Maximum Lysis Research Articles

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.

Impaired fibrinolysis in severe Covid-19 infection is detectable in early stages of the disease.

A significant degree of mortality and morbidity in Covid-19 is due to thromboembolic disease. Coagulopathy has been well described in critically unwell patients on ICU. There is less clear evidence regarding these changes at the time of presentation to the Emergency Department and the progression of disease over time. We sought to investigate whether coagulation markers can predict severity and how they change over the disease course. Patients presenting to a single University Teaching Hospital were recruited and followed up if PCR was positive. Alongside routine blood testing, Rotational Thromboelastometry (ROTEM) was performed. Outcome data was recorded for all patients, and ROTEM values were compared across outcome groups. Extem and Intem Maximum Lysis were significantly reduced in those who died or required an ICU admission, indicating a reduced ability to break down clot mass in the most critically unwell patients. Comparisons between groups demonstrated that one distinguishing feature between those who require ICU admission or die of Covid-19 compared with those who survive a hospital stay to discharge was the extent to which fibrinolysis could occur. Mortality and morbidity in Covid-19 infection appears in part driven by an inability to break down clot mass.

Hypofibrinolysis in Pediatric Patients with Veno-Occlusive Disease in Hematopoietic Stem Cell Transplantation

Background Veno-occlusive disease (VOD) is a serious complication of hematopoietic stem cell transplantation (HSCT) with a high incidence in pediatric patients (11-27%). In clinical practice, VOD is diagnosed with criteria including weight gain, ascites, hepatomegaly, hyperbilirubinemia, and refractory thrombocytopenia. To date, there are no established markers for an earlier diagnosis of VOD. The aspect of assessing hypofibrinolysis, which plays a role in VOD pathogenesis, has not been elucidated. This study aimed to detect signs of hypofibrinolysis in HSCT and VOD patients using thrombelastography parameters and to discover correlations between alterations of these parameters and established symptoms of VOD. Patients and Methods Patients admitted for HSCT to the pediatric HSCT ward at the University Children's Hospital Tübingen, Germany, between May 2020 and November 2021 were prospectively enrolled (study group). Patients who underwent minor surgical procedures or follow-up blood tests years after recovering from oncological diseases and who were otherwise healthy, were recruited as a control group. Blood samples for thrombelastography (EX and TPA test, including the coagulation time (CT), clot formation time (CFT), clot amplitude after 5 minutes (A05), maximum clot firmness (MCF), maximum lysis (ML), α-angle, and lysis time (LT)) were taken once to twice weekly in addition to daily routine blood tests in patients undergoing HSCT. Routine testing included blood counts, coagulation parameters, d-dimers, and bilirubin. VOD symptoms were monitored daily and body weight was measured twice a day. Ultrasound screenings for ascites, progredient hepatomegaly, and portal venous flow velocity were performed in case of suspected VOD. All patients received intravenous anticoagulation with 100 IU/kg and day of unfractionated heparin. Of the control group patients, 6 ml blood was collected once per patient for routine coagulation testing and thrombelastography. Results A total of 51 patients with a median age of 7.8 years were recruited in the study group. Of these patients, five (9.8%) developed VOD and received defibrotide treatment. The VOD was graded moderate (n=2; 4%), severe (n=1; 2%) or very severe (n=2; 4%). Defibrotide treatment was initiated for 4 weeks or until the resolution of symptoms in standard dosing (25 milligrams per kilogram per day (mg/kg/d)) in four doses over 2 hours). The control group consisted of 55 patients with a median age of 6.7 years. A significant difference in thrombelastographic measurements was observed when comparing the VOD to the HSCT group. The lysis time after activation of fibrinolysis in the TPA test was prolonged in the VOD group at 3 weeks after HSCT (d+14 to +21: VOD: 330±67 seconds, HSCT: 246±53 seconds, p=0.0106). Additionally, a significant difference between the VOD and control groups was observed in days +14 to +21 (control: LT 234±50 seconds, p=0.0299). For the time points of day 0 to +6 and day +7 and +13 after HSCT lysis time was also prolonged in VOD patients, but without statistical significance (LT: d0 to +6: VOD: 304±57 seconds; HSCT: 251±67 seconds, p=0.06; d+7 to +13: VOD: 278±34 seconds; HSCT: 255±70 seconds, p=0.12). It was observed that LT correlated positively with weight gain (>5%), progredient hepatomegaly, right upper quadrant pain and reduced portal venous flow velocity (d0 to d+6: weight gain: r=0.31, p=0.0351; pain: r=0.44, p=0.0018; hepatomegaly: r=0.37, p=0.0108; d+7 to +13: pain: r=0.35, p=0.0143; hepatomegaly: r=0.39, p=0.0058; d+14 to d+21: weight gain: r=0.42, p=0.0035; pain: r=0.37, p=0.0103; hepatomegaly: r=0.44, p=0.0020; reduced portal venous flow velocity: r=0.35, p= 0.0167). Conclusions The results suggest that HSCT patients and patients diagnosed with VOD show signs of hypofibrinolysis. Fibrinolysis can be assessed in a rapid and accessible way via thrombelastography. Therefore, thrombelastography might be a useful tool to facilitate early diagnosis of VOD and to identify high-risk patients. Larger studies are needed to determine specific cut-off-values. Stefanie Hammer and Michaela Döring contributed equally to this work.

Evaluation of Global Hemostasis Assays in Von Willebrand Disease Type 3 in Comparison with Mild to Moderate Hemophilia a

Introduction: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Type 3 VWD is the most severe form of this disease with a prevalence of about 0.5 to 1 per million in Western countries but is much more common in communities with high levels of consanguinity like South India. Type 3 VWD is characterised by undetectable Von Willebrand Factor and reduced Factor VIII levels. (1,2) Global hemostasis assays clot waveform analysis (CWA) [3,4] and thrombin generation test (TGT)[5,6] reflect the entire process of thrombin generation, and thus reflect the actual bleeding severity . Rotational Thromboelastogram (ROTEM) demonstrates the dynamics of clot formation over time. Global Hemostasis Assays is extensively studied in Hemophilia A(HA) patients, however there are very few studies which analysed the same in type 3 VWD in account of rarity of disease Our aim was to study ROTEM, TGT and APTT-CWA parameters in cases of vWD 3. We also aimed to compare global hemostasis parameters in vWD3 to those in mild to moderate hemophilia A with similar levels of Factor VIII. Materials and Methods: Retrospective study for a period of 2 years which included 27 cases of vWD3 (with undetectable vWF antigen) and 34 cases of Hemophilia A- 25 moderate and 9 mild deficiency (F VIII-5-10%). Complete coagulation workup was done which included International Society of Thrombosis and Hemostasis based Bleeding Assessment Tool(BAT) Score questionnaire, Bleeding time, Platelet Function Analyser 200, Prothrombin Time(PT), APTT, Ristocetin Cofactor, Von Willebrand Antigen, Factor assays, Ristocetin induced platelet aggregation and Global Hemostasis Assays-ROTEM, TGT and CWA. BAT score was done to assess severity of bleeding. TGT was performed by Calibrated Automated Thrombogram, CWA by photo-optical coagulometer and factor levels by one stage clot-based assays on ACL Top 750. Rotem was performed in whole blood by modified extem. TGT was could be done only on 16/34 Hemophilia A cases and 14/27 vWD3 cases. Statistical Analysis: For continuous data, the descriptive statistics mean, SD, and for non-normally distributed data median, IQR were done. Based on the normality of data, the parametric t-test or non-parametric Mann Whitney test was used to find the difference between groups. All tests were two-sided at alpha () =0.05 level of significance. All the statistical analysis was performed using Statistical Package for Social sciences (SPSS) version 21.0. Results: Results are summarized in Table 1Global hemostasis tests in vWD3 A.1)ROTEM- CT (Clotting Time) was prolonged in 44% cases, 55 % cases it was within normal range and shortened in rest. CFT (Clot Formation Time) and alpha angle were within normal range in 81.1% cases, prolonged in 0.3% cases and shortened in rest. MCF (Maximum Clot Firmness) was reduced in 2 cases, and normal in remaining cases. ML(Maximum Lysis) was within range for all cases. A.2) APTT CWA- Min1 was decreased in 25.9 % cases, rest 74% had within normal range. Min2 was decreased in 96% cases. Max 2 was decreased in all cases. A.3) TGT- Lag time was prolonged in 13/14 (93%) cases. ETP was increased in 7/14 cases (50%). Peak height was decreased in all cases. TTP was prolonged in 13/14 cases (93%). Start tail was prolonged in 12/14 cases.B) Comparison between vWD3 and Mild to Moderate Hemophilia Refer to table -1 APTT was significantly prolonged (P value 0.008) in HA group than vWD3, though Factor VIII was similar in both groups. CWA were comparable to HA cases. Min 2 was statistically lower in vWD3 compared to HA group between the 2 groups (P value- 0.018) ROTEM were comparable between the 2 groups. In TGT, the median lag time (min)was significantly more prolonged in vWD3 with median of 5.50(4.95-6.37) than Hemophilia A patients as seen with APTT. ETP and Peak Thrombin were not significantly different between the 2 groups. Time to peak and start tail were statistically lower among cases of vWD 3 and haemophilia A, p value being <0.05. CONCLUSION: Global hemostasis test in von Willebrand disease showed a hypocoagulable state in Thrombin Generation Tests and APTT CWA. ROTEM didn't show any significant change despite Factor VIII levels being low Tests which measure Factor VIII assay and tests which reflect Factor VIII activity as in global hemostasis assay do not show significant difference in patients who have low Factor VIII level irrespective of the diagnosis being von Willebrand disease or Hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preferential Use of Coagulation Pathways Differs between Classical Myeloproliferative Neoplasms: Results of Global Coagulation Assays with Respect to Clinical and Genetic Features

Introduction Classical Myeloproliferative Neoplasms (MPN) are hematopoietic stem cell diseases characterized by inflammation, promotion of atherosclerosis, hypercoagulability, fibrosis, and clonal evolution. Our previous studies led us to the construction of a model, called "the circulating wound", for the likely exploitation of tissue factor (TF)-dependent thrombin generation and the consequent amplification of pro-fibrotic mechanisms mediated by Protease Activated Receptors (PARs). The indirect demonstration of PARs hyperactivation is the marked reduction of platelet fibrinogen receptors (PFRs) in patients with MPNs, following the rapid conversion of functional fibrinogen to fibrin. Acetylsalicylic acid (ASA), endowed with hypoprothrombinemic and hyperfibrinolytic properties, could restore the expression of PFRs. The aim of the present study is to verify the existence of imbalances in hemostatic function in rotational thromboelastometry (ROTEM), with respect to the different classical forms of MPN, their cell counts, the presence of genetic mutations in Next Generation Sequencing (NGS), PFR expression, the use of ASA and the history of vascular events. Methods We enrolled 53 patients affected by MPNs, naïve from any therapy, with the only exception of ASA (taken by 50% of patients). We used the samples to run assays of genetics (30-gene panel in NGS - SOPHiA Myeloid Solution™, SOPHiA Genetics, Switzerland), cytofluorimetric determination of PFRs, global coagulation by rotational tromboelastometry (ROTEM® Delta, Werfen, Spain) for INTEM, EXTEM, FIBTEM parameters. Results We found that ROTEM parameters show significant divergence depending on disease type, cell number and some mutations. The results should be read in terms of "efficiency" or "preferential use" of one pathway over another, as the instrument (as expected) mostly yields values in the ranges. Essential thrombocythemia (ET) and CALR mutation exhibit preserved efficiency of both classical coagulation pathways, with significantly more contracted clot formation times (CFTs). In contrast, primary myelofibrosis (PMF) and polycythemia vera (PV) show greater imbalances in the hemostatic system. PV patients have longer CFTs (EXTEM 114 sec, p= 0.008, INTEM 82 sec, p=0.027) and selective contraction of parameters in INTEM for higher counts of platelets (Plt), white blood cells (WBC) and hemoglobin (Hb). PMF - in contrast - seems to exploit the extrinsic pathway (EXTEM) more - see figure. Multivariate analysis gives Hb levels the largest contribution to the alterations, while only Plt retain statistical significance in PMF and ET. Selected mutations are associated with changes in ROTEM parameters. Among the DTA mutations, the presence of DNMT3A shows a significant reduction in clotting time (CT) in EXTEM, while ASXL1 is associated with reduced maximum lysis (ML). EZH2 could be responsible for CFT elongations in the INTEM assay. In addition, increased expression of PFRs is associated with bleeding history and sustained FIBTEM CT under ASA prophylaxis. Conclusions In more indolent MPNs (such as ET, especially if CALR mutated) the balance and efficiency of the classical pathways are preserved. In PMFs, the hemostatic system is unbalanced toward the extrinsic pathway, confirming the link between increased fibrosis and plasma-dependent hypercoagulable states. The experiment also provides further evidence of the robustness of the "circulating wound" model. Rheological characteristics and endothelial stress in PV appear to result in increased involvement of the intrinsic pathway. Interestingly, mutations involving genes that regulate DNA methylation are confirmed to interfere with hemostatic balance. Finally, excessive restitution of PFR expression by ASA appears to be related to the history of bleeding events, making their cytofluorimetric determination a potential tool for monitoring antiplatelet prophylaxis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical Activity of Allogeneic CS1-Specific CAR T-Cells (UCARTCS1) in Multiple Myeloma

Introduction: The substantial activity of chimeric antigen receptor (CAR) T-cells in multiple myeloma (MM) has recently been shown by the success of BCMA-CAR T-cell therapy. Nevertheless the difficulties in (timely) generation of CART cells for each patient, the limited availability of manufacturing slots, and the frequent BCMAlow MM relapses after therapy urge the investigators to search for i) alternative, "off the shelf", available sources of CAR T-cells and ii) alternative target antigens than BCMA. UCARTCS1 cells are "off the shelf" allogeneic CAR T-cells derived from healthy donors targeting CS1 (SLAMF7), which is a highly attractive target, due to its overexpression on MM cells. Furthermore, the genes coding for the TCRα chain of the TCRab receptor and for CS1 are disrupted in UCARTCS1 cells using Cellectis' TALEN® gene-editing technology. These modifications aim to minimize the potential risk for allo-TCR-mediated graft-versus-host disease (GvHD) upon infusion in patients and diminish CS1-mediated fratricide. In this study, we evaluated the preclinical activity of UCARTCS1 in MM cell lines, in bone marrow (BM) samples obtained from MM patients and in a MM mouse model. We also investigated the potential impact of previous therapy and tumor characteristics on the in vitro efficacy of UCARTCS1. Methods: Luciferase-transduced MM cell lines with different expression levels of CS1 were incubated with UCARTCS1 cells or control (non-transduced CS1/TCRαβ double knock-out) T-cells at different E:T ratios for 24 hours. MM cell lysis was assessed by bioluminescence. Anti-MM activity of UCARTCS1 was also evaluated in 25 BM samples obtained from newly diagnosed (n=10; NDMM), daratumumab-naïve relapsed/refractory patients (n=10; RRMM; median of 3 prior therapies) and from daratumumab-refractory patients (n=5; DRMM; median of 3 prior therapies) in 24-hour flow cytometry-based cytotoxicity assays. The results were correlated to CS1 expression levels of MM cells, Treg frequency, and several other patient characteristics such as treatment history. Results: UCARTCS1 induced strong CS1 CAR-mediated and dose-dependent lysis of different MM cell lines expressing variable levels of CS1 (MM1s, L363, UM9, U266). UCARTCS1 cells also effectively lysed primary MM cells in a dose-dependent and CAR-mediated manner, with a mean maximal lysis of 96.9% (Figure 1). In these BM samples, UCARTCS1 cells also targeted CS1+ non-malignant hematopoietic cells, such as CD4+ T-cells, CD8+ T-cells, B cells, and NK cells, while sparing the CS1low fraction in each cell subset. However, the lysis of normal cells was less efficient than MM cell lysis, which could be explained by substantially lower CS1 expression on normal cells compared to MM cells (Figure 2). CS1 expression on MM cells and UCARTCS1 cytotoxic activity were both comparable in the different patient subgroups. In addition, baseline tumor and immune characteristics, such as the expression level of CS1 on MM cells, the tumor load (MM cell percentage in BM), or frequency of Treg cells in the BM did not affect CAR T-cell mediated cytotoxicity. Finally, UCARTCS1 exhibited durable dose-dependent anti-MM activity in mouse xenograft models after i.v. injection of CAR T-cells compared to mice treated with CAR- control T-cells. Conclusion: UCARTCS1 has potent anti-MM activity against MM cell lines and primary MM cells, as well as in a MM xenograft model. There was no difference in activity between heavily pretreated and newly diagnosed patients, indicating that mechanisms of resistance to prior therapies do not result in reduced susceptibility to UCARTCS1-mediated lysis in vitro. UCARTCS1 was also effective in samples from patients with relatively low CS1 expression, with high tumor burden, or with high Treg numbers. These data support the ongoing phase 1 clinical trial with UCARTCS1 in heavily pretreated MM patients (NCT04142619). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of the Thrombolytic and Antioxidant Activity of Leaf Extracts of Plumbago zeylanica L.

Abstract: Background: Plumbago zeylanica L. is one of the extremely accessible conventionally used herbal plants with various biological activities. However, actions of P. zeylanica L on blood clotting and other complications of blood were indisposed therapeutically studied. Therefore, the scope of the current exploration is to screen the thrombolytic, antioxidant, and cytotoxic effects of leaf extracts. Materials and Methods: Thrombolytic activity (in vitro) was assessed with clot lysis and thrombin inhibitory ability. Further, thrombolytic activity (in vivo) was evaluated by a thrombotic tail (carrageenan-induced) animal model. DPPH and nitric oxide (free radical) scavenging methods were employed to check the in vitro antioxidant property. Further cytotoxicity and acute oral toxicity were assessed for plant extract. Results: The quantitative analysis elicits the presence of the magnificent amount of the total phenolic content (96.8±7.92 mg GAE/g) and total flavonoid content (63.52±4.54 mg QE/g) on the dry weight basis. The maximum clot lysis (96.83%±0.657) of methanolic leaf extract was detected in in vitro model at 800 μg/mL in 72 hr. A strong thrombin inhibition (94.63±2.12%) effect was observed for methanol leaf extract at 2 mg/mL. In in vivo studies a significant (p< 0.001) clot lysis was achieved at the tested dose (100, 200 and 300 mg/kg). DPPH radical and nitric oxide scavenging activity showed the IC50 value of 25.47±0.51 and 56.32±0.85, respectively. The methanolic extract was found safer up to the highest lethal dose of 2000 mg/kg. Conclusion: These findings suggested that the plant leaves are comprised of significant thrombolytic properties. It could be a promising source for the existence of antioxidant and thrombolytic agents. Keywords: Plumbago zeylanica L., Thrombolytic activity, Anti-oxidant activity, Cytotoxicity, Streptokinase, DPPH radical etc.

Thromboelastometry as an Ancillary Tool for Evaluation of Coagulation Status after rFVIIa Therapy in a Pregnant Woman with Severe Hypoproconvertinemia-A Case Series and Review of the Literature.

Introduction: Factor VII (FVII) deficiency is a rare hemorrhagic diathesis. In females, heavy menstrual and postpartum bleeding can appear as a consequence of its deficiency. Supplementation of the recombinant FVIIa is widely accepted. The supplementation effect in FVII-deficient subjects is difficult to predict, and severe hemorrhage has been described even when FVII levels after supplementation were within normal ranges. The aim of this report is to present the application of thromboelastometry to control the coagulation status in a patient with severe FVII deficiency during pregnancy and delivery, supplemented by rFVIIa per protocol complicated with life-threatening venous thromboembolism. Methods: Rotational thromboelastometry (ROTEM) was performed in 16 pregnant women: in one 28 year old primigravida at 35 weeks of pregnancy with congenital FVII deficiency after rFVIIa administration and 15 healthy women at 38 gestational weeks. The results were compared. Results: The thromboelastometry results showed significant shortening of the clotting time in the extrinsic and the intrinsic pathway in the hypoproconvertinemia patient after rFVIIa administration in relation to healthy pregnant women. A significant reduction in maximum lysis of the clot after FVII supplementation was observed. Conclusions: The thromboelastometry results showed a significant hypercoagulable state with hypoproconvertinemia. Thrombotic complications after delivery might be prevented by the reduction in rFVIIa guided by thromboelastometry. Thromboelastometry performed on a pregnant woman with factor VII deficiency during the supplementation of rFVIIa in peripartum time might be helpful in order to determine an individual, effective dosage regimen of rFVIIa to ensure full correction of clotting disorders without the tendency to develop thrombosis, but further studies are needed.

In seeking diagnostic tool for laboratory monitoring of FXII-targeting agents, could assessment of rotational thromboelastometry (ROTEM) in patients with factor XII deficiency be useful?

Introduction: Targeting factor XII (FXII) is a new concept for safe thrombosis prophylaxis. Global hemostasis tests offer promise in terms of the laboratory monitoring of FXII inhibition. The present study examines selected parameters of rotational thromboelastometry (ROTEM) in patients with FXII deficiency. The objective of this study was to assess the impact of FXII deficiency on selected parameters of ROTEM, which can be significant in the laboratory monitoring of FXII inhibition. Material and methods : The study included 20 patients with FXII deficiency ≤40% and 21 volunteers free of it. Clotting time (CT), clot formation time (CFT), alpha angle (α), maximum clot firmness (MCF), and maximum lysis (ML) were recorded in ROTEM. Results: For the INTEM test, CT and CFT readings were markedly higher in FXII deficient patients than in controls. No marked differences in relation to MCF and ML were found. Conclusion: The results of ROTEM show that FXII deficiency has a great impact on the initiation and amplification of coagulation. This was confirmed by a number of marked correlations between FXII activity and certain ROTEM parameters. ROTEM tests merit further investigation as treatment control strategies in the context of FXII inhibition.

Comparison of Jugular vs. Saphenous Blood Samples, Intrarater and In-Between Device Reliability of Clinically Used ROTEM S Parameters in Dogs.

Simple SummaryViscoelastic coagulation tests such as rotational Thromboelastometry (ROTEM) have many theoretical advantages compared to traditional coagulation testing. As a point-of-care diagnostic device, ROTEM results are directly part of treatment decisions. Therefore, it is crucial to know its reliability and precision. Most recommendations for ROTEM S analyses originate from Thromboelastography (TEG), another viscoelastic coagulation assay. However, evidence about how preanalytical and analytical factors, such as sample collection technique, sample handling and the analysis itself, influence ROTEM results is scarce. Due to the absence of a gold standard method, we assessed accuracy with the coefficient of variation and intraclass correlation coefficient and examined the influence of blood collection site, as well as intrarater and in-between device variability, on ROTEM S results of clinically healthy dogs. We found significant changes between ROTEM S parameters from different blood collection sites and significant intrarater and in-between device variability. These findings were most prominent in tissue-factor-activated tests. To ensure patient safety, we therefore suggest running duplicate measurements and to interpret results obtained from tissue-factor-activated tests with caution, since some of their coefficients of variation were moderate to high.Rotational Thromboelastometry (ROTEM) allows for the global assessment of hemostasis in whole blood samples. Preanalytical and analytical factors may influence test results, and data about the reliability and reproducibility of lyophilized ROTEM tests are scarce. Therefore, the objective of this study was to evaluate the influence of blood collection site on ROTEM S parameters and to assess intrarater and in-between device variability. A total of thirty, healthy, staff-owned dogs were included. Blood collection and ROTEM analysis were performed by trained staff according to a standardized protocol. Extrinsically activated (tissue factor; Ex-TEM S), with the addition of cytochalasin for platelet inhibition (Fib-TEM S), and intrinsically activated (In-TEM) analyses were performed. Analysis of our data showed significant variability for various Ex-TEM S and Fib-TEM S parameters from different collection sites and intrarater and in-between device measurements. We conclude that serial monitoring with ROTEM should be performed on the same device, with blood always taken from the same collection site using a standardized blood sampling technique. While In-TEM S, apart from maximum lysis, showed very stable and reliable results, we suggest interpreting especially clotting and clot formation parameters from Ex-TEM S and Fib-TEM S tests with caution and using duplicate measurements to detect outliers and to prevent initiation of incorrect therapies.

Cold-stored platelets are effective in an in vitro model of massive transfusion protocol assessed by rotational thromboelastometry.

Platelets are a key component of massive transfusion in treating actively bleeding patients. While optimized for prophylactic transfusions, the effectiveness of the current standard room temperature stored platelets (RPs) in treating actively bleeding patients is not clear. Cold-stored platelets (CPs) have been shown to have superior hemostatic functions and the potential to extend shelf life. In this study, we explored the effect of using CPs versus RPs in an in vitro transfusion model based on the massive transfusion protocol. RPs or CPs were combined with RBCs and plasma in a 1:1:1 volume ratio to make transfusion packages. Whole blood was collected and then either diluted to 20% hematocrit or mixed with tPA (8.8μg/ml). By volume, 70% of transfusion package was mixed with 30% whole blood to simulate massive transfusions and analyzed by rotational thromboelastometry. Transfusion package supernatant was analyzed for PAI-1 activity as well. Both transfusion packages restored the clot characteristics of hemodiluted or hyperfibrinolytic whole blood. Specifically, only transfusion packages made with CPs significantly reduced the maximum clot lysis of hyperfibrinolytic whole blood. PAI-1 activity in CPs transfusion packages were also significantly higher. Transfusion packages containing cold-stored platelets may be able to restore the blood hemostatic profile of bleeding patients. In addition, transfusion packages made from CPs may provide additional benefit of resisting hyperfibrinolysis in bleeding patients. In trauma where post-transfusion platelet recovery is less of a concern, CPs are a viable option to restore hemostasis.

P1665: WHOLE-BLOOD ROTATIONAL THROMBOELASTOMETRY (ROTEM) AS A DIAGNOSTIC TOOL IN PATIENTS WITH BLEEDING OF UNKNOWN CAUSE (BUC)

Background: A majority of patients with a mild to moderate bleeding tendency (mild bleeding disorders, MBD) remains without a diagnosis, despite extensive laboratory investigations (bleeding of unknown cause, BUC). Patients with BUC do not differ in their bleeding phenotype to patients with an established MBD like von Willebrand disease (VWD) or platelet function defects (PFD). Whole-blood thromboelastometry (ROTEM) depicts diverse aspects of coagulation including hyper-fibrinolysis and captures the influence of erythrocytes, leucocytes and platelets to hemostasis, which might contribute to BUC. Aims: To analyze clinical risk factors for ROTEM parameters and compare results between patients with BUC and an established MBD. Methods: Patients from the Vienna bleeding biobank (VIBB, EC no. 603/2009) were analyzed (Gebhart et al, Haemophilia, 2018). Viscoelastic clot measurement was performed with non-activated ROTEM® (EM International, Munich, Germany): clotting time (CT, sec, time from analysis start until start of clot formation), clot formation time (CFT, sec, time needed to form a clot), maximum clot firmness (MCF, mm, peak amplitude of the clot) and maximal lysis (ML, %, expression of clot lysis in % of MCF). Results: ROTEM was performed in 513/714 (71.8%) patients included in the VIBB. The mean(SD) age was 41.4 (15.0) years, 425 (80.3%) were women and 252 (47.6%) patients had blood group O. In total, 318 patients had BUC, the reaiming patients hat PFD(n=122, 23.7%), VWD (n=51, 9.9%), mild clotting factor deficiencies (CFD, n=18, 3.5%) and 4 had an other bleeding disorder (0.8%). Clinical factors that were independently associated with ROTEM values in BUC patients were sex, blood group O (vs. non-O) and the bleeding score (multivariable linear regression, Table 1). BUC patients had comparable ROTEM results as patients with platelet function defects or VWD, after adjusting for blood group O and sex, whereas patients with clotting factor deficiency had longer CT and CFT, a lower MCF and higher ML (Table 2). Image:Summary/Conclusion: Hemostatic capacity measured by ROTEM is similar in patients with BUC and those with mild primary hemostatic bleeding disorders. Our data support the hemostatic imbalance in patients with BUC.

Tranexamic acid administered during cesarean delivery in high-risk patients: maternal pharmacokinetics, pharmacodynamics, and coagulation status

Tranexamic acid is frequently administered for postpartum hemorrhage. The World Health Organization recommends 1 g intravenous dosing, repeated once after 30 minutes for ongoing bleeding. Understanding the pharmacokinetics and pharmacodynamics of tranexamic acid in patients at high risk of postpartum hemorrhage may enable dosage tailoring for optimal antifibrinolysis with minimal adverse events, such as thrombosis or renal cortical necrosis. This study aimed to report tranexamic acid pharmacokinetics and pharmacodynamics after 1 g intravenous dosing during cesarean delivery in patients at risk of hemorrhage. The primary endpoint was tranexamic acid plasma concentration of >10 μg/mL, known to inhibit 80% of fibrinolysis. In addition, the correlation between patient demographics and rotational thromboelastometry coagulation changes were analyzed. In this prospective study, 20 women aged 18 to 50 years, ≥23 weeks of gestation undergoing cesarean delivery with at least 1 major (placenta previa, suspected placenta accreta spectrum, or active bleeding) or 2 minor (≥2 previous cesarean deliveries, previous postpartum hemorrhage, chorioamnionitis, polyhydramnios, macrosomia, obesity, or suspected placental abruption) risk factors for postpartum hemorrhage were recruited. The exclusion criteria were allergy to tranexamic acid, inherited thrombophilia, previous or current thrombosis, seizure history, renal or liver dysfunction, anticoagulation, or category III fetal heart tracing. Tranexamic acid 1 g was administered after umbilical cord clamping. Blood samples were drawn at 3, 7, 15, and 30 minutes and then at 30-minute intervals up to 5 hours. Plasma concentrations were evaluated as mean (standard error). Serial rotational thromboelastometry was performed and correlated with tranexamic acid plasma concentrations. The median age of participants was 37.5 years (interquartile range, 35.0-39.5), and the median body mass index was 28.6 kg/m2 (interquartile range, 24.9-35.0). The median blood loss (estimated or quantitative) was 1500 mL (interquartile range, 898.5-2076.0). Of note, 9 of 20 (45%) received a transfusion of packed red blood cells. The mean peak tranexamic acid plasma concentration at 3 minutes was 59.8±4.7 μg/mL. All patients had a plasma concentration >10 μg/mL for 1 hour after infusion. Plasma concentration was >10 μg/mL in more than half of the patients at 3 hours and fell <10 μg/mL in all patients at 5 hours. There was a moderate negative correlation between body mass index and the plasma concentration area under the curve (r=-0.49; 95% confidence interval,-0.77 to-0.07; P=.026). Rotational thromboelastometry EXTEM maximum clot firmness had a weak positive correlation with longitudinal plasma concentration (r=0.32; 95% confidence interval, 0.21-0.46; P<.001). EXTEM maximum clot lysis was 0% after infusion in 18 patients (90%), and no patient in the study demonstrated a maximum lysis of >15% at any interval from 3 minutes to 5 hours. There was no significant correlation between EXTEM clot lysis at 30 minutes and longitudinal tranexamic acid plasma concentrations (r=0.10; 95% confidence interval,-0.20 to 0.19; P=.252). After standard 1 g intravenous dosing of tranexamic acid during cesarean delivery in patients at high risk of hemorrhage, a plasma concentration of ≥10 μg/mL was sustained for at least 60 minutes. Plasma tranexamic acid levels correlated inversely with body mass index. The concurrent use of rotational thromboelastometry may demonstrate tranexamic acid's impact on clot firmness but not a hyperfibrinolysis-derived trigger for therapy.

Correlation of laboratory markers of hemostatic system activation with concentration and size of plasma extracellular microparticles in patients with COVID-19

Introduction. In recent years, much attention has been paid to the study of extracellular microparticles (microvesicles and exosomes) and their role in the pathogenesis of human diseases.The objective of this study was to determine the number and size of plasma extracellular microparticles (PEMP) in patients with severe and extremely severe COVID-19 and correlate these data with the markers of hemostasis activation, inflammation, and tissue damage.Methods and Materials. The study included 29 patients with severe and extremely severe COVID-19. Concentration and size of PEMP were determined by nanoparticle trajectory analysis (NTA). All patients underwent the complete blood count and the thromboelastometry (TEM). Hemostatic, biochemical, and immunological parameters were assessed including fibrinogen, prothrombin time, activated partial thromboplastin time, D-dimer, C-reactive protein, lactate dehydrogenase, procalcitonin, von Willebrand factor antigen, interleukin 6, and interleukin 18.Results. There were 14 patients (48.3 %) discharged from the ICU with improvement (group 1— survived patients), and 15 patients (51.7 %) with lethal outcomes (group 2 — lethal outcome); the PEMP concentration did not differ between these groups. In group 2, there were heterogeneity of PEMP population, and a tendency to the larger PEMP size (p=0.074). In all patients, the PEMP concentration correlated negatively with both prothrombin time and the number of large platelets; the size of PEMP correlated negatively with the level of von Willebrand factor antigen, and positively with the fibrinogen. In group 1, the PEMP concentration had a direct correlation with both the level of interleukin 18 and maximum clot lysis in TEM; the PEMP size had a direct correlation with the maximum clot lysis in TEM and an inverse correlation with both the level of procalcitonin and maximum clot density in TEM.Conclusion. Our study confirms the importance of the process of extracellular microparticles formation in the COVID-19 pathogenesis. Our findings are consistent with the hypothesis that the parameters of PEMP population can be predictive biomarkers of the COVID-19 severity.

Assessment of fibrinolytic status in whole blood using a dielectric coagulometry microsensor

Rapid assessment of the fibrinolytic status in whole blood at the point-of-care/point-of-injury (POC/POI) is clinically important to guide timely management of uncontrolled bleeding in patients suffering from hyperfibrinolysis after a traumatic injury. In this work, we present a three-dimensional, parallel-plate, capacitive sensor – termed ClotChip – that measures the temporal variation in the real part of blood dielectric permittivity at 1 MHz as the sample undergoes coagulation within a microfluidic channel with <10 μL of total volume. The ClotChip sensor features two distinct readout parameters, namely, lysis time (LT) and maximum lysis rate (MLR) that are shown to be sensitive to the fibrinolytic status in whole blood. Specifically, LT identifies the time that it takes from the onset of coagulation for the fibrin clot to mostly dissolve in the blood sample during fibrinolysis, whereas MLR captures the rate of fibrin clot lysis. Our findings are validated through correlative measurements with a rotational thromboelastometry (ROTEM) assay of clot viscoelasticity, qualitative/quantitative assessments of clot stability, and scanning electron microscope imaging of clot ultrastructural changes, all in a tissue plasminogen activator (tPA)-induced fibrinolytic environment. Moreover, we demonstrate the ClotChip sensor ability to detect the hemostatic rescue that occurs when the tPA-induced upregulated fibrinolysis is inhibited by addition of tranexamic acid (TXA) – a potent antifibrinolytic drug. This work demonstrates the potential of ClotChip as a diagnostic platform for rapid POC/POI assessment of fibrinolysis-related hemostatic abnormalities in whole blood to guide therapy.

In Situ Pulmonary Thrombolysis and Perfusion Lung Angiography in Severe COVID-19 Respiratory Failure.

OBJECTIVES:A proof-of-concept study using thrombolysis with catheter-directed tissue plasminogen activator (tPA) and pulmonary angiography imaging was performed to visualize perfusion deficits and reperfusion/therapeutic effects of tPA.DESIGN:A prospective, open-label, compassionate study. Descriptive statistics were presented for categorical variables and as means with sds for continuous variables. The Wilcoxon test was used to determine the differences between the two-related samples and a t test for continuous variables. Statistical significance was set at p value of less than 0.05. Agreement between observations was evaluated using the Kappa Cohen index and overall agreement using the Fleiss Kappa coefficient.SETTING:A single COVID-19 ICU of Mexico´s General Hospital Dr Eduardo Liceaga.SUBJECTS:Fifteen patients with severe Delta variant severe acute respiratory syndrome coronavirus 2 infection, 18–75 years old, requiring mechanical ventilation with a persistent Fio2 requirement of 70% or higher and Pao2/Fio2 ratio (or imputed ratio) less than 150 for more than 4 hours. The coagulation inclusion criteria were International Society on Thrombosis and Haemostasis score greater than 5, and presence of a d-dimer greater than 1,200, with viscoelastic testing using rotational thromboelastometry (Instrumentation Laboratories, Mexico City, Mexico) showing both hypercoagulability (EXTEM amplitude at 5 min > 65 FIBTEM > 30) and hypofibrinolysis (EXTEM maximum lysis < 8%).INTERVENTIONS:Catheter-directed tPA angiography and iFlow system analysis to assess pre-tPA baseline pulmonary perfusion and changes in response to thrombolysis.RESULTS:Nine patients had microvascular filling defects demonstrated by angiography, and good agreement was found with iFlow analysis (ƙ = 0.714). Statistically significant differences were identified in the area under the curve (AUC) region of interest/AUC reference tissue with and without filling defects in phase 2 DM –0.09206 (sd ± 0.16684) (p = 0.003). The Pao2/Fio2 values measured immediately and 48 hours after the procedure were significantly higher (p = 0.001 and p = 0.005, respectively). Statistically significant differences were found in d-dimer values (p = 0.007), Fio2 (p = 0.002), and oxygen saturation in arterial blood/Fio2 (p = 0.045), as well as in the number of patients who required prone positioning before, immediately after the procedure, and at 48 hours after the procedure (p = 0.002).CONCLUSIONS:Thrombolysis with catheter-directed tPA resulted in imaging evidence via pulmonary angiography and iFlow technology of improved lung perfusion in COVID-19 patients with severe respiratory failure.

Rotational thromboelastometry in critical phase of dengue infection: Association with bleeding

BackgroundThe critical phase of dengue carries a high risk of bleeding. Associations of coagulation test parameters and the risk of bleeding in the critical phase is unclear. This study examines the association of rotational thromboelastometry (ROTEM delta and ROTEM platelet) with bleeding risk of patients with dengue in the critical phase. MethodsA total of 105 patients with confirmed dengue in the critical phase were recruited, with two subsequent prospective time point analyses of ROTEM parameters and platelet count within 24 and 48 hours from the onset of the critical phase. Conventional coagulation tests were performed only at the initial time point. ResultsTwenty of 105 patients developed bleeding after onset of the critical phase. Within the first 24 hours of critical‐phase onset, platelet count, coagulation tests, and ROTEM delta were unable to differentiate patients with bleeding manifestations from those without (P < .05). Area under the curve of thrombin receptor activating peptide‐6 assay of ROTEM platelet (TRAPTEM) discriminated patients with bleeding manifestations from those without, at a cutoff value of <12.5 Ω*min at a sensitivity and specificity of 73.7%, and 60.2%. In patients who developed bleeding, the maximum lysis of extrinsic pathway of ROTEM was significantly lower in patients with severe bleeding compared to those with mild to moderate bleeding. (4.3 ± 3.4% vs 9.4 ± 7.5%; P = .01). ConclusionAn association with bleeding manifestations and TRAPTEM suggest a potential role for defective platelet aggregation in the pathogenesis of bleeding in the critical phase of dengue.

Delayed Plasma Clot Lysis in Adult Patients with Primary Immune Thrombocytopenia (ITP)

Introduction: Primary immune thrombocytopenia (ITP) is an orphan disease characterized by very low platelet counts. Patients have heterogeneous bleeding phenotypes, which are not only determined by platelet counts, also a paradoxically increased thrombotic risk has been observed.Aim: To investigate, whether the fibrinolysis inhibitors plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin are associated with impaired plasma clot lysis in primary ITP patients in comparison to non-immunologic thrombocytopenic controls (TPC) and healthy controls (HC). Furthermore, associations with bleeding severity and previous thrombotic events were investigated.Methods: Patients from the Vienna ITP biobank (EC 1843/2016), a multi-centric study including adult patients with primary ITP were investigated and compared to age- and sex-matched control groups: TPC with thrombocytopenia after chemotherapy and HC. Informed consent was obtained from all individuals before study inclusion. A clot formation and lysis assay (CLA) was performed according to the recommendations of the ISTH SSC. Platelet poor plasma samples were measured in duplicates for each patient and control. PAI-1 (PAI-1 Actibind ELISA, Technoclone, Vienna, Austria) and α2-antiplasmin by the chromogenic STA Stachrom antiplasmin assay (Diagnostic Stago, Asnieres, France) were measured. Bleeding severity was measured using the ITP-specific ITP-ISTH BAT (Rodeghiero et al. 2013).Results: In total, 37 primary ITP patients, 18 TPC and 156 healthy controls were analyzed (Table 1). Primary ITP patients had a higher BMI than HC. Bleeding severity was higher and more ITP patients had a thrombosis history compared to HC, whereas there was no difference in comparison to TPC. PAI-1 activity was highest in ITP patients, with a statistically significant difference in comparison to HC. α2-antiplasmin activity was higher in ITP patients than in TPC, whereas there was no difference in comparison to HC.After adjustment for sex, age, BMI and fibrinogen, primary ITP patients had a reduced clot formation rate (V max) and significantly delayed plasma clot lysis compared to TPC and HC (Table 2). Also, the lag phase and time to peak absorbance (TTP) were prolonged with a significant difference in comparison to HC.To investigate outliers of PAI-1 and α2-antiplasmin, we calculated cut-offs at the 75 th percentile of healthy controls (PAI-1: ≥ 3.1 U/mL, α2-antiplasmin: ≥ 107.0 %). 14 (37.8 %) ITP patients had PAI-1 levels and 10 (27.0 %) ITP patients had α2-antiplasmin activity above the cut-off. ITP patients with high PAI-1 levels had mildly delayed clot lysis in comparison to those below with a significantly lower maximal lysis rate (mLR). ITP patients with α2-antiplasmin activity above the cut-off had a significantly shorter lag phase, faster V max and shorter TTP than patients below the cut-off, whereas there was no difference in clot lysis. No differences between ITP patients above or below the respective cut-offs of PAI-1 and α2-antiplasmin regarding their bleeding severity and thrombosis incidence were observed (Table 3).Conclusion: Primary ITP patients have a tendency towards increased PAI-1 activity, which is associated with considerably delayed plasma clot lysis. Albeit an association with the bleeding score could not be identified, this impaired lysis could be seen as a counter-regulation and at least contribute to the relatively mild bleeding tendency in patients with ITP. [Display omitted] DisclosuresPabinger: CSL Behring: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Daiichi Sanchyo: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Temporal Transitions in Fibrinolysis after Trauma: Adverse Outcome Is Principally Related to Late Hypofibrinolysis.

The relationship between late clinical outcomes after injury and early dynamic changes between fibrinolytic states is not fully understood. The authors hypothesized that temporal transitions in fibrinolysis states using rotational thromboelastometry (ROTEM) would aid stratification of adverse late clinical outcomes and improve understanding of how tranexamic acid modulates the fibrinolytic response and impacts mortality. The authors conducted a secondary analysis of previously collected data from trauma patients enrolled into an ongoing prospective cohort study (International Standard Randomised Controlled Trial Number [ISRCTN] 12962642) at a major trauma center in the United Kingdom. ROTEM was performed on admission and at 24 h with patients retrospectively grouped into three fibrinolysis categories: tissue factor-activated ROTEM maximum lysis of less than 5% (low); tissue factor-activated ROTEM maximum lysis of 5 to 15% (normal); or tissue factor-activated ROTEM maximum lysis of more than 15% (high). Primary outcomes were multiorgan dysfunction syndrome and 28-day mortality. Seven-hundred thirty-one patients were included: 299 (41%) were treated with tranexamic acid and 432 (59%) were untreated. Two different cohorts with low-maximum lysis at 24 h were identified: (1) severe brain injury and (2) admission shock and hemorrhage. Multiple organ dysfunction syndrome was greatest in those with low-maximum lysis on admission and at 24 h, and late mortality was four times higher than in patients who remained normal during the first 24 h (7 of 42 [17%] vs. 9 of 223 [4%]; P = 0.029). Patients that transitioned to or remained in low-maximum lysis had increased odds of organ dysfunction (5.43 [95% CI, 1.43 to 20.61] and 4.85 [95% CI, 1.83 to 12.83], respectively). Tranexamic acid abolished ROTEM hyperfibrinolysis (high) on admission, increased the frequency of persistent low-maximum lysis (67 of 195 [34%]) vs. 8 of 79 [10%]; P = 0.002), and was associated with reduced early mortality (28 of 195 [14%] vs. 23 of 79 [29%]; P = 0.015). No increase in late deaths, regardless of fibrinolysis transition patterns, was observed. Adverse late outcomes are more closely related to 24-h maximum lysis, irrespective of admission levels. Tranexamic acid alters early fibrinolysis transition patterns, but late mortality in patients with low-maximum lysis at 24 h is not increased.

Appraisal of antioxidant potential and biological studies of bogan bail (Bougainvillea glabra) leaf extracts using different solvents

Current research work was performed to evaluate the antioxidant, antidiabetic, thrombolytic, and cytotoxic potential of Bougainvillea glabra leaf extracts with different solvents. Extraction of leaves was carried out by maceration using solvents of various polarity such as n-hexane, chloroform, ethyl acetate, n-butanol, and methanol. The highest values of total phenolic and flavonoid contents were assessed in methanolic extract, such as 58.57% and 29.41%, respectively. Antioxidant potential was measured by DPPH free radical, showing 65.16% scavenging activity in methanolic extract. Similarly, the reducing power of methanolic extract was also measured. Hemolytic activity of B. glabra leaf extracts was evaluated, and maximum percentage lysis was found as 6.86% in chloroform extract. The thrombolytic activity of B. glabra leaf extracts was evaluated against human red blood cells, and the maximum percentage of clot lysis was 59.10% in chloroform extract. Maximum antidiabetic activity (16.20%) was observed in methanolic extract. Overall, the presented results reveal that bogan bail extract is capable of being employed in nutra-pharmaceutical industry.