Evaluation of long-term frozen storage of plasma for measurement of high-density lipoprotein and its subfractions by precipitation

The concentration of high-density lipoprotein cholesterol (HDL-C) in plasma is now established as an independent risk factor for coronary heart disease, but more data are needed on the relative risk-predictive powers of different HDL subclasses. For epidemiologic and clinical purposes, isolation of HDL from other lipoproteins and separation of its two major subclasses, HDL2 and HDL3, are performed most conveniently by precipitation. Although storage of plasma is commonly necessary, little information is available on the long-term stability of HDL subclasses at different temperatures. Therefore, we quantified HDL-C, HDL2-C, and HDL3-C by dual precipitation with heparin-MnCl2/15-kDa dextran sulfate (H-M/DS) in samples of EDTA-plasma from 93 healthy subjects, after storage for one to 433 days at -20 degrees C, at -70 degrees C, or in liquid nitrogen (-196 degrees C). Fourteen samples (15%) were stored for a year or longer. At -20 degrees C, HDL-C decreased by 4.8% per year and HDL3-C decreased by 6.9% per year (P = 0.002 for both variables) relative to results obtained with samples stored in liquid nitrogen; total cholesterol, HDL2-C, and triglyceride did not change significantly at this temperature. When stored at -70 degrees C, none of the lipids showed any change relative to results obtained with liquid nitrogen. Thus, long-term storage of EDTA-plasma at -20 degrees C is unsuitable for subsequent quantification of HDL-C and its subclasses by H-M/DS dual precipitation. Storage at -70 degrees C is preferable, and is as reliable as storage in liquid nitrogen.