High-Sensitivity Troponin Assays in Clinical Diagnostics of Acute Coronary Syndrome.

Nowadays, measurement of cardiac troponins (cTn) in patient plasma is central for diagnosis of patients with acute coronary syndrome (ACS). High-sensitivity (hs) immunoassays have been developed that can very precisely record slightly elevated and rising plasma concentrations of cTn very early after onset of clinical symptoms. Algorithms integrate measurements of hs-cTn at onset of clinical symptoms of acute myocardial infarction (AMI), and 1 or 3 h after onset, to rule-in and rule-out AMI patients. More and more point-of-care (POC) cTn assays conquer the diagnostic market, but thorough clinical validation studies are required before potential implementation of such POC tests into hospital settings. This review provides an overview of the technical aspects, as well as diagnostic and prognostic use of cardiac troponins in AMI patients and in the healthy population.

Prevalence and causes of abnormal PSA recovery.

BACKGROUND: Prostate-specific antigen (PSA) test is of paramount importance as a diagnostic tool for the detection and monitoring of patients with prostate cancer. In the presence of interfering factors such as heterophilic antibodies or anti-PSA antibodies the PSA test can yield significantly falsified results. The prevalence of these factors is unknown. METHODS: We determined the recovery of PSA concentrations diluting patient samples with a standard serum of known PSA concentration. Based on the frequency distribution of recoveries in a pre-study on 268 samples, samples with recoveries <80% or >120% were defined as suspect, re-tested and further characterized to identify the cause of interference. RESULTS: A total of 1158 consecutive serum samples were analyzed. Four samples (0.3%) showed reproducibly disturbed recoveries of 10%, 68%, 166% and 4441%. In three samples heterophilic antibodies were identified as the probable cause, in the fourth anti-PSA-autoantibodies. The very low recovery caused by the latter interference was confirmed in serum, as well as heparin- and EDTA plasma of blood samples obtained 6 months later. Analysis by eight different immunoassays showed recoveries ranging between <10% and 80%. In a follow-up study of 212 random plasma samples we found seven samples with autoantibodies against PSA which however did not show any disturbed PSA recovery. CONCLUSIONS: About 0.3% of PSA determinations by the electrochemiluminescence assay (ECLIA) of Roche diagnostics are disturbed by heterophilic or anti-PSA autoantibodies. Although they are rare, these interferences can cause relevant misinterpretations of a PSA test result.

Rule-out of non-ST elevation myocardial infarction by five point of care cardiac troponin assays according to the 0 h/3 h algorithm of the European Society of Cardiology.

BACKGROUND: Point of care (POC) assays for cardiac troponins I or T (cTnI or cTnT) may accelerate the diagnosis of patients with suspected acute coronary syndrome (ACS). However, their clinical utility according to the 0 h/3 h algorithm recommended by the European Society of Cardiology (ESC) for non-ST elevation myocardial infarction (NSTEMI) is unknown. METHODS: Blood samples from 90 patients with suspected ACS were obtained at hospital admission and 3 h later. Concentrations of cTn were determined using five POC assays (AQT90 FLEX cTnI and cTnT; PATHFAST™ cTnI; Stratus CS 200 cTnI; and Triage MeterPro cTnI) and two guideline-acceptable high-sensitivity (hs) immunoassays. RESULTS: For the diagnosis of NSTEMI (n=15), AUCs for Abbott hs-cTnI and Roche hs-cTnT were 0.86 [95% confidence interval (CI), 0.75-0.96] and 0.88 (95% CI, 0.80-0.95), respectively, at admission, and 0.96 and 0.94, respectively, 3 h later. With the 99th percentile cutoff, their sensitivities were 62% and 92%, respectively, at admission, and 77% and 100%, respectively, 3 h later. The PATHFAST™ cTnI assay showed AUCs of 0.90 (95% CI, 0.82-0.97) and 0.94 (95% CI, 0.89-1.00), respectively, and sensitivities of 67% and 75% at admission and 3 h later, respectively. The other cTn POC assays had AUCs of 0.71 (95% CI, 0.53-0.89) to 0.84 (95% CI, 0.71-0.96) and 0.86 (95% CI, 0.72-0.99) to 0.87 (95% CI, 0.75-0.99) and sensitivities of 39%-50% and 62%-77% at admission and 3 h later, respectively. CONCLUSIONS: PATHFAST™ cTnI assay proved itself as comparable to ESC-guideline acceptable hs-cTn assays. The lower sensitivity of the other POC assays limits their clinical utility and would require longer follow-up monitoring of patients for the safe NSTEMI rule-out.

Circulating FABP4 is a prognostic biomarker in patients with acute coronary syndrome but not in asymptomatic individuals.

OBJECTIVE: Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS: Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS: Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS.

Clinical criteria replenish high-sensitive troponin and inflammatory markers in the stratification of patients with suspected acute coronary syndrome.

OBJECTIVES: In patients with suspected acute coronary syndrome (ACS), rapid triage is essential. The aim of this study was to establish a tool for risk prediction of 30-day cardiac events (CE) on admission. 30-day cardiac events (CE) were defined as early coronary revascularization, subsequent myocardial infarction, or cardiovascular death within 30 days. METHODS AND RESULTS: This single-centre, prospective cohort study included 377 consecutive patients presenting to the emergency department with suspected ACS and for whom troponin T measurements were requested on clinical grounds. Fifteen biomarkers were analyzed in the admission sample, and clinical parameters were assessed by the TIMI risk score for unstable angina/Non-ST myocardial infarction and the GRACE risk score. Sixty-nine (18%) patients presented with and 308 (82%) without ST-elevations, respectively. Coronary angiography was performed in 165 (44%) patients with subsequent percutaneous coronary intervention--accounting for the majority of CE--in 123 (33%) patients, respectively. Eleven out of 15 biomarkers were elevated in patients with CE compared to those without. High-sensitive troponin T (hs-cTnT) was the best univariate biomarker to predict CE in Non-ST-elevation patients (AUC 0.80), but did not yield incremental information above clinical TIMI risk score (AUC 0.80 vs 0.82, p = 0.69). Equivalence testing of AUCs of risk models and non-inferiority testing demonstrated that the clinical TIMI risk score alone was non-inferior to its combination with hs-cTnT in predicting CE. CONCLUSIONS: In patients presenting without ST-elevations, identification of those prone to CE is best based on clinical assessment based on TIMI risk score criteria and hs-cTnT.

Peripheral blood monocyte Sirt1 expression is reduced in patients with coronary artery disease.

BACKGROUND: Inflammation plays a key role in atherosclerosis. Sirt1 regulates transcription factors involved in inflammatory processes and blunts atherosclerosis in mice. However, its role in humans remains to be defined. This study was therefore designed to investigate the role of Sirt1 in the development of atherosclerosis. METHODS AND RESULTS: 48 male subjects admitted for cardiac catheterization were subdivided into healthy subjects, patients with stable coronary artery disease (CAD), and with acute coronary syndromes (ACS). Monocytes were isolated and Sirt1 mRNA levels were determined. Sirt1 gene expression was higher in healthy subjects as compared to patients with CAD or ACS (P<0.05), respectively. Interestingly, HDL levels correlated positively with Sirt1 expression. Thus, HDL from the three groups was isolated and incubated with THP-1 monocytes to determine the effects of HDL on Sirt1 protein in controlled experimental conditions. HDL from healthy subjects stimulated Sirt1 expression in THP-1 monocytes to a higher degree than HDL from CAD and ACS patients (P<0.05). Paraoxonase-1 (PON-1), a HDL-associated enzyme, showed a reduced activity in HDL isolated from CAD and ACS patients as compared to the controls (P<0.001). CONCLUSIONS: Monocytic Sirt1 expression is reduced in patients with stable CAD and ACS. Experiments on THP-1 monocytes suggest that this effect is HDL-dependent and is mediated by a reduced activity of HDL-associated enzyme PON1.

Sensible use of high-sensitivity troponin assays.

The first intracellular Ca(2+)-sensor protein to be described was the troponin complex. Only later it was -discovered that cardiac-specific isoforms of troponin I (cTnI) and troponin T (cTnT) exist, and nowadays, measurement of cardiac troponins is a corner stone in the diagnosis of patients with acute coronary syndrome (ACS). High-sensitivity (hs-) assays have been developed that can record slightly elevated plasma concentrations of cardiac troponins as early as 3 h after onset of clinical symptoms. International guidelines defined a diagnostic cut-off at cardiac troponin levels corresponding to the 99th percentile of a healthy reference population and require that hs-assays measure this concentration with an interassay coefficient of variation ≤10%. This review provides an overview of the diagnostic and prognostic use of cardiac troponins.

Antibody phage display assisted identification of junction plakoglobin as a potential biomarker for atherosclerosis.

To date, no plaque-derived blood biomarker is available to allow diagnosis, prognosis or monitoring of atherosclerotic vascular diseases. In this study, specimens of thrombendarterectomy material from carotid and iliac arteries were incubated in protein-free medium to obtain plaque and control secretomes for subsequent subtractive phage display. The selection of nine plaque secretome-specific antibodies and the analysis of their immunopurified antigens by mass spectrometry led to the identification of 22 proteins. One of them, junction plakoglobin (JUP-81) and its smaller isoforms (referred to as JUP-63, JUP-55 and JUP-30 by molecular weight) were confirmed by immunohistochemistry and immunoblotting with independent antibodies to be present in atherosclerotic plaques and their secretomes, coronary thrombi of patients with acute coronary syndrome (ACS) and macrophages differentiated from peripheral blood monocytes as well as macrophage-like cells differentiated from THP1 cells. Plasma of patients with stable coronary artery disease (CAD) (n = 15) and ACS (n = 11) contained JUP-81 at more than 2- and 14-fold higher median concentrations, respectively, than plasma of CAD-free individuals (n = 13). In conclusion, this proof of principle study identified and verified JUP isoforms as potential plasma biomarkers for atherosclerosis. Clinical validation studies are needed to determine its diagnostic efficacy and clinical utility as a biomarker for diagnosis, prognosis or monitoring of atherosclerotic vascular diseases.

High dose methylphenidate treatment in adult attention deficit hyperactivity disorder: a case report.

INTRODUCTION: Stimulant medication improves hyperactivity, inattention, and impulsivity in both pediatric and adult populations with Attention Deficit Hyperactivity Disorder (ADHD). However, data regarding the optimal dosage in adults is still limited. CASE PRESENTATION: We report the case of a 38-year-old Caucasian patient who was diagnosed with Attention Deficit Hyperactivity Disorder when he was nine years old. He then received up to 10 mg methylphenidate (Ritalin®) and 20 mg sustained-release methylphenidate (Ritalin SR®) daily. When he was 13, his medication was changed to desipramine (Norpramin®), and both Ritalin® and Ritalin SR® were discontinued; and at age 18, when he developed obsessive-compulsive symptoms, his medication was changed to clomipramine (Anafranil®) 75 mg daily. Still suffering from inattention and hyperactivity, the patient began college when he was 19, but did not receive stimulant medication until three years later, when Ritalin® 60 mg daily was re-established. During the 14 months that followed, he began to use Ritalin® excessively, both orally and rectally, in dosages from 4800-6000 mg daily. Four years ago, he was referred to our outpatient service, where his Attention Deficit Hyperactivity Disorder was re-evaluated. At that point, the patient's daily Ritalin® dosage was reduced to 200 mg daily orally, but he still experienced pronounced symptoms of, Attention Deficit Hyperactivity Disorder so this dosage was raised again. The patient's plasma levels consistently remained between 60-187 nmol/l-within the recommended range-and signs of his obsessive-compulsive symptoms diminished with fluoxetine 40 mg daily. Finally, on a dosage of 378 mg extended-release methylphenidate (Concerta®), his symptoms of Attention Deficit Hyperactivity Disorder have improved dramatically and no further use of methylphenidate has been recorded during the 24 months preceding this report. CONCLUSIONS: Symptoms of Attention Deficit Hyperactivity Disorder (ADHD) in this adult patient, who also manifested a co-occurring obsessive compulsive disorder, dramatically improved only after application of a higher-than-normal dose of methylphenidate. We therefore suggest that clinicians consider these findings in relation to their adherence to current therapeutic guidelines.

Expression of the aging gene p66Shc is increased in peripheral blood monocytes of patients with acute coronary syndrome but not with stable coronary artery disease.

OBJECTIVE: The interplay between oxidative stress and inflammation is crucial in the pathogenesis of atherosclerosis. The adaptor protein p66Shc is implicated in atherogenesis and oxidative stress related responses in animal models of diseases. However, its role in humans remains to be defined. In this study, we hypothesized that expression of p66Shc increases in peripheral blood monocytes of patients affected by acute coronary syndromes (ACS). METHODS: Male subjects aged 59±4 (mean±SD) years admitted for cardiac catheterization were subdivided in three groups: (a) no local stenosis for the control group, (b) at least one stenosis ≥75% in either left, circumflex or right coronary artery for the coronary artery disease (CAD) group or (c) ST-elevation/non-ST-elevation myocardial infarction for the ACS group. Monocytes were isolated from whole blood and p66Shc RNA levels were determined by quantitative real time PCR. RESULTS: p66Shc RNA levels were increased in ACS patients as compared to CAD (p=0.007) and controls (p=0.0249). Furthermore, malondialdehyde (MDA) and C-reactive protein (CRP) were increased in plasma of ACS patients. Levels of MDA correlated positively to p66Shc (r=0.376, p=0.01). Our data demonstrate increased p66Shc levels in monocytes of ACS but not CAD patients. CONCLUSION: This study suggests an involvement of p66Shc in the transition of a stable CAD to an ACS patient. p66Shc was associated with states of increased oxidative stress. Further work is needed to understand whether p66Shc may represent a possible pharmacological target or whether it represents an interesting novel biomarker.

Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display.

Nitrotyrosine is a posttranslational protein modification that occurs under oxidative and nitrosative stress, and plays an important role in numerous pathological conditions. To analyse nitrotyrosine formation several commercial monoclonal and polyclonal antibodies reacting with 3-nitrotyrosine have been developed which however do not work properly in all required assays. Here, antibody phage display was used to select recombinant antibodies that specifically react with nitrotyrosine in various protein contexts. Nine initial selections were carried out, using synthetic peptides, peroxynitrite-modified proteins and conjugated proteins as antigens. Four antibodies were isolated that each exhibited a characteristic binding reactivity that greatly depended on the antigens that were used for their selections. In general, the selections using small, synthetic and biotinylated peptides were the most successful approach. Subsequently, antibody 11B1 was affinity matured by error prone mutagenesis, resulting in the isolation of two antibodies, designated 47A7 and 47B1. Competition ELISA and immunoblotting after treatment with sodium dithionite further demonstrated the specificity of antibody 47B1 for nitrotyrosine. The results presented here demonstrate that antibody phage display is a useful method to isolate antibodies against posttranslational modifications, which are powerful tools in the proteomic era.

[Risk factors in atherosclerotic coronary heart disease]. / Risikofaktoren der koronaren Herzkrankheit.

Coronary heart disease (CHD), which is caused by atherosclerosis, is the most common cause of death worldwide, and the prevention of CHD is therefore an essential clinical and public task. Classical risk factors like smoking, diabetes and hypercholesterolemia, are combined in several risk scores to estimate the risk for a cardiovascular event within the next 10 years. Despite their clinical success it is important to note that current methods have limited sensitivity and a low positive predictive value. Therefore, new biomarkers need to be identified to improve risk stratification. This review describes the pathogenesis and risk stratification of CHD, and provides an overview of the most important new biomarkers and their current applications in the prevention of CHD.

A novel subtractive antibody phage display method to discover disease markers.

Today's research demands fast identification of potential diagnostic and therapeutic targets. We describe a novel phage display strategy to identify disease-related proteins that are specifically expressed in a certain (diseased) tissue or cells. Phages displaying antibody fragments are selected on complex protein mixtures in a two-step manner combining subtractive selection in solution with further enrichment of specific phages on two-dimensional Western blots. Targets recognized by the resulting recombinant antibodies are immunoaffinity-purified and identified by mass spectrometry. We used antibody fragment libraries from autoimmune patients to discover apoptosis-specific and disease-related targets. One of the three identified targets is the U1-70K protein, a marker for systemic lupus erythematosus overlap disease. Interestingly the epitope on U1-70K recognized by the selected recombinant antibody was shown to be apoptosis-dependent, and such epitopes are believed to be involved in breaking tolerance to self-antigens. The other two proteins were identified as polypyrimidine tract-binding protein-associated splicing factor (PSF)/nuclear RNA- and DNA-binding protein of 54 kDa (p54nrb) and heterogeneous ribonucleoprotein C.

Recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies.

Next to the already available mouse monoclonal and laboratory animal-derived polyclonal antibodies, recombinant antibodies offer an additional and virtually unlimited arsenal of new immunohistochemical research tools. The major advantages of recombinant antibodies are their rapid and easy generation against virtually any target. The avidity of antibody fragments can be increased by partial dimerisation. This can be achieved by fusion of CL domains derived of different species to recombinant antibody domains. The VL-linker-VH-CL constructs result in significantly lower dimerisation levels compared to the VH-linker-VL-CL antibody constructs. The most efficient dimerisation occurs with the Jun-tagged scFvs. The very large and rapidly expanding collection of recombinant antibodies already available combined with the ease of introducing various tag sequences allows for an almost unrestricted number of easily adjustable research tools. To our best knowledge we report for the first time that using CL domains derived from different species, in combination with readily available commercial secondary antibodies specific for these CL domains, provides an easy method for the application of recombinant monoclonal antibodies of various origins in immunohistochemical analyses eliminating the problem of co-staining with multiple mono- or polyclonal antibodies. Both double and triple labelling experiments can be performed successfully.

Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease.

Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent. During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on 70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest for anti-U1 snRNP-positive patients.

Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block.

Autoantibodies against amino acid 200-239 (p200) in the predicted leucine zipper region of the Ro52 protein are associated with congenital heart block, a potentially fatal condition that may affect fetuses of women with Ro52 autoantibodies. To allow detailed studies of the antibodies associated with congenital heart block, B-cell derived combinatorial antibody libraries from patients were screened for Ro52 and p200 specific antibody clones. Two human monoclonal anti-p200 antibody fragments, S3A8 and M4H1, were isolated and analysed with regard to VHand VL gene utilization, somatic mutations and binding properties. Both identified clones recognized recombinant and native intact Ro52, and reacted only with p200 in a set of related Ro52 peptides. The specificity and affinity was confirmed by biosensor measurements. Structural analysis of overlapping peptides revealed increased helicity in the p200 peptide compared to non-recognized peptides, indicating epitope conformation as essential for antibody binding. Both monoclonals produced punctate nuclear and diffuse cytoplasmic staining in human and mouse cell lines. The identified antibodies, which react specifically with the leucine zipper structure of Ro52, will be valuable in further exploration of the mechanisms operating during development of Ro52 antibody-associated congenital heart block.