Blood biomarker changes following therapeutic hypothermia in ischemic stroke.

INTRODUCTION: Therapeutic hypothermia is a promising candidate for stroke treatment although its efficacy has not yet been demonstrated in patients. Changes in blood molecules could act as surrogate markers to evaluate the efficacy and safety of therapeutic cooling. METHODS: Blood samples from 54 patients included in the EuroHYP-1 study (27 treated with hypothermia, and 27 controls) were obtained at baseline, 24 ± 2 h, and 72 ± 4 h. The levels of a panel of 27 biomarkers, including matrix metalloproteinases and cardiac and inflammatory markers, were measured. RESULTS: Metalloproteinase-3 (MMP-3), fatty-acid-binding protein (FABP), and interleukin-8 (IL-8) increased over time in relation to the hypothermia treatment. Statistically significant correlations between the minimum temperature achieved by each patient in the hypothermia group and the MMP-3 level measured at 72 h, FABP level measured at 24 h, and IL-8 levels measured at 24 and 72 h were found. No differential biomarker levels were observed in patients with poor or favorable outcomes according to modified Rankin Scale scores. CONCLUSION: Although the exact roles of MMP3, FABP, and IL-8 in hypothermia-treated stroke patients are not known, further exploration is needed to confirm their roles in brain ischemia.

Monitoring dexamethasone skin biodistribution with ex vivo MALDI-TOF mass spectrometry imaging and confocal Raman microscopy.

Two of the most promising techniques in terms of ex vivo skin imaging and quantifying are confocal Raman microscopy and MALDI-TOF mass spectrometry imaging (MALDI-TOF MSI). Both techniques were set up, and the semiquantitative skin biodistribution of previously developed dexamethasone (DEX) loaded lipomers was compared using Benzalkonium chloride (BAK) as a tracer of the nanoparticles. In MALDI-TOF MSI, DEX was derivatised with GirT (DEX-GirT) and the semiquantitative biodistribution of both DEX-GirT and BAK was successfully obtained. The amount of DEX measured by confocal Raman microscopy was higher than that measured by MALDI-TOF MSI, but MALDI-TOF MSI proved to be a more suitable technique for tracing BAK. An absorption-promoting tendency of DEX loaded in lipomers versus a free-DEX solution was observed in confocal Raman microscopy. The higher spatial resolution of confocal Raman microscopy (350 nm) with respect to MALDI-TOF MSI (50 µm) allowed to observe specific skin structures like hair follicles. Nevertheless, the faster sampling rate of MALDI-TOF-MSI, permitted the analysis of larger tissue regions. In conclusion, both techniques allowed to simultaneously analyze semiquantitative data together with qualitative images of biodistribution, which is a very helpful tool when designing nanoparticles that accumulate in specific anatomical regions.

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia.

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets.

Blood Biomarker Panels for the Early Prediction of Stroke-Associated Complications.

Background Acute decompensated heart failure (ADHF) and respiratory tract infections (RTIs) are potentially life-threatening complications in patients experiencing stroke during hospitalization. We aimed to test whether blood biomarker panels might predict these complications early after admission. Methods and Results Nine hundred thirty-eight patients experiencing ischemic stroke were prospectively recruited in the Stroke-Chip study. Post-stroke complications during hospitalization were retrospectively evaluated. Blood samples were drawn within 6 hours after stroke onset, and 14 biomarkers were analyzed by immunoassays. Biomarker values were normalized using log-transformation and Z score. PanelomiX algorithm was used to select panels with the best accuracy for predicting ADHF and RTI. Logistic regression models were constructed with the clinical variables and the biomarker panels. The additional predictive value of the panels compared with the clinical model alone was evaluated by receiver operating characteristic curves. An internal validation through a 10-fold cross-validation with 3 repeats was performed. ADHF and RTI occurred in 19 (2%) and 86 (9.1%) cases, respectively. Three-biomarker panels were developed as predictors: vascular adhesion protein-1 >5.67, NT-proBNP (N-terminal pro-B-type natriuretic peptide) >4.98 and d-dimer >5.38 (sensitivity, 89.5%; specificity, 71.7%) for ADHF; and interleukin-6 >3.97, von Willebrand factor >3.67, and d-dimer >4.58 (sensitivity, 82.6%; specificity, 59.8%) for RTI. Both panels independently predicted stroke complications (panel for ADHF: odds ratio [OR] [95% CI], 10.1 [3-52.2]; panel for RTI: OR, 3.73 [1.95-7.14]) after adjustment by clinical confounders. The addition of the panel to clinical predictors significantly improved areas under the curve of the receiver operating characteristic curves in both cases. Conclusions Blood biomarkers could be useful for the early prediction of ADHF and RTI. Future studies should assess the usefulness of these panels in front of patients experiencing stroke with respiratory symptoms such as dyspnea.

D-Dimer as Predictor of Large Vessel Occlusion in Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Improving prehospital triage of large vessel occlusion (LVO) would reduce time to reperfusion therapies. We aimed to study early predictors of LVO in acute ischemic stroke to identify candidates for endovascular treatment. METHODS: The Stroke-Chip was a prospective observational study conducted at 6 Stroke Centers in Catalonia. Blood samples were obtained in the first 6 hours from symptom onset of consecutive patients. Stroke severity was evaluated with National Institutes of Health Stroke Scale (NIHSS) and LVO was assessed. Independent association of multiple blood biomarkers with LVO was evaluated using logistic regression models adjusted by covariates. Sensitivity, specificity, and predictive values were assessed for NIHSS and the combination of NIHSS and selected serum biomarkers levels. RESULTS: One thousand three hundred eight suspected strokes were enrolled for a 17-month period. LVO was not assessed in 131 patients. One thousand one hundred seventy-seven patients were selected for analysis (mean age 69.3 years, 56% men, median baseline NIHSS of 6, and median time to blood collection 2.5 hours). LVO was detected in 262 patients. LVO patients were older, had higher baseline NIHSS, history of atrial fibrillation, and lower time from stroke onset to admission. After logistic regression analysis, D-dimer remained an independent predictor of LVO (odds ratio, 1.59 [1.31-1.92]). Specificity and positive predictive value to exclude or detect LVO were higher when using combined D-dimer levels and NIHSS score assessment rather than NIHSS alone. CONCLUSIONS: Early D-dimer levels are an independent predictor of LVO and may be useful to better optimize prehospital patient transport to the appropriate stroke center.

Peripheral inflammation preceeding ischemia impairs neuronal survival through mechanisms involving miR-127 in aged animals.

Ischemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke-induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron-specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co-existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR-127, that is highly neuronal, to be associated with increased cell death in the aged, LPS-injected ischemic mice. Target prediction tools indicate that miR-127 interacts with several basally expressed neuronal genes, and of these we verify miR-127 binding to Psmd3. Finally, we report reduced expression of miR-127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke.

A Mouse Brain-based Multi-omics Integrative Approach Reveals Potential Blood Biomarkers for Ischemic Stroke.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

Cardioembolic Ischemic Stroke Gene Expression Fingerprint in Blood: a Systematic Review and Verification Analysis.

An accurate etiological classification is key to optimize secondary prevention after ischemic stroke, but the cause remains undetermined in one third of patients. Several studies pointed out the usefulness of circulating gene expression markers to discriminate cardioembolic (CE) strokes, mainly due to atrial fibrillation (AF), while only exploring them in small cohorts. A systematic review of studies analyzing high-throughput gene expression in blood samples to discriminate CE strokes was performed. Significantly dysregulated genes were considered as candidates, and a selection of them was validated by RT-qPCR in 100 patients with defined CE or atherothrombotic (LAA) stroke etiology. Longitudinal performance was evaluated in 12 patients at three time points. Their usefulness as biomarkers for AF was tested in 120 cryptogenic strokes and 100 individuals at high-risk for stroke. Three published studies plus three unpublished datasets were considered for candidate selection. Sixty-seven genes were found dysregulated in CE strokes. CREM, PELI1, and ZAK were verified to be up-regulated in CE vs LAA (p = 0.010, p = 0.003, p < 0.001, respectively), without changes in their expression within the first 24 h after stroke onset. The combined up-regulation of these three biomarkers increased the probability of suffering from CE stroke by 23-fold. In cryptogenic strokes with subsequent AF detection, PELI1 and CREM showed overexpression (p = 0.017, p = 0.059, respectively), whereas in high-risk asymptomatic populations, all three genes showed potential to detect AF (p = 0.007, p = 0.007, p = 0.015). The proved discriminatory capacity of these gene expression markers to detect cardioembolism even in cryptogenic strokes and asymptomatic high-risk populations might bring up their use as biomarkers.

Application of an Aptamer-Based Proteomics Assay (SOMAscan™) in Rat Cerebrospinal Fluid.

The exploration of the cerebrospinal fluid (CSF) through proteomics techniques might help in the search of molecular biomarkers relevant to neurological pathologies. Aiming this, we describe here a commercially available multiplexed proteomics technology based on the use of modified aptamers (SOMAscan™ assay). From our experience in exploring the rat CSF proteome, we detail the basic principles of this oligonucleotide-based proteomics approach, as well as the main data-processing steps to obtain relative quantitative values for proteins that could discriminate among different brain conditions, as an attempt in the search of neurological biomarkers. Finally, we give some tips on performing the SOMAscan assay and key recommendations on the verification analyses of the resulting candidate biomarkers.

PATJ Low Frequency Variants Are Associated With Worse Ischemic Stroke Functional Outcome.

RATIONALE: Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome. OBJECTIVE: Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date. METHODS AND RESULTS: A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, ß=0.40, P=1.70×10-9). CONCLUSIONS: Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci.

Inflammatory molecules might become both biomarkers and therapeutic targets for stroke management.

Stroke is the fifth leading cause of death and the most frequent cause of disability worldwide. Currently, stroke diagnosis is based on neuroimaging; therefore, the lack of a rapid tool to diagnose stroke is still a major concern. In addition, therapeutic approaches to combat ischemic stroke are still scarce, since the only approved therapies are directed toward restoring blood flow to the affected brain area. However, due to the reduced time window during which these therapies are effective, few patients benefit from them; therefore, alternative treatments are urgently needed to reduce stroke brain damage in order to improve patients' outcome. The inflammatory response triggered after the ischemic event plays an important role in the progression of stroke; consequently, the study of inflammatory molecules in the acute phase of stroke has attracted increasing interest in recent decades. Here, we provide an overview of the inflammatory processes occurring during ischemic stroke, as well as the potential for these inflammatory molecules to become stroke biomarkers and the possibility that these candidates will become interesting neuroprotective therapeutic targets to be blocked or stimulated in order to modulate inflammation after stroke.

Detection of plasma MMP-9 within minutes. Unveiling some of the clues to develop fast and simple electrochemical magneto-immunosensors.

Magnetic beads (MB) have been extensively used to produce sensitive and efficient electrochemical magneto-immunosensors. However, MB effective handling requires training, and MB washing after each incubation step is time consuming and contributes to raise result variability. Consequently, most of the electrochemical magneto-immunosensors reported to date, which entailed relatively long and complex multi-step procedures, would be difficult to carry out at point-of-care (POC) settings or by laypersons. For this reason, here we targeted the development of a simplified detection path, which is fast and simple enough to be operated at a POC setting, sufficiently efficient to provide analyte quantitation comparable to classical diagnostic methods, and dependent on minimal technical requirements to facilitate method global exploitation. As a proof-of-concept, we optimized an extremely simple, fast and efficient electrochemical magneto-immunosensor for detection of matrix metalloproteinase 9 (MMP-9). To accomplish this, we optimized MB immunomodification, produced an immunomodified Poly-HRP signal amplifier, developed a single-step magneto-immunoassay, and optimized electrochemical detection using a multiplexed magnetic holder and a ready-to-use commercial substrate solution. The sensor was finally calibrated by detecting MMP-9 in clinical samples. This electrochemical magneto-immunosensor detected MMP-9 in just 12-15 min, displaying linear response between 0.03 and 2 ng mL-1 of MMP-9, limits of detection (LOD) and quantification (LOQ) of 13 pg mL-1 and 70 pg mL-1, respectively, %CV< 6%, and accurate quantification of MMP-9 in patient plasma samples. These results were comparable to those afforded by a 5-h reference ELISA that used the same antibodies, confirming the applicability of our simplified method.

Characterization of the rat cerebrospinal fluid proteome following acute cerebral ischemia using an aptamer-based proteomic technology.

The limited accessibility to the brain has turned the cerebrospinal fluid (CSF) into a valuable source that may contribute to the complete understanding of the stroke pathophysiology. Here we have described the CSF proteome in the hyper-acute phase of cerebral ischemia by performing an aptamer-based proteomic assay (SOMAscan) in CSF samples collected before and 30 min after male Wistar rats had undergone a 90 min Middle Cerebral Artery Occlusion (MCAO) or sham-surgery. Proteomic results indicated that cerebral ischemia acutely increased the CSF levels of 716 proteins, mostly overrepresented in leukocyte chemotaxis and neuronal death processes. Seven promising candidates were further evaluated in rat plasma and brain (CKB, CaMK2A, CaMK2B, CaMK2D, PDXP, AREG, CMPK). The 3 CaMK2 family-members and CMPK early decreased in the infarcted brain area and, together with AREG, co-localized with neurons. Conversely, CKB levels remained consistent after the insult and specifically matched with astrocytes. Further exploration of these candidates in human plasma revealed the potential of CKB and CMPK to diagnose stroke, while CaMK2B and CMPK resulted feasible biomarkers of functional stroke outcome. Our findings provided insights into the CSF proteome following cerebral ischemia and identified new outstanding proteins that might be further considered as potential biomarkers of stroke.

Usefulness of ADAMTS13 to predict response to recanalization therapies in acute ischemic stroke.

OBJECTIVE: We aimed to analyze ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in relation to arterial recanalization in patients treated with IV tissue plasminogen activator (tPA) and in relation to futile recanalization in patients treated with mechanical thrombectomy. METHODS: Acute ischemic stroke patients (n = 108) with documented arterial occlusions treated with IV-tPA were selected. ADAMTS13 activity was measured by ELISA in samples collected before treatment. Recanalization was assessed at 2 hours by transcranial Doppler. In 78 consecutive patients treated with endovascular thrombectomy, ADAMTS13 antigen was measured by ELISA and futile recanalization was defined as complete recanalization plus modified Rankin Scale score >2 at 3 months. Independent predictors of recanalization and futile recanalization were determined by logistic regression, adjusted by age, NIH Stroke Scale score, and time from stroke onset. RESULTS: Patients who achieved tPA-induced recanalization had higher baseline ADAMTS13 activity (78.1% [68%-88%] vs 70.1% [61%-79%], p = 0.021). In logistic regression analysis, ADAMTS13 activity >75% was an independent predictor of recanalization (odds ratio = 6.76 [1.52-30.02], p = 0.012), together with absence of early ischemic signs and Oxfordshire Community Stroke Project classification. Regarding endovascular therapies, a reduced ADAMTS13 concentration (<982 ng/mL) was an independent predictor of futile recanalization (odds ratio = 67.4 [1.4-3,282.1], p = 0.034), together with age and diabetes mellitus. The addition of ADAMTS13 to clinical predictors of tPA-induced recanalization and futile recanalization improved discrimination and reclassification (integrated discrimination improvement = 10.06% and 28.4%, net reclassification improvement = 61.0% and 107.4%, respectively). CONCLUSIONS: A reduced ADAMTS13 was associated with poor response to recanalization therapies. If confirmed in future prospective studies, a panel of blood biomarkers including ADAMTS13 might be a useful tool to guide reperfusion therapies.

Using magnetic beads and signal amplifiers to produce short and simple immunoassays: Application to MMP-9 detection in plasma samples.

Magnetic beads (MB) and signal amplifiers, such as horseradish peroxidase polymers (poly-HRP), have been used before for the production of highly sensitive immunoassays. However, most of the examples reported previously entailed long and tedious multi-step procedures, which were not necessarily shorter or simpler than classical paths such as Enzyme-Linked Immunosorbent Assay (ELISA). Here, instead of exploiting the combination of MB and poly-HRP to ameliorate sensitivity, we show that they conform a powerful tool that can be used to shorten the incubation times, which allows optimizing extremely simple, fast and efficient immunoassays with minimal technical requirements. In order to do so, here we used the highly sensitive and specific pair of antibodies of a commercial ELISA kit to optimize a magneto-ELISA for the detection of matrix metallopeptidase 9 (MMP-9). Three signal amplifiers were then tested and the best performing one was implemented in the magneto-assay to shorten the incubation times and improve assay performance. As we show, the shortened magneto-assay could be carried out in about 35 min, which included two 5-min incubations, washing, and incubation with enzyme substrate for 20 min before colorimetric detection. Moreover, the quantification of MMP-9 provided by the shortened assay in 12 plasma samples collected from patients was comparable to that generated by the 5-h ELISA, which was 8.5 times longer.

Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p < 0.05 and fold-change> 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets.

Sepsis biomarkers reprofiling to predict stroke-associated infections.

We aimed to evaluate the usefulness of sepsis biomarkers to predict stroke-associated infections. Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), mid-regional pro-adrenomedullin (MR-proADM), presepsin (sCD14), and soluble urokinase-type plasminogen activator receptor (suPAR), were explored in 125 blood samples collected at different time-points. At baseline, MR-proADM was an independent predictor of infection [>0.94pg/mL, OR=3.63 (1.16-11.33), p=0.026], as well as suPAR at 24h [>2185.8pg/mL, OR=5.81 (1.05-32.26), p=0.044]. Both MR-proADM and suPAR were raised in patients with infections throughout the first week after stroke. These results are especially relevant for MR-proADM given its early elevation, which would allow early preventive interventions.

Blood Biomarkers for the Early Diagnosis of Stroke: The Stroke-Chip Study.

BACKGROUND AND PURPOSE: Stroke diagnosis could be challenging in the acute phase. We aimed to develop a blood-based diagnostic tool to differentiate between real strokes and stroke mimics and between ischemic and hemorrhagic strokes in the hyperacute phase. METHODS: The Stroke-Chip was a prospective, observational, multicenter study, conducted at 6 Stroke Centers in Catalonia. Consecutive patients with suspected stroke were enrolled within the first 6 hours after symptom onset, and blood samples were drawn immediately after admission. A 21-biomarker panel selected among previous results and from the literature was measured by immunoassays. Outcomes were differentiation between real strokes and stroke mimics and between ischemic and hemorrhagic strokes. Predictive models were developed by combining biomarkers and clinical variables in logistic regression models. Accuracy was evaluated with receiver operating characteristic curves. RESULTS: From August 2012 to December 2013, 1308 patients were included (71.9% ischemic, 14.8% stroke mimics, and 13.3% hemorrhagic). For stroke versus stroke mimics comparison, no biomarker resulted included in the logistic regression model, but it was only integrated by clinical variables, with a predictive accuracy of 80.8%. For ischemic versus hemorrhagic strokes comparison, NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) >4.9 (odds ratio, 2.40; 95% confidence interval, 1.55-3.71; P<0.0001) and endostatin >4.7 (odds ratio, 2.02; 95% confidence interval, 1.19-3.45; P=0.010), together with age, sex, blood pressure, stroke severity, atrial fibrillation, and hypertension, were included in the model. Predictive accuracy was 80.6%. CONCLUSIONS: The studied biomarkers were not sufficient for an accurate differential diagnosis of stroke in the hyperacute setting. Additional discovery of new biomarkers and improvement on laboratory techniques seem necessary for achieving a molecular diagnosis of stroke.

C-reactive protein in the detection of post-stroke infections: systematic review and individual participant data analysis.

We conducted a systematic review and individual participant data meta-analysis to explore the role of C-reactive protein (CRP) in early detection or prediction of post-stroke infections. CRP, an acute-phase reactant binds to the phosphocholine expressed on the surface of dead or dying cells and some bacteria, thereby activating complement and promoting phagocytosis by macrophages. We searched PubMed up to May-2015 for studies measuring CRP in stroke and evaluating post-stroke infections. Individual participants' data were merged into a single database. CRP levels were standardized and divided into quartiles. Factors independently associated with post-stroke infections were determined by logistic regression analysis and the additional predictive value of CRP was assessed by comparing areas under receiver operating characteristic curves and integrated discrimination improvement index. Data from seven studies including 699 patients were obtained. Standardized CRP levels were higher in patients with post-stroke infections beyond 24 h. Standardized CRP levels in the fourth quartile were independently associated with infection in two different logistic regression models, model 1 [stroke severity and dysphagia, odds ratio = 9.70 (3.10-30.41)] and model 2 [age, sex, and stroke severity, odds ratio = 3.21 (1.93-5.32)]. Addition of CRP improved discrimination in both models [integrated discrimination improvement = 9.83% (0.89-18.77) and 5.31% (2.83-7.79), respectively], but accuracy was only improved for model 1 (area under the curve 0.806-0.874, p = 0.036). In this study, CRP was independently associated with development of post-stroke infections, with the optimal time-window for measurement at 24-48 h. However, its additional predictive value is moderate over clinical information. Combination with other biomarkers in a panel seems a promising strategy for future studies.