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REFERENCE METHODS

The CDC Lipid Reference Laboratory assigns values to CDC reference materials using the methods described below. CDC analyzes each individual pool in 12 different analytical runs and includes four replicate samples from each pool in each run. Appropriate control materials are included in each run to maintain the highest level of reliability for each reference method.

Cholesterol

Cholesterol values are assigned using the CDC reference method (modified Abell-Kendall). The cholesterol in ester form is released by saponification of a serum sample with alcoholic potassium hydroxide. This is followed by extraction with hexane, evaporation of an aliquot of the extract, and development of a chromophore at 620 nm with Liebermann-Burchard reagent (acetic anhydride, glacial acetic, and concentrated sulfuric acid). The method is calibrated using the primary reference standard, NIST SRM 911c. CDC's overall coefficient of variation (% CV) for cholesterol analysis is 1% or less (0.03879 mmol/L) (7, 8, 9).

HDL-Cholesterol

A combination of ultracentrifugation, selective precipitation with manganese and heparin, and the CDC reference method for cholesterol is used to assign HDL-cholesterol values. Ultracentrifugation of serum (105,000 x g) at a density of 1.006 kg/L for 18 hours at 18°C removes the very low-density lipoproteins that interfere with the precipitation of low-density lipoproteins with manganese and heparin. Manganese chloride and heparin are added to the bottom fraction in amounts sufficient to obtain final concentrations of 0.0456 mol Mn²⁺ and 183 UPS kU/L of heparin, which will precipitate non-HDL-C lipoproteins. After centrifugation at 1500 x g for 30 minutes at 4°C, the resulting supernate is analyzed for HDL-cholesterol by the CDC reference method for cholesterol. CDC's overall standard deviation for the HDLC analysis is at or below 1.5 mg/dL (0.03879 mmol/L) (10).

Triglyceride

Triglyceride values are assigned using the CDC reference method (chromotropic acid method). In this semi-automated method, free glycerol and phospholipids are removed using silicic acid. Triglyceride is extracted from the sample with methylene chloride. After evaporation of the methylene chloride, the triglycerides are saponified with alcoholic potassium hydroxide to release glycerol. The glycerol is then oxidized with periodate, and a reaction with chromotropic acid produces the chromophore. CDC's overall relative standard deviation for TG analysis is at or below 0.06 mmol/L (5.3097 mg/dL) (11, 12, 13, 14).

The CDC reference method for triglycerides provides a glycerol-corrected value. The method was developed at a time when procedures for preparation of reference materials resulted in high free glycerol levels. The free glycerol present in the CDC reference materials, as well as in routine clinical samples, is measured as triglyceride by non-specific methods. The current protocol for preparing CDC reference materials results in materials that have low levels of free glycerol (less than 10 mg/dL). These levels have a minimal effect on the measurement of total triglycerides and are generally not detected because of the normal analytical variability of current routine analytical methods for triglyceride. Nonetheless, standardization is best achieved through the use of glycerol-blanked methods.