CUN. CHEM. 40/10, 1915-1920 (1994) #{149} Lipids and Lipoproteuns Measurement of Apolipoproteins A-I and B During the National Health and Nutrition Examination Survey (NHANES) III Paul S. Bachorik,”2’5 Kathleen L. Lovejoy,1 Margaret D. Carroll,3 Santica M. Marcovina4 We measured apolipoproteins (apo) A-I and B by rate immunonephelometry (rate INA) during Phase 1 of the National Health and Nutrition Examination Survey (NHANES) Ill. We also made the measurements by radial immunodiffusion (RID) in a 20% subset of the samples. Aliquots of this subset were also analyzed in the Northwest Lipid Research Laboratories by fixed-time INA calibrated to the World Health Organization (WHO)-lnternational Federation of Clinical Chemistry (IFCC) First International Reference Materials for Apolipoproteins A-I and B. The CVs for the rate INA and RID measurements were: apoA-l, 4.5-7.7% and 2.5-7.6%, respectively; apoB, 2.3-5.3% and 2.3-6.4%, respectively. In NHANES Ill, rate INA values (x) canbe transformed to WHO-IFCC Reference Material-based values (y) as follows: for apoA-I, y = 0.87x + 251.8 mg/L (r = 0.93, SE510 = 0.13, SEintercept = 17, n = 708); for apoB (mg/L), y = 1.068x + 112.8 mg/L (r = 0.98, SEsiope 0.08, SEintercept = 7, n = 646). Terms: quality confrol/cholesterol/immunonephelomefly/radial immunodiffusion/coronaiy a,tery disease/standardization Indexing Apolipoproteins (apo) A-I and B, the major protein components of high-density lipoprotein (HDL) and lowdensity lipoprotein (LDL), are becoming increasingly recognized as important predictors of risk for coronary artery disease (CAD).6 Several studies have indicated that both apolipoproteins, and particularly apoB, may better identity patients with CAD than measurement of cholesterol in the corresponding lipoproteins, HDL and LDL (1-4). Although it may not yet be desirable to substitute apoA-I and apoB measurements for HDLand LDL-cholesterol measurements, apolipoprotein analysis can provide information that supplements the lipoprotein cholesterol measurements. The reasons for this are better understood for apoB: Because apoB does Departments of ‘Pediatrics and 2Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287. 3The National Center for Health Statistics, Centers for Disease Control and Prevention, Hyattsville, MD 20782. 4Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, Seattle, WA 98103. 5Author for correspondence. Fax 410-955-1276. 6Nonstandard abbreviati . apoA-I, apolipoprotein A-I; apoB, apolipoprotein B; HDL, high-density lipoproteins; LDL, low-density lipoproteins; CAD, coronary artery disease; CDC, Centers for Disease Control and Prevention; WHO, World Health Organization; IFCC, International Federation of Clinical Chemistry; NWLRL, Northwest Lipid Research Laboratories; INA, immunonephelometry; RID, radial immunodiffusion; NHANES Ill, National Health and Nutrition Examination Survey III. Received May 20, 1994; accepted June 28, 1994. Clifford L. Johnson,3 John J. Albers,4 and not exchange between lipoproteins, it is a measure of the number of apoB-containing particles, mainly LDL, in the circulation. The cholesterol composition of LDL, however, can vary. A substantial proportion of the LDL in some patients is smaller, denser, and contains less cholesteryl ester than normal (4-7). LDL-cholesterol measurements alone in such individuals can fail to detect increased concentrations of LDL, whereas such increases can be revealed by measuring apoB. The prevailing distributions of apoA-I and apoB were measured in the first phase (1988-1991) of the National Health and Nutrition Examination Survey (NHANES) Ill, a survey of a national probabffity sample of the US noninstitutionalized population. The survey was conducted from 1988 to 1994 in two 3-year phases, each of which involved a representative sample of the US population. Beth phases together also constituted a national population sample. The lipid and lipoprotein cholesterol concentrations measured during the first sampling cycle have recently been published (8, 9). Those measurements were made with standardized methods having documented accuracy with respect to Centers for Disease Control and Prevention (CDC) reference methods for lipids and lipoproteins (9). ApoA-I and B were also measured during the first phase (1988-1991), and to our knowledge this is the first national population-based assessment of the prevailing distributions of these apolipoproteins in the US. Since at the initiation of these studies there were not yet any reference methods or standardization programs for apoA-I and apoB, it was important to document how the measurements were made, their precision, and their stability during the 3-year sampling period. In addition, near the end of this period, the World Health Organization-International Federation of Clinical Chemistry (WHO-IFCC) First International Reference Materials for Apolipoproteins A-I and B were developed through the auspices of IFCC in collaboration with manufacturers involved in apolipoprotein measurements (10, 11). The Northwest Lipid Research Laboratories (NWLRL) at the University of Washington, Seattle, WA, served as the coordinating laboratory, and the CDC, Atlanta, GA, served as the consulting laboratory (12-15). These materials are intended to serve as international bases for accuracy of apoA-I and apoB measurements and are expected to lead to the development of standardization programs for the two apolipoproteins. The purpose of the present communication is to describe how the measurements were made in NHANES III and to provide information on how to relate the measurements to the new International Reference Materials. CLINICAL CHEMISTRY, Vol. 40, No. 10, 1994 1915 Materials and Methods assay time, and calculations were performed automatically by the instrument. Daily quality control was mainSample preparation. The target population, sample tained by using all the control sera indicated above collection, and sample handling procedures used for except laboratory pool 7, which had been exhausted by NHANES Ill have been previously described (8, 9). the time the rate INA method was adopted. Each pool Briefly, serum was shipped to the laboratory within 1 week of venipuncture. The samples were kept at -20#{176}C was analyzed in duplicate in each analytical run. Because the quality-control pools were analyzed only for that period, then sent to the laboratory on solid CO2 once on each RID plate and in duplicate in the rate INA and stored in the laboratory at -80#{176}C for 1-2 weeks runs, we elected to calculate the CVs for the assays with before analysis. The samples were allowed to thaw at all of the data for each method. The data in the Tables room temperature, mixed thoroughly for 30 mm on a therefore reflect the overall CVs and do not distinguish blood rotator, and then were unsealed and analyzed as between intra- and interassay components of variation. described below. Radial Immunoditfusion(RID) Fixed-Time INA ApoA-I. Immunodiffusion plates (6 g/L agarose, containing monospecific polyclonal antibody to apoA-I) were obtained from Tago, Burlingame, CA. The assay was performed as described by the manufacturer, with calibration materials supplied by the manufacturer. Briefly, sera were diluted sixfold with NaC1 (0.15 mol/ L); 5 L of the diluted specimens was added to the wells and allowed to diffuse at room temperature in a draftfree chamber for 72 h. At the end of this period, the diameters of the immunodiffusion rings were measured with a magnifying plate reader. Three-point calibration curves were used and concentrations were calculated by computer from linear-regression equations relating concentration to the squares of the ring diameters. Quality control was maintained and the precision of the assays was assessed by analyzing in each run commercially available lyophilized control sera (Omega; Technicon Instruments, Tarrytown, NY) and frozen laboratoryprepared serum pools stored at -80#{176}C. Seven pools were used during the course of the study: three Omega pools (8V2914, 9V7751, V02748) and four laboratory pools (pools 7-10). Each pool was analyzed once on each 12well plate. ApoB. Immunodiffusion plates (6 gIL agarose, containing monospecific polyclonal antibody to apoB) were obtained from Behring Diagnostics, Westwood, MA. The assay was performed according to the manufacturer’s instructions, with calibration sera supplied by the same manufacturer. The conditions of the assay, diffusion time, construction of three-point calibration curves, calculation of results, and daily quality control were as described for apoA-I. The only procedural difference from the apoA-I method was that a threefold initial dilution of the specimens was used, as indicated in the manufacturer’s instructions. The same seven serum pools indicated above were used for quality control and analyzed once on each 12-well plate to assess the precision of the apoB measurements. Samples were also sent to the Northwest Lipid Research Clinic Laboratories, where they were analyzed by fixed-time INA on another automated system (Behring Diagnostics). This was done for apoB during the last year of NHANES III, Phase 1, and during all 3 years of the sampling cycle for apoA-I. In the present communication, we have used data accumulated during the last year of the sampling cycle for both apolipoproteins for the sake of consistency, but the data for apoA-I during the earlier period were quite similar to those for the last year. Four fresh-frozen serum pools with different concentrations of apoA-I and apoB, one to be used as calibrator and three as quality-control materials, were prepared at the NWLRL as previously described (10). Target values to these pools were assigned by calibrating the fixedtime nephelometer with purified apoA-I and isolated LDL as primary reference materials as previously reported (10). Concomitantly, target values were being assigned to SP1 and SP3 preparations, which were subsequently officially recognized as the WHO-IFCC First International Reference Materials for Apolipoproteins A-I and B (14, 15). The four fresh-frozen serum pools were also analyzed against the Reference Materials and their values were confirmed. Rate ImmunonephelometricAssay (INA) ApoA-I and apoB were measured simultaneously by rate INA on a completely automated system (Array; Beckman Instruments, Brea, CA) with calibration materials supplied by the manufacturer. The assays were performed according to the manufacturer’s directions. In this system, sample dilutions, addition of reagents, 1916 CLINICAL CHEMISTRY, Vol. 40, No. 10, 1994 Results Conversionof RID to INA Values ApoA-I and apoB were measured by RID for the first 5 months of NHANES Ill; the analyses were performed by rate INA for the balance of the first phase of the survey. Since there were no available reference methods or standardization programs for apoA-I and apoB measurements during NHANES III, several procedures were established to maintain and assess the stability of the measurements over the 3-year period. First, reagents, calibration sera, and quality-control sera were purchased in large lots, generally sufficient for about a year at a time, to minimize lot-to-lot variations. Second, the same daily quality-control sera were used for both RID and rate INA measurements. Third, after changing to rate INA, -20% of the NHANES Ill subjects’ samples were analyzed by both methods for the remaining sampling period. Fourth, aliquots of this 20% subset were sent to the NWLRL for analysis by fixed-time INA during the final year of the 3-year sampling cycle. The analysis protocol for the split-sample measurements is shown in Fig. 1. To convert the RID values to equivalent rate INA values, we first compared the rate INA and RID measurements in quality-control sera. Table 1 shows this comparison for apoA-I for the period March 1989 to October 1991 (see Fig. 1). The Omega pools were supplied as lyophilized preparations and the three laboratory pools were frozen materials. The rate INA values in the six poo1s averaged -7% lower than the RID values, and the differences ranged from 3% to 18% in the various pools (Table 1). The CVs averaged -5.7% for rate INA and ranged from 4.5% to 7.7%, except for a single laboratory frozen pool (pool 8) that was excessively variable (2 1.2%). This excessive variability seemed to be a characteristic of the pool itself rather than the method because Omega pool 8V2914 analyzed at the same time did not display this variation. The CVs for the RID method averaged -4.3% and ranged from 2.5% to 7.6%. Again, the highest CV (7.6%) was observed in laboratory frozen pool 8, although the imprecision observed with this poo1 by RID was considerably less than by rate INA. The reason for this is unclear. The rate INA and RU) measurements of apoB are compared in Table 2 for the same period. Overall, the rate INA values averaged -25% lower than the RH) values, and the differences ranged from 17% to 32% in the various pools. Interestingly, the excessive variation observed for the rate INA apoA-I measurements in laboratory frozen pool 8 were not observed for the apeB measurements in this pool. The rate INA-RID differences for the laboratory frozen pools averaged -30% and were significantly greater than the 20% difference observed for the Omega lyophilized pools (P = 0.012). The CVs for the rate INA measurements averaged 3.3% and ranged from 2.3% to 5.3%. Those for the RID measurement averaged 3.8% and ranged from 2.3% to 6.4%. There were no apparent differences in precision between Fixed Time 20% Rate INA subset INA All samples RID All samples i 20% subset I 0 10 20 Time Fig. 1. Methods 30 40 (months) used for apoA-I and apoB measurement during PhaseI of NHANES III. PhaseI was conductedover36 months.RID and rateINA methodswereused inthe JohnsHopkins laboratoryand fixed-time INA was used in the NWLRL Table 1. ApoA-I measurements In qualfty-control set-a during NHANES III. RID Rate INA n Mean (SD), Mean (SD), CV, % n mg/I CV, % mg/L Omega pools 8V2914 78 1666(118) 7.1 9V7751 196 1527(118) 7.7 V02748 86 1465 (66) 4.5 Lab pools 8 57 1468(312) 21.2 9 118 1551 (71) 4.6 10 228 1501 (72) 4.7 Rate INAiRID 31 1789 (81) 75 1575 (54) 32 1780 (45) 4.5 3.4 2.5 0.93 0.97 0.82 13 1514(115) 34 1595 (55) 85 1664 (70) 7.6 3.4 4.2 0.97 0.97 0.90 Table 2. ApoB measurements In qualIty-control sara during NHANES III. RID Rate INA n Mean (SD), mg/L CV,% n Mean (SD), maJL Rate CV, % INA/RID Omegapools 8V2914 9V7751 V02748 Lab pools 8 9 10 98 685 (27) 198 587(15) 87 583(16) 53 878(47) 117 834(23) 224 856 (20) 3.9 2.6 2.7 34 73 32 827 (36) 748(17) 758 (19) 4.4 2.3 2.5 0.83 0.78 0.77 5.3 14 1264(46) 31 1228(46) 86 1191 (76) 3.6 3.7 6.4 0.69 0.68 0.72 2.8 2.3 the lyophilized pools and the laboratory pools with either method. We next compared the rate INA and RID measurements in split samples from NHANES III participants. Table 3 lists the group means and SDs for samples collected during various periods from March 1989 to October 1991. The group means and SDs for both apelipoproteins analyzed with the rate INA method remained about the same during the four periods, suggesting that these measurements were stable overtime. The same was true for the RID measurements. The constancy of the rate INA/RID ratios shown in the last column of the Table also reflect this and indicate that the relation between the two methods remained constant as well. For apoA-I, the rate INA measurements averaged 20% lower than the RID measurements overall. This difference was about threefold greater than the 7% difference that was apparent from the analysis of the lyophilized and laboratory-prepared control sera (Tables 1 and 3). The relation between the rate INA and RID measurements in participant samples was therefore not adequately reflected by measurements in control sera, with the exception of a single lyophilized pool (pool V02748, Table 1). For apoB, the rate INA measurements were 34% lower than the RID measurements overall. This value agreed well with the 30% difference reflected by measurements of the laboratory-prepared frozen control sera (Tables 2 and 3) but was significantly greater than the 20% difference observed in the Omega lyophilized CUNICAL CHEMISTRY, Vol. 40, No. 10, 1994 1917 Table 3. ApoA-I and apoB measured by INA and RID in spIlt samples from NHANES III partIcipants. Rate INA Period Mean (SD), mg/I n ApoAl Apr 1989-,Jan 1990 Jan 1990-Sep 1990 Sep 1990-Mar 1991 Mar 1991-Oct 1991 Overall ApoB Apr 1989-Jan 1990 Jan 1990-Sep 1990 Sep 1990-Mar 1991 Mar 1991-Oct 1991 Overall RID (305) (291) (280) (290) (292) 612 639 612 655 2518 1690 1746 1655 1783 1720 (352) (338) (306) (337) (337) 0.83 0.80 0.81 0.78 0.80 618 639 614 656 2527 830 857 890 844 855 (240) (247) (267) (246) (251) 615 639 608 656 2518 1236 1302 1357 1284 1294 (403) (381) (380) (350) (381) 0.69 0.66 0.65 0.66 0.67 Reference MaterIals-based values. Mean (SD), mg/L ApoA-l ApoB 1918 2526 2527 Observed rate INA 1381(292) 855 (251) Rate INA/RID 1391 1394 1348 1390 1381 617 Table 4. ApoA-l and apoB measurements by INA before and after transformation to WHO-IFCC First n Mean (SD), mg/L 639 614 656 2526 control pools (P = 0.002). Thus, for apoB the laboratory frozen control pools accurately reflected the rate INARID differences in participant samples, whereas the lyophilized pools, which are known to be inappropriate for apoB analysis (see Discussion), underestimated the differences. Based on the split-sample measurements, the linear regression equations relating the rate INA (y) and RID (x) measurements for apoA-I and apoB were: for apoA-I, y = 0.75x + 90 mg/L (r = 0.87, SE810 = 0.08, = 15, n = 2512); for apoB,y = 0.63x + 42 mgfL (r = 0.95, SE810 = 0.04, = 58, n = 2513). The NWLRL offers a standardization program to manufacturers to document that their calibration materials can be traced to the WHO-IFCC Reference Materials (12-15) and has served as coordinating laboratory for the development of these materials. For this reason, aliquots of the subset of NHANES III specimens analyzed by rate INA and RID at Johns Hopkins were also sent to the NWLRL for fixed-time INA analyses traceable to the WHO-IFCC Reference Materials. The relations between the rate INA measurements (x) made at Johns Hopkins and the WHO-IFCC Reference Materialbased fixed-time INA measurements (y) in the NWLRL were as follows: for apoA-I, y = 0.872x + 251.8 mgIL (r = 0.93, SE80p = 0.13, SEintercept = 17, n = 708); for apoB,y = 1.068x + 112.8 mg/L (r = 0.98, SE810 = 0.08, SEmter..pt = 7, n = 646). Based on these equations, the rate INA values before and after conversion to equivalent WHO-IFCC Reference Material-based values are shown in Table 4. The transformed mean apoA-I value was 5.1% higher than the observed value. For apoB, the International n WHO-IFCCS- based 1453 (254) 1028 (269) CLINICAL CHEMISTRY, Vol. 40, No. 10, 1994 DIfference, +5.2 +20.2 % transformed mean was 20.0% higher than the observed value. Discussion Here we compared RID and rate INA in both lyophilized and frozen control pools and in a random 20% subset of NHANES III samples. For apoA-I, both the lyophilized and frozen control pools indicated a -7% bias between the two methods. The pools, however, did not accurately reflect the bias in NHANES Ill subject samples, which actually averaged -20%, and remained fairly constant throughout the study. The reason for this discrepancy is not clear. Since, as judged from the control pool measurements (Tables 1 and 2) and the observed means for split-sample measurements during different periods (Table 3), both methods appeared to be reasonably stable over the 3-year period, one possibility is that the exposure of apoA-I to the antibodies used in the two methods was more uniform in the control pools than in the samples from NHANES III participants. If so, such changes may have occurred during preparation and(or) storage of the pools. Alternatively, the antibodies may have been raised against apoA-I preparations having different immunogenicities than the apoA-I in fresh samples. The precision of the assays, as judged from measurements in both the lyophilized and frozen pools, was comparable, however. For apoB, the 30% bias between rate INA and RID was accurately reflected by measurements in the frozen laboratory control pools, but was underestimated by one-third in the Omega lyophilized pools. This was not surprising, since the inadequacy of lyophilized pools for apoB measurements has been previously noted (16). Within each method, the precision of the assays in lyophilized and frozen pools was comparable; the rate INA method was somewhat more precise than the RID method. Our findings ifiustrate the difficulty in assessing the accuracy of apolipoprotein measurements based only on measurements in quality-control pools. Matrix effects, biases between methods used to assign values to the calibration sera used for different methods and to qual- ity-control pools, differences in the immunoreactivity of the apolipoproteins in quality-control sera compared with fresh samples, and other factors can lead to erroneous conclusions about the reliability of measurements in fresh specimens. During the last year of the first phase of NHANES III, aliquots of the specimens we used for the rate INA-RID comparison were sent to the NWLRL for analyses that could be referred to the newly available WHO-IFCC Reference Materials. Transformation of the rate INA values for apoA-I based on the NWLRL fixed-time INA measurements increased the concentrations in NHANES Ill specimens by only 8%, suggesting a relatively small bias between a lyophilized reference material for apoA-I and apoB (CDC 1883) and WHO Reference Materialbased calibration. For apoB, such transformation of the rate INA values increased the concentrations in NHANES III subjects by -20%, indicating a rather large discrepancy between CDC 1883 and WHO Reference Material-based calibration. Two of the important issues related to the usefulness of clinical apoA-I and apoB measurements are, first, knowledge of the prevailing concentrations of the apelipoprotein in the population and the establishment of generally agreed-upon cutoffs analogous to the National Cholesterol Education Program risk levels for LDL- and HDL-cholesterol (17), beyond which medical attention is indicated; and second, the ability to provide uniformly reliable apolipoprotein measurements regardless of where or how the measurements are made. To address the first issue, apoA-I and apoB were included in the panel of lipids and lipoproteins measured during the first phase of NHANES III. Transformation of these values to WHO-IFCC Reference Materialsbased measurements provides standardized populationbased apoA-I and apoB distributions that should facilitate the establishment of risk-based medical decision cutpoints. The second issue, the standardization of apoA-I and apoB measurements, has been under consideration for a number of years (10, 12, 18, 19). The variability of apoA-I and apoB measurements has been well documented (10, 19) and arises partly from differences in methods used in different laboratories and partly from the need for a commonly agreed-upon reference point for method calibration. CDC 1883 was developed and assigned consensus values for apoA-I and apoB. These assigned consensus values however, were later found to be low compared with results by a candidate reference method (20). The IFCC apolipoprotein standardization project led to the development of the first WHO-IFCC Reference Materials for apoA-I and apoB (14, 15) and recognition of the need to standardize the protein assays used to determine the concentrations of the reference materials themselves (21). The NWLRL now offers an apolipoprotein standardization program to manufacturers to allow them to establish the WHOIFCC Reference Materials-based apolipoprotein concentrations for their calibration pools, thereby providing a common reference point for apoA-I and apoB measurements. The WHO-IFCC Reference Materials were not available at the beginning of the NHANES III survey, and the standards provided by the manufacturer of the rate INA method we used were based on CDC pool 1883. The manufacturer eventually adopted WHO-IFCC Reference Material-based calibration during the last 2 months of NHANES III, Phase 1 (Sept.-Oct., 1991). For the purposes of data analysis, we used the original CDC 1883-based calibration for this entire study. We have also provided information to allow the transformation of the observed values to equivalent WHO-IFCC Reference Material-based values. The purpose of this study was to document the reliof apoA-I and apoB measurements made during NHANES III and to provide a link between these measurements and WHO-IFCC International Reference Material-based measurements to facilitate the comparison of the NHANES III measurements with those made in future studies that will be based on the WHO-IFCC Reference Materials. Preliminary studies suggest that the use of these materials will markedly reduce the variability of apoA-I and apoB measurements made by different laboratories with a variety of methods (10) and will allow the use of common cutpoints analogous to those for LDL- and HDL-cholesterol (17) that can be used to assess risk for CAD. ability This work was supported by Contract N01-HV-78102, National Heart, Lung and Blood Institute. We thank Carol McGeeney for preparing the manuscript. References 1. Bachorik PS, Kwiterovich P0 Jr. Apolipoprotein measurements in clinical biochemistry and their utility vis-#{224}-vis conventional assays [Review]. Clin Chiun Acta 1988;178:l-34. 2. Reunhart BA, Gani K, Arndt MR, Broste SK Apolipoproteins A-I and B as predictors of angiographically defined coronary artery disease. Arch Intern Med 1990;150:l629-33. 3. Sewdarsen M, Desai RK, Vythilingum S, Shah N, Rajput MC. 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