Investigation of the influence of xenoreactive antibodies on activation of complement and coagulation in an ex vivo perfusion animal study using porcine kidneys.

During pig-to-primate xenotransplantation or perfusion of porcine organs with human blood, a xenogeneic coagulopathy with consecutive development of thrombotic microangiopathy (TMA) can be observed. The aim of this study was to elucidate the influence of the reduction of xenoreactive natural antibodies on the coagulopathy using an ex vivo perfusion system. Thirteen perfusion experiments using landrace wild-type porcine kidneys were performed in three different experimental groups: autologous, xenogeneic, and immunoadsorption. During and after perfusion, blood and tissue samples were collected to assess markers of coagulation, complement, inflammation, and endothelial activation. Immunoadsorption prior to perfusion did not prolong perfusion time (174 min ±28) compared to xenogeneic (182 min ±22) experiments, whereas autologous perfusion was possible for maximum of 240 min in all experiments. Activation of coagulation was similar comparing perfusions after immunoadsorption (D-Dimer 24 186 µg/l ±5813; TAT 566 µg/l ±34) to xenogeneic (D-Dimer 22 175 µg/l ±7826, TAT 600 µg/l ±0) experiments. But antibody-mediated complement activation was reduced in the immunoadsorption group. TNF-alpha and markers of endothelial cell activation were lower in the immunoadsorption group compared to the xenogeneic experiments. In this ex vivo perfusion model, we observed that marked removal of xenogeneic antibodies can reduce complement activation via the classical pathway as well as endothelial cell activation and inflammation. Immunoadsorption cannot prevent the activation of the terminal complement cascade and coagulation.

Selection of an Anticalin® against the membrane form of Hsp70 via bacterial surface display and its theranostic application in tumour models.

We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD=510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro.

Preoperative Recipient Parameters Allow Early Estimation of Postoperative Outcome and Intraoperative Transfusion Requirements in Liver Transplantation.

CONTEXT: Liver transplantation is a complex intervention, and early anticipation of personnel and logistic requirements is of great importance. Early identification of high-risk patients could prove useful. We therefore evaluated prognostic values of recipient parameters commonly available in the early preoperative stage regarding postoperative 30- and 90-day outcomes and intraoperative transfusion requirements in liver transplantation. DESIGN, SETTING, AND PARTICIPANTS: All adult patients undergoing first liver transplantation at Hannover Medical School between January 2005 and December 2010 were included in this retrospective study. Demographic, clinical, and laboratory data as well as clinical courses were recorded. Prognostic values regarding 30- and 90-day outcomes were evaluated by uni- and multivariate statistical tests. Identified risk parameters were used to calculate risk scores. RESULTS: There were 426 patients (40.4% female) included with a mean age of 48.6 (11.9) years. Absolute 30-day mortality rate was 9.9%, and absolute 90-day mortality rate was 13.4%. Preoperative leukocyte count >5200/µL, platelet count <91 000/µL, and creatinine values ≥77 µmol/L were relevant risk factors for both observation periods ( P < .05, respectively). A score based on these factors significantly differentiated between groups of varying postoperative outcomes and intraoperative transfusion requirements ( P < .05, respectively). CONCLUSION: A score based on preoperative creatinine, leukocyte, and platelet values allowed early estimation of postoperative 30- and 90-day outcomes and intraoperative transfusion requirements in liver transplantation. Results might help to improve timely logistic and personal strategies.

[Competence-based Training in the "Protected Environment": From Sheltered Space to Real Life]. / Kompetenzbasierte Ausbildung im "geschützten Umfeld": vom Schonraum zum Realraum.

The idea of "learning in a protected environment" is frequently used within the framework of postgradual education in anaesthesiology. However it remains unclear and its exact meaning lies in the eye of the beholder. This paper aims to highlight the definition of the term "protected environment" and its relevance in anaesthesiology. This includes teaching and learning strategies like competence based training and simulation based training in all its variations. We conclude with an attempt to classify the wide variety of Simulators and teaching methods in use including their individual problems transferred into real life situations. Concerning the fact that this paper is supposed to be a review in medical education, its structure differs from case related papers with more medical content.

["Learning in Protected Environment"- Implementation in Continuing Medical Education]. / "Lernen im geschützten Umfeld": Implementierung in die Fort- und Weiterbildung.

Actual concepts in continuing medical education in acute or emergency medicine contain skill training as well as simulation training. Methods and mechanisms to reduce crisis, like human factor training, shared mental models or closed-loop communication are incorporated. It is unknown which training method is optimal for individual departments in hospitals or for the individual level of education of the healthcare provider. A concept we provide is the so called "learning in protected environment": this environment protects the course participants and our patients from negative consequences of a conventional hands-on training. Concurrently the participants benefit from our standardized course concepts. We achieve our goal of an optimal preparation for clinical practice by continuous re-evaluation of the content and educational objects. The implementation of a multimodal team training has to be adopted for each institution individually - methods for an implementation should be standardized. We suggest the use of the "Kern cycle" for a structured approach to curriculum development. On this foundation the combination of "learning in protected environment" and crisis training is optimal to achieve an improved patient safety in acute care.

[Simulation as a Training Method for the Professionalization of Teams]. / Simulation als Fortbildungsmethode zur Professionalisierung von Teams.

Simulation as an educational method can be applied to the training of processes, technical and non-technical skills. This article focuses on the role of simulation in crisis resource management and non-technical skills. A realistic work environment requires well-trained staff regarding simulation technology and communication. A training (unit) is divided into three sections. During the briefing the team is introduced to the scenario. Afterwards, the patient is treated by an interdisciplinary team. Communication under the pressure of action, even if one does not agree with the approach of the colleagues, should be practiced. After the scenario a structured debriefing is conducted. The trainer supervises the reflection of the teams' actions. Various methods such as "guided team self-correction", "advocacy-inquiry" and the "TeamGAINS"-approach are available for this decisive phase of the training. A safe environment is guaranteed, video recordings will never leave the training. Active experimentation, concrete experiences and accurate reflection are the key factors of success for the method simulation. Positive effects on critical incidents, resuscitation outcome and improvement of team climate can be observed after simulation training.

Simulator-Based Air Medical Training Program Christoph Life: From Concept to Course.

OBJECTIVE: Christoph Life is a simulator-based air medical training program and a new and innovative educational concept. Participants pass different scenarios with a fully equipped and movable helicopter simulator. Main focuses of the program are crew resource management (CRM) elements and team training. Information about expectations end effectiveness of the training is sparse. METHODS: During a 2-day training, participants learn CRM basics and complete various emergency medical scenarios. For evaluation, we used an anonymous questionnaire either with polar questions or a 6-coded psychometric Likert scale. The Wilcoxon test was used for statistical analysis. The significance level was set at P < .05. RESULTS: Thirteen teams of emergency physicians and specially trained paramedics underwent Christoph Life. It was evaluated largely positively and considered very helpful for daily work (5.7 ± 0.5) and avoiding mistakes (5.7 ± 0.5). The quality of participants' knowledge about CRM basics (3.5 ± 1.2 vs. 5.4 ± 0.7, P < .001), self-assessment of communication skills (4.2 ± 0.7 vs. 4.8 ± 0.8, P = .02), and active reflection of communication aspects (3.9 ± 0.9 vs. 5.5 ± 0.5, P < .001) could be strikingly increased. CONCLUSIONS: There is a considerable demand for intensified training on the part of the users. We were able to show that a simulator-based air medical training program is a helpful training tool with an obvious subjective benefit for the participants' nontechnical skills.

Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.

BACKGROUND: Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. METHODS: Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. RESULTS: Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. CONCLUSION: We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.

Recombinant human antithrombin prevents xenogenic activation of hemostasis in a model of pig-to-human kidney transplantation.

BACKGROUND: Xenogenic activation of hemostasis (XAH) represents a major hurdle for the transplantation of discordant animal organs into humans as it results in thrombotic microangiopathy (TMA). We have previously shown that recombinant human-activated protein C (rhAPC) mitigates XAH and TMA in an ex vivo model of pig-to-human kidney transplantation. However, the use of rhAPC may not be feasible in a perioperative setting due to possible bleeding complications. METHODS: Here, we explored the effects of another natural inhibitor of coagulation, human recombinant antithrombin (rhAT), in comparison with rhAPC. Unmodified porcine kidneys (n = 25) were perfused ex vivo with porcine blood, human blood, or human blood supplemented with rhAPC or rhAT. Surrogate parameters of organ survival, markers of XAH (D- Dimer, thrombin-antithrombin complex [TAT], fibrinogen, antithrombin activity, plasminogen), endothelial cell and platelet activation (E-selectin, P-selectin), platelet function tests and histological signs of TMA were evaluated. RESULTS: Perfusion was feasible for > 240 min in all experiments with autologous porcine blood, but limited to 126 ± 78 min with human blood due to increased vascular resistance. Addition of rhAT protected from TMA and allowed for perfusion times > 240 min. In addition, there were less signs of XAH with reduced release of P-selectin and overexpression of E-selectin, whereas the progressive loss of platelet function, observed during discordant perfusion, was prevented. The effect of rhAT was dose-dependent with maximum protection obtained at 3 IU/ml. CONCLUSION: In conclusion, in this ex vivo model of discordant xenotransplantation, rhAT reduced XAH and prevented TMA in doses that appear feasible for use in clinical or preclinical transplantation settings.

Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody.

Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed "TKD," comprising amino acids 450-461 (aa(450-461)) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70(+) mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.

Tuning the detection sensitivity: a model for axial backfocal plane interferometric tracking.

Backfocal plane (BFP) interferometry is a single particle tracking technique that allows one to measure minute displacements of a microscopic particle from the center of a beam's focus in three dimensions. In this Letter, we present a Fourier optics model to describe the interference effects that allow one to track the position of a particle moving along the optical axis. A detection numerical aperture is derived theoretically and confirmed experimentally, within which the interference intensity has a positive correlation with the axial position of the scatterer. For larger detection angles, the correlation is negative. The model helps to understand previously reported measurements and to optimize BFP interferometric tracking.

Characterization of the self-cleaving effector protein NopE1 of Bradyrhizobium japonicum.

NopE1 is a type III-secreted protein of the symbiont Bradyrhizobium japonicum which is expressed in nodules. In vitro it exhibits self-cleavage in a duplicated domain of unknown function (DUF1521) but only in the presence of calcium. Here we show that either domain is self-sufficient for cleavage. An exchange of the aspartic acid residue at the cleavage site with asparagine prevented cleavage; however, cleavage was still observed with glutamic acid at the same position, indicating that a negative charge at the cleavage site is sufficient. Close to each cleavage site, an EF-hand-like motif is present. A replacement of one of the conserved aspartic acid residues with alanine prevented cleavage at the neighboring site. Except for EDTA, none of several protease inhibitors blocked cleavage, suggesting that a known protease-like mechanism is not involved in the reaction. In line with this, the reaction takes place within a broad pH and temperature range. Interestingly, magnesium, manganese, and several other divalent cations did not induce cleavage, indicating a highly specific calcium-binding site. Based on results obtained by blue-native gel electrophoresis, it is likely that the uncleaved protein forms a dimer and that the fragments of the cleaved protein oligomerize. A database search reveals that the DUF1521 domain is present in proteins encoded by Burkholderia phytofirmans PsNJ (a plant growth-promoting betaproteobacterium) and Vibrio coralliilyticus ATCC BAA450 (a pathogenic gammaproteobacterium). Obviously, this domain is more widespread in proteobacteria, and it might contribute to the interaction with hosts.

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

BACKGROUND: The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. METHODS: Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. RESULTS: Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 µm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically. CONCLUSIONS: These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation.

The type III-secreted protein NopE1 affects symbiosis and exhibits a calcium-dependent autocleavage activity.

The type III-secreted proteins NopE1 and NopE2 of Bradyrhizobium japonicum contain a repeated domain of unknown function (DUF1521), which is present in a few uncharacterized proteins. A nopE1/nopE2 double mutant strain exhibited higher nodulation efficiency on Vigna radiata KPS2 than the wild type or single nopE1 or nopE2 mutants. This indicates that both proteins are effectors that functionally overlap. To test translocation into the plant cell compartment during symbiosis, NopE1 and NopE2 were fused with adenylate cyclase (cya) as reporter. A fusion with the full-length proteins or N-terminal peptides resulted in increased cAMP levels in nodules, indicating translocation. Purified NopE1 exhibited self-cleavage in the presence of Ca(2+). Two identical cleavage sites (GD'PHVD) were identified inside the DUF1521 domains. The C-terminal cleavage site was analyzed by alanine scanning. Protein variants in which aspartate or proline next to the cleavage sites was substituted displayed no cleavage. A noncleavable protein was obtained by exchange of the aspartate residues preceding both cleavage sites. Complementation analysis with the noncleavable NopE1 variant did not restore wild-type phenotype on Vigna radiata KPS2, indicating a physiological role of NopE1 cleavage in effector function.

Improved interferometric tracking of trapped particles using two frequency-detuned beams.

For most optical tweezer applications, precise and reliable tracking of the trapped particle is an important requirement. Backfocal-plane interferometry is the fastest and most accurate tracking technique if the particle displacements are limited to half of the focal width. Especially for positive axial displacements, the nonlinear detector response can lead to incorrect tracking results. Here we show how the linear detection range around the trap center can be extended by a factor of 2 to 4 in the axial direction using a second frequency-detuned tracking focus that is generated by the same laser as the optical trap. Additionally, we show how the noise in the axial signal can be decreased significantly using a second detector.

Expectations, training and evaluation of intensive care staff to an interprofessional simulation course in Germany - Development of a relevant training concept.

Objective: Increasingly, intensive care units (ICU) are operated by teams of physicians and nurses with specialist training in anaesthesia and intensive care. The aims of our study were to evaluate any prior experience, expectations and the requisites for interprofessional ICU simulation-based training (SBT), and to evaluate a newly designed training course incorporating these findings. Methods: The study was laid out as a cross-sectional study and is projected in three steps. First, questionnaires were sent out to ICU nurses and physicians from 15 different hospitals in a greater metropolitan area (> million citizens). Based upon this survey a one-day ICU simulator course designed for 12 participants (6 nurses and 6 physicians) was developed, with evaluation data from four subsequent courses being analysed. Results: In the survey 40% of nurses and 57% of the physicians had had prior exposure to SBT. Various course formats were explored with respect to duration, day of the week, and group composition. After completing the course, the majority deemed a full working day in interprofessional setting to be most appropriate (p<0.001). The scenarios were considered relevant and had a positive impact on communication, workflow and coping with stress. Conclusion: Currently SBT is not a mainstream tool used by German ICU teams for further education, and this lack of familiarity must be taken into consideration when preparing SBT courses for them. We developed a nontechnical skills training course for ICU teams which was undertaken in the setting of simulated clinical scenarios (pertinent to their work environment). The participants found the course's content to be relevant for their daily work, rated the course's impact on their workplace practices as being good and advocated for longer training sessions.

Impact of Simulator-Based Crisis Resource Management Training on Collective Orientation in Anaesthesia: Pre-Post Survey Study With Interprofessional Anaesthesia Teams.

THEORY: Individuals have different qualities, levels of willingness, and degrees of engagement for working in teams. This behaviour is termed 'Collective Orientation' (CO). Collective orientation can be trained and has a positive influence on team processes. Here, we investigated the effect of a simulator-based, Crisis Resource Management team training upon the participants' CO. HYPOTHESES: We hypothesized (1) the scales of CO and Presence for lab-based microworld research (PLBMR) are applicable to the German anaesthesia teams, (2) the CO can be influenced by means of simulation training, (3) the training effect is dependent on sex and/or profession, and (4) the change of CO depends on the perceived presence of the participants in the scenario. METHOD: In a pre-post study, 66 nurses and doctors from various anaesthetic departments took part in a 1-day training course to improve non-technical skills. The primary outcome was the mean difference between the CO measured (via questionnaires) immediately before (T1) and after (T2) training. The change was then tested for dependence upon other variables, such as sex, professional group, and immersion into the simulation scenarios. RESULTS: Collective orientation improved significantly after training (mean difference: 0.2; P < .001; dz = 0.53). Considering the subscales, affiliation increased significantly (P < .001; dz = 0.59), whereas dominance remained unchanged. Furthermore, no correlation was found regarding sex, professional group, or immersion into the simulation scenarios. CONCLUSIONS: Our study demonstrated that simulation-based training improves the participants' COs, primarily by increasing affiliation. Subjective scenario reality did not significantly influence this. Nonetheless, it remains unclear as to what factors categorically resulted in this benefit. The shared experience in the course by all team members might trigger the effects. However, further studies are needed to identify the modifiable factors that can improve teamwork attitudes.

How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach.

"How thick is your light sheet?" is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between light-sheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commonly employed measures cannot readily be applied or do not lead to easily interpretable results for the many different types of light sheet. Here, we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution by using simulation data. Unexpectedly, our analysis revealed a trade-off between better axial resolution and thinner light-sheet thickness. Our results are surprising because thicker light-sheets that provide lower image contrast have previously not been associated with better axial resolution. We conclude that classical Gaussian illumination beams should be used when image contrast is most important, and more advanced types of illumination represent a way to optimize axial resolution at the expense of image contrast.

Influence of alcohol consumption on blood coagulation in rotational thromboelastometry (ROTEM): an in-vivo study.

BACKGROUND: Twenty-five to 85% of trauma patients are under the influence of alcohol in addition to experiencing injury-related coagulation impairment. Viscoelastic point-of-care tests (thrombelastography [TEG], rotational thromboelastometry [ROTEM]) are popular tools for rapid hemostasis assessment and therapeutic decision-making in this and other settings. While alcohol affects these tests in-vitro, their specific effects in-vivo are unclear. Therefore, we evaluated the effects of alcohol ingestion on ROTEM parameters. METHODS: Twenty volunteers provided informed consent to drinking red wine, whisk(e)y, or vodka to a target blood alcohol concentration of 1 ‰ within one hour, calculated with the Widmark formula. Blood samples were collected before drinking, at a breath alcohol concentration of 0.5 ‰, and at 1.0 ‰, but no later than one hour. After each blood collection, ExTEM and FibTEM tests were performed directly "at the bedside." RESULTS: All participants had a blood alcohol concentration (BAC) of 0.00 ‰ at the beginning. The mean BACs at the second and third collection were 0.48 and 0.76 ‰, respectively. There were no significant differences in the ExTEM parameters. FibTEM measurements showed a significant difference at the A10 value (13.0 vs. 14.0 mm, P = 0.014) and a trend at the maximum amplitude (maximum clot firmness [MCF] 13.7 vs. 16.2 mm, P = 0.075). We saw no significant differences in fibrinolysis parameters and no hyperfibrinolysis in our ROTEM measurements. CONCLUSIONS: Ethanol ingestion can impair early fibrin polymerization. These results might be of special relevance in trauma and support routine application of ROTEM/TEG in such cases.

Interferometric 3D tracking of several particles in a scanning laser focus.

High-Speed tracking of several particles allows measuring dynamic long-range interactions relevant to biotechnology and colloidal physics. In this paper we extend the successful technique of 3D back-focal plane interferometry to oscillating laser beams and show that two or more particles can be trapped and tracked with a precision of a few nanometers in all three dimensions. The tracking rate of several kHz is only limited by the scan speed of the beam steering device. Several tests proof the linearity and orthogonality of our detection scheme, which is of interest to optical tweezing applications and various metrologies. As an example we show the position cross-correlations of three diffusing particles in a scanning line optical trap.