CC lab - midterm package inserts

Total Cholesterol Assay Principle

Enzymatic oxidase/peroxidase method measuring free and esterified cholesterol through formation of a colored quinoneimine dye.

HDL Cholesterol Assay Principle

Phosphotungstate-Mg2+ precipitation of VLDL and LDL, followed by enzymatic colorimetry of HDL in the supernatant.

Cholesterol Esterase Function

Hydrolyzes cholesterol esters into free cholesterol and fatty acids.

Cholesterol Oxidase Function

Oxidizes cholesterol to cholestenone, producing hydrogen peroxide (H2O2).

Peroxidase Function

Uses H2O2, 4-aminoantipyrine, and phenol to produce quinoneimine dye.

Total Cholesterol Chromophore

Quinoneimine dye.

Wavelength for Total and HDL Cholesterol

500 ± 20 nm against a reagent blank.

Total Cholesterol Standard

200 mg/dL (5.18 mmol/L).

HDL Cholesterol Standard

15 mg/dL (0.39 mmol/L).

Total Cholesterol Pipetting Scheme

10 µL sample or standard + 1.0 mL reagent.

Total Cholesterol Incubation

10 minutes at 16-25°C OR 5 minutes at 37°C.

Total Cholesterol Color Stability

At least 2 hours.

HDL Precipitating Reagent

Phosphotungstate 0.4 mmol/L and magnesium chloride 20 mmol/L.

HDL Precipitation Procedure

Mix 0.2 mL sample with 0.5 mL precipitating reagent; stand 10 minutes at room temperature; centrifuge at ≥4000 rpm for 10 minutes.

HDL Precipitation Function

Precipitates VLDL and LDL, leaving HDL in the supernatant.

HDL Sample Dilution Factor

3.5; calculated as (0.2 + 0.5 mL) / 0.2 mL.

HDL Colorimetry Pipetting Scheme

100 µL supernatant or standard + 1.0 mL cholesterol reagent.

HDL Colorimetry Incubation

20 minutes at 16-25°C OR 10 minutes at 37°C.

HDL Color Stability

At least 30 minutes.

Total Cholesterol Calculation

(Absorbance of sample / Absorbance of standard) × 200 mg/dL.

HDL Cholesterol Calculation in mg/dL

(Absorbance of sample / Absorbance of standard) × 52.5 mg/dL.

HDL Cholesterol Calculation in mmol/L

(Absorbance of sample / Absorbance of standard) × 1.36 mmol/L.

HDL Calibration Factor Derivation

15 mg/dL × 3.5 dilution factor = 52.5 mg/dL.

Total Cholesterol Dilution Rule

If the result exceeds 1000 mg/dL, dilute the sample 1:1 with distilled water and multiply the calculated result by 2.

Turbid HDL Supernatant Troubleshooting

Add 0.5 mL additional precipitating reagent, mix, and centrifuge again at ≥4000 rpm for 10 minutes; multiply the result by 1.7.

Desirable Total Cholesterol

≤200 mg/dL (≤5.20 mmol/L).

Borderline-High Total Cholesterol

200-239 mg/dL (5.20-6.21 mmol/L).

High Total Cholesterol

>240 mg/dL (>6.24 mmol/L).

Low HDL Cholesterol

≤35 mg/dL; associated with high coronary artery disease risk.

Protective HDL Cholesterol

>60 mg/dL (>1.56 mmol/L); associated with lower coronary artery disease risk.

Total Cholesterol Detection and Linearity Limits

Detection: 0.3 mg/dL; linearity: 1000 mg/dL.

HDL Cholesterol Detection and Linearity Limits

Detection: 3.0 mg/dL; linearity: 150 mg/dL.

Total Cholesterol Interference Limits

No interference up to hemoglobin 500 mg/dL, bilirubin 10 mg/dL, triglycerides 1000 mg/dL, and ascorbic acid 6.25 mg/dL.

HDL Cholesterol Interference Limits

May be affected by hemoglobin at 500 mg/dL and bilirubin at 10 mg/dL; triglycerides up to 1000 mg/dL do not interfere.

Total and HDL Cholesterol Specimen Stability

Serum or plasma; stable for 7 days at 2-8°C.

Total Cholesterol Reagent Deterioration

Turbidity, particulate matter, or reagent blank absorbance >0.200 at 500 nm using a 1 cm cuvette.

Reduced HDL Association

Associated with increased atherosclerosis, myocardial infarction, and cerebrovascular accident risk.

Low HDL-Associated Conditions

Diabetes mellitus, severe malnutrition, chronic anemia, liver disease, Tangier disease, cigarette smoking, and acute stress.

Total Cholesterol Repeatability CV

1.1% at 121 mg/dL and 0.9% at 257 mg/dL.

Total Cholesterol Reproducibility CV

1.8% at 121 mg/dL and 1.0% at 257 mg/dL.

HDL Cholesterol Repeatability CV

2.3% at 30 mg/dL and 2.0% at 55 mg/dL.

HDL Cholesterol Reproducibility CV

4.2% at 30 mg/dL and 3.2% at 55 mg/dL.