Therefore, our study investigated systematically the EPC counts in the acute, subacute, and chronic stages of ischemic stroke of different etiologies, the associated variables, and their prognostic value. Materials and Methods Patients We prospectively studied consecutive patients with a suspected ischemic stroke that were admitted to the Neurology Department at our Hospital. All the patients were included within the first 48 h after the onset of stroke. The Ethics Committee at Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) approved the study, and written informed consent was obtained from

participating patients or their legal representatives. Exclusion criteria were as follows: a previous modified Rankin scale score higher than 2; a Inhibitors,research,lifescience,medical National Institute of Health and Stroke Scale (NIHSS) score of 0; the lack of processing of the blood sample within 30 min after extraction, as this was the predefined time window to obtain reliable results. Because our laboratory Inhibitors,research,lifescience,medical could process the blood samples only during working days, we excluded those patients admitted during the selleck weekend in whom the sample could not be obtained Inhibitors,research,lifescience,medical before the 48-h limit. Endothelial progenitor cells measurement Blood samples (4 mL) were obtained by venopuncture and collected in ethylene diamine tetra acetic acid (EDTA) tubes at three time points: baseline (within 48 h from the onset

of stroke), and 7 and 90 days after the onset of stroke. Identification Inhibitors,research,lifescience,medical of EPC is typically based on the cell surface expression of the protein. It is well established that EPC are positive for the following three surface antigens: CD34 (a marker of hematopoietic stem cells), CD133 (a marker of immature hematopoietic stem cells), and KDR (a marker of endothelial protein) (Urbich and Dimmeler 2004; Werner and Nickenig 2006; Lembo et al. 2012; Paczkowska et al. 2013). We analyzed EPC by flow cytometry as previously described (Rustemeyer et al. 2006). In brief, in order to lyse erythrocytes the EDTA-blood samples were treated with BD Pharm

Lyse™ lysing solution (BD Biosciencie, San Jose). Then nucleated cells were stained with Inhibitors,research,lifescience,medical a phycoerythrincyanin-conjugated anti-CD34 monoclonal antibody (Beckman-Coulter, Marseille, France), phycoerythrin-conjugated anti-CD133 monoclonal antibody (Miltenyi-Biotec, Bergisch-Gladbach, Germany), and carboxyfluorescein-conjugated anti-KDR monoclonal antibody (R&D Systems, Wiesbaden, Germany). Isotype-matched Thymidine kinase antibodies were used as controls. After staining, the samples were fixed with 0.2% formaldehyde for 2 h and then analyzed by flow cytometry (EPICS XL). We settled on the appropriate gate for mononuclear cells based scattering light properties. Typically 300,000 total events were acquired to determinate the percentage of the CD34+/VEGF-R2+/CD133+ subpopulation in this gate. Our results are expressed as the proportion of positive cells for the three markers in relation to the total number of gated cells.