Anne Marie Dupuy

Protein biochip systems for the clinical laboratory.

Abstract Classical methods of protein analysis such as electrophoresis, ELISA and liquid chromatography are generally time-consuming, labor-intensive and lack high-throughput capacity. In addition, all existing methods used to measure proteins necessitate multiple division of the original sample and individual tests carried out for each substance, with an associated cost for each test. The chip system allows several tests to be performed simultaneously without dividing the original patient sample. This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. These emerging technologies fall into two categories: 1) spotted array-based tools, and 2) microfluidic-based tools. Miniaturized and multiplexed immunoassays allow a great deal of information to be obtained from a single sample. These analytical systems are referred to as “lab-on-a-chip” devices. This article presents current trends and advances in miniaturized multiplexed immunoassay technologies, reviewing different systems from research to point-of-care assays. We focus on a subset of chip-based assays that may be used in a clinical laboratory and are directly applicable for biomedical diagnosis. Recent advances in biochip assays combine the power of miniaturization, microfluidics, micro- to nanoparticles, and quantification. A number of applications are just beginning to be explored. The power of biochip assays offers great promise for point-of-care clinical testing and monitoring of many important analytes.

Immunoturbidimetric determination of C-reactive protein (CRP) and high-sensitivity CRP on heparin plasma. Comparison with serum determination

Abstract Determination of C-reactive protein (CRP) and high-sensitivity CRP (hs-CRP), markers of systemic inflammation and atherosclerosis risk, usually requires serum samples, while heparin plasma samples are routinely collected for routine chemistry. This study evaluates the use of heparin plasma samples for hs-CRP and CRP determinations using immunoturbidimetric methods on an Olympus system. As a result, analytical performance, including linearity, imprecision values, and determination from 185 clinical samples, are not affected by the type of sampling, validating the use of heparin plasma samples for CRP and hs-CRP determination with Olympus reagents.

Plasma brain natriuretic peptide and troponin levels in severe sepsis and septic shock: relationships with systolic myocardial dysfunction and intensive care unit mortality.

The aim of this study was to evaluate and compare brain natriuretic peptide (BNP) and cardiac troponin I (cTnI) levels as mortality prognosticator and predictor for myocardial dysfunction in severe sepsis and septic shock. Baseline clinical and biological variables were collected from 47 patients with severe sepsis or septic shock. Ventricular systolic function assessed by echocardiography was measured over a 5-day period. Both cTnI and BNP plasmatic levels were determined at intensive care unit (ICU) admission and during the following 15 days. At admission, cTnI and BNP levels were compared to those of 12 control critically ill nonseptic patients. The plasma levels of BNP and cTnI in patients with sepsis were elevated at admission and significantly higher than in the controls. Among patients with sepsis, BNP levels were significantly more elevated in nonsurvivors compared to survivors at admission and 1 day later. The cTnI levels were also significantly more elevated in nonsurvivors compared to survivors, but only at admission. From admission to day 5, patients with sepsis with left ventricular systolic dysfunction had higher BNP plasmatic concentrations than those without; differences were significant at days 3 and 4. In contrast, plasma cTnI levels were similar between the 2 groups. In critically ill patients, sepsis induces significant increase in BNP and cTnI levels. High BNP and cTnI plasma levels during ICU admission appear to be associated with poor outcome of sepsis. Time course of BNP levels seems helpful to discriminate between surviving and nonsurviving patients with sepsis and to detect myocardial dysfunction where troponin levels fail to do so.

Performance evaluation of human cytokines profiles obtained by various multiplexed-based technologies underlines a need for standardization.

Abstract Background: Multiplexed methods permit simultaneous quantification of multiple cytokines. As several manufacturers offer reagents to quantify the same cytokines on a single instrument, comparison of the distribution should be made to determine whether these data are comparable from one assay to another. Methods: We performed the quantification of cytokines in serum samples with three commercially available assays: Cytometric Bead Array (CBA), Protein Biochip Array Technology (PBAT), and Luminex Technology analysis. Using detection limit and reference range of the three commercial multiplex technologies, we evaluated: 1) the overall distribution of cytokines; and 2) the clinical impact. Results: The three cytokines, IL-1β, IL-1α and IL-4, cannot be measured by these methods because of the high number of non-detected data (>50%). By contrast, four cytokines as IL-8, VEGF, MCP-1 and EGF exhibited a low percentage of non-detected data whatever method was used. The comparison of the percentage of samples with values higher than the respective reference range of each method reported an absence of clinical concordance (Cohen’s κ-test <0.40). Conclusions: Our results highlight the lack of transferability between the three commercially available multiplex methods evaluated (CBA, PBAT and Luminex Technology). Analytical performances are adequate for longitudinal studies using a same methodology but caution should be used for comparisons between results obtained with different methods underlying a need for standardization.

Functional Vitamin E Deficiency in ApoE4 Patients with Alzheimer’s Disease

Oxidative stress has been implicated in the development of Alzheimer’s disease (AD). Consequently, antioxidant therapies including Vitamin E (VitE) supplementation for both prevention and treatment of neurodegenerative diseases currently appears to be a promising avenue of research. The aim of the present study was to examine the relationship between AD and the ApoE phenotype, lipid parameters and VitE levels in a large cohort of elderly subjects. No absolute deficit was observed in plasma VitE levels. However in AD, ApoE4 is not associated with an increase in total cholesterol (TC) and VitE levels. Moreover, our results suggest that oxidative stress-induced injury and protection by VitE in AD are related to the ApoE phenotype. Our study strongly supports the hypothesis of an impairment of lipophilic antioxidant delivery to neuronal cells in AD leading to a tissular antioxidant deficiency which could facilitate oxidative stress.

Diagnostic and prognostic value of soluble CD14 subtype (Presepsin) for sepsis and community-acquired pneumonia in ICU patients.

BackgroundThe soluble CD14 subtype, Presepsin, appears to be an accurate sepsis diagnostic marker, but data from intensive care units (ICUs) are scarce. This study was conducted to evaluate the diagnostic and prognostic value of Presepsin in ICU patients with severe sepsis (SS), septic shock (SSh) and severe community-acquired pneumonia (sCAP).MethodsPresepsin and procalcitonin (PCT) levels were determined for patients at admission to ICU. Four groups have been differentiated: (1) absence or (2) presence of systemic inflammatory response syndrome, (3) SS or (4) SSh; and 2 groups, among the patients admitted for acute respiratory failure: absence or presence of sCAP. Biomarkers were tested for diagnosis of SS, SSh and sCAP and for prediction of ICU mortality.ResultsOne hundred and forty-four patients were included: 44 SS and 56 SSh. Plasma levels of Presepsin and PCT were significantly higher in septic than in non-septic patients and in SSh as compared to others. The sepsis diagnostic accuracy of Presepsin was not superior to that of PCT (AUC: 0.75 vs 0.80). In the 72/144 patients admitted for acute respiratory failure, the capability of Presepsin to diagnose sCAP was significantly better than PCT. Presepsin levels were also predictive of ICU mortality in sepsis and in sCAP patients.ConclusionPlasma levels of Presepsin were useful for the diagnosis of SS, SSh and sCAP and may predict ICU mortality in these patients.

Kinetics of high-sensitivity cardiac troponin T or troponin I compared to creatine kinase in patients with revascularized acute myocardial infarction

Abstract Background: Cardiac biomarkers are the cornerstone of the biological definition of acute myocardial infarction (AMI). The key role of troponins in diagnosis of AMI is well established. Moreover, kinetics of troponin I (cTnI) and creatine kinase (CK) after AMI are correlated to the prognosis. New technical assessment like high-sensitivity cardiac troponin T (hs-cTnT) raises concerns because of its unclear kinetic following the peak. This study aims to compare kinetics of cTnI and hs-cTnT to CK in patients with large AMI successfully treated by percutaneous coronary intervention (PCI). Methods: We prospectively studied 62 patients with anterior AMI successfully reperfused with primary angioplasty. We evaluated two consecutive groups: the first one regularly assessed by both CK and cTnI methods and the second group by CK and hs-cTnT. Modeling of kinetics was realized using mixed effects with cubic splines. Results: Kinetics of markers showed a peak at 7.9 h for CK, at 10.9 h (6.9–12.75) for cTnI and at 12 h for hs-cTnT. This peak was followed by a nearly log linear decrease for cTnI and CK by contrast to hs-cTnT which appeared with a biphasic shape curve marked by a second peak at 82 h. There was no significant difference between the decrease of cTnI and CK (p=0.63). CK fell by 79.5% (76.1–99.9) vs. cTnI by 86.8% (76.6–92.7). In the hs-cTnT group there was a significant difference in the decrease by 26.5% (9–42.9) when compared with CK that fell by 79.5% (64.3–90.7). Conclusions: Kinetic of hs-cTnT and not cTnI differs from CK. The role of hs-cTnT in prognosis has to be investigated.

Evaluation of two automated capillary electrophoresis systems for human serum protein analysis

OBJECTIVES We compared automated capillary electrophoresis (CE) systems Capillarys 2 from Sebia and V8 from Helena Biosciences Europe (Elitech) with the semi-automated Hydrasys-Hyrys agarose gel electrophoresis (AGE) from Sebia. DESIGN AND METHODS We evaluated analytical performances and compared 129 fresh routine sera (group A) to 164 frozen pathologic samples with suspicion or antecedent of monoclonal component (MC) (group B). Immunofixation was then compared with immunotyping provided by both CE systems. RESULTS Analytical performances from both CE systems have proven suitable results for clinical use, with within-run and between-run coefficients of variation inferior or equal to 5.2% and 7.7%, respectively. A good correlation was found between AGE and CE with r-value ranging from 0.81 to 0.96 for both CE systems. We observed high MC detection sensitivities (>85%) of in electrophoretogram readings for both CE systems. MC identification using CE systems provided suitable concordance with immunofixation, although failing to detect some IgM proteins or free light chains. CONCLUSIONS Both Capillarys 2 and V8 are reliable automated CE systems for patient care.

Multi-Marker Strategy in Heart Failure: Combination of ST2 and CRP Predicts Poor Outcome

Natriuretic peptides (BNP and NT-proBNP) are recognized as gold-standard predictive markers in Heart Failure (HF). However, currently ST2 (member of the interleukin 1 receptor family) has emerged as marker of inflammation, fibrosis and cardiac stress. We evaluated ST2 and CRP as prognostic markers in 178 patients with chronic heart failure in comparison with other classical markers such as clinical established parameters but also biological markers: NT-proBNP, hs-cTnT alone or in combination. In multivariate analysis, subsequent addition of ST2 led to age, CRP and ST2 as the only remaining predictors of all-cause mortality (HR 1.03, HR 1.61 and HR 2.75, respectively) as well as of cardiovascular mortality (HR 1.00, HR 2.27 and HR 3.78, respectively). The combined increase of ST2 and CRP was significant for predicting worsened outcomes leading to identify a high risk subgroup that individual assessment of either marker. The same analysis was performed with ST2 in combination with Barcelona score. Overall, our findings extend previous data demonstrating that ST2 in combination with CRP as a valuable tool for identifying patients at risk of death.

Performances of the heart fatty acid protein assay for the rapid diagnosis of acute myocardial infarction in ED patients

OBJECTIVE We sought to evaluate the added value of heart fatty acid protein assay (HFABP) for rapid diagnosis of acute myocardial infarction in a prospective cohort of emergency department (ED) patients with acute chest pain. METHODS High-sensitivity cardiac troponin T (hs-cTnT; Roche Diagnostics, Meylan, France) and HFABP (Randox, Mauguio, France) were blindly assayed from venous blood samples obtained at admission. Diagnosis was made by 2 ED physicians using all available data and serial cardiac troponin I as the biochemical standard. Diagnostic performances of HFABP combined with hs-cTnT were assessed using logistic regression. Analysis was conducted in all patients and in patients without ST-elevation myocardial infarction. RESULTS A total of 181 patients were included (age, 61 ±17 years; male sex, 66%). Acute myocardial infarction occurred in 47 (25.9%) patients, including non-ST-elevation myocardial infarction in 31 (17.1%). The receiver operating characteristic area under the curve was 0.893 for hs-cTnT levels at presentation (95% confidence interval, 0.812-0.974) and 0.908 (95% confidence interval, 0.839-0.977) for the combination of hs-cTnT and HFABP, with no significant (P=.07). Adding HFABP to hs-cTnT increased both sensitivity and negative predictive value (NPV) for non-ST-elevation myocardial infarction diagnosis, with about 13% and 3% increase, respectively, leading to a sensitivity of 97% and an NPV of 99%. CONCLUSION The assessment of HFABP at ED admission adds incremental value to initial hs-cTnT. The increase of sensitivity and NPV without sacrificing the specificity and positive predictive value in patients with chest pain with noncontributive electrocardiogram could potentially allow safe and early rule out of acute myocardial infarction without the need for further serial troponin testing.