Reference Measurement Procedures
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 measurement procedure.
Cholesterol values are assigned using the CDC reference measurement procedure (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).
A combination of ultracentrifugation, selective precipitation with manganese and heparin, and the CDC reference measurement procedure 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 measurement procedure for cholesterol. CDC's overall standard deviation for the HDLC analysis is at or below 1.5 mg/dL (0.03879 mmol/L) (10).
The CDC LDL-C RMP is the reference point for LDL-C recommended by the NCEP Lipoprotein Measurement Working Group.4 The CDC reference procedure for LDL-C is performed exactly like the CDC HDL-C RMP described above, but it is expanded to include analysis of the beta-quantification ultracentrifuge bottom fraction (d >1.006 Kg/L) by the cholesterol RMP. The beta-quantification calculation is: LDL-C = Bottom fraction cholesterol – HDL-C. The CDC HDL-C/LDL-C reference procedure is based on the beta quantification ultracentrifuge procedure used by the Lipid Research Clinics program but differs in centrifugation temperature (18°C instead of 10°C) and density of saline overlay (0.195 M instead of 0.15 M). Also, because serum is used instead of EDTA plasma, a lower concentration of heparin manganese reagent appropriate for precipitation of serum is used (10). This method has also been recognized by the JCTLM as an international RMP of higher order.
The current operational definition of LDL-C based on physical separations (ultracentrifugation and chemical precipitation) contains the mixture of lipoprotein cholesterol, which was defined as "LDL-C" by NCEP and includes some Lp(a) cholesterol, IDL-C, and the core LDL cholesterol (10, 13). The operational definition of LDL-C also incorporates the operational definition of HDL-C, with the tacit assumption that the specificity in the precipitation step is sufficient to adequately and consistently separate the continuum of LDL lipoprotein particles from the continuum of HDL lipoprotein particles.
CDC's Lipid Reference Laboratory has completed development of a gas chromatography isotope dilution mass spectrometric (GC-IDMS) method to measure total glycerides. Since most laboratories measure "triglycerides" as total glycerides, CDC believes it is appropriate to switch to mass spectrometry to generate target values for our reference materials used to standardize total glycerides. In brief, aliquots of diluted serum are fortified with isotopically labeled 13C3-glycerol, thoroughly mixed and hydrolyzed with alcoholic/KOH at 60°C for 1 hr. After hydrolysis, the solvent is evaporated under nitrogen and the residue containing free and hydrolyzed glycerol is derivatized with acetic anhydride in the presence of pyridine. The derivatized product is then extracted with ethyl acetate and the organic layer is removed and dried under nitrogen at 60°C. Finally, the dried glycerol acetate derivative is reconstituted in methanol and analyzed by GC-MS (11).
Designated Comparison Methods
Apolipoprotein A-I (apo A-I) and apolipoprotein B (apo B) target values are assigned by Northwest Lipid Metabolism and Diabetes Research Laboratories using Siemens-Behring reagent on a BNII analyzer. The assay is calibrated with the WHO/IFCC reference materials for apo A-I and apo B and this assay as well as the value assignment protocol is the same used during the IFCC Standardization of analytical methods for the measurement of apo A-I and apo B. The assay precision is monitored by three quality controls samples with low, medium, high apo A-I and apo B levels. These fresh-frozen pools are in-house prepared with assigned values traceable to the WHO/IFCC reference materials (16, 17, 18, 19, 20).