Development of a Paper-based Hematocrit Test and a Lateral Flow Assay to Detect Critical Fibrinogen Concentrations Using a Bottom-Up Pyramid Workflow Approach.

Fibrinogen is a coagulation factor in human blood and the first one to reach critical levels in major bleeding. Hypofibrinogenemia (a too low fibrinogen concentration in blood) poses great challenges to first responders, clinicians, and healthcare providers since it represents a risk factor for exsanguination and massive transfusion requirements. Thus, the rapid assessment of the fibrinogen concentration at the point of care has gained considerable importance in preventing and managing major blood loss. However, in whole blood measurements, hematocrit variations affect the amount (volume fraction) of plasma that passes the detection zone. In an attempt to accurately determine realistic critical levels of fibrinogen (<1.5 mg/mL) in patients needing immediate treatment and medical interventions, we have developed novel diagnostic systems capable of estimating hematocrit and critical fibrinogen concentrations. A lateral flow assay (LFA) for the detection of fibrinogen has been developed by establishing a workflow employing rapid characterization methods to streamline LFA development. The integration of two detection lines enables (i) the identification of fibrinogen (first line) present in the sample and (ii) the determination of the clinically critical fibrinogen concentrations below 1.5 mg/mL (second line). Furthermore, the paper-based separation of blood cells from plasma provides a semiquantitative estimate of the hematocrit by analyzing the fractions. Initial validation of the point-of-care (PoC) hematocrit test revealed good comparability to a standard laboratory method. The developed diagnostic systems have the ability to accelerate decision-making in cases with major bleeding.

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients.

Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.

Management of severe peri-operative bleeding: Guidelines from the European Society of Anaesthesiology and Intensive Care: Second update 2022.

BACKGROUND: Management of peri-operative bleeding is complex and involves multiple assessment tools and strategies to ensure optimal patient care with the goal of reducing morbidity and mortality. These updated guidelines from the European Society of Anaesthesiology and Intensive Care (ESAIC) aim to provide an evidence-based set of recommendations for healthcare professionals to help ensure improved clinical management. DESIGN: A systematic literature search from 2015 to 2021 of several electronic databases was performed without language restrictions. Grading of Recommendations, Assessment, Development and Evaluation (GRADE) was used to assess the methodological quality of the included studies and to formulate recommendations. A Delphi methodology was used to prepare a clinical practice guideline. RESULTS: These searches identified 137 999 articles. All articles were assessed, and the existing 2017 guidelines were revised to incorporate new evidence. Sixteen recommendations derived from the systematic literature search, and four clinical guidances retained from previous ESAIC guidelines were formulated. Using the Delphi process on 253 sentences of guidance, strong consensus (>90% agreement) was achieved in 97% and consensus (75 to 90% agreement) in 3%. DISCUSSION: Peri-operative bleeding management encompasses the patient's journey from the pre-operative state through the postoperative period. Along this journey, many features of the patient's pre-operative coagulation status, underlying comorbidities, general health and the procedures that they are undergoing need to be taken into account. Due to the many important aspects in peri-operative nontrauma bleeding management, guidance as to how best approach and treat each individual patient are key. Understanding which therapeutic approaches are most valuable at each timepoint can only enhance patient care, ensuring the best outcomes by reducing blood loss and, therefore, overall morbidity and mortality. CONCLUSION: All healthcare professionals involved in the management of patients at risk for surgical bleeding should be aware of the current therapeutic options and approaches that are available to them. These guidelines aim to provide specific guidance for bleeding management in a variety of clinical situations.

Role of DOAC plasma concentration on perioperative blood loss and transfusion requirements in patients with hip fractures.

BACKGROUND: There is an ever-increasing number of hip fracture (HF) patients on direct oral anticoagulants (DOAC). The impact of DOAC plasma level prior to HF surgery on perioperative blood loss and transfusion requirements has not been investigated so far. MATERIALS AND METHODS: In this retrospective study of HF patients on DOACs admitted to the AUVA Trauma Center Salzburg between February 2015 and December 2021. DOAC plasma levels were analysed prior to surgery. Patients were categorized into four DOAC groups: Group A < 30 ng/mL, Group B 30-49 ng/mL, Group C 50-79 ng/mL, and Group D ≥ 80 ng/mL. Haemoglobin concentration was measured upon admission, prior to surgery, after ICU/IMC admission, and on day 1 and 2 post-surgery. Difference in the blood loss via drains, transfusion requirements and time to surgery were compared. RESULTS: A total of 155 subjects fulfilled the predefined inclusion criteria. The median age of the predominantly female patients was 86 (80-90) years. Haemoglobin concentration in Group D was lower upon admissions but did not reach statistical significance. The decrease in haemoglobin concentration over the entire observation time was comparable between groups. Blood transfusion requirements were significantly higher in Group D compared to Group A and B (p = 0.0043). Time to surgery, intra- and postoperative blood loss via drains were not different among groups. CONCLUSION: No strong association between the DOAC plasma levels and perioperative blood loss was detected. Higher transfusion rates in patients with DOAC levels ≥ 80 ng/mL were primarily related to lower admission haemoglobin levels. DOAC concentration measurement is feasible and expedites time to surgery.

Conventional and Pro-Inflammatory Pathways of Fibrinolytic Activation in Non-Traumatic Hyperfibrinolysis.

Hyperfibrinolysis (HF) frequently occurs after severe systemic hypoperfusion during major trauma and out-of-hospital cardiac arrest (OHCA). In trauma-induced HF, hypoperfusion, the activation of protein C (APC), and the release of tissue plasminogen activator (t-PA) have been identified as the driving elements of premature clot breakdown. The APC pathway also plays a role in inflammatory responses such as neutrophil extracellular trap formation (NETosis), which might contribute to lysis through cleavage of fibrin by neutrophil elastases. We investigated whether the APC and the plasminogen pathway were general drivers of HF, even in the absence of a traumatic incident. Additionally, we were interested in inflammatory activation such as the presence of NETs as potential contributing factors to HF. A total of 41 patients with OHCA were assigned to a HF and a non-HF group based on maximum lysis (ML) in thromboelastometry. Thrombin-antithrombin (TAT)-complex, soluble thrombomodulin (sTM), APC-PC inhibitor complex, t-PA, PAI-1, t-PA-PAI-1 complex, plasmin-antiplasmin (PAP), d-dimers, neutrophil elastase, histonylated DNA (hDNA) fragments, and interleukin-6 were assessed via immunoassays in the HF group vs. non-HF. APC-PC inhibitor complex is significantly higher in HF patients. Antigen levels of t-PA and PAI-1 do not differ between groups. However, t-PA activity is significantly higher and t-PA-PAI-1 complex significantly lower in the HF group. Consistent with these results, PAP and d-dimers are significantly elevated in HF. HDNA fragments and neutrophil elastase are not elevated in HF patients, but show a high level of correlation, suggesting NETosis occurs in OHCA as part of inflammatory activation and cellular decay. Just as in trauma, hypoperfusion, the activation of protein C, and the initiation of the plasminogen pathway of fibrinolysis manifest themselves in the HF of cardiac arrest. Despite features of NETosis being detectable in OHCA patients, early pro-inflammatory responses do not appear be associated with HF in cardiac arrest.

Factor XIII Measurement and Substitution in Trauma Patients after Admission to an Intensive Care Unit.

Trauma patients admitted to an intensive care unit (ICU) may potentially experience a deficiency of coagulation factor thirteen (FXIII). In this retrospective cohort study conducted at a specialized trauma center, ICU patients were studied to determine the dependency of FXIII activity levels on clinical course and substitution with blood and coagulation products. A total of 189 patients with a median injury severity score (ISS) of 25 (16−36, IQR) were included. Abbreviated injury scores for extremities (r = −0.38, p < 0.0001) but not ISS (r = −0.03, p = 0.45) showed a negative correlation with initial FXIII levels. Patients receiving FXIII concentrate presented with a median initial FXIII level of 54 (48−59)% vs. 88 (74−108)%, p < 0.0001 versus controls; they had fewer ICU-free days: 17 (0−22) vs. 22 (16−24), p = 0.0001; and received higher amounts of red blood cell units: 5 (2−9) vs. 4 (1−7), p < 0.03 before, and 4 (2−7) vs. 1 (0−2), p < 0.0001 after FXIII substitution. Matched-pair analyses based on similar initial FXIII levels did not reveal better outcome endpoints in the FXIII-substituted group. The study showed that a low initial FXIII level correlated with the clinical course in this trauma cohort, but a substitution of FXIII did not improve endpoints within the range of the studied FXIII levels. Future prospective studies should investigate the utility of FXIII measurement and lower threshold values of FXIII, which trigger substitution in trauma patients.

Operability of a Resonance-Based Viscoelastic Haemostatic Analyzer in the High-Vibration Environment of Air Medical Transport.

Trauma and bleeding are associated with a high mortality, and most of these deaths occur early after injury. Viscoelastic haemostatic tests have gained increasing importance in goal-directed transfusion and bleeding management. A new generation of small-sized and thus portable ultrasound-based viscoelastic analysers have been introduced in clinical practice. We questioned whether a promising candidate can be used in emergency helicopters, with a focus on the susceptibility to vibration stress. We investigated whether the high vibration environment of an emergency helicopter would affect the operability of an ultrasound-based viscoelastic analyser and would yield reproducible results in flight and on the ground. We drew blood from 27 healthy volunteers and performed simultaneous analyses on two TEG 6s. Each measurement was performed in-flight on board an Airbus H135 emergency helicopter and was repeated on the ground, close to the flight area. Results from both measurements were compared, and the recorded tracings and numeric results were analysed for artifacts. Vibratometric measurements were performed throughout the flight in order to quantify changes in the magnitude and character of vibrations in different phases of helicopter operation. The high vibration environment was associated with the presence of artifacts in all recorded tracings. There were significant differences in citrated Kaolin + Heparinase measurements in-flight and on the ground. All other assays increased in variability but did not show significant differences between the two time points. We observed numerous artifacts in viscoelastic measurements that were performed in flight. Some parameters that were obtained from the same sample showed significant differences between in-flight and on-ground measurements. Performing resonance-based viscoelastic tests in helicopter medical service is prone to artifacts. However, a 10 min delay between initiation of measurement and take-off might produce more reliable results.

Multiplate Platelet Function Testing upon Emergency Room Admission Fails to Provide Useful Information in Major Trauma Patients Not on Platelet Inhibitors.

Platelet dysfunction is a suggested driver of trauma-induced coagulopathy. However, there is still a paucity of data regarding the impact of injury pattern on platelet function and the association of platelet dysfunction on transfusion requirements and mortality. In this retrospective cohort study, patients were grouped into those with isolated severe traumatic brain injury (TBI group), those with major trauma without TBI (MT group), and a combination of both major trauma and traumatic brain injury (MT + TBI group). Platelet function was assessed by whole blood impedance aggregometry (Multiplate®, MP). Three different platelet activators were used: adenosine-diphosphate (ADP test), arachidonic acid (ASPI test), and thrombin activated peptide-6 (TRAP test). Blood transfusion requirements within 6 h and 24 h and the association of platelet dysfunction on mortality was investigated. A total of 328 predominantly male patients (75.3%) with a median age of 53 (37-68) years and a median ISS of 29 (22-38) were included. No significant difference between the TBI group, the MT group, and the MT + TBI group was detected for any of the investigated platelet function tests. Unadjusted and adjusted for platelet count, the investigated MP assays revealed no significant group differences upon ER admission and were not able to sufficiently predict massive transfusion, neither within the first 6 h nor for the first 24 h after hospital admission. No association between platelet dysfunction measured by MP upon ER admission and mortality was observed. Conclusion: Injury pattern did not specifically impact platelet function measurable by MP. Platelet dysfunction upon ER admission measurable by MP was not associated with transfusion requirements and mortality. The clinical relevance of platelet function testing by MP in trauma patients not on platelet inhibitors is questionable.

Regional anaesthesia in patients on antithrombotic drugs: Joint ESAIC/ESRA guidelines.

BACKGROUND: Bleeding is a potential complication after neuraxial and peripheral nerve blocks. The risk is increased in patients on antiplatelet and anticoagulant drugs. This joint guideline from the European Society of Anaesthesiology and Intensive Care and the European Society of Regional Anaesthesia aims to provide an evidence-based set of recommendations and suggestions on how to reduce the risk of antithrombotic drug-induced haematoma formation related to the practice of regional anaesthesia and analgesia. DESIGN: A systematic literature search was performed, examining seven drug comparators and 10 types of clinical intervention with the outcome being peripheral and neuraxial haematoma. Grading of Recommendations, Assessment, Development and Evaluation (GRADE) was used for assessing the methodological quality of the included studies and for formulating recommendations. A Delphi process was used to prepare a clinical practice guideline. RESULTS: Clinical studies were limited in number and quality and the certainty of evidence was assessed to be GRADE C throughout. Forty clinical practice statements were formulated. Using the Delphi-process, strong consensus (>90% agreement) was achieved in 57.5% of recommendations and consensus (75 to 90% agreement) in 42.5%. DISCUSSION: Specific time intervals should be observed concerning the adminstration of antithrombotic drugs both prior to, and after, neuraxial procedures or those peripheral nerve blocks with higher bleeding risk (deep, noncompressible). These time intervals vary according to the type and dose of anticoagulant drugs, renal function and whether a traumatic puncture has occured. Drug measurements may be used to guide certain time intervals, whilst specific reversal for vitamin K antagonists and dabigatran may also influence these. Ultrasound guidance, drug combinations and bleeding risk scores do not modify the time intervals. In peripheral nerve blocks with low bleeding risk (superficial, compressible), these time intervals do not apply. CONCLUSION: In patients taking antiplatelet or anticoagulant medications, practitioners must consider the bleeding risk both before and after nerve blockade and during insertion or removal of a catheter. Healthcare teams managing such patients must be aware of the risk and be competent in detecting and managing any possible haematomas.

Thromboelastometry fails to detect autoheparinization after major trauma and hemorrhagic shock.

BACKGROUND: Heparan sulfate is an integral component of the glycocalyx that provides an anticoagulant layer close to the endothelium. Hypoperfusion, inflammation, and sympathoadrenal activation following major trauma result in glycocalyx shedding and subsequent release of heparan sulfate into the bloodstream. The possible anticoagulant effect of this "autoheparinization" has been suggested as a potential driver of trauma-induced coagulopathy. We investigated whether thromboelastometry can be used to detect trauma-induced autoheparinization. METHODS: This study comprised three parts. First, in a retrospective clinical study of 264 major trauma patients, the clotting time (CT) in the intrinsic activation (INTEM) and intrinsic activation plus heparinase (HEPTEM) assays were evaluated upon emergency room admission. Second, in an in vivo experimental rat model of hemorrhagic-traumatic shock, the release of heparan sulfate was investigated with INTEM and HEPTEM analyses of whole blood. Third, in vitro spiking of whole blood from healthy volunteers was undertaken to assess the effects of clinically relevant quantities of heparan sulfate and heparin on CT in the INTEM and HEPTEM assays. RESULTS: In the first part, severe injury and hemorrhagic shock was not associated with any increases in INTEM CT versus HEPTEM CT. Part 2 showed that an approximate threefold increase in heparan sulfate resulting from hemorrhagic traumatic shock in rats did not prolong INTEM CT, and no significant differences between INTEM CT and HEPTEM CT were observed. Third, spiking of whole blood with heparan sulfate had no impact on INTEM CT, whereas heparin elicited significant prolongation of INTEM CT. CONCLUSION: Despite structural similarity between heparan sulfate and heparin, the amounts of heparan sulfate shed in response to trauma did not exert an anticoagulant effect that was measurable by the intrinsically activated CT in thromboelastometry. The extent to which heparan sulfate contributes to trauma-induced coagulopathy has yet to be elucidated. LEVEL OF EVIDENCE: Prognostic and Epidemiologic; Level III.

Impact of Idarucizumab and Andexanet Alfa on DOAC Plasma Concentration and ClotPro® Clotting Time: An Ex Vivo Spiking Study in A Cohort of Trauma Patients.

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro® Russell's viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers' blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

High Interleukin-6 Plasma Concentration upon Admission Is Predictive of Massive Transfusion in Severely Injured Patients.

Severe bleeding remains a prominent cause of early in-hospital mortality in major trauma patients. Thus, prompt prediction of patients at risk of massive transfusion (MT) is crucial. We investigated the ability of the inflammatory marker interleukin (IL)-6 to forecast MT in severely injured trauma patients. IL-6 plasma levels were measured upon admission. Receiver operating characteristic curves (ROCs) were calculated, and sensitivity and specificity were determined. In this retrospective study, a total of 468 predominantly male (77.8%) patients, with a median injury severity score (ISS) of 25 (17-34), were included. The Youden index for the prediction of MT within 6 and 24 h was 351 pg/mL. Patients were dichotomized into two groups: (i) low-IL-6 < 350 pg/mL and (ii) high-IL-6 ≥ 350 pg/mL. IL-6 ≥ 350 pg/mL was associated with a lower prothrombin time index, a higher activated partial thromboplastin time, and a lower fibrinogen concentration compared with IL-6 < 350 pg/mL (p <0.0001 for all). Thromboelastometric parameters were significantly different between groups (p <0.03 in all). More patients in the high-IL-6 group received MT (p <0.0001). The ROCs revealed an area under the curve of 0.76 vs. 0.82 for the high-IL-6 group for receiving MT in the first 6 and 24 h. IL-6 ≥ 350 pg/mL predicted MT within 6 and 24 h with a sensitivity of 45% and 58%, respectively, and a specificity of 89%. IL-6 ≥ 350 pg/mL appears to be a reasonable early predictor for coagulopathy and MT within the first 6 and 24 h intervals. Large-scale prospective studies are warranted to confirm these findings.

Management of Coagulopathy in Bleeding Patients.

Early recognition of coagulopathy is necessary for its prompt correction and successful management. Novel approaches, such as point-of-care testing (POC) and administration of coagulation factor concentrates (CFCs), aim to tailor the haemostatic therapy to each patient and thus reduce the risks of over- or under-transfusion. CFCs are an effective alternative to ratio-based transfusion therapies for the correction of different types of coagulopathies. In case of major bleeding or urgent surgery in patients treated with vitamin K antagonist anticoagulants, prothrombin complex concentrate (PCC) can effectively reverse the effects of the anticoagulant drug. Evidence for PCC effectiveness in the treatment of direct oral anticoagulants-associated bleeding is also increasing and PCC is recommended in guidelines as an alternative to specific reversal agents. In trauma-induced coagulopathy, fibrinogen concentrate is the preferred first-line treatment for hypofibrinogenaemia. Goal-directed coagulation management algorithms based on POC results provide guidance on how to adjust the treatment to the needs of the patient. When POC is not available, concentrate-based management can be guided by other parameters, such as blood gas analysis, thus providing an important alternative. Overall, tailored haemostatic therapies offer a more targeted approach to increase the concentration of coagulation factors in bleeding patients than traditional transfusion protocols.

Sufficient Thrombin Generation Despite 95% Hemodilution: An In Vitro Experimental Study.

Guidelines for the treatment of severe bleeding comprise viscoelastic-test-guided use of coagulation factor concentrates as part of their recommendations. The aim of this study is to investigate the effects of substituting fibrinogen, prothrombin complex concentrate, and a combination of both on conventional coagulation tests, viscoelastic test results, and thrombin generation. Blood was drawn from seven healthy volunteers to obtain platelet-free plasma, which later was diluted by replacing 40%, 60%, 80%, 90%, 95%, and 99% with a crystalloid solution. The diluted samples were spiked with fibrinogen concentrate, prothrombin complex concentrate, a combination of both, or a corresponding amount of crystalloid solution. Up to a dilution level of 95%, viscoelastically determined clotting time was significantly shorter in the group substituted with fibrinogen only in comparison with the additional use of prothrombin complex concentrate. Clot firmness and endogenous thrombin potential remained at relatively stable values up to a dilution level of 95% with the substitution of fibrinogen but not prothrombin complex concentrate. Substitution of prothrombin complex concentrate led to an excessive overshoot of thrombin generation. The results of our study question currently propagated treatment algorithms for bleeding patients that include the use of prothrombin complex concentrate for patients without former intake of oral anticoagulants. Even in severely bleeding patients, thrombin generation might be sufficient to achieve adequate hemostasis.

Comparison of fresh frozen plasma vs. coagulation factor concentrates for reconstitution of blood: An in vitro study.

BACKGROUND: Many trauma centres have adopted the administration of fixed ratios of packed red blood cells (PRBCs), platelet concentrates and fresh frozen plasma (FFP) for bleeding patients. However, the haemostatic efficacy of this concept is not well proven. OBJECTIVE: Our objective was to characterise the haemostatic profile of different ratios (2 : 1 : 1, 1 : 1 : 1 and 1 : 1 : 2) of PRBCs, platelet concentrates and FFP in comparison with coagulation factor concentrates (fibrinogen and/or prothrombin complex concentrate). DESIGN: An in vitro study. SETTING: Research laboratories of the department of transfusion medicine, Linz, Austria. MATERIALS: Whole blood donations from a total of 20 male volunteers. INTERVENTION: Reconstitution of blood at different ratios of PRBCs, platelet concentrates and FFP or coagulation factor concentrates. MAIN OUTCOME MEASURES: Cell count, conventional and thromboelastometric coagulation parameters, single coagulation factor activities as well as endogenous thrombin potential. RESULTS: Fibrinogen levels and haematocrit were lower in the FFP group at any ratio compared with the concentrate-based groups (P < 0.0001). Reconstitution of blood with FFP at different ratios resulted in haematocrit or fibrinogen levels that were borderline with regard to recommended substitution triggers (haematocrit 41 ±â€Š2% and fibrinogen 1.5 ±â€Š0.3 g l at the 2 : 1 : 1 ratio vs. 21 ±â€Š1% and 2.1 ±â€Š0.4 g l respectively at the 1 : 1 : 2 ratio). Compared with FFP at any ratio, maximum clot firmness showed higher values in the groups using fibrinogen concentrate (P < 0.0001), whereas endogenous thrombin potential revealed higher values in the groups using prothrombin complex concentrate (P < 0.0001). CONCLUSION: Use of coagulation factor concentrates for the reconstitution of blood allows for delivery of a higher haematocrit and a higher fibrinogen content compared with FFP. However, prothrombin complex concentrate might result in an unnecessary excess of thrombin generation. Clinical studies are warranted to further investigate these in vitro findings.

Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry.

Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.

Concentrated lyophilized plasma used for reconstitution of whole blood leads to higher coagulation factor activity but unchanged thrombin potential compared with fresh-frozen plasma.

BACKGROUND: During massive hemorrhage, it is recommended to transfuse red blood cells, platelet concentrate, and fresh-frozen plasma in a ratio close to 1:1:1. To avoid the thawing process of fresh frozen plasma, lyophilized plasma (LP) is increasingly used. Evidence is limited on the activity of coagulation factors in reconstituted blood using LP and concentrated LP versions. STUDY DESIGN AND METHODS: Whole blood from ten healthy volunteers was separated into red blood cell, fresh frozen plasma, and platelet concentrate units. Aliquots of red blood cells and plasma concentrate were mixed with either fresh frozen plasma (200 mL) or LP at reconstitution ratios of 2:1:1, 1:1:1, and 1:1:2. LP was used either at the recommended standard volume of 200 mL (LP200) or was more concentrated at volumes of 100 and 50 mL (LP100 and LP50, respectively). The hemostatic capacity of each reconstituted whole blood sample was tested with blood cell counts, standard coagulation tests, factor activity, thrombin generation, and viscoelastic assays. RESULTS: Hematocrit, platelet counts, and fibrinogen levels of the three ratios were similar between FFP200 and LP200 units but were lower compared with the corresponding ratios in LP100 and LP50 units. The activity of procoagulant and anticoagulant factors increased linearly with the increasing plasmatic fraction and, at 1:1:2 ratio, was significantly higher in LP50 units compared with FFP200 and LP200 units. Thrombin generation was similar throughout the four plasma groups at any ratio. CONCLUSIONS: Decreasing the dilution volume of LP facilitates reaching higher hematocrit and coagulation protein levels without a relevant increase in thrombin generation. This is due to preserved balance between procoagulant and anticoagulant factors in the concentrated LP preparations.