BACKGROUND: The utility of insulin-like growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. We sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals.

METHODS: We measured IGF-1, IGF binding protein 3 (IGFBP-3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) ages 22.2 (5.2) years [mean (SD)]. We estimated between-subject and within-subject variances by mixed–effects ANOVA.

RESULTS: Within-subject variance accounted for 32% to 36% and 4% to 13% of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11% to 21% for the IGF axis markers and from 13% to 15% for the collagen markers. The index of individuality for the IGF axis markers was 0.66–0.76, and for the collagen markers, 0.26–0.45. For each marker, individuals with initial extreme measured values tended to regress toward the population mean in subsequent repeated measurements. We developed a Bayesian model to estimate the long-term probable value for each marker.

CONCLUSIONS: These results indicate that in healthy individuals the within-subject variability was greater for IGF-1 than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping.

BACKGROUND: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC/tandem MS procedures in a method comparison with an ID-gas chromatography (GC)/MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine.

METHODS: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC/tandem MS) and quadruplicate (ID-GC/MS) in independent runs.

RESULTS: The testosterone concentrations by ID-GC/MS were 0.2–4.4 nmol/L (women), 0.2–2.0 nmol/L (hypogonadal man), and 10.1–31.3 nmol/L (normogonadal men). For ID-GC/MS, the CV was nearly constant, with a median of 1.0%; for ID-LC/tandem MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90–1.11, -0.055–0.013 nmol/L, and 0.993–0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC/MS between -9.6 and 0.4%.

CONCLUSIONS: This study demonstrated fairly good accuracy and standardization of the tested ID-LC/tandem MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC/tandem MS procedures.

BACKGROUND: Adequate vitamin D status is important for bone growth and mineralization and has been implicated in the regulation of autoimmunity, metabolic function, and cancer prevention. There are no reports of population-based studies on the vitamin D status of Canadian youth, a population with mandatory fortification of foods.

METHODS: We measured plasma 25-hydroxyvitamin D [25(OH)D], the best indicator of vitamin D status, in a school-based cross-sectional sample of representative French Canadian youth (n = 1753) ages 9, 13, and 16 years living in Québec (latitude: (45°–48°N). Blood samples were collected from January to May 1999. We defined 25(OH)D deficiency as ≤27.5 nmol/L, hypovitaminosis as ≤37.5 nmol/L, and optimal as >75.0 nmol/L.

RESULTS: More than 93% of youth in each age and sex group had suboptimal 25(OH)D concentrations. The prevalence of 25(OH)D deficiency increased with age in both sexes (P < 0.0001). It was 2%, 3%, and 13% in 9-, 13-, and 16-year-old boys and 2%, 8%, and 10% in 9-, 13-, and 16-year-old girls. Girls with higher body mass index and girls from households with lower income had lower 25(OH)D concentrations. These effects were not observed in boys.

CONCLUSIONS: Inadequate vitamin D status is a potentially serious public health problem among children and adolescents in Québec. Youth living at high latitudes in countries with and without mandatory fortification of vitamin D are likely at heightened risk of 25(OH)D deficiency. These results call for renewed efforts to ensure adequate vitamin D intake among growing children and adolescents.

BACKGROUND: Glycohemoglobin (GHB), reported as hemoglobin (Hb) A_1c, is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA_1c measurement in the presence of HbE and/or HbD traits.

METHODS: We evaluated 23 HbA_1c methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA_1c results with the use of ±10% relative bias at 6% and 9% HbA_1c as evaluation limits.

RESULTS: Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively.

CONCLUSIONS: Some current HbA_1c methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers' instructions do not provide adequate information for making correct decisions about reporting results.

BACKGROUND: A serum ceruloplasmin concentration below 0.20 g/L is conventionally considered as one of the major diagnostic criteria for Wilson disease. This decision threshold has not been fully validated for its diagnostic characteristics, however. In this study, we evaluated various decision thresholds of serum ceruloplasmin concentration in the diagnosis of Wilson disease based on genotype-verified Wilson disease patients, carriers, and normal individuals.

METHODS: Serum ceruloplasmin concentration was measured by a nephelometric method in 57 Wilson disease patients and 71 family members (49 heterozygotes and 22 wild-type homozygotes), a validation group of 25 subjects clinically suspected of Wilson disease, and 690 normal individuals. We performed ROC analysis using Analyze-it software and confirmed the genotypes by direct DNA sequencing of ATP7B.

RESULTS: Serum ceruloplasmin concentrations <0.20, 0.14, and 0.10 g/L showed positive predictive values of 48.3%, 100%, and 100%, respectively, and negative predictive values of 98.7%, 97.1%, and 91.9%. In the validation group, a serum ceruloplasmin threshold of 0.14 g/L rendered 100% sensitivity and specificity. Forty of 690 healthy subjects had serum ceruloplasmin concentrations <0.20 g/L; however, these 40 individuals had normal genotypes by DNA sequencing, and none of the 40 had ceruloplasmin concentrations <0.14 g/L.

CONCLUSIONS: The diagnostic accuracy for Wilson disease using a serum ceruloplasmin concentration of 0.14 g/L as the local decision threshold was better than that using a threshold of 0.20 g/L. We suggest that laboratories providing ceruloplasmin assays determine decision thresholds based on local populations.

BACKGROUND: Hypertriglyceridemia is a cardiovascular risk factor. Apolipoprotein C-III (apoC-III) is an important determinant of the catabolic rate of triglyceride-rich lipoproteins. The aim of this study was to investigate the prognostic value of plasma apoC-III concentrations for cardiovascular mortality.

METHODS: We performed this prospective study in 2244 subjects ages 49–77 years who participated in the Hoorn Study. During a mean follow-up of 15 years, 504 individuals died: 231 of cardiovascular disease, 180 of cancer, and 93 of other causes. Cardiovascular disease risk factors and plasma apoC-III concentrations were measured at baseline.

RESULTS: The age- and sex-adjusted plasma apoC-III concentration was prospectively associated with cardiovascular mortality (P < 0.001). After adjustment for traditional risk factors, including fasting triglycerides, the hazard ratio (95% CI) for cardiovascular death between the highest and the lowest quartile of apoC-III was 1.85 (1.02–3.38). High concentrations of apoC-III did not appear to be associated with noncardiovascular mortality.

CONCLUSIONS: In this general population cohort, a high apoC-III concentration in plasma, independently of fasting triglycerides and other traditional risk factors, predicts cardiovascular mortality.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.

METHODS: 3β-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.

RESULTS: In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00–0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.

CONCLUSIONS: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.

BACKGROUND: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation (IDF)/IFCC Consensus Statement on the worldwide standardization of HbA1c states that "HbA1c results are to be reported world-wide in IFCC units and derived NGSP units, using the IFCC-NGSP master equation."

METHODS: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs.

RESULTS: For NGSP and Mono-S, slope, intercept, and derived percentage HbA1c at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA1c in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA1c (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S).

CONCLUSIONS: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the IFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/IDF/IFCC Consensus Statement on HbA1c measurement.

BACKGROUND: The emergence of type 2 diabetes in young populations has mirrored a rising prevalence of obesity and insulin resistance during childhood and adolescence. At the same time, the role of adipokines as links between obesity and insulin resistance is becoming more appreciated. We sought to establish age- and sex-specific distributions of metabolic correlates of insulin resistance in healthy prepubertal children.

METHODS: We collected fasting blood samples from a contemporary cohort of 307 British children at ages 5, 6, 7, and 8 years and measured insulin, glucose, triglycerides, total and HDL cholesterol, urate, glycohemoglobin, sex hormone–binding globulin (SHBG), leptin, and adiponectin. We used homeostasis model assessment (HOMA 2) to estimate insulin sensitivity (HOMA-%S) and β-cell function (HOMA-%B). Anthropometric measures included body mass index.

RESULTS: Body mass index increased from age 5 to 8 years (P < 0.001). HOMA-%B decreased (P < 0.001) and HOMA-%S increased (P < 0.05), but glucose also increased (P < 0.001) whereas glycohemoglobin decreased (P < 0.001). Consistent with the rise in insulin sensitivity, HDL cholesterol increased (P < 0.001) and triglycerides decreased (NS), whereas adiponectin decreased (P = 0.02). The patterns were similar in boys and girls, although girls were less insulin sensitive throughout. Accordingly, triglycerides tended to be higher in the girls, and HDL cholesterol and SHBG lower.

CONCLUSIONS: The metabolic disturbances associated with insulin resistance appear to be more advanced in girls. Markers of metabolic health improve in both sexes from 5 to 8 years, despite rising adiposity.

BACKGROUND: Increased N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations are associated with increased cardiovascular mortality in chronic hemodialysis patients. Previous studies focused on prevalent dialysis patients and examined single measurements of NT-proBNP in time.

METHODS: We measured NT-proBNP concentrations in 2990 incident hemodialysis patients to examine the risk of 90-day and 1-year mortality associated with baseline NT-proBNP concentrations. In addition, we calculated the change in concentrations after 3 months in a subset of 585 patients to examine the association between longitudinal changes in NT-proBNP and subsequent mortality.

RESULTS: Increasing quartiles of NT-proBNP were associated with a monotonic increase in 90-day [quartile 1, referent; from quartile 2 to quartile 4, hazard ratio (HR) 1.7–6.3, P < 0.001] and 1-year (quartile 1, referent; from quartile 2 to quartile 4, HR 1.7–4.9, P < 0.001) all-cause mortality. After multivariable adjustment, these associations remained robust. When examined using a multivariable fractional polynomial, increased NT-proBNP concentrations were associated with increased 90-day (HR per unit increase in log NT-proBNP 1.5, 95% CI 1.3–1.7) and 1-year (HR per unit increase in log NT-proBNP 1.4, 95% CI 1.3–1.5) all-cause mortality. In addition, patients with the greatest increase in NT-proBNP after 3 months of dialysis had a 2.4-fold higher risk of mortality than those with the greatest decrease in NT-proBNP.

CONCLUSIONS: NT-proBNP concentrations are independently associated with mortality in incident hemodialysis patients. Furthermore, the observation that longitudinal changes in NT-proBNP concentrations were associated with subsequent mortality suggests that monitoring serial NT-proBNP concentrations may represent a novel tool for assessing adequacy and guiding therapy in patients initiating hemodialysis.

BACKGROUND: The availability of commutable calibrator materials may ease considerably the task of harmonizing assay results and ensuring their traceability to reference procedures. We sought to verify the commutability of potential calibrator materials and evaluate their effectiveness in harmonizing LDH results by 2 measurement methods.

METHODS: We measured LDH in 109 serum samples and 31 materials, including frozen serum pools (with either normal or abnormal isoenzyme patterns), commercial stabilized materials, and the ERM-AD453/IFCC reference material. We assayed LDH activity with the IFCC reference procedure and with 2 commercial methods, 1 using the lactate-to-pyruvate (LP) reaction, and the other the pyruvate-to-lactate (PL) reaction. We selected a commutable material, with LDH value assigned by the reference procedure, as a calibrator for recalculating the results for patient sera by both LP and PL, thereby making them traceable to the IFCC reference procedure.

RESULTS: Original values for patient sera (n = 109) by the 2 commercial methods showed a mean (SD) PL/LP ratio of 1.97 (0.03); this ratio changed to 1.06 (0.02) after recalculation of results. Linear regression of PL vs LP recalibrated values gave y = 1.108x – 9.7. At the clinically important concentration of 250 U/L (upper reference limit), the systematic difference between methods was 6.8%, which met our proposed quality specifications for inaccuracy and total error.

CONCLUSIONS: By properly selecting the calibrator, the results of serum LDH measurement by 2 different methods may be harmonized and made traceable to the selected highest (reference) metrological level.

BACKGROUND: The area under the receiver operating characteristics curve (AUC) is widely used to measure the diagnostic accuracy of noninvasive fibrosis indices. However, use of the AUC assumes a binary gold standard, whereas fibrosis staging is based on an ordinal scale and also depends on the distribution of fibrosis stages in the study sample. We explored other fibrosis staging accuracy measures designed for ordinal gold standards, the C-statistic and the Obuchowski measure.

METHODS: We performed a simulation study to assess the bias in estimating the accuracy measures when the distribution of fibrosis stages in the study sample do not fit the reference distribution in the population to which the indices are applied. We also estimated the type I error of the tests comparing these measures in 2 samples with different distributions of fibrosis stages. We illustrated the practical use of these measures by reanalyzing real data.

RESULTS: Compared with the AUC or the C-statistic, the Obuchowski measure showed limited bias when the distribution of fibrosis stages in the study sample differed from the reference distribution. The type I error was strongly inflated with the AUC or the C-statistic but was preserved in the Obuchowski measure. When we compared noninvasive indices on real data, AUC analysis led to discordant results depending on how the fibrosis stages were grouped together. One single conclusion was drawn from the analysis based on the Obuchowski measure.

CONCLUSIONS: We recommend using the Obuchowski measure for assessing the diagnostic accuracy of noninvasive indices of fibrosis.

BACKGROUND: Individuals with severe deficiency in serum ₁-antitrypsin (AAT) concentrations are at high risk for developing chronic obstructive pulmonary disease (COPD), whereas those carrying the PI*MZ genotype are at slightly increased risk. Testing appropriate subgroups of the population for AAT deficiency (AATD) is therefore an important aspect of COPD prevention and timely treatment. We decided to perform an exhaustive investigation of SERPINA1 gene variants in individuals from the general population with a moderately reduced serum AAT concentration, because such information is currently unavailable.

METHODS: We determined the Z and S alleles of 1399 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) with serum AAT concentrations <113 mg/dL and submitted 423 of these samples for complete exon 2->5 sequencing.

RESULTS: We found that 900 of 1399 samples (64%), carried the normal PI*MM genotype, whereas 499 samples (36%) carried at least 1 SERPINA1 deficiency variant. In the subpopulations in which AAT concentrations ranged from 103 to 113 and from 93 to 103 mg/dL, individuals with the PI*MM genotype represented the majority (86.5% and 53.8%, respectively). The PI*MS genotype was predominant (54.9%) in the AAT range of 83 to 93 mg/dL, whereas PI*MZ represented 76.4% in the AAT range of 73 to 83 mg/dL.

CONCLUSIONS: This analysis provided a detailed molecular definition of intermediate AATD, which would be helpful in the diagnostic setting.

BACKGROUND: Cellular markers help identify different components of a pathological process and may contribute to the diagnosis, prognostic assessment, and management of patients with suspected syndromes. Flow cytometry can be used to accurately assess markers of platelet and leukocyte activation and cellular aggregation in whole blood. To use cell markers as predictors of disease requires that they be measured reliably and show modest within-individual, day-to-day variation.

METHODS: We used whole blood flow cytometry to analyze monocyte and platelet markers in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study. We estimated laboratory variability using 20 split samples, process variation using replicate blood tubes taken from 112 subjects, and biologic plus process variation using replicate blood samples taken 4–8 weeks apart from 55 people.

RESULTS: For most analytes, the laboratory CV was <10% (mean 3.6%, range 0%–14.5%) and reliability was excellent (75% of analytes had R > 0.90). Reliability coefficients based on repeat-visit data indicated substantial to high repeatability (R > 0.60) for CD14, Toll-like receptor (TLR)-2, CD162, CD61, CD41, CD62P, CD154, and platelet–leukocyte aggregates. In contrast, TLR-4, CD45, myeloperoxidase (MPO), and cyclooxygenase (COX)-2 had slight to moderate repeat visit reliability.

CONCLUSIONS: The high repeatability results for selected platelet and monocyte markers indicate that they can be reliably measured in multicenter studies with delayed sample processing, provided that rigorous standardization of sample collection, shipping, and flow cytometry procedures is applied.

BACKGROUND: Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL).

METHODS: After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2–540.0 Å. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis.

RESULTS: Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92).

CONCLUSIONS: The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.