A multicenter comparison of established and emerging cardiac biomarkers for the diagnostic evaluation of chest pain in the emergency department.

BACKGROUND: The aim of this study is to assess the role of novel biomarkers for the diagnostic evaluation of acute coronary syndrome (ACS). METHODS: Among 318 patients presenting to an emergency department with acute chest discomfort, we evaluated the diagnostic value of 5 candidate biomarkers (amino terminal pro-B-type natriuretic peptide [NT-proBNP], ischemia modified albumin, heart fatty acid binding protein, high-sensitivity troponin I [hsTnI], and unbound free fatty acids [FFAu]) for detecting ACS, comparing their results with that of conventional troponin T (cTnT). RESULTS: Sixty-two subjects (19.5%) had ACS. The sensitivity and negative predictive values of NT-proBNP (73%, 90%) and hsTnI (57%, 89%) were higher than that of cTnT (22%, 84%). Unbound free fatty acids had the highest overall combination of sensitivity (75%), specificity (72%), and negative predictive values (92%) of all the markers examined. A significant increase in the C-statistic for cTnT resulted from the addition of results for NT-proBNP (change 0.09, P = .001), hsTnI (change 0.13, P < .001), and FFAu (change 0.15, P < .001). In integrated discrimination improvement and net reclassification improvement analyses, NT-proBNP, hsTnI, and FFAu added significant diagnostic information to cTnT; when changing the diagnostic criterion standard for ACS to hsTnI, FFAu still added significant reclassification for both events and nonevents. In serial sampling (n = 180), FFAu added important reclassification information to hsTnI. CONCLUSION: Among emergency department patients with symptoms suggestive of ACS, neither ischemia modified albumin nor heart fatty acid binding protein detected or excluded ACS, whereas NT-proBNP, hsTnI, or FFAu added diagnostic information to cTnT. In the context of hsTnI results, FFAu measurement significantly reclassified both false negatives and false positives at baseline and in serial samples.

Stacking due to ionic transport number mismatch during sample sweeping on microchips.

Sample stacking can occur in isoconductive buffer systems as a result of ion transport mismatches that cause changes in buffer conductivity during electrophoresis. Fluorescence imaging was used to examine this effect in the sweeping of hydrophobic dyes with sodium dodecyl sulfate (SDS) on microchips. Imaging revealed the occurrence of a stacking effect in a sodium borate buffer system in which the sample buffer and SDS-containing run buffer had the same initial conductivity. Injected sample plugs were first swept by SDS micelles and the swept band was then stacked at the trailing end of the sample zone. This effect is due to changes in conductivity at both the front and back interfaces of the injected sample plug and can be modeled by moving boundary equations. Maximum signal enhancements of 86-, 160- and 560-fold were obtained for Rhodamine 560, Rhodamine B and Rhodamine 6G, respectively, by the combination of sweeping and stacking within a 1 cm section of microchannel. Based on sample sweeping/stacking and manipulation of the electric field polarity, a method of trapping and concentrating analyte from multiple injections was also demonstrated.

Detection of exercise-induced ischemia by changes in B-type natriuretic peptides.

OBJECTIVES: The purpose of this study was to examine the effect of exercise-induced ischemia on levels of B-type natriuretic peptide (BNP) and its inactive N-terminal fragment (NT-pro-BNP)and to determine whether measurement of these peptides can improve the diagnostic accuracy of exercise testing. BACKGROUND: The ability of exercise testing to detect coronary artery disease (CAD) is limited by modest sensitivity and specificity. B-type natriuretic peptides (NT-pro-BNP and BNP) are released by ventricular myocytes in response to wall stress. We hypothesized that exercise-induced ischemia results in increased wall stress and triggers release of NT-pro-BNP and BNP. METHODS: A total of 74 patients with known CAD, normal left ventricular function, and normal resting levels of NT-pro-BNP and BNP who were referred for exercise testing with radionuclide imaging, and 21 healthy volunteers, were enrolled. Blood was drawn before and after maximal exercise and analyzed for NT-pro-BNP and BNP. RESULTS: Of the patients with CAD, 40 had ischemia on perfusion images and 34 did not. Median post-exercise increases in NT-pro-BNP and BNP (DeltaNT-pro-BNP and DeltaBNP) were approximately four-fold higher in the ischemic group than in the nonischemic group (DeltaNT-pro-BNP 14.5 vs. 4 pg/ml, p < 0.0001; DeltaBNP 36.5 vs. 7.5 pg/ml, p < 0.0001). In volunteers, median DeltaNT-pro-BNP was almost identical to that of the nonischemic patient group. At equal specificity to the electrocardiogram (ECG) (58.8%), the sensitivities of DeltaNT-pro-BNP and DeltaBNP for detecting ischemia were 90% and 80%, respectively; in contrast, the sensitivity of the exercise ECG was 37.5%. CONCLUSIONS: Measurement of exercise-induced increases in BNPs more than doubles the sensitivity of the exercise test for detecting ischemia with no loss of specificity.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease.

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne's disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a large degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD-negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested using commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads using laser-induced fluorescence. Antigen-coated magnetic beads treated with the bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples with the microfluidic system were compared to those analyzed with the flow cytometer, a high level of correlation (linear regression, r(2)=0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD.

Can exercise-induced changes in B-type natriuretic peptides be used to detect cardiac ischemia?

BACKGROUND: We reviewed the current medical literature that pertained to the question of whether myocardial ischemia triggers the release of B-type natriuretic peptides (BNPs) and, in particular, whether transient exercise-induced ischemia can be detected by the measurement of changes in these biomarkers. BNPs are well-established as markers of left ventricular dysfunction, particularly heart failure. There is accumulating evidence that various conditions with the common denominator of myocardial ischemia are also associated with increased circulating levels of these peptides. METHODS AND RESULTS: Recently published methods and results, which includes our published and unpublished data, were reviewed. CONCLUSION: The results show that exercise-induced ischemia or its associated regional wall-motion abnormalities trigger the release of BNPs and that the measurement of plasma levels of N-terminal pro brain natriuretic peptide and BNP before and immediately after symptom-limited exercise can distinguish patients with and without ischemia with a high degree of accuracy.

Preconcentration of proteins on microfluidic devices using porous silica membranes.

Fluorescently labeled proteins were electrophoretically concentrated on microfabricated devices prior to separation and laser-induced fluorescence detection on the same device. The proteins were concentrated using a porous silica membrane between adjacent microchannels that allowed the passage of buffer ions but excluded larger migrating molecules. Concentrated analytes were then injected into the separation column for analysis. Two basic microchip designs were tested that allowed sample concentration either directly in the sample injector loop or within the microchannel leading from the sample reservoir to the injector. Signal enhancements of approximately 600-fold were achieved by on-chip preconcentration followed by SDS-CGE separation. Preconcentration for CE analysis in both coated and uncoated open channels was also demonstrated. Fluorescently labeled ovalbumin could be detected at initial concentrations as low as 100 fM by using a combination of field-amplified injection and preconcentration at a membrane prior to CE in coated channels.