Hypothermia Induced Impairment of Platelets: Assessment With Multiplate vs. ROTEM-An In Vitro Study.

Introduction: This experimental in vitro study aimed to identify and characterize hypothermia-associated coagulopathy and to compare changes in mild to severe hypothermia with the quantitative measurement of rotational thromboelastometry (ROTEM) and multiple-electrode aggregometry (MULTIPLATE). Methods: Whole blood samples from 18 healthy volunteers were analyzed at the target temperatures of 37, 32, 24, 18, and 13.7°C with ROTEM (ExTEM, InTEM and FibTEM) and MULTIPLATE using the arachidonic acid 0.5 mM (ASPI), thrombin receptor-activating peptide-6 32 µM (TRAP) and adenosine diphosphate 6.4 µM (ADP) tests at the corresponding incubating temperatures for coagulation assessment. Results: Compared to baseline (37°C) values ROTEM measurements of clotting time (CT) was prolonged by 98% (at 18°C), clot formation time (CFT) was prolonged by 205% and the alpha angle dropped to 76% at 13.7°C (p < 0.001). At 24.0°C CT was prolonged by 56% and CFT by 53%. Maximum clot firmness was only slightly reduced by ≤2% at 13.7°C. Platelet function measured by MULTIPLATE was reduced with decreasing temperature (p < 0.001): AUC at 13.7°C -96% (ADP), -92% (ASPI) and -91% (TRAP). Conclusion: Hypothermia impairs coagulation by prolonging coagulation clotting time and by decreasing the velocity of clot formation in ROTEM measurements. MULTIPLATE testing confirms a linear decrease in platelet function with decreasing temperatures, but ROTEM fails to adequately detect hypothermia induced impairment of platelets.

Efficacy of prehospital administration of fibrinogen concentrate in trauma patients bleeding or presumed to bleed (FIinTIC): A multicentre, double-blind, placebo-controlled, randomised pilot study.

BACKGROUND: Trauma-induced coagulopathy (TIC) substantially contributes to mortality in bleeding trauma patients. OBJECTIVE: The aim of the study was to administer fibrinogen concentrate in the prehospital setting to improve blood clot stability in trauma patients bleeding or presumed to bleed. DESIGN: A prospective, randomised, placebo-controlled, double-blinded, international clinical trial. SETTING: This emergency care trial was conducted in 12 Helicopter Emergency Medical Services (HEMS) and Emergency Doctors' vehicles (NEF or NAW) and four trauma centres in Austria, Germany and Czech Republic between 2011 and 2015. PATIENTS: A total of 53 evaluable trauma patients aged at least 18 years with major bleeding and in need of volume therapy were included, of whom 28 received fibrinogen concentrate and 25 received placebo. INTERVENTIONS: Patients were allocated to receive either fibrinogen concentrate or placebo prehospital at the scene or during transportation to the study centre. MAIN OUTCOME MEASURES: Primary outcome was the assessment of clot stability as reflected by maximum clot firmness in the FIBTEM assay (FIBTEM MCF) before and after administration of the study drug. RESULTS: Median FIBTEM MCF decreased in the placebo group between baseline (before administration of study treatment) and admission to the Emergency Department, from a median of 12.5 [IQR 10.5 to 14] mm to 11 [9.5 to 13] mm (P = 0.0226), but increased in the FC Group from 13 [11 to 15] mm to 15 [13.5 to 17] mm (P = 0.0062). The median between-group difference in the change in FIBTEM MCF was 5 [3 to 7] mm (P < 0.0001). Median fibrinogen plasma concentrations in the fibrinogen concentrate Group were kept above the recommended critical threshold of 2.0 g l-1 throughout the observation period. CONCLUSION: Early fibrinogen concentrate administration is feasible in the complex and time-sensitive environment of prehospital trauma care. It protects against early fibrinogen depletion, and promotes rapid blood clot initiation and clot stability. TRIAL REGISTRY NUMBERS: EudraCT: 2010-022923-31 and ClinicalTrials.gov: NCT01475344.

Hypothermia-Associated Coagulopathy: A Comparison of Viscoelastic Monitoring, Platelet Function, and Real Time Live Confocal Microscopy at Low Blood Temperatures, an in vitro Experimental Study.

INTRODUCTION: Hypothermia has notable effects on platelets, platelet function, fibrinogen, and coagulation factors. Common laboratory techniques cannot identify those effects, because blood samples are usually warmed to 37°C before analysis and do not fully reflect the in vivo situation. Multiple aspects of the pathophysiological changes in humoral and cellular coagulation remain obscure. This in vitro experimental study aimed to compare the measurements of thromboelastometry (TEM), multiple-electrode aggregometry (MEA) and Real Time Live Confocal Imaging for the purpose of identifying and characterizing hypothermia-associated coagulopathy. METHODS: Blood samples were drawn from 18 healthy volunteers and incubated for 30 min before being analyzed at the target temperatures (37, 32, 24, 18, and 13.7°C). At each temperature thromboelastometry and multiple-electrode aggregometry were measured. Real Time Live Confocal Imaging was performed at 4, 24, and 37°C. The images obtained by Real Time Live Confocal Imaging were compared with the functional results of thromboelastometry and multiple-electrode aggregometry. RESULTS: Thromboelastometry standard parameters were impaired at temperatures below baseline 37°C (ANOVA overall effect, p < 0.001): clotting time was prolonged by 27% at 13.7°C and by 60% at 18°C (p < 0.044); clot formation time was prolonged by 157% (p < 0.001). A reduction in platelet function with decreasing temperatures was observed (p < 0.001); the area under the curve at 13.7°C was reduced by 96% (ADP test), 92% (ASPI test), and 91% (TRAP test) of the baseline values. Temperature-associated changes in coagulation were visualized with Real Time Live Confocal Imaging. Molecular changes such as the temperature-associated decrease in the fibrin network are paralleled by cellular effects like the lesser activity of the platelets as a result of decreased temperature. The maximum clot firmness (MCF) in TEM only changed slightly within the temperature range tested. CONCLUSION: The inhibitory effects of temperature on clot formation were visualized with Real Time Live Confocal Microscopy and compared with standard point-of-care testing. Inhibition of clotting factors and impaired platelet function are probably a result of hypothermia-induced impairment of thrombin. Measurement of MCF in TEM does not fully concur with Real Time Live Confocal Microscopy or MEA in hypothermia.

A Prospective Pilot Trial to Assess the Efficacy of Argatroban (Argatra®) in Critically Ill Patients with Heparin Resistance.

The current study aims to evaluate whether prophylactic anticoagulation using argatroban or an increased dose of unfractionated heparin (UFH) is effective in achieving the targeted activated partial thromboplastin time (aPTT) of more than 45 s in critically ill heparin-resistant (HR) patients. Patients were randomized either to continue receiving an increased dose of UFH, or to be treated with argatroban. The endpoints were defined as achieving an aPTT target of more than 45 s at 7 h and 24 h. This clinical trial was registered on clinicaltrials.gov (NCT01734252) and on EudraCT (2012-000487-23). A total of 42 patients, 20 patients in the heparin and 22 in the argatroban group, were included. Of the patients with continued heparin treatment 55% achieved the target aPTT at 7 h, while only 40% of this group maintained the target aPTT after 24 h. Of the argatroban group 59% reached the target aPTT at 7 h, while at 24 h 86% of these patients maintained the targeted aPTT. Treatment success at 7 h did not differ between the groups (p = 0.1000), whereas at 24 h argatroban showed significantly greater efficacy (p = 0.0021) than did heparin. Argatroban also worked better in maintaining adequate anticoagulation in the further course of the study. There was no significant difference in the occurrence of bleeding or thromboembolic complications between the treatment groups. In the case of heparin-resistant critically ill patients, argatroban showed greater efficacy than did an increased dose of heparin in achieving adequate anticoagulation at 24 h and in maintaining the targeted aPTT goal throughout the treatment phase.

Response patterns of routinely measured inflammatory and coagulatory parameters in sepsis.

BACKGROUND: Sepsis is characterized by a pro-inflammatory and pro-coagulatory shift which can induce life-threatening complications. Close monitoring and risk stratification of sepsis patients is crucial for proper treatment and consequently patient outcome. Therefore, this study focuses on the response patterns of inflammatory and coagulatory parameters used in clinical routines to estimate the course of sepsis. METHODS: A total of 1,110 patients diagnosed with sepsis were retrospectively analyzed to identify response patterns for risk stratification of routine parameters measured at the peak level of C-reactive protein. Cluster analysis was used and the differences in the patient characteristics and 28-day survival were assessed. Cox proportional hazards regression model for survival stratified by the clusters was performed. RESULTS: The analyses revealed the parameters to have five distinct response patterns. These clusters reflect the etiology as well as the course of sepsis associated with different mortalities. Here, impairment of the liver plays a crucial role in the ability to appropriately respond to sepsis. Of the routinely measured parameters, C-reactive protein and antithrombin seem to be unspecific for stratification of septic patients. Adjusted for the individual clusters, survival was associated with an increase in fibrinogen (p = 0.0042), platelets (p = 0.0003) and PT (p = 0.001) as well as a decrease in leukocytes (p = 0.034). CONCLUSIONS: This study reveals that patients have distinct response patterns of inflammatory and coagulatory parameters depending on disease etiology. These patterns are associated with different mortalities although the patients have similar levels of C-reactive protein. Independently of the type of response, good coagulatory capacity seems to be crucial for patient survival.

Influence of factor XII deficiency on activated partial thromboplastin time (aPTT) in critically ill patients.

FXII deficiency results in spontaneous prolongation of activated partial thromboplastin time (aPTT), which is widely used to monitor thromboprophylaxis. Misinterpretation of spontaneously prolonged aPTT may result in omission of thromboembolic treatment or even unnecessary transfusion of blood products. This retrospective analysis was performed to calculate a threshold level of FXII resulting in aPTT prolongation. 79 critically ill patients with spontaneous prolongation of aPTT were included. A correlation analysis and a ROC curve for aPTT prolongation predicted by FXII level were created to find the FXII threshold level. Prolongation of aPTT was associated with disease severity. A significant inverse proportionality between FXII and aPTT was seen. A ROC curve for aPTT prolongation, predicted by FXII level (AUC 0.85; CI 0.76-0.93), revealed a FXII threshold level of 42.5%. Of our patients 50.6% experienced a FXII deficiency, in 80.0% of whom we found aPTT to be prolonged without a significantly higher bleeding rate. The FXII deficiency was more common in patients with higher SAPS3 scores, septic shock, transfusion of red blood cells and platelet concentrates as well as in patients receiving renal replacement therapy. Patients with a FXII deficiency and prolonged aPTT less often received anticoagulatory therapy although they were more severely ill. The rate of thromboembolic events was higher in these patients although the difference was not statistically significant. Of all patients with spontaneous aPTT prolongation 50.6% had a FXII level of 42.5% or less. Those patients received insufficient thromboembolic prophylaxis.

Reversing anticoagulation in the hemorrhaging patient.

PURPOSE OF REVIEW: Anticoagulants in general, but especially the relatively new direct oral anticoagulants and platelet inhibitors, pose a great challenge for physicians in the hemorrhaging patient. The aim of the present review is to provide an overview on recent studies dealing with the reversal of anticoagulation in the hemorrhaging patient and to describe our therapeutic emergency strategy for those patients. RECENT FINDINGS: A specific antidote for dabigatran is already on the market and antidotes for the direct and indirect factor Xa inhibitors are in development. Moreover, bleeding under platelet inhibitors remains critical with very little evidence on effective reversal strategies. SUMMARY: To reverse anticoagulation in the hemorrhaging patient, specific antidotes should be the first option if available, followed by four-factor prothrombin complex concentrate (PCC), activated PCC and recombinant activated factor seven as the emergency strategy. Fibrinogen concentrate, antifibrinolytics and oral charcoal, respectively, can be considered as an additional measure. Massive blood loss and thrombocytopenia should be treated independently according to the respective, local guidelines for (massive) transfusion of blood and blood products.

Antithrombin deficiency is associated with mortality and impaired organ function in septic pediatric patients: a retrospective study.

BACKGROUND: Sepsis remains a major problem in intensive care medicine. It is often accompanied by coagulopathies, leading to thrombotic occlusion of small vessels with subsequent organ damage and even fatal multi-organ failure. Prediction of the clinical course and outcome-especially in the heterogeneous group of pediatric patients-is difficult. Antithrombin, as an endogenous anticoagulant enzyme with anti-inflammatory properties, plays a central role in controling coagulation and infections. We investigated the relationship between antithrombin levels and organ failure as well as mortality in pediatric patients with sepsis. METHODS: Data from 164 patients under the age of 18, diagnosed with sepsis, were retrospectively reviewed. Antithrombin levels were recorded three days before to three days after peak C-reactive protein to correlate antithrombin levels with inflammatory activity. Using the concept of developmental haemostasis, patients were divided into groups <1 yr and ≥1 yr of age. RESULTS: In both age groups, survivors had significantly higher levels of antithrombin than did deceased patients. An optimal threshold level for antithrombin was calculated by ROC analysis for survival: 41.5% (<1 yr) and 67.5% (≥1 yr). The mortality rate above this level was 3.3% (<1 yr) and 9.5% (≥1 yr), and below this level 41.7% (<1 yr) and 32.2% (≥1 yr); OR 18.8 (1.74 to 1005.02), p = 0.0047, and OR 4.46 (1.54 to 14.89), p = 0.003. In children <1 yr with antithrombin levels <41.5% the rate of respiratory failure (66.7%) was significantly higher than in patients with antithrombin levels above this threshold level (23.3%), OR 6.23 (1.23 to 37.81), p = 0.0132. In children ≥1 yr, both liver failure (20.3% vs 1.6%, OR 15.55 (2.16 to 685.01), p = 0.0008) and a dysfunctional intestinal tract (16.9% vs 4.8%, OR 4.04 (0.97 to 24.08), p = 0.0395) occurred more frequently above the antithrombin threshold level of 67.5%. CONCLUSION: In pediatric septic patients, significantly increased mortality and levels of organ failure were found below an age-dependent antithrombin threshold level. Antithrombin could be useful as a prognostic marker for survival and occurrence of organ failure in pediatric sepsis.

Four-factor prothrombin complex concentrate improves thrombin generation and prothrombin time in patients with bleeding complications related to rivaroxaban: a single-center pilot trial.

BACKGROUND: Direct oral anticoagulants (DOACs) pose a great challenge for physicians in life-threatening bleeding events. The aim of this study was to test the efficacy of reversing the DOAC rivaroxaban using four-factor PCC (prothrombin complex concentrate), a non-specific reversing agent. METHODS: Patients with life-threatening bleeding events during rivaroxaban treatment were included and administered 25 U kg-1 of PCC. Blood samples were collected immediately prior to as well as after PCC treatment at predefined time intervals. The primary endpoint was defined as the difference in thrombin generation (TG) parameters ETP (endogenous thrombin potential) and Cmax (peak thrombin generation) prior to and ten minutes subsequent to PCC treatment. RESULTS: Thirteen patients, of whom the majority suffered from intra-cranial haemorrhage (ICH) or subdural haemorrhage (SDH), were included and administered PCC. The results show that the ETP (TG) significantly (p = 0.001) improved by 68% and Cmax (TG) by 54% (p = 0.001) during PCC treatment. In addition, the Quick value (prothrombin time: QuickPT) significantly improved by 28% and the activated partial thromboplastin time (aPTT) was decreased by 7% ten minutes after PCC administration. Cmax was reduced at baseline, but not ETP, aPTT or QuickPT. Lag time until initiation (TG, tlag), thromboelastometry clotting time (CTEXTEM) and time to peak (TG, tmax) correlated best with measured rivaroxaban levels and were out of normal ranges at baseline, but did not improve after PCC administration. In 77% of the patients bleeding (ICH/SDH-progression) ceased following PCC administration. During the study three participants passed away due to other complications not related to PCC treatment. The possibility of thrombosis formation was also evaluated seven days after administering PCC and no thromboses were found. CONCLUSIONS: This study shows that use of PCC improved ETP, Cmax, QuickPT and aPTT. However, of these parameters, only Cmax was reduced at the defined baseline. It can be concluded that CTEXTEM, tlag and tmax correlated best with the measured rivaroxaban levels. The study drug was found to be safe. Nonetheless, additional studies specifically targeting assessment of clinical endpoints should be performed to further confirm these findings. CLINICAL TRIAL REGISTRATION: EudraCT trial No. 2013-004484-31.

Aviscumine, a recombinant ribosomal inhibitor, increases the antitumor activity of natural killer cells.

Aviscumine, a recombinant lectin I, has been identified as an immunomodulatory agent within a new class of ribotoxic stress-inducing anticancer substances that have demonstrated efficacy in phase I/II trials. The aim of the present study was to elucidate the presumed effect of aviscumine on enhancing human natural killer (NK) cell antitumor cytotoxicity. To measure the effect of aviscumine on human NK cell cytotoxicity, chromium-51-release assays against K-562 cells were performed with isolated NK cells from the whole blood of 34 healthy volunteers. Two effector-to-target cell ratios (12.5:1 and 25:1) were used by two independent investigators with a focus on the concentration-dependent effect (0.5 vs. 1 ng/ml aviscumine), reproducibility (first vs. second investigator) and the specificity of the effect by comparison to a heat-inactivated aliquot and interleukin 2 (IL-2) stimulation (10 ng/ml). The mediation of the effect via degranulation was demonstrated by flow cytometric analyses of CD107α expression. Statistics were performed with SPSS using Student's t-tests for normally distributed data. Aviscumine induced a significant and reproducible, concentration-dependent increase in NK cell cytotoxicity (n=22; P<0.01 for both concentrations and ratios), which was also demonstrated when administered in combination with IL-2 (n=12; 12.5:1 ratio, P<0.001; 25:1 ratio, P=0.025) and when compared with the heat-inactivated aliquots (n=12; 12.5:1, P=0.004; 25:1 ratio, P=0.007). The mediation of its effect via interferon γ degranulation was demonstrated by significantly enhanced CD107α expression (n=7; P=0.005). Taken together, the results indicate that aviscumine induced an increase in NK cell anticancer cytotoxicity. These results highlight its clinical potential as an immunostimulatory agent, particularly with regard to combined use with chemotherapeutics or immune checkpoint inhibitors. However, further studies are required.

FITC-linked Fibrin-Binding Peptide and real-time live confocal microscopy as a novel tool to visualize fibrin(ogen) in coagulation.

Background and Aim: Although fibrinogen has been established as a key player in the process of coagulation, many questions about the role of fibrinogen under specific conditions remain. Confocal microscopic assessment of clot formation, and in particular the role that fibrinogen plays in this process, is commonly investigated using pre-labeled fibrinogen. This has a number of drawbacks, primarily that it is impossible to stain fibrin networks after their formation. The aim of the present study is to present an alternative for conveniently post-staining fibrin in a wide range of models/situations, in real time and with high resolution. Methods: We combined a peptide known to bind fibrin and linked it to fluorescein isothiocyanate (FITC), creating the FITC-Fibrin-Binding Peptide (FFBP). We subsequently tested its fibrin-binding capability in vitro under static conditions, as well as under simulated flow, using real-time live confocal microscopy. Results: Fibrin stained with FFBP overlaps with fibrin stained with fibrinogen pre-labeled with Alexa Fluor 647 following coagulation induction. In contrast to pre-labeled fibrinogen, FFBP also stains already formed fibrin networks. By combining FFBP with real-time live confocal microscopy even the visualization of single fibrin fibers is possible. Conclusions: These data indicate that FFBP is a valid and valuable tool for real-time live confocal assessment of clot formation. Relevance for patients: Our findings present a valuable alternative for the visualization of fibrin even after its formation, and we believe this approach will be particularly valuable for future investigations of important, but previously overlooked, aspects of clot formation.

Increase in antibody-dependent cellular cytotoxicity (ADCC) in a patient with advanced colorectal carcinoma carrying a KRAS mutation under lenalidomide therapy.

The failure of EGFR inhibitors in colorectal tumors with KRAS mutations requires the development of alternative treatment strategies for this patient subgroup. Among the hallmarks of cancer the disturbed immunosurveillance and cancer immune evasion have become emerging targets for cancer therapy. Due to their pleiotropic functions immunomodulatory drugs (IMiDs) are interesting agents for combination therapies in solid tumors. However, their possible contribution and a way of monitoring their biological effects have yet to be revealed. In a heavily pretreated patient with advanced colorectal cancer carrying mutations in APC and KRAS genes, we show an early metabolic response and enhanced NK cell activity to monotherapy with lenalidomide. After subsequent lenalidomide/cetuximab combination treatment, the patient had progressive disease. At the same time a reduced performance status, complicated by febrile neutropenia, occurred, as well as a slight increase in metabolic activity. Concordantly NK cell activity dropped back to baseline. Thus, laboratory measurements and metabolic response assessment correlated with clinical conditions. This case report describes the feasibility and potential of a functional assessment of patient derived immune competent cells in combination with functional imaging for the detection of a biological response.

Middle to Late Eocene paleoenvironmental changes in a marine transgressive sequence from the northern Tethyan margin (Adelholzen, Germany).

The northern Tethyan margin is a key region for determining environmental changes associated with the collision of continental and oceanic tectonic plates and Alpine orogeny. Herein we investigated Middle to Late Eocene neritic to bathyal sediments deposited during an interval of unstable climatic conditions. In order to quantify paleoenvironmental changes, we developed a detailed age model based on biozonations of planktic foraminifera, calcareous nannoplankton, and larger benthic foraminifera. The section at Adelholzen covers the almost complete Lutetian Stage (calcareous nannoplankton zones NP15a-16, planktic foraminifera zones E8-11, shallow benthic (foraminifera) zones SBZ13-15) and large parts of the Priabonian Stage (NP18-20, E14/15), while the intermediate Bartonian Stage (NP17) is completely missing. Foraminiferal, calcareous nannoplankton, and macrofossil assemblages were analyzed for changes in paleo-water depth, mixing and stratification, paleo-primary productivity (pPP), food supply, and bottom water oxygenation. Paleo-water depth estimates range from 50 m (middle neritic, early Lutetian) to nearly 500 m (upper bathyal, late Priabonian). The combination of assemblage composition, planktic and benthic foraminiferal accumulation rates, and derived parameters (carbon-flux to sea floor, pPP) enabled us to identify a series of distinct paleoceanographic events of at least regional significance. Such events are characterized by considerable changes in primary productivity or reduced bottom water ventilation. Calculated pPP-values indicate oligotrophic conditions throughout.