by CHOD-PAP method (enzymatic photometric method). Measurement of serum cholesterol is useful in screening the lipid status of individuals to detect atherosclerotic risks and in monitoring the response to lipid-lowering measures. It is also useful in the diagnosis and classification of hyperlipidemias. Other conditions such as hepatic and thyroid diseases also influence cholesterol levels.

2. Principle

Cholesterol esters are hydrolyzed by cholesterol esterase to produce free cholesterol and fatty acids. Hydrogen peroxide is produced from the oxidation of cholesterol by cholesterol oxidase. In a coupled reaction catalyzed by peroxidase (POD), red quinoneimine dye is formed from 4-aminoantipyrine, phenol and hydrogen peroxide. The absorbance of this dye at 500 nm is proportional to the cholesterol concentration in the sample (Trinder’s reaction).

Cholesterol ester --(Chol. esterase)--> Cholesterol + Fatty acids
Cholesterol --(Chol. oxidase)--> 2 H₂O₂ + Cholesten-4-en-3-one
2 H₂O₂ + 4-Aminoantipyrine + Phenol --(POD)--> Red quinoneimine + H₂O (Red dye)
  

3. Performance Specifications

• Linearity: Up to 1000 mg/dL of serum
• Measurement range: 1 – 1000 mg/dL
• Sensitivity: Minimum detection limit = 1 mg/dL
• Specificity: Cholesterol oxidase is not totally specific; analogs may also be oxidized but occur rarely in serum.

4. Primary Sample

1. Use only plasma as specimen.
2. Collect 4 mL venous blood in a heparin vacutainer tube.
3. Centrifuge at 2500 rpm for 10 minutes.
4. Do not use lysed or contaminated samples.
5. Process within 3 hours of collection; otherwise recollect sample.

5. Container and Additive

Use plain or heparin vacutainer tubes for sample collection.

6. Instrument

Semi-auto analyzer.

7. Reagents / Consumables

• 4-Aminoantipyrine
• Phenol
• Cholesterol esterase
• Cholesterol oxidase buffer (pH 6.8)
• Cholesterol standard (200 mg/dL in alcohol)

8. Procedure

1. Switch on the analyzer and flush tubing with 2% detergent for 2 min, then with distilled water for 2 min.
2. Select “PROC”, choose test number, and press ENTER.
3. Check reagent blank (absorbance < 0.300). If higher, discard reagent.
4. Run test samples and record readings.
5. If sample is icteric or lipemic, dilute 1:10 with distilled water and multiply result × 10.

Assay Conditions:

• Type: End-point
• Wavelength: 510 nm
• Temperature: 37 °C
• Incubation time: 5 min
• Reagent volume: 1000 µL
• Sample volume: 10 µL
• Standard concentration: 200 mg/dL

9. Interference

Avoid hemolyzed or icteric samples. Over-time reagent discoloration (light pink) is acceptable, but discard if absorbance > 0.3 OD vs distilled water at 510 nm.

10. Calculation of Results

Sample Concentration (mg/dL) = (Sample Absorbance × Standard Concentration) / Standard Absorbance

11. Biological Reference Range

• Serum: 135 – 220 mg/dL
• Desirable: ≤ 200 mg/dL
• Borderline high: 200 – 240 mg/dL
• High risk: > 240 mg/dL
• Critical/Alert: > 300 mg/dL

12. Laboratory Interpretation

Hypercholesterolemia occurs in hypothyroidism (Myxedema), nephrotic syndrome, atherosclerosis, arteriosclerosis, uncontrolled diabetes, and obstructive jaundice. Hypocholesterolemia is seen in hyperthyroidism and acanthocytosis.

13. Potential Sources of Variability

• Lysed plasma specimens may yield falsely elevated results.
• Plasma cholesterol is stable for 7 days at room temperature and 6 months at –20 °C.
• Do not use turbid reagents; this indicates contamination.

14. References

1. Richmond W. Clin Chem 1973; 19:1350.
2. Tarbutton PN, Gunter CR. Clin Chem 1974; 20:724.
3. Allain CC et al. Clin Chem 1974; 20:470.
4. Richmond W. Scand J Clin Lab Invest 1972; 29 (Suppl 26): Abst 3.25.
5. Young DS et al. 1975; 21D.