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Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes - A new assay suitable for bench and bedside conditions. - Thrombosis research
Platelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood.Platelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively.The sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-Î± and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support.Using the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting.Copyright Â© 2015 Elsevier Ltd. All rights reserved.
Individual Organ Failure and Concomitant Risk of Mortality Differs According to the Type of Admission to ICU - A Retrospective Study of SOFA Score of 23,795 Patients. - PloS one
Organ dysfunction or failure after the first days of ICU treatment and subsequent mortality with respect to the type of intensive care unit (ICU) admission is poorly elucidated. Therefore we analyzed the association of ICU mortality and admission for medical (M), scheduled surgery (ScS) or unscheduled surgery (US) patients mirrored by the occurrence of organ dysfunction/failure (OD/OF) after the first 72h of ICU stay.For this retrospective cohort study (23,795 patients; DIVI registry; German Interdisciplinary Association for Intensive Care Medicine (DIVI)) organ dysfunction or failure were derived from the Sequential Organ Failure Assessment (SOFA) score (excluding the Glasgow Coma Scale). SOFA scores were collected on admission to ICU and 72h later. For patients with a length of stay of at least five days, a multivariate analysis was performed for individual OD/OF on day three.M patients had the lowest prevalence of cardiovascular failure (M 31%; ScS 35%; US 38%), and the highest prevalence of respiratory (M 24%; ScS 13%; US 17%) and renal failure (M 10%; ScS 6%; US 7%). Risk of death was highest for M- and ScS-patients in those with respiratory failure (OR; M 2.4; ScS 2.4; US 1.4) and for surgical patients with renal failure (OR; M 1.7; ScS 2.7; US 2.4).The dynamic evolution of OD/OF within 72h after ICU admission and mortality differed between patients depending on their types of admission. This has to be considered to exclude a systematic bias during multi-center trials.
Tricuspid annular plane systolic excursion (TAPSE) predicts poor outcome in patients undergoing acute pulmonary embolectomy. - Heart, lung and vessels
Right ventricular failure remains a major cause of mortality during acute pulmonary embolism. Right ventricular function can be assessed with transesophageal echocardiography. However, due to the complex right ventricular anatomy, only a few echocardiographic parameters are reliable and easily obtainable intraoperatively. Tricuspid annular plane systolic excursion is a validated parameter of global right ventricular function.Data from 81 patients with acute pulmonary embolus undergoing pulmonary embolectomy were evaluated. Transesophageal echocardiography derived parameters of right ventricular function were obtained and compared to tricuspid annular plane systolic excursion measurements. Patients were then divided into two groups (TAPSEÂ < 18 mm and â‰¥18 mm).The patient population consisted of 46 males and 35 females, mean age 61.0 Â± 12.9 years. Patients in the TAPSE <18 mm group had significantly larger diastolic (p=0.0015) and systolic (p=0.0031) right ventricular diameters, lower right ventricular fractional area changeÂ (p=0.0065) and greater degrees of tricuspid regurgitation (p=0.0001) compared to patients with TAPSE â‰¥18 mm. In addition, all patients who needed intraoperative cardiopulmonary resuscitation (11/81) or died intraoperatively (8/81) belonged to the TAPSE <18 mm group. Logistic regression analysis confirmed TAPSE <18 mm as an independent risk factor for intraoperative cardiopulmonary resuscitation and death.Transesophageal echocardiography derived TAPSE is easily obtainable and correlates well with other standardized parameters of right ventricular function. TAPSE <18 mm is an independent predictor of intraoperative cardiopulmonary resuscitation and death in patients undergoing emergent pulmonary embolectomy.
Impella 5.0 Support in INTERMACS II Cardiogenic Shock Patients Using Right and Left Axillary Artery Access. - Artificial organs
The catheter-based Impella 5.0 left ventricular assist device (LVAD) is a powerful and less invasive alternative for patients in cardiogenic shock. The use of this device as a primary mechanical circulatory support strategy in INTERMACS II patients should be evaluated. From April 2014 to August 2014, eight Impella 5.0 devices were implanted in seven patients via the axillary artery access (six right and two left). We analyzed the outcome of the four patients in whom the Impella 5.0 device was implanted for the purpose of primary stabilization of cardiogenic shock (INTERMACS II). The remaining three patients had a contraindication for a permanent LVAD and received the device for prolonged weaning from extracorporeal life support (ECLS) system. The implantation of the Impella 5.0 was technically successful in all patients and resulted in the stabilization of the clinical situation. All four patients could be bridged to a long-term device (nâ€‰=â€‰3) or to cardiac recovery (nâ€‰=â€‰1). In one patient, 2 days of ECLS support was necessary because of pump thrombosis after 31 days of Impella 5.0 support. One patient with bronchopneumonia had the Impella 5.0 exchanged from the right to the left axillary artery after 22 days of support because of the progressive loss of purge flow and the need for longer bridging to a permanent LVAD. The last patient was supported for giant-cell myocarditis for 22 days and bridged to cardiac recovery. All patients were transferred to the intensive care unit with the Impella device in place. In INTERMACS II situations, the implantation of the Impella 5.0 via the right or left axillary access allowed additional time for decision making. Early patient mobilization, including walking with the Impella device in place, optimized the conditions for either weaning or the implantation of a permanent LVAD. This novel technique of left axillary approach leads to more flexibility in the case of anatomical- or device-related contraindications to right-side access, or when the device needs to be exchanged while continuous support is necessary.Copyright Â© 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Effect of remote ischemic preconditioning on kidney injury among high-risk patients undergoing cardiac surgery: a randomized clinical trial. - JAMA
No interventions have yet been identified to reduce the risk of acute kidney injury in the setting of cardiac surgery.To determine whether remote ischemic preconditioning reduces the rate and severity of acute kidney injury in patients undergoing cardiac surgery.In this multicenter trial, we enrolled 240 patients at high risk for acute kidney injury, as identified by a Cleveland Clinic Foundation score of 6 or higher, between August 2013 and June 2014 at 4 hospitals in Germany. We randomized them to receive remote ischemic preconditioning or sham remote ischemic preconditioning (control). All patients completed follow-up 30 days after surgery and were analyzed according to the intention-to-treat principle.Patients received either remote ischemic preconditioning (3 cycles of 5-minute ischemia and 5-minute reperfusion in one upper arm after induction of anesthesia) or sham remote ischemic preconditioning (control), both via blood pressure cuff inflation.The primary end point was the rate of acute kidney injury defined by Kidney Disease: Improving Global Outcomes criteria within the first 72 hours after cardiac surgery. Secondary end points included use of renal replacement therapy, duration of intensive care unit stay, occurrence of myocardial infarction and stroke, in-hospital and 30-day mortality, and change in acute kidney injury biomarkers.Acute kidney injury was significantly reduced with remote ischemic preconditioning (45 of 120 patients [37.5%]) compared with control (63 of 120 patients [52.5%]; absolute risk reduction, 15%; 95% CI, 2.56%-27.44%; Pâ€‰=â€‰.02). Fewer patients receiving remote ischemic preconditioning received renal replacement therapy (7 [5.8%] vs 19 [15.8%]; absolute risk reduction, 10%; 95% CI, 2.25%-17.75%; Pâ€‰=â€‰.01), and remote ischemic preconditioning reduced intensive care unit stay (3 days [interquartile range, 2-5]) vs 4 days (interquartile range, 2-7) (Pâ€‰=â€‰.04). There was no significant effect of remote ischemic preconditioning on myocardial infarction, stroke, or mortality. Remote ischemic preconditioning significantly attenuated the release of urinary insulinlike growth factor-binding protein 7 and tissue inhibitor of metalloproteinases 2 after surgery (remote ischemic preconditioning, 0.36 vs control, 0.97 ng/mL2/1000; difference, 0.61; 95% CI, 0.27-0.86; Pâ€‰<â€‰.001). No adverse events were reported with remote ischemic preconditioning.Among high-risk patients undergoing cardiac surgery, remote ischemic preconditioning compared with no ischemic preconditioning significantly reduced the rate of acute kidney injury and use of renal replacement therapy. The observed reduction in the rate of acute kidney injury and the need for renal replacement warrants further investigation.German Clinical Trials Register Identifier: DRKS00005333.
Platelet Gi protein GÎ±i2 is an essential mediator of thrombo-inflammatory organ damage in mice. - Proceedings of the National Academy of Sciences of the United States of America
Platelets are crucial for hemostasis and thrombosis and exacerbate tissue injury following ischemia and reperfusion. Important regulators of platelet function are G proteins controlled by seven transmembrane receptors. The Gi protein GÎ±(i2) mediates platelet activation in vitro, but its in vivo role in hemostasis, arterial thrombosis, and postischemic infarct progression remains to be determined. Here we show that mice lacking GÎ±(i2) exhibit prolonged tail-bleeding times and markedly impaired thrombus formation and stability in different models of arterial thrombosis. We thus generated mice selectively lacking GÎ±(i2) in megakaryocytes and platelets (Gna(i2)(fl/fl)/PF4-Cre mice) and found bleeding defects comparable to those in global GÎ±(i2)-deficient mice. To examine the impact of platelet GÎ±(i2) in postischemic thrombo-inflammatory infarct progression, Gna(i2)(fl/fl)/PF4-Cre mice were subjected to experimental models of cerebral and myocardial ischemia/reperfusion injury. In the model of transient middle cerebral artery occlusion stroke Gna(i2)(fl/fl)/PF4-Cre mice developed significantly smaller brain infarcts and fewer neurological deficits than littermate controls. Following myocardial ischemia, Gna(i2)(fl/fl)/PF4-Cre mice showed dramatically reduced reperfusion injury which correlated with diminished formation of the ADP-dependent platelet neutrophil complex. In conclusion, our data provide definitive evidence that platelet GÎ±(i2) not only controls hemostatic and thrombotic responses but also is critical for the development of ischemia/reperfusion injury in vivo.
Influence of intraaortic balloon pump counterpulsation on transesophageal echocardiography derived determinants of diastolic function. - PloS one
Intraaortic balloon pump counterpulsation (IABP) is often used in patients with acute coronary syndrome for its favourable effects on left ventricular (LV) systolic function and coronary perfusion. However, the effects of IABP on LV diastolic function have not been comprehensively investigated. Acute diastolic dysfunction has been linked to increased morbidity and mortality. The aim of this study was to examine the influence of IABP on LV diastolic dysfunction using standard TEE derived parameters.Intraoperative TEE was performed in 10 patients (mean age 65 Â± 11 yrs) undergoing urgent coronary artery bypass graft surgery (CABG), who had received an IABP preoperatively. TEE derived measures of diastolic dysfunction included early to late transmitral Doppler inflow velocity ratio (E/A), deceleration time (Dt), pulmonary venous systolic to diastolic Doppler velocity ratio (S/D), transmitral propagation velocity (Vp), and the ratio of early to late mitral annular tissue Doppler velocities (e'/a'). Statistical analyses included the Wilcoxon Sign-Rank test, and a p<0.05 was considered significant.Transmitral inflow E/A ratios increased significantly from 0.86 to 1.07 (p < 0.05), while Dt decreased significantly from 218 to 180 ms (p < 0.05) with the use of IABP. Significant increases in Vp (34 cm/s to 43 cm/s; p < 0.05), and e'/a' (0.58 to 0.71; p < 0.05) suggested a favourable influence of intraaortic counterpulsation on diastolic function.The use of perioperative IABP significantly improves TEE derived parameters of diastolic function consistent with a favourable impact on LV relaxation in cardiac surgery patients undergoing CABG.
Preoperative interleukin-22 values add valuable information for outcome prediction following orthotopic liver transplantation: a preliminary study. - Annals of transplantation : quarterly of the Polish Transplantation Society
Recent findings support the idea that interleukin (IL)-22 serum levels are related to disease severity in end-stage liver disease. Existing scoring systems--Model for End-Stage Liver Disease (MELD), Survival Outcomes Following Liver Transplantation (SOFT) and Pre-allocation-SOFT (P-SOFT)--are well-established in appraising survival rates with or without liver transplantation. We tested the hypothesis that IL-22 serum levels at transplantation date correlate with survival and potentially have value as a predictive factor for survival.MELD, SOFT, and P-SOFT scores were calculated to estimate post-transplantation survival. Serum levels of IL-22, IL-6, IL-10, C-reactive protein (CRP), and procalcitonin (PCT) were collected prior to transplantation in 41 patients. Outcomes were assessed at 3 months, 1 year, and 3 years after transplantation.IL-22 significantly correlated with MELD, P-SOFT, and SOFT scores (Rs 0.35, 0.63, 0.56 respectively, p<0.05) and with the discrimination in post-transplantation survival. IL-6 showed a heterogeneous pattern (Rs 0.40, 0.63, 0.57, respectively, p<0.05); CRP and PCT did not correlate. We therefore added IL-22 serum values to existing scoring systems in a generalized linear model (GLM), resulting in a significantly improved outcome prediction in 58% of the cases for both the P-SOFT (p<0.01) and SOFT scores (p<0.001).Further studies are needed to address the concept that IL-22 serum values at the time of transplantation provide valuable information about survival rates following orthotopic liver transplantation.
Basic life support is effectively taught in groups of three, five and eight medical students: a prospective, randomized study. - BMC medical education
Resuscitation is a life-saving measure usually instructed in simulation sessions. Small-group teaching is effective. However, feasible group sizes for resuscitation classes are unknown. We investigated the impact of different group sizes on the outcome of resuscitation training.Medical students (nâ€‰=â€‰74) were randomized to courses with three, five or eight participants per tutor. The course duration was adjusted according to the group size, so that there was a time slot of 6 minutes hands-on time for every student. All participants performed an objective structured clinical examination before and after training. The teaching sessions were videotaped and resuscitation quality was scored using a checklist while we measured the chest compression parameters with a manikin. In addition, we recorded hands-on-time, questions to the tutor and unrelated conversation.Results are displayed as median (IQR). Checklist pass rates and scores were comparable between the groups of three, five and eight students per tutor in the post-test (93%, 100% and 100%). Groups of eight students asked fewer questions (0.5 (0.0 - 1.0) vs. 3.0 (2.0 - 4.0), pâ€‰<â€‰.001), had less hands-on time (2:16 min (1:15 - 4:55 min) vs. 4:07 min (2:54 - 5:52 min), pâ€‰=â€‰.02), conducted more unrelated conversations (17.0â€‰Â±â€‰5.1 and 2.9â€‰Â±â€‰1.7, pâ€‰<â€‰0.001) and had lower self-assessments than groups of three students per tutor (7.0 (6.1 - 9.0) and 8.2 (7.2 - 9.0), pâ€‰=â€‰.03).Resuscitation checklist scores and pass rates after training were comparable in groups of three, five or eight medical students, although smaller groups had advantages in teaching interventions and hands-on time. Our results suggest that teaching BLS skills is effective in groups up to eight medical students, but smaller groups yielded more intense teaching conditions, which might be crucial for more complex skills or less advanced students.
Inhibition of neogenin dampens hepatic ischemia-reperfusion injury. - Critical care medicine
Liver ischemia and reperfusion injury is a common source of significant morbidity and mortality following liver transplantation, hemorrhagic shock, or major hepatic surgery. Based on studies showing a critical role for the neuronal guidance receptor neogenin (Neo1) outside the nervous system in mediating tissue adaption during acute inflammation, we hypothesized that Neo1 enhances hepatic ischemia and reperfusion injury.Animal study.University-based experimental laboratory.Wid-type, neogenin deficient and chimeric mice.Neogenin expression was evaluated during inflammatory stimulation in vitro and during ischemia and reperfusion injury in vivo, intravital microscopy performed to study intravascular flow characteristics. The extent of liver injury was evaluated using histology, serum levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. The functional role of Neo1 during liver IR was evaluated in mice with gene targeted repression of neogenin (Neo1-/-), bone marrow chimeric animals and controls. In addition, functional inhibition of neogenin was performed using antibody injection.We observed an induction of Neo1 during inflammation in vitro and ischemia and reperfusion in vivo. Intravital microscopy demonstrated a decreased ability of Neo1 leukocytes to attach to endothelial vascular wall during inflammation. Subsequent studies in Neo1 mice showed attenuated serum levels of lactate dehydrogenase, aspartate, alanine, and proinflammatory cytokines during hepatic ischemia and reperfusion injury. This was associated with improved hepatic histology scores. Studies in chimeric animals demonstrated that the hematopoietic Neo1 expression to be crucial for the observed results. Treatment with an anti-Neo1 antibody resulted in a significant reduction of experimental hepatic ischemia and reperfusion injury, involving attenuated variable of lactate dehydrogenase, alanine, aspartate, and cytokine levels.These data provide a unique role for Neo1 in the development of hepatic ischemia and reperfusion injury and identified Neo1 as a potential target to prevent liver dysfunction in the future.
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