Comparison between activated clotting time and anti-activated factor X activity for the monitoring of unfractionated heparin therapy in patients with aortic aneurysm undergoing an endovascular procedure.

OBJECTIVE: Current guidelines recommend administration of unfractionated heparin (UFH) and measurement of activated clotting time (ACT) during endovascular procedures. The aim of this study was to compare ACT and anti-activated factor X (anti-Xa) measurements for monitoring of UFH therapy during an aortic endograft procedure and to assess the association of peak ACT and peak anti-Xa activity with periprocedural bleeding. METHODS: We retrospectively studied 104 patients with aortic aneurysm undergoing endovascular procedures with repeated coagulation measurements. After a UFH bolus, further UFH doses were given according to ACT (target range, ≥250 seconds) in clinical routine, and in parallel to each ACT (Hemochron; Accriva Diagnostics, Newport Beach, Calif) measurement, we determined anti-Xa activity (HemosIL Liquid anti-Xa; Instrumentation Laboratory, Bedford, Mass). UFH redosing was solely based on the ACT measurements. We defined periprocedural bleeding as a drop in hemoglobin level ≥3 g/dL or red blood cell transfusion within 24 hours. RESULTS: After the initial UFH bolus (median, 67 IU/kg body weight), ACT and anti-Xa measurements showed a weak correlation (rs, 0.46; P < .001). Median ACT was 233 seconds (range, 127-374 seconds; interquartile range [IQR], 204-257 seconds); median anti-Xa activity was 1.0 IU/mL (range, 0.5-2.0 IU/mL; IQR, 0.9-1.2 IU/mL). Only 31% of the patients had an ACT value ≥250 seconds, whereas all patients had an anti-Xa activity ≥0.5 IU/mL. Accordingly, ACT triggered redosing of UFH frequently. Consequently, we saw a median total UFH use of 90 IU/kg during the procedure, a median peak ACT of 255 seconds (IQR, 234-273 seconds), and a median peak anti-Xa activity of 1.2 IU/mL (IQR, 1.0-1.4 IU/mL). Periprocedural bleeding occurred in 40 (38%) patients. Peak ACT ≥250 seconds was not associated with bleeding (odds ratio, 1.05; 95% confidence interval, 0.41-2.70; P = .952), whereas peak anti-Xa activity ≥1.2 IU/mL was independently associated with bleeding (odds ratio, 4.95; 95% confidence interval, 1.82-13.48; P = .002). Moreover, no periprocedural thromboembolic event occurred. CONCLUSIONS: In this retrospective study of patients with aortic aneurysm undergoing an endovascular procedure, ACT and anti-Xa measurements showed poor correlation; only increased peak anti-Xa activity was independently associated with periprocedural bleeding, not increased ACT. Our findings also suggest that monitoring of UFH therapy with anti-Xa during aortic endograft procedures may reduce total UFH use. We further speculate that this approach could reduce periprocedural bleeding.

The heart matters in diabetes: 10-Year outcomes of peripheral artery disease.

OBJECTIVES: Mortality rates at 10 years are higher in diabetic patients with chronic lower extremity peripheral arterial disease than in non-diabetic peripheral arterial disease patients. We tested the hypothesis that the predictors of mortality differ between diabetic and non-diabetic peripheral arterial disease patients. METHODS: We studied 331 consecutive patients who were <75 years of age, symptomatic for peripheral arterial disease, and admitted to a tertiary care hospital. Our cohort included 216 patients without diabetes mellitus and 115 with diabetes mellitus. The outcome measure was all-cause mortality at 10 years post-admission. RESULTS: Mortality rates at 10 years were 29% among non-diabetic peripheral arterial disease patients and 58% among diabetic peripheral arterial disease patients. We identified the following independent predictors of death in the 216 peripheral arterial disease patients without diabetes: age ≥65 years (risk ratio: 2.15; 95% confidence interval: 1.28-3.59), ankle brachial index <0.60 mmHg/mmHg (risk ratio: 1.88; 95% confidence interval: 1.14-3.08), history of peripheral arterial disease-specific intervention (risk ratio: 1.81; 95% confidence interval: 1.10-2.97), and high-sensitivity C-reactive protein ≥5.0 mg/L (risk ratio: 2.11; 95% confidence interval: 1.28-3.47). For the 115 peripheral arterial disease patients with diabetes, independent predictors of mortality were as follows: age ≥65 years (risk ratio: 1.72; 95% confidence interval: 1.05-2.83) and amino-terminal pro-B-type natriuretic peptide ≥125 ng/L (risk ratio: 2.10; 95% confidence interval: 1.22-3.60). CONCLUSION: In this study, the predictors of death at 10 years differed between peripheral arterial disease patients with and without diabetes. Among the biomarkers tested, high-sensitivity C-reactive protein was independently associated with outcomes in non-diabetic patients, whereas amino-terminal pro-B-type natriuretic peptide was an independent predictor of death in patients with diabetes. Our findings suggest that in future studies, risk assessment and treatment strategies should be differentially applied to the two peripheral arterial disease subgroups.

Prognostic Value of Inflammatory and Cardiovascular Biomarkers for Prediction of 90-Day All-Cause Mortality after Acute Ischemic Stroke-Results from the Linz Stroke Unit Study.

BACKGROUND: Early outcome prediction after acute ischemic stroke is of great interest. The aim of our study was to evaluate the prognostic value of blood biomarkers in patients with acute ischemic stroke. METHODS: We measured interleukin-6 (IL-6), d-dimer, amino-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity cardiac troponin T, and soluble ST2 plasma concentrations within 24 h after admission to our stroke unit in 721 consecutive acute ischemic stroke patients. End point was 90-day all-cause mortality. RESULTS: During follow-up 81 patients died (11%). In univariate Cox proportional hazards regression analyses with the biochemical markers dichotomized according to median values, all baseline blood biomarkers were strong prognostic markers. However, in the multivariate analysis after adjustment for several clinical variables and the NIH Stroke Scale (NIHSS), only NIHSS >3 [risk ratio (RR) 7.87, 95% CI, 3.61-17.16; P < 0.001], IL-6 > 7 pg/mL (RR 4.09, 95% CI, 2.02-8.29; P < 0.001), and NT-proBNP >447 ng/L (RR 4.88, 95% CI, 2.41-9.88; P < 0.001) remained independent predictors. Using a simple multimarker approach combining these 3 complementary markers, we demonstrated that patients with increased NIHSS, IL-6, and NT-proBNP had the poorest outcome with a mortality rate of 38%, whereas no patient with negative readings for all 3 markers died during follow-up. CONCLUSIONS: In this large cohort of patients with acute ischemic stroke, IL-6 and NT-proBNP at admission were strong and independent prognostic markers for 90-day all-cause mortality, and provided complementary prognostic information to the routinely used stroke severity score NIHSS.

Diagnostic and prognostic accuracy of galectin-3 and soluble ST2 for acute heart failure.

BACKGROUND: We aimed to compare head-to-head the diagnostic and prognostic capabilities of galectin-3, soluble ST2 (sST2) and B-type natriuretic peptide (BNP) for heart failure (HF) in an emergency setting. METHODS: We studied 251 consecutive patients with dyspnoea as a chief compliant presenting to an emergency department. The diagnosis of HF was based on the Framingham score for HF plus echocardiographic evidence of systolic or diastolic dysfunction. All-cause mortality was assessed at one year. Plasma concentrations of galectin-3 and BNP were measured with two commercially available assays from Abbott Diagnostics, plasma concentrations of sST2 were quantified with the Presage ST2 assay. The diagnostic and prognostic accuracies of galectin-3, sST2 and BNP were assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: Of the 251 patients, 137 had dyspnoea attributable to acute HF and 114 had dyspnoea attributable to other reasons. BNP had a higher area under the curve (AUC) for the diagnosis of HF (0.92; 95% CI, 0.87-0.95) than galectin-3 (0.57; 95% CI, 0.51-0.64) and sST2 (0.63; 95% CI, 0.56-0.69). Of the 137 patients with acute HF, 41 died and 96 survived during follow up. The AUC of BNP for the prediction of one-year all-cause mortality in HF patients (0.72; 95% CI, 0.63-0.79) was not different from the AUCs of galectin-3 (0.70; 95% CI, 0.62-0.78) and sST2 (0.75; 95% CI, 0.67-0.82). CONCLUSIONS: In this study, galectin-3, sST2 and BNP were equally useful for the prediction of one-year all-cause mortality in patients with acute HF. However, in contrast to BNP, galectin-3 and sST2 were not useful as an aid in the diagnosis of acute HF in short of breath patients presenting to an emergency department.

Interleukin 6, galectin 3, growth differentiation factor 15, and soluble ST2 for mortality prediction in critically ill patients.

PURPOSE: The aim of this study was to compare the prognostic value of interleukin 6 (IL-6), galectin 3, growth differentiation factor 15 (GDF-15), and soluble ST2 (sST2) in an unselected cohort of critically ill patients. METHODS: During a study period of 1 year, we recruited 530 consecutive patients admitted to a medical intensive care unit of a tertiary care hospital. We examined a combination of inflammatory, renal, and cardiac biomarkers for the prediction of 90-day all-cause mortality. RESULTS: During follow-up, 118 patients died (22%). In univariate analyses, increased IL-6, galectin 3, GDF-15, and sST2 plasma concentrations at baseline were strong prognostic markers. However, in the multivariate models, only IL-6 and sST2 remained independent biomarkers adding additional prognostic information to the routinely used Simplified Acute Physiology Score (SAPS) II. Using a simple multimarker approach, patients with increased SAPS II, IL-6, and sST2 (ie, SAPS II >35, IL-6 >32.3pg/mL, and sST2 >103ng/mL) had the poorest outcome. CONCLUSIONS: In this heterogeneous group of critically ill patients, only SAPS II, IL-6, and sST2 remained independent and additive prognostic markers for 90-day all-cause mortality. A combination of the SAPS II with the 2 complementary biomarkers might provide a valuable tool for risk stratification of critically ill patients.

Mortality rates at 10 years are higher in diabetic than in non-diabetic patients with chronic lower extremity peripheral arterial disease.

Patients with lower extremity peripheral artery disease (PAD) have a substantially increased risk for mortality as compared to healthy individuals. We aimed to evaluate the risk for all-cause mortality in PAD patients and in healthy controls during a 10-year follow-up period. Our hypothesis was that the mortality rates at 10 years would differ in diabetic and non-diabetic PAD patients. Our study group consisted of 331 consecutive patients with symptomatic PAD <75 years of age admitted to a tertiary care hospital, including 216 patients without diabetes and 115 with diabetes. Control subjects without atherosclerotic disease were matched to the patients in a 1:1 design by sex, age, and diabetes mellitus status. The outcome measure was all-cause mortality at 10 years. Mortality rates at 10 years were 29% in non-diabetic PAD patients versus 14% in age- and sex-matched non-diabetic controls (risk ratio (RR), 2.31; 95% confidence interval (CI), 1.54-3.47; p<0.001), and 58% in diabetic PAD patients versus 19% in age- and sex-matched diabetic controls (RR, 4.06; 95% CI, 2.67-6.18; p<0.001). Further, PAD patients with diabetes had a significantly increased risk for death within 10 years than did the non-diabetic PAD patients (RR, 2.51; 95% CI, 1.72-3.66; p<0.001). Diabetes was independently associated with outcome, and was the strongest predictor of death in multivariate Cox proportional hazards regression. We conclude that mortality rates at 10 years differ in PAD patients <75 years old with and without diabetes. Our findings suggest that future studies should apply distinct risk assessment strategies in the two PAD subgroups.

Reference values of galectin-3 and cardiac troponins derived from a single cohort of healthy blood donors.

BACKGROUND: Here we describe the determination of upper reference limits (URL) for galectin-3, high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT) in a single cohort of healthy blood donors using routine assays. METHODS: For this reference value study, we used a cohort of 402 consecutive blood donors (64% were male and 36% were female). The median individuals' age was 35.0 years (range, 18.0-64.4). Individuals of this reference population were free of cardiovascular disease, diabetes mellitus, renal disease, cancer, current infection and chronic inflammatory disease. Plasma concentrations of galectin-3 were measured with the "routine Galectin-3" assay (Abbott Diagnostics), of hs-cTnI with the "STAT High Sensitive Troponin-I" assay (Abbott Diagnostics), and of hs-cTnT with the "Troponin T hs" assay (Roche Diagnostics). URLs were calculated by using a non-parametric percentile method. RESULTS: The 97.5th percentile URL for galectin-3 was 16 ng/mL in males and 17 ng/mL in females; the 99 th percentile URL for hs-cTnI was 39 ng/L in males and 24 ng/L in females; and the 99 th percentile URL for hs-cTnT was 14 ng/L in males and 11 ng/L in females. Those individuals with hs-cTnI values ≥ 15 ng/L (n=8) were different from those individuals with hs-cTnT values ≥ 10 ng/L (n=7). Of the 402 individuals, none had galectin-3 values below the limit of detection (LOD, <1.0 ng/mL), 290 (72%) had hs-cTnI values below the LOD (i.e., 1.9 ng/L), and 359 (89%) had hs-cTnT values below the LOD (i.e., 5.0 ng/L). CONCLUSION: Plasma concentrations of galectin-3, hs-cTnI and hs-cTnT and corresponding 99 th percentile URLs were rather low in our cohort of healthy blood donors compared with previously published data. In our reference population, analyte plasma concentrations above the LOD were detectable in 100% of the individuals with the Abbott galectin-3 assay, but only in less than 50% for both the Abbott hs-cTnI assay and the Roche hs-cTnT assay.

Analytical and clinical evaluation of a rapid quantitative lateral flow immunoassay for measurement of soluble ST2 in human plasma.

BACKGROUND: Soluble ST2 (sST2) is gaining growing interest as a biomarker in heart failure. So far, the ELISA-format is widely used for commercially available ST2 assays, which hampers their use in clinical routine. Recently, a rapid quantitative lateral flow immunoassay for the measurement of sST2 in human plasma has been developed. METHODS: We evaluated precision and linearity of the ASPECT-PLUS ST2 test, and performed an analytical and clinical assay comparison with the MBL and the PRESAGE ST2 ELISAs. We measured sST2 with these three assays in a clinical cohort of 251 consecutive patients with acute dyspnea as the chief compliant (i.e., 137 patients with dyspnea attributable to heart failure and 114 patients with dyspnea attributable to other reasons). RESULTS: Within-run and total coefficients of variation of the ASPECT-PLUS ST2 test were < 17% and the assay was linear across its measurement range. We found a constant and proportional bias between the MBL ST2 assay, the PRESAGE ST2 assay and the ASPECT-PLUS ST2 test, respectively. However, at the proposed cut-off of 35 ng/mL, sST2 results obtained with the PRESAGE ST2 assay and the ASPECT-PLUS ST2 test were similar. Testing clinically, the three assays deemed equally useful for the diagnosis of heart failure (AUC, 0.670 for the MBL ST2 assay vs. 0.626 for the PRESAGE ST2 assay vs. 0.630 for the ASPECT-PLUS ST2 test) and for the prediction of 1-year mortality in dyspnoeic patients (AUC, 0.743 for the MBL assay vs. 0.742 for the PRESAGE ST2 assay vs. 0.752 for the ASPECT-PLUS ST2 test). CONCLUSION: The ASPECT-PLUS test meets the analytical requirements for point-of-care testing. Test results of the ASPECT-PLUS ST2 and the PRESAGE ST2 methods were comparable at the proposed cut-off, and the diagnostic/prognostic capabilities of the three methods were similar.

Plasma concentrations of novel cardiac biomarkers before and after hemodialysis session.

OBJECTIVES: Biomarkers are useful for establishing disease severity or prognosis in patients with chronic kidney disease. The aim of our study was to determine the plasma concentrations of novel cardiovascular biomarkers in patients on chronic hemodialysis in the context of published upper reference limits (URL) of these biomarkers; and to compare the plasma concentrations of those same analytes before and after hemodialysis session. DESIGN AND METHODS: Plasma concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP), mid-regional pro-A-type natriuretic peptide (MR-proANP), mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1), C-terminal pro-arginine vasopressin (CT-proAVP, also known as Copeptin) and soluble ST2 (sST2) were measured in 28 patients before and after dialysis session. Of the 28 patients with conventional hemodialysis, 24 had low-flux hemofiltration and 4 had high-flux hemodiafiltration. RESULTS: Median plasma concentrations of the biomarkers obtained before hemodialysis were as follows: NT-proBNP, 11,307ng/L (URL, 500ng/L); MR-proANP, 778pmol/L (URL, 250pmol/L); MR-proADM, 2.57nmol/L (URL, 0.52nmol/L); median CT-proET-1, 252pmol/L (URL, 75pmol/L); median CT-proAVP, 142pmol/L (URL, 19pmol/L); and median sST2, 27ng/mL (URL, 50ng/mL). Median relative analyte changes after low-flux vs. high-flux dialysis compared to predialysis values were +19% vs. -43% for NT-proBNP; +7% vs. -45% for MR-proANP; -2% vs. -63% for MR-proADM; -19% vs. -61% for CT-proET-1; +13% vs. -64% for CT-proAVP; and +2% vs. +3% for sST2. CONCLUSIONS: Plasma concentrations of the investigated biomarkers were markedly increased in chronic hemodialysis patients (with the exception of sST2). After hemodialysis session, analyte concentrations (with the exception of sST2) decreased significantly using a high-flux membrane but not if using a low-flux membrane.

Association of the biomarkers soluble ST2, galectin-3 and growth-differentiation factor-15 with heart failure and other non-cardiac diseases.

BACKGROUND: The biomarkers soluble ST2 (sST2), galectin-3, and growth-differentiation factor-15 (GDF-15) provide prognostic information in patients with heart failure (HF). The aim of this study was to evaluate to which extent plasma concentrations of these biomarkers are increased in HF compared with diverse non-cardiac conditions such as infectious disease or chronic kidney disease. METHODS: We recruited 15 patients in each of the following clinical categories: HF without co-morbidity, pneumonia without co-morbidity, chronic obstructive pulmonary disease (COPD) without co-morbidity, HF and a co-morbidity of pneumonia, renal disease without co-morbidity, and sepsis. We used 22 healthy individuals as control group. In each of the 112 study participants, we measured plasma concentrations of sST2 (Presage assay), galectin-3 (Abbott assay) and GDF-15 (Roche assay). RESULTS: Compared to controls, the median sST2 concentration was ~2.5-fold increased in HF, ~3.5-fold in pneumonia, ~5.0-fold in COPD, ~5.8-fold in HF+pneumonia, and ~70-fold in sepsis (p<0.001 for all). sST2 was not significantly increased in renal disease. Compared to controls, the median galectin-3 concentration was ~1.5-fold increased in HF, ~1.4-fold in pneumonia, ~2.4-fold in HF+pneumonia, ~2.5-fold in renal disease, and ~2.7-fold in sepsis (p<0.001 for all). Galectin-3 was not significantly increased in COPD. Compared to controls, the median GDF-15 concentration was ~4.4-fold increased in HF, ~5.4-fold in pneumonia, ~2.1-fold in COPD, ~8.3-fold in HF+pneumonia, ~5.1-fold in renal disease, and ~27-fold in sepsis (p<0.001). In the 112 study participants, correlation analyses revealed a relatively strong association between galectin-3 and GDF-15 (correlation coefficient, 0.739; p<0.001). CONCLUSION: Because increased plasma concentrations of sST2, galectin-3, and GDF-15 are not specific for a distinct disease group, the three biomarkers are not useful for diagnostic purposes. The results of our study are novel with respect to sST2, galectin-3 and GDF-15 as markers of inflammatory diseases and should encourage further studies.

Mortality rates and mortality predictors in patients with symptomatic peripheral artery disease stratified according to age and diabetes.

OBJECTIVE: Atherosclerotic peripheral arterial disease (PAD) is one of the most prevalent, morbid, and mortal diseases. The aim of this study was to evaluate mortality rates of patients with atherosclerotic PAD stratified according to age and diabetes and to determine predictors of death. METHODS: We studied 487 patients with symptomatic PAD consecutively admitted to the hospital. This cohort included the following four patient subgroups: (1) 216 patients with PAD <75 years of age without diabetes mellitus; (2) 115 patients with PAD < 75 years of age with diabetes mellitus; (3) 102 patients with PAD ≥ 75 years of age without diabetes mellitus; and (4) 54 patients with PAD ≥ 75 years of age with diabetes mellitus. Control subjects without atherosclerotic disease were matched to the patients with PAD in a 1:1 design by sex, age (± 2 years), and diabetes mellitus status. Outcome measure was all-cause mortality at 5 years. RESULTS: Mortality rates at 5 years were 10% in nondiabetic patients with PAD < 75 years of age (vs 5% in control subjects; risk ratio [RR], 2.15; 95% confidence interval [CI], 1.60-4.34); 23% in diabetic patients with PAD < 75 years of age (vs 7% in control subjects; RR, 3.53; 95% CI, 1.80-6.91); 38% in nondiabetic patients with PAD ≥ 75 years of age (vs 22% in control subjects; RR, 2.08; 95% CI, 1.26-3.44); and 52% in diabetic patients with PAD ≥ 75 years of age. Applying multivariate Cox proportional hazards regression analyses (with cardiovascular risk factors, coexisting atherosclerotic disease, clinical stage of PAD, and several biochemical markers as predictor variables), we found the following independent predictors of outcome: in the 216 nondiabetic patients with PAD < 75 years of age, high-sensitivity C-reactive protein (hs-CRP) (RR, 3.04; 95% CI, 1.48-6.26); in the 115 diabetic patients with PAD < 75 years of age, amino-terminal pro-B-type natriuretic peptide (NT-proBNP) (RR, 2.63; 95% CI, 1.65-4.19); in the 102 nondiabetic patients with PAD ≥ 75 years of age, critical limb ischemia (RR, 3.70; 95% CI, 1.82-7.52) and NT-proBNP (RR, 1.93; 95% CI, 1.32-2.82); and in the 54 diabetic patients with PAD ≥ 75 years of age, hs-CRP (RR, 2.61; 95% CI, 1.45-4.67) and NT-proBNP (RR, 3.31; 95% CI, 1.96-5.60). CONCLUSIONS: Mortality rates at 5 years varied considerably among patients with PAD stratified according to age and diabetes. Predictors of death differed among the four patient subgroups in this study and included critical limb ischemia, hs-CRP, and NT-proBNP. Our results might help to develop future strategies for optimized treatment of hospitalized patients with symptomatic PAD.

Increased soluble ST2 predicts long-term mortality in patients with stable coronary artery disease: results from the Ludwigshafen risk and cardiovascular health study.

BACKGROUND: Soluble suppression of tumorigenicity 2 (sST2) has emerged as a strong prognostic biomarker in patients with heart failure and myocardial infarction. The aim of this study was to evaluate the long-term prognostic value of sST2 in patients with stable coronary artery disease (CAD). METHODS: sST2 plasma concentrations were measured in 1345 patients with stable CAD referred for coronary angiography at a single tertiary care center. The primary endpoint was all-cause mortality. RESULTS: During a median follow-up time of 9.8 years, 477 (36%) patients died. The median sST2 plasma concentration at baseline was significantly higher among decedents than survivors (21.4 vs 18.5 ng/mL; P < 0.001). In multivariate Cox proportional hazards regression analysis, sST2 was an independent predictor of all-cause mortality (risk ratio 1.16 per 1-SD increase in log-transformed values; 95% CI 1.05-1.29; P = 0.004). In the same multivariate analysis, amino-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) were also independent predictors, whereas galectin-3 was not. Patients with sST2 in the highest quartile (>24.6 ng/mL) displayed a 2-fold increased risk of death in univariate analysis, which was attenuated but remained significant in a fully adjusted model (risk ratio 1.39; 95% CI 1.10-1.76; P = 0.006). Further analysis showed that the prognostic impact of sST2 was additive to NT-proBNP and hs-cTnT. Using a multibiomarker approach combining these 3 complementary makers, we demonstrated that patients with all 3 biomarkers in the highest quartiles had the poorest outcome. CONCLUSIONS: In this cohort of patients with stable CAD, increased sST2 was an independent predictor of long-term all-cause mortality and provided complementary prognostic information to hs-cTnT and NT-proBNP.

Analytical characterization and clinical evaluation of an enzyme-linked immunosorbent assay for measurement of afamin in human plasma.

BACKGROUND: Comparative proteomics has recently identified afamin, the newest member of the albumin gene family, as a potential biomarker for ovarian cancer. The aim of this study was the analytical and clinical evaluation of a sandwich enzyme-linked immunosorbent assay for the determination of afamin in human plasma. METHODS: We evaluated precision, linearity, and detection limit of the assay, analyte stability and biological variability, determined reference values and quantified afamin concentrations in various diseases. RESULTS: Within-run and total coefficients of variation were <10%. The method was linear across the tested measurement range. Detection limit was 7 mg/L for the assay. The analyte was stable for 24 h at room temperature, for 48 h at 4°C, and for at least one year at -20°C and -80°C. The reference change value for healthy individuals was 24%. Age- and sex-independent reference values in healthy blood donors were 45-99 mg/L (median 68 mg/L). In the clinical assay evaluation afamin plasma concentrations were modestly decreased in patients with heart failure. Patients with pneumonia or sepsis exhibited markedly decreased afamin plasma concentrations. However, patients with chronic renal disease or chronic obstructive pulmonary disease showed no difference in afamin plasma concentrations as compared to healthy individuals. Correlation analyses revealed an inverse association between afamin and inflammatory biomarkers. CONCLUSIONS: The afamin assay meets quality specifications for laboratory medicine. The results of the clinical assay evaluation revealed novel insights with respect to afamin as a potential negative acute phase protein and should encourage further studies.

Genetic determinants of the ankle-brachial index: a meta-analysis of a cardiovascular candidate gene 50K SNP panel in the candidate gene association resource (CARe) consortium.

BACKGROUND: Candidate gene association studies for peripheral artery disease (PAD), including subclinical disease assessed with the ankle-brachial index (ABI), have been limited by the modest number of genes examined. We conducted a two stage meta-analysis of ∼50,000 SNPs across ∼2100 candidate genes to identify genetic variants for ABI. METHODS AND RESULTS: We studied subjects of European ancestry from 8 studies (n=21,547, 55% women, mean age 44-73 years) and African American ancestry from 5 studies (n=7267, 60% women, mean age 41-73 years) involved in the candidate gene association resource (CARe) consortium. In each ethnic group, additive genetic models were used (with each additional copy of the minor allele corresponding to the given beta) to test each SNP for association with continuous ABI (excluding ABI>1.40) and PAD (defined as ABI<0.90) using linear or logistic regression with adjustment for known PAD risk factors and population stratification. We then conducted a fixed-effects inverse-variance weighted meta-analyses considering a p<2×10(-6) to denote statistical significance. RESULTS: In the European ancestry discovery meta-analyses, rs2171209 in SYTL3 (ß=-0.007, p=6.02×10(-7)) and rs290481 in TCF7L2 (ß=-0.008, p=7.01×10(-7)) were significantly associated with ABI. None of the SNP associations for PAD were significant, though a SNP in CYP2B6 (p=4.99×10(-5)) was among the strongest associations. These 3 genes are linked to key PAD risk factors (lipoprotein(a), type 2 diabetes, and smoking behavior, respectively). We sought replication in 6 population-based and 3 clinical samples (n=15,440) for rs290481 and rs2171209. However, in the replication stage (rs2171209, p=0.75; rs290481, p=0.19) and in the combined discovery and replication analysis the SNP-ABI associations were no longer significant (rs2171209, p=1.14×10(-3); rs290481, p=8.88×10(-5)). In African Americans, none of the SNP associations for ABI or PAD achieved an experiment-wide level of significance. CONCLUSIONS: Genetic determinants of ABI and PAD remain elusive. Follow-up of these preliminary findings may uncover important biology given the known gene-risk factor associations. New and more powerful approaches to PAD gene discovery are warranted.

Prognostic value of soluble ST2 in an unselected cohort of patients admitted to an intensive care unit - The Linz Intensive Care Unit (LICU) study.

BACKGROUND: Soluble ST2 (sST2) has emerged as a prognostic biomarker in patients with heart disease. We tested the hypothesis that sST2 is an independent predictor of mortality in patients admitted to an intensive care unit (ICU). METHODS: We performed measurements of sST2 plasma concentrations in 530 consecutive patients admitted to a medical ICU of a tertiary care hospital during a study period of one year. The patients recruited during the first six months were used for the derivation cohort (n=274) and the patients recruited during the second six months were used for the validation cohort (n=256). The endpoint was defined as 90-day all-cause mortality. RESULTS: In the derivation cohort, sST2 was higher among decedents (n=56; median, 146 U/mL) than survivors (n=218; median 42 U/mL, p<0.001). In multivariate Cox proportional-hazard regression analysis (offering age, sex, BMI, APACHE II score, SAPS II, CRP, IL-6, PCT, creatinine, total cholesterol, albumin, hs-cTnT, BNP and sST2 as independent variables), sST2 was a significant predictor of mortality (risk ratio 1.48, 95% CI 1.15-1.90; p=0.002 per 1 SD increase in log transformed values). In this statistical model, only sST2 and SAPS II contributed independently to mortality prediction. We further observed an additive effect of an sST2 plasma concentration of >84 U/mL and an increased SAPS II for mortality prediction. The findings from the derivation cohort were confirmed in the independent validation cohort. In those patients with a length of stay of >48 h at the ICU (n=225), sST2 obtained two days after baseline measurement had a better capability than baseline sST2 to predict mortality. CONCLUSIONS: In an unselected cohort of patients admitted to the ICU, sST2 was an independent predictor of 90-day all-cause mortality and added prognostic information to the SAPS II.

Proguanylin and prouroguanylin--assay evaluation and clinical analyte characterization.

BACKGROUND: The biomarkers proguanylin and prouroguanylin are members of the natriuretic peptide family. The aim of this study was to evaluate two commercially available assays for proguanylin and prouroguanylin and to further characterize both analytes in terms of important clinical features. METHODS: We evaluated precision and linearity of the BioVendor human proguanylin and prouroguanylin ELISAs. In order to characterize both analytes, we tested in vitro analyte stabilities at -80 °C, and determined biological variability and reference values for proguanylin and prouroguanylin. RESULTS: Within-run and total coefficients of variation were <10% for the BioVendor proguanylin and prouroguanylin assays. Both methods were linear across the tested measurement ranges. The analytes proguanylin and prouroguanylin were stable for at least 2 months at -80 °C. With respect to biological variability, the reference change values (RCV) were 27% and 59% for proguanylin and prouroguanylin, respectively. For proguanylin, age-independent reference values were 4.0-13.4 ng/mL in males and 4.6-16.3 ng/mL in females. For prouroguanylin, age- and sex-independent reference values were 2.1-11.2 ng/mL. CONCLUSION: The BioVendor human proguanylin ELISA and the BioVendor human prouroguanylin ELISA meet the needs of quality specifications of laboratory medicine. The results of the characterization of both analytes provide essential information for further clinical studies.

Soluble ST2 is not independently associated with androgen and estrogen status in healthy males and females.

BACKGROUND: Soluble ST2 (sST2) plasma concentrations are significantly higher in healthy men than in healthy women. The reason for the sex-specific difference of sST2 plasma concentrations is not established. The aim of this study was to evaluate the association of sST2 with sex-hormones in healthy males and females separately. METHODS: We recruited 528 consecutive blood donors and measured plasma concentrations of sST2 and several sex-hormones (i.e., total testosterone, estradiol, sex hormone-binding globulin, follicle-stimulating hormone, and luteinizing hormone). Of the 528 blood donors, 338 were male and 190 were female. For data analysis, we further divided the group of females into the subgroups of pre- and postmenopausal women using the age of 50 years as a proxy for menopause. RESULTS: In non-parametric Spearman's correlation analyses, we found a weak association between sST2 and total testosterone (r(s)+0.126, p=0.021) and also between sST2 and estradiol (r(s)+0.117, p=0.032) in males. In females <50 years of age (n=158) and ≥50 years of age (n=32), respectively, we did not detect any significant association between sST2 and sex-hormones. As a result of multiple linear regression analyses (calculated with log sST2 as dependent variable and log of age and all sex-hormones as explanatory variables), there was no independent association between sST2 and any of the sex-hormones neither in males nor in females. CONCLUSIONS: In the present study cohort we did not find an independent association of sST2 with sex-hormones in healthy males and females. Therefore, the reason for the sex-specific difference of sST2 plasma concentrations still remains unclear.

Long-term stability of soluble ST2 in frozen plasma samples.

OBJECTIVE: The aim of this study was to investigate the long-term in vitro stability of soluble ST2 (sST2). DESIGN AND METHODS: EDTA plasma samples were drawn from 15 individuals with various diseases. The Presage ST2 assay was used for measurement of sST2 concentrations directly after blood collection and after storing plasma samples for 18 months at -20 degrees C and -80 degrees C. The default criterion for analyte stability was set at 95%. RESULTS: sST2 concentrations in the 15 individuals ranged from 12 U/mL to 140 U/mL. Directly after blood collection, the mean (+/-SD) sST2 concentration was 51+/-37 U/mL, and absolute analyte recoveries were 50+/-35 U/mL and 51+/-34 U/mL after storage of samples for 18 months at -20 degrees C and -80 degrees C, respectively. Relative analyte recoveries after 18 months of storage at -20 degrees C and -80 degrees C were 99+/-5% and 101+/-7%. CONCLUSION: sST2 is stable for at least 1.5 years in plasma samples stored at -20 degrees C and -80 degrees C.