Stroke Biomarkers Research Articles

Assessing the Quality and Reproducibility of a Proteomic Platform for Clinical Stroke Biomarker Discovery

The aim of this study was to investigate the quality and reproducibility of mass spectra derived from a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) platform in a patient population undergoing carotid endarterectomy. Plasma samples were either digested with trypsin or left undigested, fractionated with either C18 or weak cation exchange (WCX) columns and analysed by MALDI-TOF MS. Quality of mass spectra for each method was assessed by baseline correction (lower area under the curve ratio indicating higher quality) and signal-to-noise ratio. Mean coefficient of variation (CV%) assessed reproducibility between repeated experiments and methods. Identified mass peak intensity differences were assessed for consistency across repeated experiments. Plasma from six patients was analysed. The quality of mass spectra was significantly better when derived from digested plasma fractionated by either WCX or C18 methods compared to undigested plasma fractionated by WCX (analysis of variance, p < 0.05). Mean CV% for repeated experiments was 18% and 28% for WCX and C18 fractionated digested plasma, respectively. A small number of differences in mass peak intensities were consistently observed in repeated experiments. Repeated experiments are required to confidently identify non-random mass peak intensity differences as putative plasma biomarkers that merit further investigation.

Blood Biomarkers in Cardioembolic Stroke

One promising field in neurovascular diseases investigation is the use of biomarkers to guide stroke etiology diagnosis and classification. Since treatment differs among etiologic subtypes and nowadays many patients receive a diagnosis of undetermined stroke, biomarkers might become an important additional diagnostic tool. In this review we update current knowledge about biomarkers related with cardioembolic stroke etiology (such as BNP and D-dimer proteins, or PITX2 and ZFHX3 genes), that in the future, might allow rapidly guiding other diagnostic tests and accelerating the onset of an optimal secondary prevention.

Editorial [Hot topic: Cardioembolic Infarction: A Renewed Topic of Interest (Guest Editor: Adria Arboix)

Cardioembolic infarction accounts for one quarter of all cerebral infarcts and is the most severe subtype of ischemic stroke. The rate of in-hospital death may be as high as 27% and a few patients are symptom-free at discharge from the hospital. Major cardiac sources of embolism are well-established and cardioembolic stroke is largely preventable. However, once stroke due to cardiac embolism has occurred, the likelihood of recurrence is relatively high (up to 10% for early embolic recurrence). Therefore, efforts at primary prevention for major-risk cardioembolic sources and secondary prevention of recurrence are very important. Although large series of patients with cardioembolic infarction have been reported in the literature, some clinical aspects of the natural course of the disease are still poorly defined. The objective of this supplement is to present an update review of relevant aspects related to clinical manifestations, biological characteristics, prognostic implications and treatment of cardioembolic stroke, in order to contribute to improve the quality of care and outcome of acute stroke patients. The most relevant aspects of cardioembolic infarction are discussed in the following 11 chapters: 1)epidemiology and risk factors of cerebral ischemia and ischemic heart diseases: similarities and differences; 2) clinical and prognostic features of cardioembolic stroke; 3) patent oval foramen in cerebral infarction; 4) cardiac workup of ischemic stroke; 5) complex atheromatosis of the aortic arch in cerebral infarction; 6) blood biomarkers in cardioembolic stroke; 7) heart failure in acute ischemic stroke; 8) cerebrovascular disease as a complication of cardiac transplantation; 9) antithrombotic therapy in cardiac embolism; 10) thrombolytic therapy in acute stroke; and 11) angiogenesis, neurogenesis and neuroplasticity in ischemic stroke. Cerebral cardioembolism is an important topic in the frontier between cardiology and vascular neurology, occurs frequently in daily practice, and has a high impact for patients, health care systems and society. It is for all these reasons that an update on cardioembolic infarction is now fully justified. It is worth mentioning that a team of excellent professionals belonging to the Expert Committee on Cerebrovascular Diseases of the Catalonian Society of Neurology and with proven experience in the field have contributed to the different chapters. My gratitude to all contributors for their support of this project and particularly to Dr. Jianyi Zhang, Editor-in-Chief of the Current Cardiology Reviews series. We hope that clinicians involved in the care of patients with cardioembolic infarction and interested in this topic will profit this publication and find this resource valuable and useful.

MicroRNAs in Cerebral Ischemia

Pathogenesis of cerebral ischemia has so far been described in the context of proteins and the pathways that they regulate. The discovery of biomarkers has also been focussed mainly on proteins and to some extent on the mRNAs that encode them. The knowledge on the role of microRNAs in understanding the pathogenesis of cerebral ischemia is still at its infancy. In this study, using rat models subjected to middle cerebral artery occlusion, we have profiled the microRNAs at different reperfusion times (0 to 48h) to understand the progression of cerebral ischemia. We have also attempted to correlate the expression of microRNAs to treatment with an NMDA antagonist (MK801) and to protein expression with the hope of demonstrating the potential use of microRNAs as early biomarkers of stroke.

Discriminative Capacity of Biomarkers for Acute Stroke in the Emergency Department

Acute ischemic stroke remains largely a clinical diagnosis. To assess the potential of several biomarkers to distinguish acute ischemic stroke from mimics in the emergency department (ED). In this prospective study, 63 patients with suspected acute stroke were enrolled. Blood samples were collected at ED presentation and assayed for B-type natriuretic peptide, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP-9), D-dimer, and protein S100B. Final diagnosis of stroke was rendered by blinded independent stroke experts after review of all clinical, imaging, and conventional laboratory data during admission. Logistic regression and bootstrapping models were used to evaluate the association between biomarker values and acute stroke. Thirty-four patients had a final diagnosis of stroke and 29 with mimics. The initial ED values of CRP, MMP-9, and S100B (C-indices of 0.808, 0.811, and 0.719, respectively) and the National Institutes of Health Stroke Scale (NIHSS) (C-index 0.887) predicted acute cerebral ischemia. CRP levels added discriminative value over clinical variables alone in the diagnosis of stroke. When the levels of CRP were added to the NIHSS, the combination was highly predictive of stroke (bootstrap mean C-index 0.951, 90% Confidence Interval 0.903-0.991, likelihood test p = 0.004). Biomarker testing with CRP and potentially MMP-9 and S100B, may add valuable and time-sensitive diagnostic information in the early evaluation of patients with suspected stroke in the ED. Future prospective evaluations are necessary to validate the diagnostic capability of these biomarkers for acute ischemic stroke in the ED before they should be considered for use in clinical practice.

Serum Cholinesterase Activities Distinguish between Stroke Patients and Controls and Predict 12-Month Mortality

To date there is no diagnostic biomarker for mild stroke, although elevation of inflammatory biomarkers has been reported at early stages. Previous studies implicated acetylcholinesterase (AChE) involvement in stroke, and circulating AChE activity reflects inflammatory response, since acetylcholine suppresses inflammation. Therefore, carriers of polymorphisms that modify cholinergic activity should be particularly susceptible to inflammatory damage. Our study sought diagnostic values of AChE and Cholinergic Status (CS, the total capacity for acetylcholine hydrolysis) in suspected stroke patients. For this purpose, serum cholinesterase activities, butyrylcholinesterase-K genotype and inflammatory biomarkers were determined in 264 ischemic stroke patients and matched controls during the acute phase. AChE activities were lower (P<0.001), and butyrylcholinesterase activities were higher in patients than in controls (P=0.004). When normalized to sampling time from stroke occurrence, both cholinergic parameters were correlated with multiple inflammatory biomarkers, including fibrinogen, interleukin-6 and C-reactive protein (r=0.713, r=0.607; r=0.421, r=0.341; r=0.276, r=0.255; respectively; all P values<0.001). Furthermore, very low AChE activities predicted subsequent nonsurvival (P=0.036). Also, carriers of the unstable butyrylcholinesterase-K variant were more abundant among patients than controls, and showed reduced activity (P<0.001). Importantly, a cholinergic score combining the two cholinesterase activities discriminated between 94.3% matched pairs of patients and controls, compared with only 75% for inflammatory measures. Our findings present the power of circulation cholinesterase measurements as useful early diagnostic tools for the occurrence of stroke. Importantly, these were considerably more distinctive than the inflammatory biomarkers, albeit closely associated with them, which may open new venues for stroke diagnosis and treatment.

Biomarkers in stroke

Although rare, childhood stroke has become a problem of great interest, as new modalities for treatment and prevention are being integrated into practice. Obviously, much of our approach begins with an appreciation of adult practice, given the vastly higher rate of the problem in adults. Adult data suggest that there is a role for the measurement of biomarkers of inflammation and hypercoaguability in categorizing patients and predicting their response to treatment. This has not been examined systematically in children. In this issue of The Journal, a group from Denver provides us with information from a single-center study of 50 children with acute stroke. Although acute changes in most markers were identified, the one of greatest interest is that of D-dimer. Significant elevations of this marker were most dramatically elevated in the group of children whose stroke was determined to be cardio-embolic in origin. Although this study will need to be confirmed by larger, likely multicenter, ones, it suggests that biomarkers such as these may prove useful in the classification of childhood stroke. One day, they may prove important in targeting therapy. Biomarkers of Hypercoagulability and Inflammation in Childhood-Onset Arterial Ischemic StrokeThe Journal of PediatricsVol. 156Issue 4PreviewTo test the hypothesis that acute elevations of biomarkers of hypercoagulability and inflammation are common in children with arterial ischemic stroke (AIS), particularly among etiologic subtypes that carry an increased risk of recurrent stroke. Full-Text PDF

Stroke Biomarkers: Progress and Challenges for Diagnosis, Prognosis, Differentiation, and Treatment

Stroke is a devastating condition encompassing a wide range of pathophysiological entities that include thrombosis, hemorrhage, and embolism. Current diagnosis of stroke relies on physician clinical examination and is further supplemented with various neuroimaging techniques. A single set or multiple sets of blood biomarkers that could be used in an acute setting to diagnosis stroke, differentiate between stroke types, or even predict an initial/reoccurring stroke would be extremely valuable. We discuss the current classification, diagnosis, and treatment of stroke, focusing on use of novel biomarkers (either solitary markers or multiple markers within a panel) that have been studied in a variety of clinical settings. The current diagnosis of stroke remains hampered and delayed due to lack of a suitable mechanism for rapid (ideally point-of-care), accurate, and analytically sensitive biomarker-based testing. There is a clear need for further development and translational research in this area. Potential biomarkers identified need to be transitioned quickly into clinical validation testing for further evaluation in an acute stroke setting; to do so would impact and improve patient outcomes and quality of life.

Biomarkers of Ischemic Stroke

The diagnosis and management of patients with ischemic stroke is primarily based on clinical assessment in conjunction with imaging tests. Development of molecular biomarkers as additional tools to support a clinical diagnosis, identify patients at risk of disease, and help guide patient treatment and prognosis would be of great value. Currently, no such biomarkers are used in the management of patients with ischemic stroke; however, several promising proteomic and genomic markers have been identified, as presented in this review.

Proteomic analysis in ischemic stroke

脳梗塞は死亡，身体障害の主な原因疾患であり，社会に及ぼす影響は大きい．後遺症を減らすためには，できるだけ早く診断し，血栓溶解療法などの適切な治療を行うことが重要である．脳梗塞の診断や進行・再発，薬剤反応性の予測が血液検査により可能となれば，早期から最適な治療選択が可能となり有用と考えられる．血漿タンパク質はダイナミックレンジが広く微量タンパク質の測定は困難であったが，質量分析器の発達によりプロテオミクス技術は格段に進歩し，血漿タンパク質の網羅的なハイスループット解析が可能となっている．われわれは脳梗塞患者における血漿タンパク質の変化を経時的，網羅的に探索し，種々のタンパク質が急性期から慢性期まで変動していることを明らかにした(Research for Biomarkers in Ischemic Stroke, REBIOS)．バイオインフォマティクスの手法を用いることで診断，予測マーカーを発見し，創薬ターゲットとなるタンパク質を発掘することが可能になると期待される．

Intense correlation between protein-conjugated acrolein and primary Sjögren's syndrome

We recently found that an increased plasma concentration of protein-conjugated acrolein is a good biomarker for stroke. Therefore we determine whether the concentration of protein-conjugated acrolein is increased in saliva from patients with primary Sjögren's syndrome. Stimulated whole-mixed saliva was collected from 10 patients and 13 control subjects. The concentration of protein-conjugated acrolein in saliva and plasma was measured by either Western blotting or enzyme-linked immunosorbent assay. The concentration of protein-conjugated acrolein, especially albumin-conjugated acrolein, was greatly increased in saliva from patients with primary Sjögren's syndrome (p<0.001). The concentration of protein-conjugated acrolein was inversely correlated with the flow rate of saliva. The results indicate that the concentration of protein-conjugated acrolein, a marker of cell or tissue damage, in saliva is well correlated with seriousness of primary Sjögren's syndrome.

Abstract 5081: Circulating Microparticles as Potential Biomarkers for Primary Intracerebral Hemorrhage Stroke

Introduction: Primary intracerebral hemorrhage (ICH) stroke is a serious complication of uncontrolled hypertension. Recent clinical studies have shown that circulating microparticles (MPs) from endothelia (EMPs), platelets (PMPs) and endothelial progenitor cells (EPC-MPs) are potential biomarkers for cardiovascular diseases. However, the role of circulating MPs in ICH stroke is unknown. Methods and Results: The human renin and angiotensinogen transgenic (R+A+) and the control (R−A−) mice (male, 8 –10 weeks) were fed with normal diet or high salt diet (8% NaCl, HSD) plus nitric oxide synthase inhibitor L-NAME (120 mg/kg/day) in drinking water for two weeks. Mean arterial pressure (MAP) was measured using telemetric probes. Circulating MPs were identified by membrane markers, CD62E (for EMPs), CD62P (for PMPs), and CD34 and VEGFR2 (for EPC-MPs) using flow cytometry. The severity of ICH was determined by histological analysis of total bleeding area. The lumen/wall in the middle cerebral artery (MCA) was histologically measured. Results (Table and Figure ) showed: The R+A+ mice were hypertensive and had higher basal level of circulating PMPs and EMPs with no difference in EPC-MPs; After fed with HSD and L-NAME, R+A+ mice had severe hypertension and 50% of them developed ISH stroke within two weeks; There was a further increase of EMPs and a decrease of EPC-MPs in R+A+ mice with ICH stroke; There were strong correlations of EMPs (r=0.44~0.61) and EPC-MPs (r=−0.52~0.78) with MAP, MCA lumen/wall and total area of bleeding sites. Conclusion: Circulating EPC-MPs and EMPs could serve as useful biomarkers for uncontrolled hypertension, MCA remodeling and ICH stroke. Table. Blood pressure, circulating MPs and MCA lumen/wall in R+A+ and R−A− mice fed with normal diet or HSD and L-NAME for two weeks Fig. Hemorrhages in the brain (A, D: brainstem: B, E: cerebellum; C,F: basal ganglia) and MCA remodeling, A–C; R+A+ mice fed with HSD+L-NAME; D–F: R−A− mice fed with HSD+L-NAME; G: R−A− mice fed with normal diet, H: R−A− mice fed with HSD+L-NAME; I: R+A+ mice fed with HSD+L-NAME

Some Notes on Visinin-Like Protein 1 and Alzheimer Disease

We wish to comment and add some potentially useful information on certain aspects of the report of Lee and collaborators, which was recently published in Clinical Chemistry (1). In an earlier study, the authors had proposed visinin-like protein 1 [VILIP-11 or VSNL1; VSNL1 gene (visinin-like 1)] as a potential biomarker for stroke because they had detected this intracellular calcium-binding protein in cerebrospinal fluid (CSF) in a rat model of stroke and in the plasma of patients after stroke (2). The group extended these findings to Alzheimer disease (AD) in a second publication (1). VILIP-1 concentrations were significantly altered in the CSF of AD patients; hence, the authors concluded that VILIP-1 might also be a useful novel biomarker for AD-related brain injury. Lee et al. put forward the hypothesis that measures of VILIP-1 might reflect neuronal injury with subsequent release of the intracellular protein VILIP-1 into the CSF. The authors stated that both Aβ …

Serum free hemoglobin as a novel potential biomarker for acute ischemic stroke

Currently, no practical biomarker is available for the diagnosis of acute ischemic stroke. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has being applied extensively to analyze large biomolecules such as proteins. The technique is likely to be of remarkable value as indicators of systemic processes such as atherosclerosis and stroke. The aim of this study is to identify potential protein biomarkers for ischemic stroke diagnosis utilizing MALDI-TOF MS. Serum samples obtained from acute ischemic stroke patients (n = 47) and controls (n = 34) were analyzed by MALDI-TOF MS. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS), subtype by the Trial of Org 10172 in Acute Stroke Treatment (TOAST), functional outcome by the modified Rankin Scale (mRS) and infarct volume by the diffusion-weighted images. Risk factors and routine laboratory data of the stroke patients were registered prospectively. The peaks of hemoglobin (Hb) alpha-chain and beta-chain were differentially expressed between stroke patients and controls (p < 0.0001). Hb ions were detected in the samples collected from 33 (70.2%) stroke patients and 5 (14.7%) controls. The sensitivity is 70.2% and the specificity is 85.3%. Among stroke patients, there is no significant correlation (p > 0.05) between Hb peaks and the NIHSS, TOAST, mRS, stroke risk factors, infarct volume, infarct location and laboratory data. Serum free Hb may serve as a novel potential biomarker for the diagnosis of acute ischemic stroke. The clinical value of this potential biomarker may be clarified by further studies quantifying serum free Hb levels.

Potential biomarkers for the diagnosis of stroke

Stroke is common, with a high rate of disability and death, and current therapies are both highly time sensitive and carry some risk, making rapid diagnosis important. Many cases of stroke are difficult to diagnose clinically during the acute phase, and there are limitations to the ability of diagnostic imaging to help with the necessary speed. A reliable and valid biomarker would be invaluable for this common clinical situation, as it has been with myocardial infarction. A large number of molecules have been evaluated for this role, both in the laboratory and in patients, but the results to date have been disappointing. In this article, we review the operative concepts in the search for a diagnostic biomarker of stroke, a few of the promising candidates and the current challenges to validation.

Serologic and Genetic Biomarkers for Stroke

This article reviews 2 studies that have used RNA as a biomarker for stroke and that exemplified the progress in and complexity of such studies, as well as discusses the potential use of biomarkers as predictors of stroke outcome and recovery.

Magnetic resonance imaging biomarkers of ischemic stroke: Criteria for the validation of primary imaging biomarkers

Ischemic stroke is associated with a high rate of disability and death. Establishing valid biomarkers could help accelerate the approval of promising new therapies for stroke. Whereas many serum biomarkers have been evaluated, possible imaging biomarkers of stroke lack validation. Magnetic resonance imaging (MRI) is a very sensitive technique to study acute stroke and MRI parameters have been established to assess the outcome of acute stroke. This review reassesses the criteria for the validation of MRI biomarkers of acute ischemic stroke (MRI-BAS). Seven criteria were used to review the validity of the main MRI-BAS: vascular status, lesion volume, reversibility on diffusion-weighted imaging, perfusion alteration, penumbra studied with diffusion-perfusion mismatch, clinical-diffusion mismatch, diffusion-angiography mismatch and hemorrhagic transformation. We analyzed the definitions of these biomarkers and the extent to which each fulfills the criteria for validation and found that few MRI-BAS have been fully validated. Further studies should help to improve the validation of current MRI-BAS and develop new biomarkers.

What Kind of Stroke Is It?

The search for protein biomarkers in peripheral blood to aid in the diagnosis of stroke and the prediction of prognosis after stroke and risk of future stroke is an active area of investigation. Although some markers, such as S100b and neuron-specific enolase, have been shown to have prognostic value in ischemic stroke (1)(2), no biochemical markers have proven to be really useful in routine clinical practice. In this issue of Clinical Chemistry , Nybo and associates present similarly discouraging data for osteoprotegerin as a potential stroke biomarker (3). Why are we continuing to see lack of demonstrated clinical utility in studies for stroke biomarkers? Should the search for biomarkers for stroke be abandoned? Some answers may lie in the deceptively simple question, what is a stroke? Stroke is currently defined as a disruption of brain function due to a vascular insult, with symptoms lasting 24 h or more. This definition, now old, is in flux and in truth, our understanding of stroke has evolved tremendously with the evolution of new diagnostic technology. MRI has allowed us to show that ischemic stroke damage can occur with complete resolution of symptoms well before a 24-h time limit. Transient ischemic attack may be caused by …

Serum S100B, brain edema, and hematoma formation in a rat model of collagenase-induced hemorrhagic stroke

S100B, a 21-kD Ca(2+) binding protein expressed in Schwann cells and astroglia, has often been reported as a promising biomarker for ischemic stroke. In addition to ischemic stroke, the peripheral S100B level may also be useful as a biomarker for intracerebral hemorrhage (ICH). However, the kinetics and characterization of peripheral S100B in patients or experimental animal models with ICH have not been carefully examined. The present study investigated the kinetics and characteristics of the serum S100B level in a rat collagenase-induced ICH model. The serum S100B kinetics and the time-course of brain edema and hematoma formation were examined. Then, the correlations between the elevated serum S100B level and brain edema or hematoma formation were investigated. A transient elevation of serum S100B that peaked at 6 h after ICH induction was observed. The single measurement of serum S100B at 6 h after ICH induction was significantly correlated with brain edema formation and the maximal extent of the hematoma volumes. These results suggest the significance of serum S100B as a biomarker of brain damage resulting from ICH.

Increased Left Atrial Volume Index: Potent Biomarker for First-Ever Ischemic Stroke

To establish the incidence and correlation of increased left atrial volume index (LAVI) in patients with first-ever ischemic stroke. Using our institution's epidemiological database, we defined a cohort of 432 patients (cases) who underwent transthoracic echocardiography within 60 days of first ischemic stroke between January 1, 1985, and December 31, 1994. Left atrial volume was measured with the biplane area-length method, indexed to body surface area (LAVI, expressed as mL/m(2)). The control group consisted of 416 community residents who underwent transthoracic echocardiography as participants in a stroke risk factor study. Increased LAVI was defined as 28 mL/m(2) or higher. Survival in patients was compared with expected survival among white Minnesotans and was further modeled as a function of age, sex, LAVI, and clinical risk factors. Among the included 306 patients, 230 (75%) had increased LAVI (mean+/-SD, 49+/-21 mL/m(2)). Patients with increased LAVI were older than those with normal LAVI (mean+/-SD age, 76+/-11 vs 71+/-13 years; P=.003) and had more cardiovascular risk factors (mean+/-SD number, 1.8+/-0.07 vs 1.3+/-0.89; P<.001). Mean LAVI was higher in cases than in age- and sex-matched controls (P<.001). At 5-year follow-up, cases showed excess mortality compared with age-matched controls (P=.001). After variables were adjusted for age, sex, and clinical risk factors, LAVI was independently associated with mortality. A useful index for prediction of adverse cardiovascular events, LAVI might also predict first ischemic stroke and subsequent mortality.