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The study of modern lipid chemistry began in the 17th and 18th centuries with early observations by Robert Boyle, Poulletier de la Salle, Antoine François de Fourcroy and others. The 19th century chemist, Chevreul, identified several fatty acids, suggested the name 'cholesterine' for the fatty substance in gallstones, coined the word 'glycerine', and showed that fats were comprised of glycerol and fatty acids. The 20th century brought many advances in the understanding of lipoprotein structure and function, and explored relationships between lipoproteins and disease states. The development of the ultracentrifuge and other lipoprotein separation techniques, and reagents for accurate, standardized quantitative measurement have steadily increased our understanding of the important role of lipoprotein metabolism in both healthy and disease states.

C-reactive protein (CRP), when measured by a highly sensitive method, is a measure of lowgrade, chronic inflammation and is an independent risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). CRP also has the capacity to interact with other risk factors to increase the risk for T2D and CVD. Population distributions divided into tertiles provide the capacity to predict onset of T2D and associated CVD. Preanalytical as well as analytical sources of variation in high-sensitivity CRP (hsCRP) measurements need to be standardized in order for CRP results to be optimally useful. The Centers for Disease Control and Prevention and the American Heart Association have issued guidelines for clinical usefulness of hsCRP measurements. The Centers for Disease Control and Prevention has taken steps to standardize hsCRP assays by evaluating secondary reference materials to be used by manufacturers to calibrate their assays.

Biomarkers of nutritional status provide alternative measures of dietary intake. Like the error and variation associated with dietary intake measures, the magnitude and impact of both biological (preanalytical) and laboratory (analytical) variability need to be considered when one is using biomarkers. When choosing a biomarker, it is important to understand how it relates to nutritional intake and the specific time frame of exposure it reflects as well as how it is affected by sampling and laboratory procedures. Biological sources of variation that arise from genetic and disease states of an individual affect biomarkers, but they are also affected by nonbiological sources of variation arising from specimen collection and storage, seasonality, time of day, contamination, stability and laboratory quality assurance. When choosing a laboratory for biomarker assessment, researchers should try to make sure random and systematic error is minimized by inclusion of certain techniques such as blinding of laboratory staff to disease status and including external pooled standards to which laboratory staff are blinded. In addition analytic quality control should be ensured by use of internal standards or certified materials over the entire range of possible values to control method accuracy. One must consider the effect of random laboratory error on measurement precision and also understand the method's limit of detection and the laboratory cutpoints. Choosing appropriate cutpoints and reducing error is extremely important in nutritional epidemiology where weak associations are frequent. As part of this review, serum lipids are included as an example of a biomarker whereby collaborative efforts have been put forth to both understand biological sources of variation and standardize laboratory results.

Accurate cholesterol and lipoprotein measurements have provided dependable and powerful basic risk factors for cardiovascular disease. A battery of total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, and triglycerides (TG) is recommended in the initial evaluation for classification of patients (based on lipids) into highly desirable, desirable, borderline, high, and very high lipid risk factor for cardiovascular disease. Treatment is based largely on the LDL cholesterol measurement result of the patient. The risk factor score of a patient greatly increases when other risk factors for cardiovascular disease exist, along with increased lipid risk factors. Attainment of the needed acceptable accurate lipid and lipoprotein measurements depends upon prevention or control of multiple sources of errors or variation that can exist in preanalytic, analytic, and postanalytic stages of determination of the reported result. Highly important is to control nonfasting, posture, diet, and alcohol intake in the preanalytic part, elimination of matrix effects and use of accurate calibrators in the analytic part, and check for transcription errors in preparation of reports in the postanalytic part of the measurement of lipids.

Background: The US National Cholesterol Education Program has recently released the third report of the Adult Treatment Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Incorporating new evidence and more consistent with other international intervention programs, these more complex guidelines will considerably expand indications for treatment. The implications for clinical laboratories are summarized in this report. Content: LDL-cholesterol (LDL-C) remains the major focus for classification and treatment, whereas diabetes, the presence of multiple risk factors, including the metabolic syndrome, and increased triglycerides (TGs), will now require more intensive management. For screening, a fasting lipoprotein profile is recommended, adding LDL-C and TGs to the previous measurements of total cholesterol and HDL-cholesterol (HDL-C). Lowering the cutpoints defining optimal LDL-C [100 mg/dL (2.58 mmol/L)] and normal TGs [150 mg/dL (1.70 mmol/L)] and raising the cutpoint for low HDL-C to 40 mg/dL (1.03 mmol/L) will select more patients for treatment. A new marker, non-HDL-C, becomes a secondary target in treating high TGs. Conclusions: Laboratories will need to adjust reporting formats and interpretations and can expect more requests for tests to characterize secondary causes of dyslipidemia, e.g., diabetes, and for the so-called “emerging risk factors”, e.g., lipoprotein(a), homocysteine, and C-reactive protein.

Background: Accurate and precise measurement of blood cholesterol plays a central role in the National Cholesterol Education Program’s strategy to reduce the morbidity and mortality attributable to coronary heart disease. Matrix effects hamper the ability of manufacturers to adequately calibrate and validate traceability to the National Reference System for Cholesterol (NRS/CHOL). CDC created the Cholesterol Reference Method Laboratory Network (CRMLN) to improve cholesterol measurement by assisting manufacturers of in vitro diagnostic products with validation of the traceability of their assays to the NRS/CHOL.Methods: CRMLN laboratories established the CDC cholesterol reference method (modification of the Abell-Levy-Brodie-Kendall chemical method) and are standardized using CDC frozen serum reference materials. CRMLN laboratories use common quality-control materials and participate in monthly external performance evaluations conducted by CDC. The CRMLN performance criteria require member laboratories to agree with CDC within ± 1.0% and maintain a CV ≤2.0%.Results: From 1995 to 2000, the CRMLN laboratories met the accuracy criterion 97% of the time and the precision criterion 99% of the time. During this time period, the CRMLN maintained an average bias to CDC of 0.01% and an average collective CV of 0.33%.Conclusions: CDC established the CRMLN as the first international reference method laboratory network. The CRMLN assists manufacturers in the validation of the calibration of their diagnostic products so that clinical laboratories can measure blood cholesterol more reliably. The CRMLN can serve as a model for other clinical analytes where traceability to a hierarchy of methods is needed and matrix effects of the field methods with processed calibrators or reference materials are present.

In 1998 the AACC celebrates its 50th anniversary. Thus, we are honored to call attention to the outstanding professional contributions of the journal Clinical Chemistry over the years by pointing out the citation classics that have been published in its pages. By the end of 1995, 31 articles in Clinical Chemistry had been cited more than 300 times (1). Thirty of these articles were still being cited in 1995, and some are still cited to the present day. These 31 papers are distributed in multiple areas of biological sciences, and in particular: lipids, 8 papers (2)(3)(4)(5)(6)(7)(8)(9); endocrinology, 2 papers; clinical enzymology, 4 papers; intermediary metabolism, 6 papers; renal function, 2 papers; cancer, 1 paper; and calcium metabolism, 3 papers. Publications on methodology research and laboratory applications of lipid, lipoprotein, and apolipoprotein measurements in clinical investigations and epidemiologic trials have had a major impact on research of lipid metabolism and cardiovascular medicine (2)(3)(4)(5)(6)(7)(8)(9). The combined efforts of epidemiologists, clinicians, and laboratorians have produced classic publications on the understanding of lipid metabolic disorders, have stimulated development of new novel equipment and methodology, and have helped to establish the development of beneficial preventive and control measures for diseases of the heart and blood vessels. The cited lipid papers published in Clinical Chemistry included mainly the original papers on measurement of lipoproteins, enzymatic measurements of cholesterol and triglycerides, and immunoassays of apolipoproteins. The method used most widely today for estimation of the concentration of LDL-cholesterol (LDL-C) in both clinical and research laboratories arose from the publication of Friedewald et al. (2) that showed that the serum concentration of LDL-C can be estimated from available established measurements …

We examine the effect of systematic bias and random error, quality control, and intraperson biological variation on the National Cholesterol Education Program (NCEP) clinical classifications for reported lipid measurements. We consider misclassification to occur if a true lipid homeostatic set point is within a desirable range but the reported lipid value is in a high-risk range, or if a true lipid homeostatic set point is in a high-risk range but the reported lipid value is in a desirable range. To evaluate the overall adequacy of the NCEP guidelines to ensure correct patient classification, we construct operating characteristic curves for total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. We demonstrate that if laboratories are meeting the NCEP guidelines for inherent bias and analytic precision and are using standard quality-control (QC) procedures incorporating at least two QC samples per analytical run from each of two QC pools (for a total of 4 QC samples), the current NCEP guidelines are adequate to ensure (probability >0.90) correct patient classifications regardless of the size of the systematic bias of the laboratory or increased random analytic error. Thus we suggest that at least two concentrations of QC material be included in the QC scheme to ensure that the measurement system is operating within desired specifications across the entire range of desirable and high-risk lipid concentrations and to ensure with high probability that patients are correctly classified.

We read with concern the recent paper by Fallest-Strobl, Olafsdottir, Wiebe, and Westgard (1) in which the authors state, “… the NCEP recommendations fail to adequately consider the quality control requirements necessary to detect medically important systematic errors.” We also note that Westgard, Petersen, and Wiebe (2) published a similar paper in 1991 questioning the adequacy of the NCEP recommendations for total cholesterol. We appreciate the importance of the issues addressed in these two papers and find no fault in the majority of the derivations. We do notice, however, that the authors did not address the conditional nature of their calculations. That is, the OPSpecs charts do not take into account the fact that a patient result will only be reported when the quality control (QC) run associated with the patient specimen is considered to be in control. Thus, the OPSpecs charts relate to the conditional probability that a measured patient result will exceed a specified limit, not to the joint probability of patient misclassification. For misclassification to occur, the measured patient result must not only exceed the specified limit, but the QC procedure must also fail to indicate an out-of-control condition. Without taking into account the probability that the QC procedure will fail to detect an out-of-control condition, the authors do not accomplish their stated intention to evaluate the NCEP recommendations with regard to “quality control procedures that are necessary to detect unstable operation” (2). Incorporating QC performance into the OPSpecs charts requires inclusion of the probability that a patient result will be reported on the basis of the QC outcome of the analytic run in which the patient specimen was measured. To illustrate our point, consider the intended 0.95 probability of correct classification chosen by the authors. This conditional probability does not reflect the overall likelihood of correct … bAuthor for correspondence.

Cholesterol and triglyceride standardization procedures have been used extensively and continuously since the 1950s. Definitive and Reference Methods, as well as primary and secondary standards, have been developed and maintained as the basis for evaluating the accuracy of results by various methods in many laboratories. But, although standardization efforts for apolipoprotein A-I and B measurements have been reported in detail in the scientific literature, much less has been reported in the area of total and lipoprotein cholesterol and triglyceride standardization efforts. Standardized cholesterol and triglyceride concentrations, determined in multiple large epidemiological and clinical studies, have been instrumental to the National Cholesterol Education Program panels that have assessed the lipoprotein values associated with risk of coronary disease, and have determined the cutpoints that are now used extensively by physicians to guide diagnosis and treatment of individual patients.

The lipid and lipoprotein parameters which are predominantly measured and effectively comprise the traditional lipoprotein profile include total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, and triglyceride. Total cholesterol is accepted as the initial entry point in a case finding approach such as that recommended by the National Cholesterol Education Program (NCEP). HDL cholesterol, known to be a strong inverse predicator of risk, is an additional measurement to total cholesterol to improve risk assessments. The evidence for triglyceride association remains mixed: although strong associations are found in some studies, the evidence as an independent risk factor is still incomplete. Triglyceride is therefore measured primarily for LDL estimation. Final classification and potential intervention is ultimately based on the measurement of LDL cholesterol. Reliability in the measurement of total cholesterol, HDL, LDL, and triglyceride is especially important if the uniform decision points established by the NCEP are to be properly implemented. Attention must be placed on controlling preanalytical sources of variation, which can account for as much as 60% of the total measurement variability. The major analytical source of error comes from matrix effects, which results in problems of proper analytical calibration. Instrument system calibration should be verified by a comparison with an accuracy base using fresh patient specimens. CDC has established a network of reference method laboratories to provide access to these lipid and lipoprotein accuracy bases.

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.

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 biological variation 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.

Since scientists recognized that preanalytical sources of variation often constitute more than 60% of the total variation in reported lipid measurements, they have been concerned about both the effects of these sources and ways to minimize their impact on lipid measurement. Sampling techniques, behavioral factors, metabolic disorders, and disease status are major preanalytical sources of variation. Lipid analysis of serial specimens from one person can minimize the effect of preanalytical sources on the reported lipid test result. A procedure using relative range can determine the number of specimens needed to assure that the total intraindividual coefficient of variation is

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.

Background: Heart disease and stroke continue to be the leading causes of death in the US. As a result, investigators continue to look for new and emerging biomarkers of disease risk. Because many of these emerging biomarkers are not as well documented as those of conventional lipid and lipoprotein risk factors, their value in clinical practice needs to be critically appraised and appropriate guidelines developed for their proposed use. Content: The National Academy of Clinical Biochemistry (NACB) convened a multidisciplinary expert panel to develop laboratory medicine practice guidelines for a selected subset of these emerging risk factors as applied in a primary prevention setting of heart disease and stroke. The NACB expert panel selected lipoprotein subclasses and particle concentration, lipoprotein(a), apolipoproteins A-I and B, high sensitivity C-reactive protein (hsCRP), fibrinogen, white blood cell count, homocysteine, B-type natriuretic peptide (BNP), N-terminal proBNP (NT-proBNP), and markers of renal function as biomarkers that fell within the scope of these guidelines. Conclusions: Based on a thorough review of the published literature, only hsCRP met all of the stated criteria required for acceptance as a biomarker for risk assessment in primary prevention.

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.

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.

This article summarizes epidemiological studies of inflammation markers, particularly C-reactive protein, and cardiovascular disease as of early 2002. Gaps in the research and the public health practice implications are also discussed. Although considerable work has been published since this review was completed, the perspectives and issues presented are still useful in evaluating the use of inflammation markers for risk stratisfication and prevention.

The journal Clinical Chemistry has played a major role in facilitating the progress that has occurred in lipid and lipoprotein testing over the past 50 years. In celebration of the 50th anniversary of the Journal, this Special Report highlights five selected areas of highly significant accomplishments during the past 50 years contributed by key researchers, some whose careers have spanned the entire history of modern lipid analysis and research. An overview of the history of lipid analysis from nonspecific strong acid methods to modern enzymatic assays, fully integrated lipoprotein methods, and the remarkable instrumentation developed by industry is presented by G. Russell Warnick. Dr. Bill Friedewald, whose name is associated with the widely recognized calculation of LDL-cholesterol (1), will relate the genesis of this important development. His 1972 report in Clinical Chemistry (1) has become the most cited ever in the Journal, with more than 6000 citations. Dr. Richard Havel developed an ultracentrifugal method in 1955 that continues to be the common citation for preparative isolation of the lipoproteins (2). The Lipid Research Clinic (LRC)1 laboratories, in addition to assaying for cholesterol and triglycerides, separated VLDL from the bottom layer of LDL and HDL for HDL analyses by ultracentrifugation, following this method. Dr. Havel, who is still active in lipid research, provides his views on the history of the lipoproteins and separation methodology. Drs. Gerald Cooper and Gary Myers present the story of how the CDC/National Heart Lung and Blood Institute (CDC-NHLBI) Lipid Standardization Program was developed over a period of nearly 50 years. The LRC program also focused attention on the need for standardization of lipid and lipoprotein measurements. Dr. Cooper of the CDC, one of the co-authors of the standardization article (3), has been a leader in the standardization field for 52 years. The CDC standardization effort, …

Background: Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed. Methods: We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum. Results: All materials had similar imprecision; CVs for the undiluted materials were 2.1–3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays. Conclusions: All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.

The US National Cholesterol Education Program has recently released the third report of the Adult Treatment Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Incorporating new evidence and more consistent with other international intervention programs, these more complex guidelines will considerably expand indications for treatment. The implications for clinical laboratories are summarized in this report.LDL-cholesterol (LDL-C) remains the major focus for classification and treatment, whereas diabetes, the presence of multiple risk factors, including the metabolic syndrome, and increased triglycerides (TGs), will now require more intensive management. For screening, a fasting lipoprotein profile is recommended, adding LDL-C and TGs to the previous measurements of total cholesterol and HDL-cholesterol (HDL-C). Lowering the cutpoints defining optimal LDL-C [100 mg/dL (2.58 mmol/L)] and normal TGs [150 mg/dL (1.70 mmol/L)] and raising the cutpoint for low HDL-C to 40 mg/dL (1.03 mmol/L) will select more patients for treatment. A new marker, non-HDL-C, becomes a secondary target in treating high TGs.Laboratories will need to adjust reporting formats and interpretations and can expect more requests for tests to characterize secondary causes of dyslipidemia, e.g., diabetes, and for the so-called "emerging risk factors", e.g., lipoprotein(a), homocysteine, and C-reactive protein.

We wanted to ascertain whether the current format of lipid laboratory reports seemed adequate to promote identification and treatment of patients with dyslipidemia. In a random survey of lipid laboratory reports from 25 laboratories, we found great inconsistencies among reporting formats and contents. Fewer than half the laboratories correctly reported the ranges for cholesterol, only 4 correctly reported ranges for high-density lipoprotein cholesterol, only 2 correctly reported ranges for triglycerides, and none presented low-density lipoprotein cholesterol ranges in terms of risk factors for coronary heart disease. Reports typically were disjointed and difficult to read. The current practice of reporting results for lipid panels is confusing and does not follow the National Cholesterol Education Program (NCEP) guidelines. We recommend that reporting of results be standardized, and a “model” standardized report is presented herein, based on consensus from a team of experts. The standardized report uses current recommendations for ranges, follows the flowcharts of the NCEP guidelines, and takes the patient’s clinical condition (the number of risk factors and the presence of coronary heart disease) into consideration. Standardizing lipid reports should decrease confusion and perhaps increase application of the guidelines and patient compliance with treatment.

Reference methods for lipids, lipoproteins, and apolipoproteins have been developed for use as part of an accuracy base for institutional, national, or international reference systems. A widely accepted reference method exists only for total cholesterol. Well described interim or institutional in-house reference methods have been established for the other lipids, lipoproteins, and apolipoproteins. The major criteria for a reference method are 1) scientific basis, 2) sound principles, 3) available calibration and control materials, 4) traceability to a definitive method or a point of reference, and 5) applicability to reference materials that provide traceability to clinical methods and transferability to other reference laboratories. The total cholesterol reference method of the U.S. National Reference System demonstrates how a reference method can be developed and applied. Reference methods now available can lead to an accepted international accuracy base for the clinically useful lipid, lipoprotein, and apolipoprotein measurements.

In Reply. —Since early 1990, when suggestions were made that oat bran probably possesses a cholesterol-lowering effect because it replaces dietary fats, numerous researchers have reported experiments in which they found oat bran to have an intrinsic hypocholesterolemic capacity. 1-4 Additional studies have reported that other types of fiber possess a similar intrinsic capacity to lower total and LDL cholesterol. It is timely for Dr Koval to call attention to this recent accumulating information on the inherent cholesterol-lowering effect of oat fiber and to point out that the mechanism of total cholesterol— and LDL cholesterol-lowering is not simply by replacement of dietary saturated fats. The soluble fraction of β-glucan has been identified as the hypocholesterolemic component in oat cereals. Oat, guar gum, and barley fiber appear to be the most effective fiber sources for lowering blood cholesterol levels in hypercholesterolemic men. Oat bran supplementation can decrease total and LDL cholesterol

Objective. —To describe the magnitude and impact of the major biological and analytical sources of variation in serum lipid and lipoprotein levels on risk of coronary heart disease; to present a way to qualitatively estimate the total intraindividual variation; and to demonstrate how to determine the number of specimens required to estimate, with 95% confidence, the "true" underlying total cholesterol value in the serum of a patient. Data Sources. —Representative references on each source of variation were selected from more than 300 reviewed publications, most published within the past 5 years, to document current findings and concepts. Most articles reviewed were in English. Study Selections. —Studies on biological sources of variation were selected using the following criteria: representative of published findings, clear statement of either significant or insignificant results, and acquisition of clinical and laboratory data under standardized conditions. Representative results for special populations such as women and children are reported when results differ from those of adult men. Data Extraction. —References were selected based on acceptable experimental design and use of standardized laboratory lipid measurements. Data Synthesis. —The lipid levels considered representative for a selected source of variation arose from quantitative measurements by a suitably standardized laboratory. Statistical analysis of data was examined to assure reliability. The proposed method of estimating the biological coefficient of variation must be considered to give qualitative results, because only two or three serial specimens are collected in most cases for the estimation. Conclusions. —Concern has arisen about the magnitude, impact, and interpretation of preanalytical as well as analytical sources of variation on reported results of lipid measurements of an individual. Preanalytical sources of variation from behavioral, clinical, and sampling sources constitute about 60% of the total variation in a reported lipid measurement of an individual. A technique is presented to allow physicians to qualitatively estimate the intraindividual biological variation of a patient from the results of two or more specimens reported from a standardized laboratory and to determine whether additional specimens are needed to meet the National Cholesterol Education Program recommendation that the intraindividual serum total cholesterol coefficient of variation not exceed 5.0. A National Reference Method Network has been established to help solve analytical problems. ( JAMA . 1992;267:1652-1660)

We evaluated a lyophilized serum preparation for use as a candidate Reference Material for apolipoproteins (apo) A-I and B. An international collaborative study was conducted with 28 participating laboratories, selected on the basis of participation and demonstrated expertise in a 1983 survey of apolipoproteins A-I and B. The analytical suitability of the material was established by confirming linearity of its dose-response curves over a desired concentration range and demonstrating that its response curves paralleled those for fresh sera. Differences in dilution-adjusted mass units ascribable to the five analytical methods used by the various laboratories constituted only 1% of the total variation for apo A-I, but 32% for apo B. The dominant source of error, however, for both apo A-I and B was the variability among laboratories, rather than variability among methods and antisera. The assigned consensus mass-concentration units based on study data are 1.124 g/L for apo A-I, 0.589 g/L for apo B. For these estimates the coefficients of variation were 13% and 27%, respectively. These findings on the proposed Reference Material meet the requirements suggested by the World Health Organization's Expert Committee on Biological Standards for a candidate WHO Reference Preparation.

We report here results of a worldwide survey of apolipoprotein (apo) A1 and B measurements. A lyophilized pool of human serum was developed for the survey to test its suitability as a candidate Reference Material. Seventy-five laboratories from 18 countries responded to invitations to participate. Using several methods, they evaluated the mass concentration of apo A1 and B in the proposed Reference Material. The bias between laboratories and between analytical methods was then characterized. Fifty-five laboratories reported results before the deadline on measurement of at least one apolipoprotein by one method. Variation in apo A1 and B among collaborators constituted the largest source of error in the study (68% of the total variation for apo A1, 73% for apo B). Variation among method means was not significant for apo A1, but for apo B it constituted 20% of the total variation. Differences due to vials or in replicate measurements within vials were found to constitute 6% or less of the total variability. Within-laboratory, among-vial, and among-replicate CV values were less than 5% for the study. These results suggest that future standardization efforts can be successful.

Plasma levels of lipids, lipoprotein-cholesterol and three major apolipoproteins (ApoA-I, A-II and C-II) were studied in 318 black and white males and females randomly sampled in Evans County, Georgia. Black-white differences in lipid and lipoprotein-cholesterol concentrations were observed, with low-density fractions higher in whites and high-density fractions higher in blacks. Plasma levels of ApoA-I but not ApoA-II were higher in blacks than in whites and in females than in males. ApoC-II concentrations were lower in black than in white men and women. Black-white differences in atherogenic lipoprotein fractions were statistically explained (in the sense of association, not necessarily of causal process) by the differences in ApoC-II concentrations between the race groups. Black-white differences in anti etherogenic high-density lipoprotein-cholesterol were greater than statistically predicted by differences in ApoA lipoprotein levels. The findings are indicative of black-white differences in lipoprotein composition.

Conflicting reports on the effect of smoking and coffee drinking on lipoproteins prompted us to study the combined effect of these two associated, widely prevalent habits in 361 persons randomly sampled from the Evans County cohort. Low-density lipoprotein (LDL) cholesterol levels were significantly higher among persons who smoked cigarettes and consumed five or more cups of coffee per day than among nonsmokers who abstained from coffee. Conversely, high-density lipoprotein (HDL) cholesterol was higher in persons who did not smoke or drink coffee than in coffee-consuming smokers. However, this trend was not statistically significant. Triglycerides and very low-density lipoprotein (VLDL) cholesterol were highest among smokers who drank five or more cups of coffee per day, but these differences did not reach statistical significance. Lipoprotein cholesterol levels were adjusted for age, sex and body mass. Smoking and coffee drinking interact in affecting LDL and total cholesterol, but coffee drinking alone did not appear to affect blood lipids.

Local antisera (LA) were compared with a common interim reference antiserum (IRA) to examine the antiserum component of the among-laboratory variation in an international collaborative study with 28 laboratories evaluating a candidate international Reference Material (apo-RM) for apolipoproteins A-I (apo A-I) and B (apo B). Measurement of the relative concentration of lyophilized preparations differed by less than 1% for LA and IRA. The percentage of the total variation in measurement of the concentration of apo-RM that was contributed by antisera among laboratories was 5% and 8% for apo A-I and B, respectively. Estimated differences from overall mean concentrations for the five different immuno-methods were greater for apo B (range: +22% to -23.5%) than for apo A-I (range +14% to -14%), but were similar within a method for LA and IRA. The results indicated that antisera are not a major source of error among laboratories and, indeed, are responsible for relatively little of the total variability.

The Evans County Cardiovascular Disease Survey has demonstrated a higher prevalence and incidence of coronary heart disease in white than in black men. Prior studies by us have disclosed similar associations of CHD with the risk factors of elevated blood pressure, serum cholesterol, and smoking in Evans County as have been reported elsewhere. The lower frequencies of CHD in black than in white males are present, controlling for the standard risk factors in univariate and in multivariate logistic risk function analyses. Black-white differences in total serum triglyceride and in cholesterol within lipoprotein fractions at identical total serum cholesterol levels are described in this report. Significantly higher LDL cholesterol and total triglycerides are present in whites, and higher HDL cholesterol was found in blacks in an age, sex, and total serum cholesterol matched comparison. The determinants of the black-white differences in lipid and lipoprotein fraction distributions are still to be elucidated, but in this sample, lipid differences are not explained by social characteristics, such as occupation and social class. The black-white lipoprotein fraction differences in Evans County are consistent with a negative, coronary risk factor role of elevated HDL cholesterol, which has been postulated based on other experimental and prevalence studies.

Quality control was emphasized in the screening and clinic laboratory during the early stages of the trial to cover all aspects of screening, and in the later stages to cover local testing used to monitor the participant in the clinic and to guarantee collection of valid specimens to be shipped to the Central Laboratory. Special attention throughout the trial was focused on techniques for collection of specimens, since it was recognized that analytical results could not be better than the quality of the specimens. Training courses, on-site visits, and newsletters were used to sensitize the staff of the clinic laboratory about the necessity to follow protocol. Any monitoring evidence of carelessness, use of deteriorated supplies, failure to follow safety rules, and deviation from directions in the MRFIT Manual of Operations resulted in memoranda from the Coordinating Center or the Central Laboratory.

The acquisition of quality biochemical data in the MRFIT was enhanced by writing detailed protocols, instituting strict rules for collecting and shipping specimens, setting high goals for analytical performance, using reliable means to transfer data, employing dedicated and competent analysts, and having a director of the Central Laboratory who believed in and enforced an effective quality control system. A Laboratory Quality Control Subcommittee was formed to work with this director in solving laboratory problems, with the Coordinating Center in conducting appropriate monitoring, with the Quality Control Committee in documenting the quality of data and investigating any laboratory problems, and with other committees of the directors and Program Office in responding to any questions raised about laboratory aspects of the trial. All of these efforts culminated in reliable biochemical data.

SummaryThe electrophoretic patterns of 2 human kala-azar sera revealed the presence in high concentration of a unique, abnormal component migrating with the γ globulin of the slowest mobility. The kala-azar sera, negative to serological tests for syphilis, provide instances which show that the presence of hyperproteinemia, hyperglobulinemia or hypergammaglobulinemia cannot be used to prove or disprove the specificity of positive serologic reactions for syphilis.

SummaryInfluenza virus B (Lee strain) increases to small degree in the chorio-allantoic fluid of 11-day-old chick embryos incubated for 60 hours at 39°C after inoculation with the virus. Increase was great at 37°C and still greater at 35°C. The pH of the chorio-allantoic fluid of the same embryos descended into the acid region at 39°C; at 37° C the pH remained essentially above 7.0; and at 35°C the reaction was maintained well in the alkaline region. These relations suggest the possibility that the extent of increase of the virus in the chorio-allantoic fluid may be influenced more by the pH of the fluid at the various temperatures than by the temperature per se.