Multiplexed Analysis of Biomarkers Related to Obesity and the Metabolic Syndrome in Human Plasma, Using the Luminex-100 System

Clinical Chemistry 51:7 1102 1109 (2005) Molecular Diagnostics and Genetics Multiplexed Analysis of Biomarkers Related to Obesity and the Metabolic Syndrome in Human Plasma, Using the Luminex-100 System Mine Y. Liu, 1 Antonios M. Xydakis, 2 Ron C. Hoogeveen, 1 Peter H. Jones, 1 E. O Brian Smith, 3 Kathleen W. Nelson, 4 and Christie M. Ballantyne 1* Background: The complex pathology of disease has sparked the development of novel protein expression profiling techniques that require validation in clinical settings. This study focuses on multiplexed analyses of adipocytokines and biomarkers linked to the metabolic syndrome, diabetes, and cardiovascular disease. Methods: Multiplexed immunoassays using fluorescent microspheres and the Luminex-100 system were performed on plasma from 80 obese patients (40 with the metabolic syndrome) before and after 6 8 weeks of diet-induced weight loss. Leptin, insulin, C-peptide, monocyte chemoattractant protein-1 (MCP-1), eotaxin, interleukin-8 (IL-8), tumor necrosis factor- (TNF- ), and IL-6 concentrations measured with multiplex panels from 3 different manufacturers were compared with results from commercial ELISAs. Detection limits and between- and within-run imprecision were determined for each analyte. Bland Altman analysis was used to determine agreement between multiplexed immunoassays and ELISAs. Results: Correlation between the Luminex multiplexed assays and ELISAs was good for leptin (Linco), insulin (Linco), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) with correlation coefficients of 0.711 0.895; fair for eotaxin (Upstate) and C-peptide (Linco) with correlation coefficients of 0.496 0.582; and poor for 1 Section of Atherosclerosis, Department of Medicine, 2 Division of Endocrinology, Diabetes and Metabolism, and 3 Section of Nutrition, Department of Pediatrics, Baylor College of Medicine, Houston, TX. 4 Methodist Wellness Services, The Methodist Hospital, Houston, TX. *Address correspondence to this author at: Baylor College of Medicine, 6565 Fannin, M.S. A-601, Houston, TX 77030. Fax 713-798-3057; e-mail cmb@bcm.tmc.edu. Received December 17, 2004; accepted April 6, 2005. Previously published online at DOI: 10.1373/clinchem.2004.047084 TNF-, IL-8, and IL-6 (Linco, Biosource, Upstate, and R&D) with correlation coefficients of 0.107 to 0.318. Within- and between-run imprecision values for the multiplex method were generally <15%. Relative changes in plasma leptin and insulin concentrations after diet-induced weight loss were similar whether assessed by multiplex assay or ELISA. Conclusion: Although this technology appears useful in clinical research studies, low assay sensitivity and poor correlations with conventional ELISA methods for some analytes with very low plasma concentrations should be considered when using the Luminex platform in clinical studies. 2005 American Association for Clinical Chemistry Obesity has reached epidemic proportions in the United States; 30% of US adults are obese, and the percentage is increasing (1). Obesity increases the risk for the metabolic syndrome, diabetes, hypertension, atherosclerosis, and thrombosis (2 4). The National Cholesterol Education Program Adult Treatment Panel III (ATP III) 5 defined the metabolic syndrome using clinical criteria for abdominal obesity, triglyceride and HDL-cholesterol concentrations, blood pressure, and fasting glucose (5), and 20% of adults in the United States meet these clinical criteria (6). Novel technologies such as gene expression arrays have identified a large number of molecules that have altered expression in adipose tissue and may be relevant to the development of the metabolic syndrome, diabetes, and cardiovascular disease with obesity. In contrast to the ability to simultaneously examine the concentrations of thousands of mrna transcripts, plasma concentrations of 5 Nonstandard abbreviations: ATP III, Adult Treatment Panel III; MCP-1, monocyte chemoattractant protein-1; TNF-, tumor necrosis factor- ; IL, interleukin; and NIBSC, National Institute for Biological Standards and Control. 1102

Clinical Chemistry 51, No. 7, 2005 1103 cytokines, chemokines, or other biomarkers are usually measured one at a time by ELISA. Measurement of plasma concentrations of each putative biomarker with individual ELISAs incurs considerable time, cost, and sample volume, which thus limits the ability to systematically examine the effects of a clinical intervention. Multiplexed proteomics research may offer a major advance for clinical research. Protein microarrays are the most commonly used for simultaneous determination of multiple proteins in a biological fluid. The technique uses primary antibodies as the immobilized probe on a solid surface, and protein antigens labeled with fluorophores with or without bound secondary antibodies are recognized and detected. However, binding of antibodies and antigens to a solid support can cause denaturation or drying of proteins. The Luminex-100 bead-based system is a recently developed platform that provides multiplexing in a solution phase and thus is particularly flexible and nondestructive for protein analysis. Each set of up to 100 uniquely color-coded polystyrene microspheres is anchored with a different capture antibody. The use of detection antibodies labeled with the fluorochrome R- phycoerythrin allows quantification of antigen antibody reactions that occur on the microsphere surface by measurement of the relative fluorescence intensity. The system is capable of measuring potentially up to 100 analytes simultaneously in a small sample volume (25 50 L). In theory, the Luminex platform may also provide a wider dynamic range than conventional ELISA methods because of the greater linear range of fluorescence intensity compared with absorbance. The increased dynamic movement during antibody antigen reactions that occurs in solution also may increase its assay efficiency (7 11). The purpose of this study was to compare, in a clinical research study, several commercially available Luminex multiplex panels with conventional commercial ELISAs for measurement, in human plasma, of biomarkers associated with obesity and inflammation. Materials and Methods participants From September 2001 to September 2002, a total of 80 obese individuals [56 women and 24 men; mean (SD) age, 47.1 (0.9) years; mean (SD) body mass index, 38.3 (0.7) kg/m 2 ] were enrolled in a protein-sparing, very low calorie diet-induced weight loss program. Written informed consent was obtained from each participant. The weight loss program was approved by the Baylor College of Medicine Institutional Review Board (12). All participants were obese (body mass index 30 kg/m 2 ), and one-half had the metabolic syndrome based on the ATP III criteria (5). Analytes were measured in plasma at baseline for all 80 participants and after 4 6 weeks of rapid weight loss for the 40 individuals with metabolic syndrome (mean weight loss, 7%). During weight loss, participants were not started on any medication that would influence glucose tolerance, insulin secretion, or insulin sensitivity. collection and storage of blood samples Blood samples were collected by venipuncture from the participants into EDTA-containing Vacutainer Tubes after an overnight fast. The freshly drawn blood was centrifuged at 3000g for 20 min at 4 C. Plasma was then separated and subsequently stored at 70 C in small aliquots. All analyses were performed on frozen plasma samples. luminex multiplex assays Multianalyte profiling was performed on the Luminex- 100 system and the XY Platform (Luminex Corporation). Calibration microspheres for classification and reporter readings as well as sheath fluid were also purchased from Luminex Corporation. Acquired fluorescence data were analyzed by the MasterPlex TM QT software (Ver. 1.2; MiraiBio, Inc.). Plasma concentrations of leptin, insulin, and C-peptide were determined by the Linco Human Endocrine 3-Plex Panel (Linco Research, Inc.). Chemokines [monocyte chemoattractant protein-1 (MCP-1) and eotaxin] were measured by both the Biosource Chemokine 5-Plex Panel (Biosource International, Inc.) and the Upstate Beadlyte Human Cytokine 8-Plex Panel (Upstate USA, Inc.). Cytokines [tumor necrosis factor- (TNF- ), interleukin-8 (IL-8), and IL-6] were analyzed by the Linco Human Cytokine 6-Plex Panel (Linco), the Upstate Beadlyte Human Cytokine 8-Plex Panel (Upstate), and the R&D Fluorokine MAP Human Cytokine 6-Plex Panel (R&D Systems, Inc.). All analyses were performed according to the manufacturers protocols. ELISAs All corresponding ELISAs were performed with commercially available reagent sets. Plasma leptin and C-peptide were analyzed by use of the Human Leptin ELISA Kit and the Human C-Peptide ELISA Kit (Linco). Insulin was assayed by the 1-2-3 Ultrasensitive Human Insulin ELISA (Alpco Diagnostics, American Laboratory Products Company). MCP-1 and eotaxin were measured by the Quantikine Human Eotaxin Immunoassay and Quantikine Human MCP-1 Immunoassay (R&D Systems). TNF- and IL-6 were measured by the Quantikine HS Human TNF- Immunoassay and the Quantikine HS Human IL-6 Immunoassay (R&D Systems). The circulating IL-8 concentration was measured by the Biosource Human IL-8 Immunoassay (Biosource International). detection limits and recovery study To determine the detection limits for the Luminex assays, 3 SD were added to the mean fluorescence intensity of 2 zero calibrator replicates. The respective analyte concentrations were then calculated from the calibration curve. Similarly, to evaluate the detection limits for the ELISAs, 3 SD were added to the mean absorbance value of 2 zero

1104 Liu et al.: Luminex Multiplexed Analysis of Human Plasma calibrator replicates, and the respective analyte concentrations were calculated from the calibration curves. The recombinant protein MCP-1 and IL-6 calibrators provided with the ELISA and Luminex reagents were added into normal healthy human plasma. The plasma was manufacturer pooled with specific concentrations of lipids requested (lot no. BC0208A; Pacific Biometrics, Inc.). statistical analysis Statistical analyses were performed with SPSS data analysis software (Ver. 11.5; SPSS Inc.). Analyze-it software (Ver. 1.71; Analyze-it Software Ltd.) was used for Deming regression and Passing Bablok analyses for method comparisons to account for imprecision in both the ELISA and Luminex methods. The Wilcoxon signed-rank test was used to analyze the effect of rapid weight loss on leptin and insulin. Bland Altman analyses (13) were performed to calculate limits of agreement and systematic errors, and the results were plotted with y as the value of the difference ( ) between Luminex and ELISA, x as the mean of the Luminex and ELISA values, and the upper and lower limits defined by the mean 2 SD. Results detection limits of luminex assays and ELISAs A comparison of detection limits for the Luminex assays and the ELISAs is shown in Table 1. Some analyte concentrations were also converted to the National Institute for Biological Standards and Control (NIBSC)/WHO international reference standard unit (IU/L) if calibrations of the assays were performed by the manufacturers. In the Linco Endocrine multiplex panel, leptin and C- peptide had significantly lower detection limits by the Luminex assay. The Alpco ultrasensitive insulin ELISA was more sensitive for detection of insulin than the Linco Endocrine multiplex. Two chemokine panels (Biosource and Upstate) were chosen to compare with ELISAs for the analysis of MCP-1 and eotaxin. Because the MCP-1 assay in the Biosource panel was not yet calibrated against the WHO standard and no WHO standard for eotaxin is currently available, it was not possible to compare the detection limits of the Biosource panel with ELISAs. Unlike the Biosource panel, the Upstate multiplex panel has a significantly higher detection limit for MCP-1 than the ELISA from R&D. Cytokines TNF-, IL-8, and IL-6 in 3 different multiplex panels from different manufacturers (Linco, Upstate, and R&D) were compared with ELISAs. In general, the ELISAs tended to have lower detection limits. percentage of samples with undetectable MCP-1, TNF-, IL-6, and IL-8 concentrations in the luminex assays and ELISAs We compared the percentage of samples in which 4 representative analytes could not be detected by the Luminex and ELISA methods; the results are shown in Table 2. For the ELISAs, generally the percentage of samples with undetectable analyte concentrations was 0%, except for IL-8 in the Biosource assay, which is not a high-sensitivity assay. For the Luminex assay, in the Upstate multiplex panel, nearly 50% of samples had undetectable concentrations of IL-6 and TNF- ; in the R&D multiplex panel, nearly 100% of samples had undetectable IL-6, TNF-, and IL-8; and in the Biosource multiplex panel, 61% of samples had undetectable IL-8. assay precision of luminex and ELISA methods The between- and within-run imprecision, as the CV (CV SD/mean), for the Luminex assays is shown in Table 1 of the Data Supplement (available at http:// www.clinchem.org/content/vol51/issue7/) and in Table 3. The Upstate multiplex panel and 3 plasma pools from patients with metabolic syndrome with low, medium, and high concentrations of added analyte were chosen to test the between-run precision. The 2 normal control plasma samples (QA and QC) were not selected because they were undetectable by the multiplex panel. The MCP-1 between-run imprecision, determined from 7 multiplex Table 1. Comparison of detection limits for the Luminex multiplexed assays and the individual ELISAs. Multiplex panels Analytes Luminex ELISA ELISA manufacturer Linco Endocrine Leptin 0.13 g/l 1.39 g/l Linco Insulin 0.31 miu/l 0.14 miu/l Alpco C-peptide 0.037 g/l 0.132 g/l Linco Biosource Chemokine MCP-1 2.35 ng/l 2.57 ng/l R&D Eotaxin 4.37 ng/l 3.80 ng/l R&D Upstate Chemokine MCP-1 20.5 ng/l (0.023 kiu/l) 2.57 ng/l (0.002 kiu/l) R&D Eotaxin 27.72 ng/l 3.80 ng/l R&D Linco Cytokine TNF- 1.61 ng/l (0.249 kiu/l) 0.092 ng/l (0.0028 kiu/l) R&D IL-8 2.52 ng/l (0.026 kiu/l) 0.26 ng/l (0.0018 kiu/l) Biosource Upstate Cytokine TNF- 0.065 ng/l (0.0019 kiu/l) 0.092 ng/l (0.0028 kiu/l) R&D IL-8 0.70 ng/l (0.0003 kiu/l) 0.26 ng/l (0.0018 kiu/l) Biosource IL-6 0.75 ng/l (0.1029 kiu/l) 0.068 ng/l (0.0084 kiu/l) R&D R&D Cytokine TNF- 1.53 ng/l (0.052 kiu/l) 0.092 ng/l (0.0028 kiu/l) R&D IL-6 0.977 ng/l (0.1280 kiu/l) 0.068 ng/l (0.00843 kiu/l) R&D

Clinical Chemistry 51, No. 7, 2005 1105 Table 2. Samples with undetectable concentrations of MCP-1, TNF-, IL-6, and IL-8 in the ELISAs and the Luminex multiplexed assays. IL-6 TNF- MCP-1 IL-8 Assay manufacturer % undetectable n % undetectable n % undetectable n % undetectable n ELISAs R&D 0 40 0 40 0 120 NA a NA Biosource NA NA NA NA 40.5 40 Luminex system Upstate 47.4 154 51.7 114 10.3 154 0 120 R&D 100 40 95.2 40 NA NA 100 40 Linco 16.7 40 7.1 40 NA NA 21.4 40 Biosource NA NA NA NA 2.4 40 61 40 a NA, not applicable. assays with each sample analyzed in duplicate, was 15%. The within-run precision represents the mean values of 3 samples with low, medium, and high concentrations of the analyte that were analyzed 20 times in 1 assay. The within-run CVs for all analytes ranged from 6.6% to 12% and were within the acceptable limit. The between- and within-run precision of the ELISAs are shown in Tables 1 and 3 of the online Data Supplement. The between-run CV was determined from 2 plasma control samples (QA and QC) because they were detectable by the high-sensitivity ELISAs. For each sample, the CV was determined from the means of 6, 8, and 5 assays for MCP-1, TNF-, and IL-6, respectively, and were analyzed in duplicate. The within-run precision was calculated from the mean CVs of n samples analyzed in duplicate. Overall, the within-run CVs were within reasonable limits, ranging from 5.0% to 15%, except for IL-6 (21%). For the high-sensitivity ELISAs, the between-run imprecision generally indicated a 15% CV of random errors, and the within-run imprecision showed a 15% CV of random errors. Table 3. Within-run assay imprecision. Analytes Within-run CV, % n Multiplexed panels Linco Endocrine Leptin 11 120 Insulin 6.6 120 C-Peptide 10 80 Biosource Chemokine MCP-1 12 40 Eotaxin 10 40 Upstate Chemokine MCP-1 8.1 40 Eotaxin 11 40 ELISAs Linco Leptin 5.9 120 Alpco Insulin 15 40 Linco C-Peptide 10 40 R&D MCP-1 5.4 120 R&D Eotaxin 5.0 120 validation of the luminex method in human plasma We chose ELISAs for comparison with the newer Luminex technique because the ELISA is a generally accepted method for analyzing biomarkers in human serum or plasma. Two methods of comparison between Luminex assays and ELISAs were carried out. We first used linear correlation to evaluate the relationship between Luminex assays and ELISAs. We then applied the Bland Altman method (13) to assess the agreement between the 2 measurement methods. Correlation. The results of the Deming regression analysis comparing the Luminex and ELISA methods are shown in Table 4. The correlation coefficient indicates scatter of the measured data about the Deming regression line, and it is determined by the precision of the 2 assay methods. Table 4 shows the results for the Linco endocrine panel. The correlation coefficients (R) were 0.812, 0.895, and 0.582 for leptin, insulin, and C-peptide, respectively. The correlation coefficient for C-peptide was 0.67 after omission of the 2 outliers (defined as values outside 3 SD). In the Deming regression analysis, the slope indicates a proportional error that might have originated from incomplete recovery in one of the methods or from different accuracies of the calibrators used. The slopes were 1.28, 2.38, and 0.68 for leptin, insulin, and C-peptide, respectively. The smallest proportional error was for leptin, i.e., the values obtained from the 2 assay methods were the least different. The intercept indicates a systematic error that might be caused by interference, such as insufficient blank compensation. The intercepts were 2.26 g/l, 3.03 kiu/l, and 1.83 g/l for leptin, insulin, and C-peptide, respectively. The smallest systematic error was for C- peptide. The correlation coefficients were 0.263 and 0.899 for MCP-1 in the Biosource panel with and without the 2 outliers, respectively; outliers were defined as values outside 3 SD. For eotaxin, the correlation coefficient was 0.711 for the Biosource panel. The slopes for MCP-1

1106 Liu et al.: Luminex Multiplexed Analysis of Human Plasma Table 4. Deming regression results for comparison of the multiplexed assays performed on the Luminex systems (y) and individual ELISAs (x). Slope Intercept Analyte a Deming S y x R Coefficient SE 95% CI b Mean SE 95% CI Leptin ( g/l) 9.90 0.812 1.28 1.28 1.11 1.44 2.26 3.19 4.05 to 8.57 Insulin (miu/l) 5.62 0.895 2.38 0.19 1.99 2.77 3.03 3.47 10.06 to 4.00 C-Peptide ( g/l) 2.32 0.582 0.68 0.15 0.37 0.99 1.83 1.20 4.25 to 0.59 Eotaxin (ng/l) 20.44 0.711 7.71 1.29 5.10 10.32 49.85 80.64 213.55 to 113.86 MCP-1 c (ng/l) With outliers 110.14 0.263 40.44 25.09 10.49 to 91.36 7829.64 6421.28 20 865.54 to 5206.25 Without outliers 49.99 0.899 4.97 0.42 4.11 5.82 79.60 109.36 142.90 to 302.09 a The manufacturers of the assays for the Luminex multiplex systems (y) and the individual ELISAs (x) were as follows: for leptin, Linco for both; for insulin, Linco and Alpco, respectively; for C-peptide, Linco for both; for eotaxin, Biosource and R&D, respectively; and for MCP-1, Biosource and R&D, respectively. b CI, confidence interval. c Outliers are defined as values outside 3 SD of the mean for 2 replicates of the zero calibrators. without the outliers and for eotaxin were 4.97 and 7.71 for the Biosource panel, respectively, and the intercepts were 79.60 and 49.85 ng/l, respectively. Passing Bablok analysis revealed both proportional and constant bias for MCP-1 and eotaxin (data not shown). We also analyzed the correlations between the Luminex assays and ELISAs for the cytokines TNF-, IL-8, and IL-6. The correlation coefficients were 0.104 and 0.107 for TNF- and 0.266 and 0.318 for IL-8 in the Linco and Upstate panels, respectively. The correlation coefficient was 0.298 for IL-6 in the Upstate panel. Overall, the correlations between Luminex assays and ELISAs for the 3 cytokines analyzed were poor in all the multiplex panels. Bland Altman analysis. The difference between the Luminex assay and the ELISA was plotted against the mean of the Luminex assay and ELISA for each plasma sample measured. The Bland Altman plots for several analytes are shown in Fig. 1. The Bland Altman analysis was carried out only for (a) analytes that showed good correlation and used the same calibrators for calibration curves in both the Luminex assays and ELISAs (leptin, insulin, and C-peptide) or (b) assays already calibrated against the WHO standards (MCP-1 and insulin). The Bland Altman plots for leptin, insulin, and C- peptide in the Linco endocrine panel and MCP-1 in the Upstate panel are shown in Fig. 1. The systematic differences between the 2 assay methods as calculated by the Bland Altman analysis were 11.0 g/l, 13.8 kiu/l, 3.9 g/l, and 0.21 kiu/l for leptin, insulin, C-peptide, and MCP-1, respectively. The 2 methods agreed fairly well for leptin values 50 g/l. Above 50 g/l, however, the Luminex assays gave both higher and lower values than the ELISA; the discrepancy for some measurements was as large as 75%. For leptin, 7.5% of the patients had values that differed by 2 SD. Agreement between 2 methods is generally acceptable if 5% of values differ by 2 SD. For insulin, the 2 assays agreed very well at concentrations 30 miu/l, but above 30 miu/l, the Luminex assay gave higher values than the ELISA, although only 2.5% of the values differed by 2 SD. For C-peptide, most of the Luminex measurements gave lower values than the ELISA (mean difference, 3.9 g/l). At concentrations 4 g/l, the 2 methods agreed very well, and only 2.5% of the values differed by 2 SD. For MCP-1 in the Biosource panel, the 2 methods agreed well at concentrations 0.5 kiu/l, and 5.5% of patients had values that differed by 2 SD. For insulin, C-peptide, and MCP-1, the only measurements that differed by 2 SD were the extreme highest values of the analytes for patients in this study. recovery study Recovery experiments detect inaccuracy of a method resulting from systematic errors. The results of the recovery study for MCP-1 in both the ELISA and Luminex assay are shown in Table 2 of the online Data Supplement. The recombinant protein calibrators provided with the ELISA and the Luminex assay were added at 3 different concentrations in pooled normal healthy human serum for measurement of recovery with 10 replicates or more. The 3 concentrations (32, 128, and 512 ng/l) of MCP-1 were selected because they were commonly seen in our study in healthy individuals and in patients with metabolic syndrome. The pools containing added Luminex calibrator were measured by the Luminex method, and the pools containing the added ELISA calibrator were measured by ELISA. As shown in Table 2 of the online Data Supplement, for the pools containing 128 and 512 ng/l MCP-1, the ELISA gave mean recoveries of 98.8% and 102.1%, respectively. However, in the Luminex assay, the mean recoveries of MCP-1 were 58.2%, 49.1%, and 54.6%, respectively, for the 3 concentrations. effects of active weight loss on leptin and insulin in individuals with the metabolic syndrome The patients with metabolic syndrome lost 7% of their initial weight [8.0 (0.55) kg, or 17.6 (1.2) pounds] after 4 6

Clinical Chemistry 51, No. 7, 2005 1107 Fig. 1. Bland Altman analysis showing the agreement between Luminex assays and ELISAs for the measurement of leptin (A), insulin (B), C-peptide (C), and MCP-1 (D). The y axis in each panel represents the difference ( ) between Luminex and ELISA values, and the x axis represents the average of the Luminex and ELISA values. Solid lines are mean values; dotted lines are 2 SD. For leptin (A), the mean difference is 11.0 g/l (2 SD 29.7 g/l); for insulin (B), the mean difference is 13.8 miu/l (2 SD 37.4 miu/l); for C-peptide (C), the mean difference is 3.9 g/l (2 SD 6.8 g/l); and for MCP-1 (D), the mean difference is 0.21 kiu/l (2 SD 0.99 kiu/l). weeks of the diet-induced weight loss program. Plasma concentrations of leptin and insulin at baseline and after weight loss were measured by both the Luminex and ELISA methods, and the results were compared. Mean (SD) leptin concentrations measured by ELISA were significantly higher before weight loss compared with after weight loss [36.3 (21.5) vs 19.8 (18.7) g/l; n 40; P 0.001, Wilcoxon signed-rank test]. The Luminex assay also showed a significant mean (SD) difference after weight loss [47.4 (25.6) vs 27.6 (22.1) g/l; n 40; P 0.001]. Insulin concentrations were also significantly higher before weight loss than after weight loss whether measured by ELISA [19.2 (17.3) vs 9.4 (7.8) miu/l; n 13; P 0.039] or the Luminex assay [37.4 (41.1) vs 14.7 (16.0) miu/l; n 13; P 0.028]. The percentage changes in leptin and insulin after weight loss are shown in Fig. 1 of the online Data Supplement. The median percentage changes in leptin were 54% and 46% as measured by the ELISA and Luminex assay, respectively, and the median percentage changes in insulin were 41% and 56%, respectively. comparison of MCP-1 between healthy controls and metabolic syndrome patients with 4 or more risk factors We compared plasma MCP-1 concentrations in healthy controls (n 14) and metabolic syndrome patients with 4 or more risk factors (n 20). The MCP-1 concentrations measured by ELISA and the Luminex assay are shown in Fig. 2 of the online Data Supplement. The mean (SD) MCP-1 concentrations measured by ELISA were significantly higher in the metabolic syndrome patients than in healthy controls [536.5 (316.1) vs 263.3 (96.5) ng/l; P 0.001, Wilcoxon signed-rank test]. The median MCP-1 values measured by ELISA were 414.6 and 231.4 ng/l for patients and controls, respectively. The results obtained with the Luminex assay also indicated that the mean (SD) MCP-1 concentrations in the metabolic syndrome patients

1108 Liu et al.: Luminex Multiplexed Analysis of Human Plasma were significantly higher than those in healthy controls [83.5 (101.1) vs 20.4 (8.6) ng/l; P 0.001, Wilcoxon signed-rank test]. In the Luminex assay, the median MCP-1 values were 63.9 and 18.4 ng/l for patients and controls, respectively. Discussion Multiplex immunoassays using a small sample volume could facilitate clinical research in complex disorders such as the metabolic syndrome and could be extremely helpful in the evaluation of changes in biomarkers with therapeutic interventions. The detection limits shown in Table 1 allow for a potentially high degree of detectability in the patients samples. However, because the focus of this study was to compare the validity of Luminex-based assays vs traditional ELISAs as a tool for clinical research, the use of functional sensitivity is a more meaningful index than limit of detection, and in our clinical study, many cytokines were not detectable in a high percentage of patient plasma samples by the Luminex multiplex method. The inability to detect these analytes by the multiplex method may be explained in part by interferences, such as heterophilic antibodies, that are present in complex sample matrices such as plasma. The within-run imprecision (CV) values obtained for the multiplex method were generally 15%, whereas the between-run imprecision for the multiplex method for MCP-1 was 15%. The large interassay variations seen with MCP-1 should be considered when using multiplex panels for this analyte (and potentially others) in clinical research, for example, by measuring all samples in 1 run (or at least all the samples from a specific individual before and after an intervention), and by including control sample pools in all runs. The poor correlations between the Luminex assay and ELISAs for the 3 cytokines analyzed in our study are most likely attributable to the extremely low plasma concentrations of these cytokines and the presence in blood of interfering substances such as heterophilic antibodies, which are known to interfere with immunoassays (14, 15). Furthermore, our results show an increase in variability at lower concentrations for both methods (Luminex and ELISA), which also may partially explain the poor correlations between the 2 methods. Our studies demonstrate that in our laboratory the multiplexed assays for leptin (Linco), insulin (Linco), MCP-1 (Biosource and Upstate), and eotaxin (Biosource) showed good correlations of 0.7 (correlation coefficients ranging from 0.711 to 0.895); eotaxin (Upstate) and C- peptide (Linco) showed fair correlations of 0.5 0.7 (correlation coefficients ranging from 0.496 to 0.582); and TNF-, IL-8, and IL-6 (Linco, Biosource, Upstate, and R&D) showed very poor correlations ( 0.5) with ELISAs (correlation coefficients ranging from 0.107 to 0.318). The changes in plasma concentrations of leptin and insulin after diet-induced weight loss showed similar percentage reductions as assessed by multiplex assays or ELISA (Fig. 1 of the online Data Supplement). The relative difference in plasma concentrations of MCP-1 between controls and metabolic syndrome patients also were similar in the 2 methods (Fig. 2 of the online Data Supplement). The total error acceptable for an assay depends on the clinical setting in which the assay is being used. For example, in clinical research, studies are frequently designed to examine the association of plasma concentrations of analytes with the disease phenotype (in this case, obese patients with the metabolic syndrome vs lean controls) and the impact of a therapeutic intervention on the disease state (such as weight loss). In the present study, measurements of MCP-1 by ELISA or Luminex assay provided similar information about the relative increase in plasma concentrations associated with obesity and the metabolic syndrome compared with lean controls, and the relative changes in leptin and insulin concentrations before and after weight loss were similar by either ELISA or Luminex assay. However, the total error of an assay that is acceptable to assess relative change in a clinical research setting may be far greater than what is acceptable in a clinical practice setting, which is focused on diagnosis and treatment of disease. Our study has important differences in design compared with previous reports that have validated multiplexed immunoassays, which measured IgG antibodies (16), lipopolysaccharide-stimulated human plasma samples (8), or supernatant from stimulated cells (17). A recent study reported the use of Luminex-based multiplex analysis to measure 9 cytokines in sera from 30 children infected by rotavirus and 9 healthy control children, but the investigators did not validate their findings against conventional ELISAs (18). A different study used dried blood, spotted on paper, from children with cerebral palsy and showed rather poor correlation between the Linco and Upstate multiplex panels (19). In conclusion, although this technology appears useful for analytes present in a wide range of concentrations in plasma, low detectability and poor correlations with conventional ELISA methods for some analytes when present in very low concentrations in plasma should be considered when using the multiplexed assay platform in clinical studies. Furthermore, improvement in the detection limits for some of the analytes that have very low plasma concentrations and are currently commercially available for Luminex-based multiplex assays is crucial for their use in analysis of unstimulated blood samples. Issues such as the effects of interfering heterophilic antibodies, efficiency of capture antibody-coupling techniques, development of high-sensitivity multiplex assays, and optimization of capture/detection antibody pairing need to be studied further to improve the efficacy of this technology for use in clinical studies. Manufacturers should calibrate their secondary standards to the primary NIBSC/WHO standards, consistent with the recommendations for immunoassay standardization described in the report of Wadhwa

Clinical Chemistry 51, No. 7, 2005 1109 and Thorpe (20). However, our data, as well as results from a growing number of research studies, demonstrate that multiplex technology may be useful in clinical research to measure a large number of analytes to examine the association with a clinical phenotype and the effects of therapeutic interventions, and that this technology may be particularly useful when sample volume is limited, such as in large epidemiologic studies and clinical trials. This study was supported by Applied Technology Grant No. 004949-0093-2001 from the state of Texas and an American Diabetes Association Research Award. The lipid laboratory is supported by donations from George and Cynthia Mitchell, Nijad Fares, and Jeffrey Hines. The Upstate Beadlyte Human Cytokine 8-Plex Panel was kindly provided by Upstate USA, Inc. (Lake Placid, NY), The IL-8 ELISA was a gift from Biosource International, Inc. (Camarillo, CA). We also appreciate the technical assistance and useful discussions of Dr. Laurie Stephen of Upstate USA, Inc., and the helpful comments of Henry Pownall, PhD, of the Section of Atherosclerosis, Baylor College of Medicine. References 1. Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and trends in obesity among US adults, 1999 2000. JAMA 2002; 288:1723 7. 2. Moller DE, Flier JS. Insulin resistance mechanisms, syndromes, and implications. N Engl J Med 1991;325:938 48. 3. Hubert HB, Feinleib M, McNamara PM, Castelli WP. Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham Heart Study. Circulation 1983;67:968 77. 4. Loskutoff DJ, Samad F. The adipocyte and hemostatic balance in obesity. Studies of PAI-1. Arterioscler Thromb Vasc Biol 1998;18: 1 6. 5. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285: 2486 97. 6. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 2002;287:356 9. 7. Tarnok A, Hambsch J, Chen R, Varro R. Cytometric bead array to measure six cytokines in twenty-five microliters of serum. Clin Chem 2003;49:1000 2. 8. Prabhakar U, Eirikis E, Davis HM. Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay. J Immunol Methods 2002;260:207 18. 9. Kellar KL, Kalwar RR, Dubois KA, Crouse D, Chafin WD, Kane BE. Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants. Cytometry 2001;45:27 36. 10. Vignali DAA. Multiplexed particle-based flow cytometric assays. J Immunol Methods 2000;243:243 55. 11. Oliver KG, Kettman JR, Fulton RJ. Multiplexed analysis of human cytokines by use of the FlowMetrix system. Clin Chem 1998;44: 2057 60. 12. Case CC, Jones PH, Nelson K, Smith EO, Ballantyne CM. Impact of weight loss on the metabolic syndrome. Diabetes Obes Metab 2002;4:407 14. 13. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307 10. 14. Marks V. False-positive immunoassay results: a multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries. Clin Chem 2002;48:2008 16. 15. Kaplan IV, Levinson SS. When is a heterophile antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem 1999;45:616 8. 16. Biagini RE, Sammons DL, Smith JP, MacKenzie BA, Striley CA, Semenova V, et al. Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins. Clin Diagn Lab Immunol 2004;11: 50 5. 17. de Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol 2003;10:133 9. 18. Jiang B, Snipes-Magaldi L, Dennehy P, Keyserling H, Holman RC, Bresee J, et al. Cytokines as mediators for or effectors against rotavirus disease in children. Clin Diagn Lab Immunol 2003;10: 995 1001. 19. Nelson KB, Grether JK, Dambrosia JM, Walsh E, Kohler S, Satyanarayana G, et al. Neonatal cytokines and cerebral palsy in very preterm infants. Pediatr Res 2003;53:600 7. 20. Wadhwa M, Thorpe R. Cytokine immunoassays: recommendations for standardisation, calibration and validation. J Immunol Methods 1998;219:1 5.