Showing papers in "Clinical Chemistry in 2012"

Analytical Characteristics of High-Sensitivity Cardiac Troponin Assays

Abstract: BACKGROUND: Cardiac troponins I (cTnI) and T (cTnT) have received international endorsement as the standard biomarkers for detection of myocardial injury, for risk stratification in patients suspected of acute coronary syndrome, and for the diagnosis of myocardial infarction. An evidence-based clinical database is growing rapidly for high-sensitivity (hs) troponin assays. Thus, clarifications of the analytical principles for the immunoassays used in clinical practice are important. CONTENT: The purpose of this mini-review is ( a ) to provide a background for the biochemistry of cTnT and cTnI and ( b ) to address the following analytical questions for both hs cTnI and cTnT assays: ( i ) How does an assay become designated hs? ( ii ) How does one realistically define healthy (normal) reference populations for determining the 99th percentile? ( iii ) What is the usual biological variation of these analytes? ( iv ) What assay imprecision characteristics are acceptable? ( v ) Will standardization of cardiac troponin assays be attainable? SUMMARY: This review raises important points regarding cTnI and cTnT assays and their reference limits and specifically addresses hs assays used to measure low concentrations (nanograms per liter or picograms per milliliter). Recommendations are made to help clarify the nomenclature. The review also identifies further challenges for the evolving science of cardiac troponin measurement. It is hoped that with the introduction of these concepts, both laboratorians and clinicians can develop a more unified view of how these assays are used worldwide in clinical practice.

Determination of 19 Cardiac Troponin I and T Assay 99th Percentile Values from a Common Presumably Healthy Population

Abstract: BACKGROUND: Between-assay comparability of 99th percentiles for cardiac troponin concentrations has not been assessed systematically in a single population for a large number of assays. METHODS: We determined 99th percentiles for 19 cardiac troponin assays in heparin plasma samples from a population of 272 and 252 presumably healthy males and females, respectively. The assays evaluated included 1 cardiac troponin T (cTnT) assay from Roche and 18 cTnI assays from Abbott, Alere, Beckman, bioMerieux, Instrumentation Laboratory, Ortho-Clinical Diagnostics, Singulex, Siemens, and Roche. Five of these assays were categorized as high-sensitivity, 9 as sensitive-contemporary, and 5 as point-of-care (POC) assays. RESULTS: For high-sensitivity cTnI (hs-cTnI) assays 99th percentiles varied from 23 to 58 ng/L. At least 80% of individuals had measurable hs-cTnI, whereas only 25% had measurable high-sensitivity cTnT. All high-sensitivity cardic troponin assays had 99th percentiles that were 1.2–2.4-fold higher in males than females. For the 9 sensitive-contemporary cTnI assays, 99th percentiles varied from 12 to 392 ng/L, and only the Beckman assay gave measurable concentrations in a substantial portion of the population (35% vs ≤6% for the others). Seven of these 9 assays had 1.3–5.0-fold higher 99th percentiles for males than females. For 5 cTnI POC assays, 99th percentiles varied from <10 to 40 ng/L. The Instrumentation Laboratory assay gave measurable results in 27.8% of study participants vs ≤6% for the others. Correlations were generally poor among assays. CONCLUSIONS: Among cardiac troponin assays 99th percentile concentrations appear to differ. High-sensitivity assays provide measurable cardiac troponin results in a substantially greater fraction of presumably healthy individuals.

Serum MicroRNA Expression Profile as a Biomarker in the Diagnosis and Prognosis of Pancreatic Cancer

Abstract: BACKGROUND: Detection of pancreatic cancer (PaC), particularly at early stages, remains a great challenge owing to lack of specific biomarkers. We sought to identify a PaC-specific serum microRNA (miRNA) expression profile and test its specificity and sensitivity as a biomarker in the diagnosis and prognosis of PaC. METHODS: We obtained serum samples from 197 PaC cases and 158 age- and sex-matched cancer-free controls. We screened the differentially expressed serum miRNAs with Illumina sequencing by synthesis technology using pooled serum samples followed by RT-qPCR validation of a large number of samples arranged in multiple stages. We used risk score analysis to evaluate the diagnostic value of the serum miRNA profiling system. To assess the serum miRNA–based biomarker accuracy in predicting PaC, we performed additional double-blind testing in 77 PaC cases and 52 controls and diagnostic classification in 55 cases with clinically suspected PaC. RESULTS: After the selection and validation process, 7 miRNAs displayed significantly different expression levels in PaC compared with controls. This serum 7-miRNA–based biomarker had high sensitivity and specificity for distinguishing various stages of PaC from cancer-free controls and also accurately discriminated PaC patients from chronic pancreatitis (CP) patients. Among the 7 miRNAs, miR-21 levels in serum were significantly associated with overall PaC survival. The diagnostic accuracy rate of the serum 7-miRNA profile was 83.6% in correctly classifying 55 cases with clinically suspected PaC. CONCLUSIONS: These data demonstrate that the serum 7-miRNA–based biomarker can serve as a novel noninvasive approach for PaC diagnosis and prognosis.

State-of-the-Art Vitamin D Assays: A Comparison of Automated Immunoassays with Liquid Chromatography–Tandem Mass Spectrometry Methods

Abstract: BACKGROUND: Vitamin D testing is increasing worldwide. Recently several diagnostic manufacturers including Abbott and Siemens have launched automated 25-hydroxy vitamin D (25OH-D) immunoassays. Furthermore, preexisting assays from DiaSorin and Roche have recently been modified. We compared the performance of 5 automated immunoassays, an RIA and 2 liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods. METHODS: Aliquots of 170 randomly selected patient samples were prepared and 25OH-D was measured by 2 LC-MS/MS methods, an RIA (DiaSorin), and automated immunoassays from Abbott (Architect), DiaSorin (LIAISON), IDS (ISYS), Roche (E170, monoclonal 25OH-D3 assay), and Siemens (Centaur). Within-run and between-run imprecision were evaluated by measurement of 5 replicates of 2 serum pools on 5 consecutive days. RESULTS: The LC-MS/MS methods agreed, with a concordance correlation coefficient (CCC) of 0.99 and bias of 0.56 μg/L (1.4 nmol/L). The RIA assay showed a performance comparable to LC-MS/MS, with a CCC of 0.97 and a mean bias of 1.1 μg/L (2.7 nmo/L). All immunoassays measured total 25OH-D (including D3 and D2), with the exception of the Roche assay (D3 only). Among the immunoassays detecting total 25OH-D, the CCCs varied between 0.85 (Abbott) to 0.95 (LIAISON). The mean bias ranged between 0.2 μg/L (0.5 nmol/L) (LIAISON) and 4.56 μg/L (11.4 nmol/L) (Abbott). The Roche 25OH-D3 assay demonstrated small mean bias \[−2.7 μg/L (−6.7 nmol/L)\] \[−2.7 μg/L (−6.7 nmol/L)\] but a low CCC of just 0.66. Most assays demonstrated good intra- and interassay precision, with CV <10%. CONCLUSIONS: Automated immunoassays demonstrated variable performance and not all tests met our minimum performance goals. It is important that laboratories be aware of the limitations of their assay.

Closing the Gaps in Pediatric Laboratory Reference Intervals: A CALIPER Database of 40 Biochemical Markers in a Healthy and Multiethnic Population of Children

Abstract: BACKGROUND: Pediatric healthcare is critically dependent on the availability of accurate and precise laboratory biomarkers of pediatric disease, and on the availability of reference intervals to allow appropriate clinical interpretation. The development and growth of children profoundly influence normal circulating concentrations of biochemical markers and thus the respective reference intervals. There are currently substantial gaps in our knowledge of the influences of age, sex, and ethnicity on reference intervals. We report a comprehensive covariate-stratified reference interval database established from a healthy, nonhospitalized, and multiethnic pediatric population. METHODS: Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer. RESULTS: Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers. CONCLUSIONS: The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

Accuracy of 6 Routine 25-Hydroxyvitamin D Assays: Influence of Vitamin D Binding Protein Concentration

Abstract: BACKGROUND: Recent recognition of its broad pathophysiological importance has triggered an increased interest in 25-hydroxyvitamin D [25(OH)D]. By consequence, throughput in 25(OH)D testing has become an issue for clinical laboratories, and several automated assays for measurement of 25(OH)D are now available. The aim of this study was to test the accuracy and robustness of these assays by comparing their results to those of an isotope dilution/online solid-phase extraction liquid chromatography/tandem mass spectrometry (ID-XLC-MS/MS) method. We put specific focus on the influence of vitamin D–binding protein (DBP) by using samples with various concentrations of DBP. METHODS: We used 5 automated assays (Architect, Centaur, iSYS, Liaison, and Elecsys), 1 RIA (Diasorin) preceded by extraction, and an ID-XLC-MS/MS method to measure 25(OH)D concentrations in plasma samples of 51 healthy individuals, 52 pregnant women, 50 hemodialysis patients, and 50 intensive care patients. Using ELISA, we also measured DBP concentrations in these samples. RESULTS: Most of the examined 25(OH)D assays showed significant deviations in 25(OH)D concentrations from those of the ID-XLC-MS/MS method. As expected, DBP concentrations were higher in samples of pregnant women and lower in samples of IC patients compared to healthy controls. In 4 of the 5 fully automated 25(OH)D assays, we observed an inverse relationship between DBP concentrations and deviations from the ID-XLC-MS/MS results. CONCLUSIONS: 25(OH)D measurements performed with most immunoassays suffer from inaccuracies that are DBP concentration dependent. Therefore, when interpreting results of 25(OH)D measurements, careful consideration of the measurement method is necessary.

Resolvins D1, D2, and Other Mediators of Self-Limited Resolution of Inflammation in Human Blood following n-3 Fatty Acid Supplementation

Abstract: BACKGROUND: Resolvins and protectins are families of local lipid mediators generated from the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) during self-limited resolution of inflammation. We aimed to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay to measure these lipid mediators in human blood following n-3 fatty acid supplementation and to determine whether the blood collection method affects their measured concentration. METHODS: Blood samples from 20 healthy volunteers enrolled in an n-3 fatty acid supplementation trial were collected in EDTA, heparin, or citrate, or prepared as serum after volunteers had undergone 3 weeks of supplementation. Plasma or serum was purified by solid-phase chromatography and analyzed with LC-MS/MS. RESULTS: The assay identified 18R/S-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid (18R/S-HEPE); 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17R/S-HDHA); 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (RvD1); 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoicacid (17R-RvD1); 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (RvD2); 10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoicacid (10S,17S-DiHDHA); and 10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (protectin D1, PD1). The limits of detection and quantification were 3 pg and 6 pg on-column, respectively. The pathway precursors 18R/S-HEPE and 17R/S-HDHA, but not the resolvins, were lower in serum compared with plasma. After n-3 fatty acid supplementation, mean (SD) EDTA plasma concentrations were: 18R/S-HEPE 386 (56) pg/mL, 17R/S-HDHA 365 (65) pg/mL, RvD2 26 (4) pg/mL, RvD1 31 (5) pg/mL, and 17R-RvD 161 (7) pg/mL. 10S,17S-DiHDHA and PD1 concentrations were below the limit of quantification. CONCLUSIONS: This is the first study reporting 17R/S-HDHA, RvD1, and RvD2 concentrations measured in human blood following oral n-3 fatty acid supplementation. The concentrations of the antiinflammatory lipid mediators RvD1 and RvD2 were within the biological range known to have antiinflammatory and proresolving activities in isolated human leukocytes and in in vivo studies in mice.

Emerging Biomarkers in Heart Failure

Abstract: BACKGROUND: Until recently, biomarker testing in heart failure (HF) syndromes has been viewed as an elective supplement to diagnostic evaluation of patients suspected to suffer from this condition This approach to the use of biomarker testing contrasts with other cardiovascular diagnoses such as acute myocardial infarction, for which biomarkers are integral to disease process definition, risk stratification, and in some cases treatment decision making CONTENT: In this review we consider various perspectives on the evaluation of biomarkers in HF In addition, we examine recent advances in the understanding of established biomarkers in HF (such as the natriuretic peptides), the elucidation of novel biomarkers potentially useful for the evaluation and management of patients with HF, and the growing understanding of important and relevant comorbidities in HF We also review candidate biomarkers from a number of classes: ( a ) myocyte stretch, ( b ) myocyte necrosis, ( c ) systemic inflammation, ( d ) oxidative stress, ( e ) extracellular matrix turnover, ( f ) neurohormones, and ( g ) biomarkers of extracardiac processes, such as renal function SUMMARY: Novel applications of established biomarkers of HF as well as elucidation and validation of emerging assays for HF syndromes have collectively led to a growing interest in the more widespread use of such testing in patients affected by the diagnosis

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction

Abstract: BACKGROUND: Rapid and correct diagnosis of acute myocardial infarction (MI) has an important impact on patient treatment and prognosis. We compared the diagnostic performance of high-sensitivity cardiac troponin T (hs-cTnT) and cardiac enriched microRNAs (miRNAs) in patients with MI. METHODS: Circulating concentrations of cardiac-enriched miR-208b and miR-499 were measured by quantitative PCR in a case-control study of 510 MI patients referred for primary mechanical reperfusion and 87 healthy controls. RESULTS: miRNA-208b and miR-499 were highly increased in MI patients (>105-fold, P < 0.001) and nearly undetectable in healthy controls. Patients with ST-elevation MI (n = 397) had higher miRNA concentrations than patients with non–ST-elevation MI (n = 113) ( P < 0.001). Both miRNAs correlated with peak concentrations of creatine kinase and cTnT ( P < 10−9). miRNAs and hs-cTnT were already detectable in the plasma 1 h after onset of chest pain. In patients who presented <3 h after onset of pain, miR-499 was positive in 93% of patients and hs-cTnT in 88% of patients ( P = 0.78). Overall, miR-499 and hs-cTnT provided comparable diagnostic value with areas under the ROC curves of 0.97. The reclassification index of miR-499 to a clinical model including several risk factors and hs-cTnT was not significant ( P = 0.15). CONCLUSIONS: Circulating miRNAs are powerful markers of acute MI. Their usefulness in the establishment of a rapid and accurate diagnosis of acute MI remains to be determined in unselected populations of patients with acute chest pain.

Influence of Population Selection on the 99th Percentile Reference Value for Cardiac Troponin Assays

Abstract: OBJECTIVE: We sought to determine the effect of patient selection on the 99th reference percentile of 2 sensitive and 1 high-sensitivity (hs) cardiac troponin assays in a well-defined reference population. METHODS: Individuals >45 years old were randomly selected from 7 representative local community practices. Detailed information regarding the participants was collected via questionnaires. The healthy reference population was defined as individuals who had no history of vascular disease, hypertension, or heavy alcohol intake; were not receiving cardiac medication; and had blood pressure 60 mL · min−1 · (1.73 m2)−1, and normal cardiac function according to results of echocardiography. Samples were stored at −70 °C until analysis for cardiac troponin I (cTnI) and cardiac troponin T (cTnT) and N-terminal pro-B–type natriuretic peptide. RESULTS: Application of progressively more stringent population selection strategies to the initial baseline population of 545 participants until the only individuals who remained were completely healthy according to the study criteria reduced the number of outliers seen and led to a progressive decrease in the 99th-percentile value obtained for the Roche hs-cTnT assay and the sensitive Beckman cTnI assay but not for the sensitive Siemens Ultra cTnI assay. Furthermore, a sex difference found in the baseline population for the hs-cTnT ( P = 0.0018) and Beckman cTnI assays ( P < 0.0001) progressively decreased with more stringent population selection criteria. CONCLUSIONS: The reference population selection strategy significantly influenced the 99th percentile reference values determined for troponin assays and the observed sex differences in troponin concentrations.

Absolute and Relative Kinetic Changes of High-Sensitivity Cardiac Troponin T in Acute Coronary Syndrome and in Patients with Increased Troponin in the Absence of Acute Coronary Syndrome

Abstract: BACKGROUND: We evaluated kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) in patients with acute coronary syndrome (ACS) and patients with hs-cTnT increases not due to ACS to rule in or rule out non–ST-segment elevation myocardial infarction (STEMI). METHODS: hs-cTnT was measured serially in consecutive patients presenting to the emergency department. Patients with ACS who had at least 2 hs-cTnT measurements within 6 h and non-ACS patients with hs-cTnT concentrations above the 99th percentile value (14 ng/L) were enrolled to compare absolute and relative kinetic changes of hs-cTnT. RESULTS: For discrimination of non-STEMI (n = 165) in the entire study population (n = 784), the absolute δ change with the ROC-optimized value of 9.2 ng/L yielded an area under the curve of 0.898 and was superior to all relative δ changes ( P < 0.0001). The positive predictive value for the absolute δ change was 48.7%, whereas the negative predictive value was 96.5%. In a specific ACS population with exclusion of STEMI (n = 342), the absolute δ change with the ROC-optimized value of 6.9 ng/L yielded a positive predictive value of 82.8% and a negative predictive value of 93.0%. In comparison to the ≥20% relative δ change, the ROC-optimized absolute δ change demonstrated a significantly added value for the entire study population and for the ACS cohort (net reclassification index 0.331 and 0.499, P < 0.0001). CONCLUSIONS: Absolute δ changes appear superior to relative δ changes in discriminating non-STEMI. A rise or fall of at least 9.2 ng/L in the entire study population and 6.9 ng/L in selected ACS patients seems adequate to rule-out non-STEMI. However, δ-values are useful to rule-in non-STEMI only in a specific ACS population.

Circulating Epithelial Cells in Patients with Benign Colon Diseases

Abstract: BACKGROUND: Detection of circulating tumor cells (CTCs) in the peripheral blood is a rapidly developing research field with clear clinical implications for the staging and monitoring of cancer patients. Current CTC assays, including the US Food and Drug Administration–cleared CellSearch® system, typically use markers [e.g., cytokeratins (CKs), the transmembrane protein EpCAM (epithelial cell adhesion molecule)] that are expressed on normal and malignant epithelial cells but not on the surrounding normal leukocytes. METHODS: We enrolled 53 patients with benign colon diseases (e.g., diverticulosis, benign polyps, Crohn disease, ulcerative rectocolitis, colonic endometriosis) and analyzed their peripheral blood with 2 previously validated CTC assays: the epithelial immunospot (EPISPOT) assay and the CellSearch system. The EPISPOT assay detects only viable, CK19-releasing CTCs that were enriched by depletion of CD45+ leukocytes, whereas the CellSearch system detects CK-positive CTCs after positive EpCAM-based immunomagnetic enrichment. RESULTS: In patients with benign colon diseases, positive events that met the criteria for “tumor cells” were detected with both the CellSearch system (11.3%) and the CK19-EPISPOT assay (18.9%), whereas no positive events were detected in samples from healthy volunteers. Positive events were detected most frequently in patients with diverticulosis and Crohn disease. All positive events lacked expression of CD45, a common leukocyte antigen. CONCLUSIONS: These results indicate that patients with benign inflammatory colon diseases in particular can harbor viable circulating epithelial cells that are detectable with current CTC assays. This finding points to the need for further molecular characterization of circulating epithelial cells and has important implications for the use of CTC testing.

Beyond Diagnostic Accuracy: The Clinical Utility of Diagnostic Tests

Abstract: Like any other medical technology or intervention, diagnostic tests should be thoroughly evaluated before their introduction into daily practice. Increasingly, decision makers, physicians, and other users of diagnostic tests request more than simple measures of a test's analytical or technical performance and diagnostic accuracy; they would also like to see testing lead to health benefits. In this last article of our series, we introduce the notion of clinical utility, which expresses--preferably in a quantitative form--to what extent diagnostic testing improves health outcomes relative to the current best alternative, which could be some other form of testing or no testing at all. In most cases, diagnostic tests improve patient outcomes by providing information that can be used to identify patients who will benefit from helpful downstream management actions, such as effective treatment in individuals with positive test results and no treatment for those with negative results. We describe how comparative randomized clinical trials can be used to estimate clinical utility. We contrast the definition of clinical utility with that of the personal utility of tests and markers. We show how diagnostic accuracy can be linked to clinical utility through an appropriate definition of the target condition in diagnostic-accuracy studies.

Metabolomics and Cardiovascular Biomarker Discovery

Abstract: BACKGROUND: Metabolomics, the systematic analysis of low molecular weight biochemical compounds in a biological specimen, has been increasingly applied to biomarker discovery. CONTENT: Because no single analytical method can accommodate the chemical diversity of the entire metabolome, various methods such as nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) have been employed, with the latter coupled to an array of separation techniques including gas and liquid chromatography. Whereas NMR can provide structural information and absolute quantification for select metabolites without the use of exogenous standards, MS tends to have much higher analytical sensitivity, enabling broader surveys of the metabolome. Both NMR and MS can be used to characterize metabolite data either in a targeted manner or in a nontargeted, pattern-recognition manner. In addition to technical considerations, careful sample selection and study design are important to minimize potential confounding influences on the metabolome, including diet, medications, and comorbitidies. To this end, metabolite profiling has been applied to human biomarker discovery in small-scale interventions, in which individuals are extremely well phenotyped and able to serve as their own biological controls, as well as in larger epidemiological cohorts. Understanding how metabolites relate to each other and to established risk markers for diseases such as diabetes and renal failure will be important in evaluating the potential value of these metabolites as clinically useful biomarkers. SUMMARY: Applied to both experimental and epidemiological study designs, metabolite profiling has begun to highlight the breadth of metabolic disturbances that accompany human disease. Experimental work in model systems and integration with other functional genomic approaches will be required to establish a causal link between select biomarkers and disease pathogenesis.

Established and Emerging Markers of Kidney Function

Abstract: BACKGROUND: The kidney performs a multitude of essential functions to maintain homeostasis. In clinical medicine, glomerular filtration rate (GFR) provides the best index of overall kidney function, and proteinuria adds additional information on renal and nonrenal prognosis. Several novel biomarkers of kidney injury and function are under investigation. CONTENT: Plasma creatinine concentration is the most widely used measure for estimation of GFR. Plasma cystatin C and β-trace protein may eventually prove to be superior to creatinine. GFR may be measured directly by use of exogenous filtration markers, although their role is primarily limited to the research setting. Real-time, noninvasive measurement of GFR by using fluorescently labeled markers may be available in the future. Novel biomarkers of tubular injury such as neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, liver-type fatty acid binding protein, N-acetyl-β-(D)-glucosaminidase, and interleukin-18 may enable the early detection of acute kidney injury before or in the absence of a change in GFR. SUMMARY: A variety of methods are available to assist clinicians in the assessment of kidney function and injury. Ongoing investigation will help determine the utility of several new markers and clarify their role in the care of patients with and at risk for kidney disease.

Digital PCR Analysis of Maternal Plasma for Noninvasive Detection of Sickle Cell Anemia

Abstract: BACKGROUND: Cell-free fetal DNA (cffDNA) constitutes approximately 10% of the cell-free DNA in maternal plasma and is a suitable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). The objective of this study was to determine the feasibility of using digital PCR for NIPD in pregnancies at risk of sickle cell anemia. METHODS: Minor-groove binder (MGB) TaqMan probes were designed to discriminate between wild-type hemoglobin A and mutant (hemoglobin S) alleles encoded by the HBB (hemoglobin, beta) gene in cffDNA isolated from maternal plasma samples obtained from pregnancies at risk of sickle cell anemia. The fractional fetal DNA concentration was assessed in male-bearing pregnancies with a digital PCR assay for the Y chromosome–specific marker DYS14. In pregnancies with a female fetus, a panel of biallelic insertion/deletion polymorphism (indel) markers was developed for the quantification of the fetal DNA fraction. We used digital real-time PCR to analyze the dosage of the variant encoding hemoglobin S relative to that encoding wild-type hemoglobin A. RESULTS: The sickle cell genotype was correctly determined in 82% (37 of 45) of male fetuses and 75% (15 of 20) of female fetuses. Mutation status was determined correctly in 100% of the cases (25 samples) with fractional fetal DNA concentrations >7%. The panel of indels was informative in 65% of the female-bearing pregnancies. CONCLUSIONS: Digital PCR can be used to determine the genotype of fetuses at risk for sickle cell anemia. Optimization of the fractional fetal DNA concentration is essential. More-informative indel markers are needed for this assay's comprehensive use in cases of a female fetus.

Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

Abstract: BACKGROUND: A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS: We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS: A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS: Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

Distribution and Clinical Correlates of the Interleukin Receptor Family Member Soluble ST2 in the Framingham Heart Study

Abstract: BACKGROUND: Soluble ST2 (sST2) is a cardiac biomarker whose concentration rises in response to myocardial strain. Increased sST2 concentrations may predict adverse outcomes in patients with heart failure and myocardial infarction. Because sST2 was largely undetectable with first-generation assays in ambulatory individuals, there are few data regarding its distribution and correlates in community-based populations. METHODS: We measured sST2 using a highly sensitive ELISA in 3450 Framingham Heart Study participants who attended a routine examination. We used multivariable linear regression models to identify covariates associated with sST2 in the general sample. We obtained a reference sample (n = 1136) by excluding individuals with prevalent coronary disease, heart failure, atrial fibrillation, diabetes, hypertension, obesity, valvular disease, left ventricular systolic dysfunction, and pulmonary and renal dysfunction. We used empiric and quantile regression techniques to estimate the 2.5th, 50th, 97.5th, and 99th quantiles. RESULTS: In the general sample (mean age 59 years, 55% women), systolic blood pressure ( P = 0.006), antihypertensive medication use ( P = 0.03), and diabetes ( P < 0.001) were associated with sST2 concentrations. In the reference sample (mean age 55, 59% women), male sex ( P < 0.0001) and older age ( P = 0.004) were predictive of higher sST2 concentrations. Quantile and empirical methods were used to define the reference intervals. Using the empirical approach, upper 99% percentile values in different age groups ranged from 46.6 to 64.4 μg/L in men and 36.7 to 53.0 μg/L in women. CONCLUSIONS: In a well-characterized, community-based cohort, values for sST2 differ between men and women, increase with age, and are associated with diabetes and hypertension.

Troponin T percentiles from a random population sample, emergency room patients and patients with myocardial infarction.

Abstract: BACKGROUND: High-sensitivity cardiac troponin T (cTnT) assays detect small clinically important myocardial infarctions (MI) but also yield higher rates of false-positive results owing to increased concentrations sometimes present in patients without MI. Better understanding is needed of factors influencing the 99th percentile of cTnT concentrations across populations and the frequency of changes in cTnT concentrations >20% often used in combination with increased cTnT concentrations for diagnosis of MI. METHODS: cTnT percentiles were determined by use of the Elecsys® hscTnT immunoassay (Modular® Analytics E170) in a random population sample, in emergency room (ER) patients, and in patients with non–ST-elevation MI (NSTEMI). Changes in cTnT concentrations were determined in hospitalized patients without MI. RESULTS: The 99th cTnT percentile in a random population sample (median age, 65 years) was 24 ng/L. In ER patients 65 years old it was 82 ng/L and highly age dependent. In hospitalized patients without MI the 97.5th percentile for change in the cTnT concentration was 51%–67%. cTnT remained below the 99th percentile (12 ng/L) in 1% of patients with NSTEMI until 8.5 h after symptom onset and 6 h after ER arrival. CONCLUSIONS: Age >65 years was the dominant factor associated with increased cTnT in ER patients. This age association was more prominent in ER patients than in a random population sample. Changes in serial cTnT concentrations >20% were common in hospitalized patients without MI.

Diagnostic accuracy of plasma glial fibrillary acidic protein for differentiating intracerebral hemorrhage and cerebral ischemia in patients with symptoms of acute stroke.

Abstract: BACKGROUND: Glial fibrillary acidic protein (GFAP) is a biomarker candidate indicative of intracerebral hemorrhage (ICH) in patients with symptoms of acute stroke. GFAP is released rapidly in the presence of expanding intracerebral bleeding, whereas a more gradual release occurs in ischemic stroke. This study determined the diagnostic accuracy of plasma GFAP in a prospective multicenter approach. METHODS: Within a 1-year recruitment period, patients suspected of having acute (symptom onset <4.5 h before admission) hemispheric stroke were prospectively included into the study in 14 stroke centers in Germany and Switzerland. A blood sample was collected at admission, and plasma GFAP was measured by use of an electrochemiluminometric immunoassay. The final diagnosis, established at hospital discharge, was classified as ICH, ischemic stroke, or stroke mimic. RESULTS: The study included 205 patients (39 ICH, 163 ischemic stroke, 3 stroke mimic). GFAP concentrations were increased in patients with ICH compared with patients with ischemic stroke [median (interquartile range) 1.91 μg/L (0.41–17.66) vs 0.08 μg/L (0.02–0.14), P < 0.001]. Diagnostic accuracy of GFAP for differentiating ICH from ischemic stroke and stroke mimic was high [AUC 0.915 (95% CI 0.847–0.982), P < 0.001]. A GFAP cutoff of 0.29 μg/L provided diagnostic sensitivity of 84.2% and diagnostic specificity of 96.3% for differentiating ICH from ischemic stroke and stroke mimic. CONCLUSIONS: Plasma GFAP analysis performed within 4.5 h of symptom onset can differentiate ICH and ischemic stroke. Studies are needed to evaluate a GFAP point-of-care system that may help optimize the prehospital triage and management of patients with symptoms of acute stroke.

Association of Growth Differentiation Factor-15 with Coronary Atherosclerosis and Mortality in a Young, Multiethnic Population: Observations from the Dallas Heart Study

Abstract: BACKGROUND: Growth differentiation factor 15 (GDF-15) is produced by cardiomyocytes and atherosclerotic lesions under stress conditions. Although higher circulating GDF-15 concentrations are associated with mortality across a spectrum of cardiovascular conditions, the relationship of GDF-15 with atherosclerosis and mortality in the general population remains undefined. METHODS: We measured plasma GDF-15 in 3219 participants of the Dallas Heart Study, a population sample of adults ages 30–65 years (55% women, 49% black). GDF-15 was analyzed in prespecified categories ( 10 Agatston units), increased CAC (CAC ≥100 Agatston units) by electron beam computed tomography, and mortality through a median 7.3 years of follow-up (120 deaths, 48 cardiovascular deaths). RESULTS: Increasing GDF-15 associated with older age, black race, hypertension, diabetes, smoking, left ventricular (LV) mass/body surface area, and worse renal function ( P 10 (odds ratio 2.1; 95% CI 1.2–3.7; P = 0.01), CAC ≥100 (odds ratio 2.6; 95% CI 1.4–4.9; P = 0.002), all-cause mortality (hazard ratio 3.5; 95% CI 2.1–5.9, P < 0.0001), and cardiovascular mortality (hazard ratio 2.5; 95% CI 1.1–5.8, P = 0.03). Adding log GDF-15 to fully adjusted models modestly improved the c statistic ( P = 0.025), the integrated discrimination index (0.028; P < 0.0001) and the category-less net reclassification index (0.42; P = 0.002). These findings remained significant with further adjustment for high-sensitivity C-reactive protein, N-terminal pro–B-type natriuretic peptide, and cardiac troponin T. CONCLUSIONS: GDF-15 is independently associated with subclinical coronary atherosclerosis and mortality, and its potential role for risk stratification in the general population merits further evaluation.

25-Hydroxyvitamin D: A Difficult Analyte

Abstract: In their article in the current issue of Clinical Chemistry , Farrell et al. (1) describe the latest in a number of studies (2, 3) highlighting the method-related variability in serum 25-hydroxyvitamin D (25-OHD)2 results. There is common agreement that 25-OHD is a “difficult” analyte. That quality is generally ascribed to its hydrophobic nature, its existence in several different molecular forms, its tight binding to vitamin D– binding protein (VDBP) and, until recently, the absence of reference materials or a reference measurement procedure (RMP) against which assays could be standardized. This last problem was mitigated to some extent with the introduction in 2009 of the NIST Standard Reference Materials (SRM 972 and SRM 2972) and the acceptance of the NIST and University of Ghent liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays as RMPs (4). Unfortunately, 3 of the 4 SRM 972 reference materials are either spiked with exogenous metabolites or diluted with equine serum, characteristics that render these reference materials unsuitable for many immunoassays (5, 6). The history of 25-OHD methodology could serve as a case study of the consequences of transferring a rigorous but labor-intensive method from the unhurried atmosphere of the research laboratory to the bustle of a routine clinical laboratory having to meet tight deadlines. The pioneering competitive protein-binding (CPB) method of Haddad and Chyu (7) involved solvent extraction and chromatography, with the results of every sample corrected for procedural losses. Subsequent attempts at simplifying a CPB method by omitting the chromatography stage proved unsuccessful (8), and an automated version (Nichols Advantage) introduced in 2004 was withdrawn in 2006. The successive abandonment of sample extraction, chromatography, and correction for procedural losses in immunoassays has undoubtedly contributed to the inconsistencies reported by Farrell et al., as have differences in assay standardization. Nevertheless, results submitted to …

Detection of Microdeletion 22q11.2 in a Fetus by Next-Generation Sequencing of Maternal Plasma

Abstract: BACKGROUND: Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA. We examined a clinically relevant genomic region, chromosome 22q11.2, the location of a series of well-characterized deletion anomalies that cause 22q11.2 deletion syndrome. METHODS: We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region. RESULTS: The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%–18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples. CONCLUSIONS: Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.

Comprehensive One-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing

Abstract: BACKGROUND: Mitochondrial diseases are clinically and genetically heterogeneous, with variable penetrance, expressivity, and differing age of onset. Disease-causing point mutations and large deletions in the mitochondrial genome often exist in a heteroplasmic state. Current molecular analyses require multiple different and complementary methods for the detection and quantification of mitochondrial DNA (mtDNA) mutations. We developed a novel approach to analyze the mtDNA in 1 step. METHODS: The entire human mitochondrial genome was enriched by a single amplicon long-range PCR followed by massively parallel sequencing to simultaneously detect mtDNA point mutations and large deletions with heteroplasmic levels of the mutations and variants quantified. QC samples were designed and analyzed along with each sample. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity. RESULTS: Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing. The deep coverage allowed the detection and quantification of heteroplasmy at every single nucleotide position of the 16 569-bp mitochondrial genome. Moreover, the method also detected large deletions with the breakpoints mapped. CONCLUSIONS: This “deep” sequencing approach provides a 1-step comprehensive molecular analysis of the whole mitochondrial genome for patients in whom a mitochondrial disease is suspected.

Specificity Characteristics of 7 Commercial Creatinine Measurement Procedures By Enzymatic and Jaffe Method Principles

Abstract: BACKGROUND: Standardized calibration does not change a creatinine measurement procedure's susceptibility to potentially interfering substances. METHODS: We obtained individual residual serum or plasma samples (n = 365) from patients with 19 different disease categories associated with potentially interfering substances and from healthy controls. Additional sera at 0.9 mg/dL (80 μmol/L) and 3.8 mg/dL (336 μmol/L) creatinine were supplemented with acetoacetate, acetone, ascorbate, and pyruvate. We measured samples by 4 enzymatic and 3 Jaffe commercially available procedures and by a liquid chromatography/isotope dilution/mass spectrometry measurement procedure against which biases were determined. RESULTS: The number of instances when 3 or more results in a disease category had biases greater than the limits of acceptability was 28 of 57 (49%) for Jaffe and 14 of 76 (18%) for enzymatic procedures. For the aggregate group of 59 diabetes samples with increased β-hydroxybutyrate, glucose, or glycosylated hemoglobin (Hb A1c), the enzymatic procedures had 10 biased results of 236 (4.2%) compared with 89 of 177 (50.3%) for the Jaffe procedures, and these interferences were highly procedure dependent. For supplemented sera, interferences were observed in 11 of 24 (46%) of groups for Jaffe and 8 of 32 (25%) of groups for enzymatic procedures and were different at low or high creatinine concentrations. CONCLUSIONS: There were differences in both magnitude and direction of bias among measurement procedures, whether enzymatic or Jaffe. The influence of interfering substances was less frequent with the enzymatic procedures, but no procedure was unaffected. The details of implementation of a method principle influenced its susceptibility to potential interfering substances.

Role of ST2 in Non–ST-Elevation Acute Coronary Syndrome in the MERLIN-TIMI 36 Trial

Abstract: OBJECTIVE: We investigated the prognostic performance of ST2 with respect to cardiovascular death (CVD) and heart failure (HF) in patients with non–ST-elevation acute coronary syndrome (NSTE-ACS) in a large multinational trial. BACKGROUND: Myocytes that are subjected to mechanical stress secrete ST2, a soluble interleukin-1 receptor family member that is associated with HF after STE-ACS. METHODS: We measured ST2 with a high-sensitivity assay in all available baseline samples (N = 4426) in patients enrolled in the Metabolic Efficiency With Ranolazine for Less Ischemia in the Non–ST-Elevation Acute Coronary Syndrome Thrombolysis in Myocardial Infarction 36 (MERLIN-TIMI 36), a placebo-controlled trial of ranolazine in NSTE-ACS. All events, including cardiovascular death and new or worsening HF, were adjudicated by an independent events committee. RESULTS: Patients with ST2 concentrations in the top quartile (>35 μg/L) were more likely to be older and male and have diabetes and renal dysfunction. ST2 was only weakly correlated with troponin and B-type natriuretic peptide. High ST2 was associated with increased risk for CVD/HF at 30 days (6.6% vs 1.6%, P < 0.0001) and 1 year (12.2% vs 5.2%, P < 0.0001). The risk associated with ST2 was significant after adjustment for clinical covariates and biomarkers (adjusted hazard ratio CVD/HF 1.90, 95% CI 1.15–3.13 at 30 days, P = 0.012; 1.51, 95% CI 1.15–1.98 at 1 year, P = 0.003), with a significant integrated discrimination improvement ( P < 0.0001). No significant interaction was found between ST2 and ranolazine ( P interaction = 0.15). CONCLUSIONS: ST2 correlates weakly with biomarkers of acute injury and hemodynamic stress but is strongly associated with the risk of HF after NSTE-ACS. This biomarker and related pathway merit further investigation as potential therapeutic targets for patients with ACS at risk for cardiac remodeling.

Interpreting Cardiac Troponin Results from High-Sensitivity Assays in Chronic Kidney Disease without Acute Coronary Syndrome

Abstract: BACKGROUND: Quantification and comparison of high-sensitivity (hs) cardiac troponin I (cTnI) and cTnT concentrations in chronic kidney disease (CKD) have not been reported. We examined the associations between hs cTnI and cTnT, cardiovascular disease, and renal function in outpatients with stable CKD. METHODS: Outpatients (n = 148; 16.9% with prior myocardial infarction or coronary revascularization) with an estimated glomerular filtration rate (eGFR) of <60 mL · min−1· (1.73 m2)−1 had serum cTnI (99th percentile of a healthy population = 9.0 ng/L), and cTnT (99th percentile = 14 ng/L) measured with hs assays. Left ventricular ejection fraction (LVEF) and mass were assessed by echocardiography, and coronary artery calcification (CAC) was determined by computed tomography. Renal function was estimated by eGFR and urine albumin/creatinine ratio (UACR). RESULTS: The median (interquartile range) concentrations of cTnI and cTnT were 6.3 (3.4–14.4) ng/L and 17.0 (11.2–31.4) ng/L, respectively; 38% and 68% of patients had a cTnI and cTnT above the 99th percentile, respectively. The median CAC score was 80.8 (0.7–308.6), LV mass index was 85 (73–99) g/m2, and LVEF was 58% (57%–61%). The prevalences of prior coronary disease events, CAC score, and LV mass index were higher with increasing concentrations from both hs cardiac troponin assays ( P < 0.05 for all). After adjustment for demographics and risk factors, neither cardiac troponin assay was associated with CAC, but both remained associated with LV mass index as well as eGFR and UACR. CONCLUSIONS: Increased hs cTnI and cTnT concentrations are common in outpatients with stable CKD and are influenced by both underlying cardiac and renal disease.

Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures.

Abstract: BACKGROUND: Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of multiple-reaction monitoring–mass spectrometry (MRM-MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use MRM-MS with established immunoassays to measure endogenous proteins has not been reported. METHODS: We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution MRM-MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and MRM-MS assays were then compared with commercially available immunoassays for each of the 6 analytes. RESULTS: Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution MRM-MS approaches showed correlations with immunoassays of r = 0.61–0.96. The MRM-MS approaches had acceptable reproducibility (<13% CV) and linearity ( r ≥0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. CONCLUSIONS: Because peak area ratios measured in multiplexed MRM-MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that MRM-MS could be used to replace immunoassays in a variety of settings.

Rosuvastatin, Proprotein Convertase Subtilisin/Kexin Type 9 Concentrations, and LDL Cholesterol Response: the JUPITER Trial

Abstract: BACKGROUND: Although statin therapy is known to increase concentrations of PCSK9, whether this effect is related to the magnitude of LDL reduction is uncertain. This study was undertaken to understand the extent of this effect and examine the relationship between PCSK9 and LDL cholesterol (LDL-C) reduction. METHODS: We measured plasma PCSK9 concentrations by ELISA at baseline and at 1 year in 500 men and 500 women participating in the Justification for Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial that randomly allocated participants to rosuvastatin 20 mg daily or placebo. We also evaluated rs11591147, a single nucleotide polymorphism known to have an impact on plasma PCSK9 concentrations. RESULTS: At baseline, median (interquartile range) PCSK9 concentrations were higher in women \[73 (62–90)] ng/mL than in men [69 (57–81) ng/mL\] ( P < 0.005). During 1 year, there was no change in PCSK9 concentrations in the placebo arm, suggesting stability in time. In contrast, the rosuvastatin increased PCSK9 by 35% in women \[101 (82–117) ng/mL] and 28% in men [89 (71–109) ng/mL\] ( P < 0.0001). Among those allocated to rosuvastatin, greater reductions in LDL-C were associated with greater increases in PCSK9 on both absolute and relative scales ( r = −0.15, P < 0.0005). Furthermore PCSK9 (rs11591147) did not alter the magnitude of LDL-C reduction associated with rosuvastatin use. CONCLUSIONS: In this randomized trial, rosuvastatin increased plasma concentration of PCSK9 in proportion to the magnitude of LDL-C reduction; the LDL-C response to statin could not be inferred by PCSK9 concentrations.

Quantifying the Accuracy of a Diagnostic Test or Marker

Abstract: BACKGROUND: Inrecentyears,increasingfocushasbeen directed to the methodology for evaluating (new) tests or biomarkers. A key step in the evaluation of a diagnostic test is the investigation into its accuracy. CONTENT: We reviewed the literature on how to assess the accuracy of diagnostic tests. Accuracy refers to the amount of agreement between the results of the test under evaluation (index test) and the results of a reference standard or test. The generally recommended approach is to use a prospective cohort design in patients who are suspected of having the disease of interest, in which each individual undergoes the index and same referencestandardtests.Thisapproachpresentsseveral challenges, including the problems that can arise with theverificationoftheindextestresultsbythepreferred reference standard test, the choice of cutoff value in case of a continuous index test result, and the determination of how to translate accuracy results to recommendations for clinical use. This first in a series of 4 reports presents an overview of the designs of singletest accuracy studies and the concepts of specificity, sensitivity, posterior probabilities (i.e., predictive values)forthepresenceoftargetdisease,ROCcurves,and