The Multiple Risk Factor Intervention Trial was a randomized primary prevention trial to test the effect of a multifactor intervention program on mortality from coronary heart disease (CHD) in 12,866 high-risk men aged 35 to 57 years. Men were randomly assigned either to a special intervention (SI) program consisting of stepped-care treatment for hypertension, counseling for cigarette smoking, and dietary advice for lowering blood cholesterol levels, or to their usual sources of health care in the community (UC). Over an average follow-up period of seven years, risk factor levels declined in both groups but to a greater degree for the SI men. Mortality from CHD was 17.9 deaths per 1,000 in the SI group and 19.3 per 1,000 In the UC group, a statistically nonsignificant difference of 7.1% (90% confidence interval, -15% to 25%). Total mortality rates were 41.2 per 1,000 (SI) and 40.4 per 1,000 (UC). Three possible explanations for these findings are considered: (1) the overall intervention program, under these circumstances, does not affect CHD mortality; (2) the intervention used does affect CHD mortality, but the benefit was not observed in this trial of seven years' average duration, with lower-than-expected mortality and with considerable risk factor change in the UC group; and (3) measures to reduce cigarette smoking and to lower blood cholesterol levels may have reduced CHD mortality within subgroups of the SI cohort, with a possibly unfavorable response to antihypertensive drug therapy in certain but not all hypertensive subjects. This last possibility was considered most likely, needs further investigation, and lends support to some preventive measures while requiring reassessment of others.

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.

Biologic intraindividual variation (CVb) is a major source of inaccuracy in current lipid and lipoprotein measurements. Metaanalysis has been used to estimate the average CVb of serum total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), and triglyceride (TG). These CVb values are larger than the National Cholesterol Education Program-accepted and -proposed analytic (CVa) goals. Measuring serial specimens reduces the error in determination of the mean concentration used in classification of the patient by cutoff points. We show (a) a convenient technique, based on the relative range, to qualitatively estimate and interpret biologic variation of TC, HDLC, LDLC, and TG, and (b) the number of serial specimens required to meet a total variation goal for measurements of mean lipid and lipoprotein values. A total variation goal has been selected that can be met by two serial specimens for a majority of individuals.

Using accelerated Arrhenius-type short-term and long-term temporal studies, we evaluated the storage life of a stabilized, liquid-frozen reference material (SLRM) for human apolipoprotein B (apo B) developed by the International Federation of Clinical Chemistry. As measured by our candidate reference RIA, the concentrations of immunoreactive apo B in the SLRM showed pronounced degradation with exposure to increasing temperatures over time. The SLRM was stable for as long as 1 year when stored at - 70 degrees C, but its immunoreactive apo B declined by < 10% when stored at 4 degrees C for 10 months. Using radial immunodiffusion and an ELISA to assess the equivalency of measured mass for the accelerated thermal stability of the SLRM, we found a loss of immunoreactive apo B similar to that measured by RIA. Analyzing the same samples by liquid immunoprecipitation (nephelometry) resulted in the amount of apo B present being overestimated, especially in samples held for long periods. By using different immunological methods to evaluate this thermally aged SLRM, we demonstrated that its measured behavior varies depending on the method of quantitation.

To obtain the best estimates of the average intraindividual biological variability (CVb) in the concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), and triglyceride serum lipids in a person’s blood, we evaluated results from 30 studies published from 1970 to 1992. The usually more applicable random-effects model estimated an average CVb of 6.1% for TC, 7.4% for HDLC, 9.5% for LDLC, and 22.6% for triglyceride. Composite estimates of the average CVb from all evaluated published studies by different models of estimation ranged from 6.0% to 6.4% for TC, from 6.2% to 7.5% for HDLC, from 7.0% to 9.6% for LDLC, and from 22.4% to 22.9% for triglyceride. Two important factors influenced the reported biologicalvariation of the study subjects: (a) the magnitude of the variability of the analytical method used and (b) the design characteristics of the study-primarily the number of subjects, the sampling interval, and the number of measurements per subject. For TC, we found a statistically significant positive correlation between the reported mean CVb and both the number of study subjects and the analytical variation. For TC and LDLC we estimate CVb as a function of the study design features. The number of patient specimens required to obtain reliable estimates for serum lipid concentrations are determined from the CVb and the current analytical variation.

Previous comparisons between the Reference and Definitive Methods for measuring serum cholesterol have demonstrated a small but persistent positive bias in the Reference Method, averaging about +1.6%. Here we describe the results of further investigations designed to better characterize the nature of this bias. Analysis of a well-characterized model serum sample (SRM 909) suggests that more than half of the difference in cholesterol values determined by the two methods is the result of small contributions from cholesterol precursor sterols and phytosterols, which are also measured for the Reference Method. An additional significant contribution may be from cholesterol oxidation products, particularly 7-hydroxycholesterol isomers, which are active in the Liebermann-Burchard reaction. The 7-hydroxycholesterol in SRM 909, most of which appeared to be already present in the serum rather than formed during saponification, may account for as much as 20% of the observed difference between the methods. Contributions from other possible sources, including impurities in the cholesterol standard and incomplete saponification of cholesteryl esters, are very small. Because the observed bias is both quite small and consistent among samples, the cholesterol Reference Method continues to meet all of the requirements generally expected for a dependable and effective Reference Method.

In 1987 a collaborative study was initiated with 140 laboratories worldwide to evaluate the effects of analytical method and lyophilization on the measurement of different concentrations of apolipoproteins (apo) A-I and B in four lyophilized serum pool samples. This survey confirmed that the lyophilized apo Reference Material of the International Union of Immunological Societies (IUIS) is useful for apo A-I assays as an international serum-based reference material, because among-method variation is negligible. The apo A-I concentration value of 1.24 g/L is now assigned to the IUIS Reference Material (CDC 1883) by a Centers for Disease Control RIA in-house reference method. Use of lyophilized serum preparation as a reference material for some modes of apo B measurement is questionable because of lyophilization and matrix effects. Both radial immunodiffusion and liquid immunoprecipitin methods demonstrated bias in measured apo B concentrations, compared with overall method-weighted means values on the IUIS Reference Material. Because of the uncertainty associated with LDL primary standard, protein analysis, and concentration differences among analytical methods, assigning a single apo B concentration value to the IUIS Reference Material appears inadvisable at present.

In 1989 the Committee on Apolipoproteins of the International Federation of Clinical Chemistry and the Centers for Disease Control conducted an international survey of total-protein measurements of isolated low-density lipoproteins (LDL) and delipidated high-density lipoproteins (HDL), and of their relationships to the National Institute of Standards and Technology (NIST) bovine serum albumin (BSA) Standard Reference Material (SRM). Most of the 93 apolipoprotein laboratories surveyed use the Lowry total-protein method. Results reported with the LDL preparations demonstrated a large bias and variation among methods; those with delipidated HDL were not as great, but were similar to those for BSA. Performance improved appreciably with use of the Lowry-sodium dodecyl sulfate method and the NIST BSA SRM for protein measurement. The total CVs, including among-laboratory and within-laboratory errors, averaged approximately 50% and 23% for LDL, 17% and 11% for HDL, and 18% and 11% for BSA solutions by all methods and by the Lowry methods, respectively. Regardless of the methods used, greater variability of the protein measurements was seen with the normally occurring LDL than with the nonlipoprotein BSA or delipidated HDL. The mean CV values for all samples among laboratories averaged between 10% and 15% with the modified Lowry methods; the biuret method gave the highest among-laboratory CV, 34%; the Kjeldahl had the lowest, 7.7%. Use of the same methodology and primary nonapolipoprotein standard is essential for comparability of protein results for apolipoprotein primary standard solutions. This is especially true for apolipoprotein B, because its inherent properties and lability make protein analysis difficult. This study supports the use of a standardized selected Lowry-sodium dodecyl sulfate method traceable to quantitative amino acid analysis as a point of reference for determining the protein concentration of primary calibration reference materials for apolipoproteins.

Several recent studies to evaluate the performance of laboratory instruments have shown that with some instrument systems processed (lyophilized, frozen, and stabilized) materials exhibit matrix effects that cause the assay for cholesterol to respond differently for them than for patient specimens. To understand this phenomenon better the College of American Pathologists, Northfield, III, and the Centers for Disease Control, Atlanta, Ga, have conducted a collaborative study with 44 laboratories where 16 instruments manufactured by nine companies are evaluated. The purposes were to assess measurement variation on several reference materials used for standardizing total cholesterol measurements and to evaluate a new stabilized liquid serum as a potential reference material. Lypophilized, frozen, fresh-frozen, and stabilized materials at three concentrations were measured for total cholesterol. The results show that the average coefficient of variation of measured total cholesterol for all instruments, laboratories, vials, and replicates is 3.6% to 4.1% for each of the materials measured (excluding the results for one instrument). For one instrument, however, significant bias was found on the stabilized liquid serum material. Results from the fresh-frozen materials indicate that the instrument systems evaluated allow laboratories to attain the National Cholesterol Education Program analytical performance goals.

The Centers for Disease Control (CDC), in collaboration with the National Heart, Lung and Blood Institute, has supported programs for standardization of lipid measurements for more than 30 years. The cornerstone of these lipid standardization programs has been an accuracy base of lipid reference materials and methods developed by the CDC. Through its efforts with manufacturers of diagnostic products and specialized lipid research laboratories, CDC′s standardization programs have become a national resource for improving accuracy in the measurement of cholesterol and other lipids, lipoproteins, and apolipoproteins.

Using data from the National Health and Nutrition Examination Survey (NHANES II) 1976-1980, we demonstrate how cross-sectional total serum cholesterol surveillance data can be used by an individual to assess current and future personal cholesterol risk status. We propose statistical models, based on a person's current measured cholesterol level and the relationship between cross-sectional age and cholesterol percentile estimates, that will allow prediction of future cholesterol levels or the age at which specified cholesterol risk levels will be reached if no cholesterol-altering intervention is taken. These models incorporate the observed variation in the NHANES II data and expected intraperson biological variation and intralaboratory analytical variation. We illustrate the adequacy of the models using data from the longitudinal Framingham Study.

Two international apolipoprotein A-1 and B (apoA-1 and apoB) surveys were conducted in 1983 and 1986 with use of a lyophilized serum measured by 55 and 91 participants, respectively. The chief source of variability in both surveys was among laboratories, but among-method variation was significant for apoB. Comparing the 1986 with the 1983 findings, we saw a 60% decrease in apoA-1 variability among laboratories, and a similar decrease (53%) was found for apoB. Evaluation of individual measurements suggests that some collaborators may have adjusted their calibrators toward the consensus values published in 1983.