Fibrinolysis in trauma patients: wide variability demonstrated by the Lysis Timer.

Hyperfibrinolysis contributes to the pathophysiology of trauma-induced coagulopathy. At present, systematic administration of tranexamic acid (TXA) is recommended in all patients in the early phase of trauma. However, there is some debate regarding whether TXA is beneficial in all trauma patients. A rapid and accurate tool to diagnose hyperfibrinolysis may be useful for tailoring TXA treatment. We conducted a proof-of-concept study of consecutive adult trauma patients. A first blood sample was obtained at the time of pre-hospital care (T1). Patients received 1 g of TXA after T1. A second sample was obtained on arrival at the emergency unit (T2). We examined coagulation, fibrin and fibrinogen formation and degradation. Fibrinolysis was assessed by determining tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor 1 (PAI-1) activity and global fibrinolysis capacity assay using a device developed by Hyphen BioMed: the Lysis Timer (GFC/LT). The study population consisted of 20 patients (42 ± 21 years, index of severity score 32 ± 21). Both coagulation and fibrinolysis were altered at T1. GFC/LT values exhibited hyperfibrinolysis only in five patients. Principal component analysis carried out at T1 showed two main axes of alteration. The major axis was related to coagulation, altered in all patients, while the second axis was related to fibrinolysis. GFC/LT was mainly influenced by PAI-1 activity while fibrin monomers were related to the severity of trauma. At T2, GFC/LT exhibited the marked effect of TXA on clot lysis time. In conclusion, GFC/LT demonstrated huge variation in the fibrinolytic response to trauma.

Hemostatic Effects of Tranexamic Acid in Cesarean Delivery: An Ancillary Study of the TRAAP2 Study.

BACKGROUND: Fibrinolysis activation during delivery contributes to postpartum hemorrhage (PPH). Clot lysis time studied with the global fibrinolytic capacity device (GFC/LT) is a functional test which rapidly assesses fibrinolytic profile. Tranexamic acid (TXA) is an efficient antifibrinolytic therapy. METHODS: We prospectively studied fibrinolysis and coagulation in 33 women included in the TRAAP2 trial, which aimed to assess the impact of TXA in preventing PPH following a cesarean delivery. TXA or placebo was randomly administered after childbirth as part of the TRAAP2 trial's protocol. Fibrinolytic (GFC/LT, plasma concentration of fibrinolysis activators and inhibitors) and hemostatic parameters were assayed at three sample times (TREF [T-reference] after anesthesia, T15 and T120minutes after TXA, or placebo administration). RESULTS: All cesarean deliveries were elective. In the placebo group, the clot lysis time assessed with GFC/LT significantly decreased between TREF and T120, indicating an activated fibrinolysis (44 [interquartile range, IQR: 40-48] vs. 34 [IQR: 30-36] minutes, p<0.001). In both TXA and placebo groups, significant fluctuations of the plasmatic concentrations of fibrinolytic mediators were noticed over time, suggesting fibrinolysis activation. Clot lysis time measured by GFC/LT was significantly increased in women of the TXA group as compared with those in the placebo group at T15 (120 [120-120] vs. 36 [34-41] minutes, p<0.001) and T120minutes (113 [99-120] vs. 34 [30-36] minutes, p<0.001) after drug administration, indicating a decreased in fibrinolysis in those women. CONCLUSION: GFC/LT evidenced fibrinolysis activation during cesarean delivery, linked to a decrease in fibrinolytic inhibitors. GFC/LT revealed a significant antifibrinolytic effect of TXA compared with placebo.

Comparison of Two Thrombin Generation Methods, CAT and ST-Genesia, in Liver Transplant Patients.

BACKGROUND: During liver transplantation (LT), thrombin generation (TG) is altered. The most frequently used assay for TG is the Calibrated Automated Thrombogram (CAT). It is designed for series of plasmas and is semi-automated. Complete automation has led to a new device, the ST-Genesia, enabling quantitative standardized TG evaluation. OBJECTIVE: The aim of this observational study was to compare the TG results of the CAT and the ST-Genesia on frozen-thawed plasma samples prepared from the blood of LT patients. PATIENTS AND METHODS: Poor platelet plasma aliquots were prepared from blood samples from six LT patients selected to get the whole range of TG and were assessed with CAT (recombinant human tissue factor [TF] concentration 5 pm) and with ST-Genesia Bleedscreen assay (BS, using 'low' recombinant human TF concentration) and Thromboscreen assay (TS, using 'medium' recombinant human TF concentration). The TG parameters studied were: lag time, peak, time to peak, endogenous thrombin potential, velocity index and start tail. RESULTS: BS and TS did not differ significantly from each other whatever the parameter studied, whereas most of the CAT parameters were significantly different from those obtained with BS and TS. Hierarchical clustering analysis of the different parameters of TG showed three homogeneous groups. One cluster gathered TG quantitative parameters from ST-Genesia. A second cluster gathered all the kinetic parameters. The last cluster isolated the quantitative parameters of CAT. CONCLUSION: In patients undergoing LT, TG performed with CAT and with ST-Genesia provided different results, for unknown reasons.

Fibrinolysis during liver transplantation: analysis by the Thrombodynamics method.

An issue in orthotopic liver transplantation (OLT) is the diagnosis of hyperfibrinolysis. The Thrombodynamics-4D assay (TD4D) is a videomicroscopy system allowing the dynamic analysis of fibrin clot. Fibrinolysis is highlighted by a change in clot intensity. The aim of this observational study was to evaluate the TD4D as a tool to diagnose fibrinolysis during OLT. Thirty consecutive patients were included. We studied a subset of 41 samples from 13 patients who demonstrated hyperfibrinolysis during OLT by global fibrinolytic capacity studied by the Lysis Timer (GFC/LT) and/or euglobulin clot lysis time (ECLT) and/or EXTEM maximum lysis (EXTEM ML) on ROTEM. Three samples exhibited fibrinolysis. They exhibited significantly shorter ECLT, higher lysis on EXTEM graphs, shorter GFC/LT clot lysis time and higher t-PA activity values. After adding urokinase, 13 samples exhibited fibrinolysis. In conclusion, TD4D allows the dynamic analysis of fibrin clot formation and lysis. It only recognises the most severe forms of hyperfibrinolysis during OLT.

Lysis Timer: a new sensitive tool to diagnose hyperfibrinolysis in liver transplantation.

AIMS: Diagnosis of hyperfibrinolysis in orthotopic liver transplantation (OLT) remains challenging. Euglobulin clot lysis time (ECLT) is not adapted to clinical situations. ROTEM is specific but seldom sensitive to hyperfibrinolysis. The Lysis Timer assesses 'Global Fibrinolytic Capacity' in citrated plasma (GFC/LT). GFC/LT associates reagents for in vitro triggering of the clot (thrombin and calcium) and its lysis (tissue-plasminogenactivator (t-PA)), turbidity signal acquisition by the Lysis Timer, and dedicated software converting the digital signal into an optical curve. A visual check of the curves was systematic to ascertain the lysis time values calculated by the software. The primary aim of this prospective observational study was to evaluate the ability of GFC/LT to recognise hyperfibrinolysis during OLT. The secondary aim was to compare its results with ROTEM maximum lysis (EXTEM ML) and with standard laboratory tests. METHODS: Thirty consecutive adult patients undergoing OLT were included (NCT03012633). Standard laboratory tests, ROTEM, GFC/LT, ECLT and fibrinolysis parameters were assayed at five sample times. RESULTS: GFC/LT was correlated with ECLT, plasmin activator inhibitor 1 antigen and activity and t-PA activity (r=0.490, 0.681, 0.643 and -0.359, respectively). Hyperfibrinolysis was defined as ECLT ≤60 min. Receiver operating characteristic curve analysis showed that GFC/LT with a threshold of 31 min detected hyperfibrinolysis with a sensitivity of 0.88 (95% CI 0.73 to 0.96), a specificity of 0.68 (95% CI 0.56 to 0.78) and an area under the curve (AUC) of 0.85 (95% CI 0.74 to 0.94). EXTEM ML >12% did not detect hyperfibrinolysis (sensitivity 0.38 (95% CI 0.24 to 0.55), specificity 0.95 (95% CI 0.86 to 0.99) and AUC 0.60 (95% CI 0.46 to 0.75)). CONCLUSIONS: GFC/LT recognised hyperfibrinolysis during OLT with a significant agreement with the other tests of fibrinolysis. TRIAL REGISTRATION NUMBER: NCT03012633.

Genetic background of type I protein C deficiency in Finland.

In contrast to other populations the usually rare type II form of protein C deficiency is as common in Finland as type I deficiency. We recently reported that a single mutation explained virtually all cases of type II protein C deficiency in Finland, indicating strong founder effect. We now investigated in the same population the genetic background of type I protein C deficiency. Thirty-eight apparently unrelated families were studied. They represent the vast majority of all families with type I deficiency in Finland. A genetic defect was identified in 23 (61%) families who carried 13 different mutations. Only three of the 13 mutations have been reported in other populations. Unlike in type II deficiency, considerable heterogeneity in mutations was found in type I deficiency. Our results indicate interesting differences in mutational histories of these two different forms of protein C deficiency in Finland.

Diagnosis accuracy of a new challenger for thrombosis exclusion, the Stratus CS DDMR.

BACKGROUND: The diagnostic accuracy of a new Point of Care, rapid and quantitative D-dimer assay (Stratus CS DDMR from Dade Behring) was evaluated. METHODS: Vidas test from bioMerieux was used as reference method in 279 patients recruited from a management study in progress in our institution. RESULTS: Both assays show comparable reproducibility (2.9% with the Stratus CS DDMR and 4.4% with the D-dimer Vidas in the cut-off range) and good correlation (R(2)=0.9057). The overall test performance as assessed by the area under the curve of the ROC curves is 0.801 for the Stratus CS DDMR assay and 0.798 for the D-dimer Vidas assay. By using assay regression curves, likelihood ratios or test agreement approaches, a 440-450 ng/ml value is evidenced as the threshold value for the Stratus CS DDMR, which nears at best the performances of the D-dimer Vidas 500 or 550 ng/ml threshold value. This proposed exclusion value ensures a 95% sensitivity and a 45% specificity. The few false negative results using the two assays only evidenced sub-popliteal thromboses, that would not have been considered as having thrombosis if an above-the-knee test had been performed. In these conditions, sensitivity would have been 100%. CONCLUSIONS: A new quantitative D-dimer assay, the Stratus CS DDMR, demonstrated performances comparable with those of the DD Vidas test.

Haemostaseome-associated SNPs: has the thrombotic phenotype a greater influence than ethnicity? GMT study from Aquitaine including Basque individuals.

The Genetic Markers for Thrombosis (GMT) study compared the relative influence of ethnicity and thrombotic phenotype regarding the distribution of SNPs implicated in haemostasis pathophysiology ("haemostaseome"). We assessed 384 SNPs in three groups, each of 480 subjects: 1) general population of Aquitaine region (Southwestern France) used as control; 2) patients with venous thromboembolism from the same area; and 3) autochthonous Basques, a genetic isolate, who demonstrate unusual characteristics regarding the coagulation system. This study sought to evaluate i) the value of looking for a large number of genes in order to identify new genetic markers of thrombosis, ii) the value of investigating low risk factors and potential preferential associations, iii) the impact of ethnicity on the characterisation of markers for thrombosis. We did not detect any previously unrecognised SNP significantly associated with thrombosis risk or any preferential associations of low-risk factors in patients with thrombosis. The sum of Ï°² values for our 110 significant SNPs demonstrated a smaller genetic distance between patients and controls (321 cumulated Ï°² value) than between Basques and controls (1,570 cumulated Ï°² value). Hence, our study confirms the genetic particularity of Basques especially regarding a significantly lower expression of the non-O blood group (p< 0.0004). This is mitigated by a higher prevalence of factor II Leiden (p< 0.02) while factor V Leiden prevalence does not differ. Numerous other differences covering a wide range of proteins of the haemostaseome may result in an overall different genetic risk for venous thromboembolism.

Rivaroxaban and apixaban in orthopaedics: is there a difference in their plasma concentrations and anticoagulant effects?

The aim of this study was to improve knowledge of what happens in the coagulation of orthopaedic patients under rivaroxaban and apixaban, in order to finalize and cross-validate effective measurement methods and to provide arguments for helping to reference one or the other drug in our central pharmacy. One hundred and two patients undergoing total hip or knee replacement were included. Half of them received rivaroxaban and the other half received apixaban. Blood samples (n = 244 with each drug) were taken at Cmax preoperatively and twice a week, apart from the day of the patient's discharge, when Ctrough concentration was targeted. Routine coagulation parameters, and functional and liquid chromatography tandem mass spectrometry assays for measurement of circulating concentrations were studied. The LC-MS/MS assay and the functional assays carried out in patients under routine conditions were highly correlated, apart from low concentrations (<30 ng/ml), which were affected by the variable individual potential to inhibit the exogenous bovine Xa used in the functional assays. After 1 week of treatment, the drugs differed: Cmax and Ctrough were closer when apixaban was taken twice daily (83 ±â€Š39 and 58 ±â€Š17 ng/ml) than with rivaroxaban taken once a day (113 ±â€Š67 and 13 ±â€Š20 ng/ml). Rivaroxaban had a greater influence on routine coagulation tests and reduced the maximum thrombin concentration more efficiently, as assessed by the thrombin generation test. Although rivaroxaban and apixaban present apparently similar constant rates, they exhibit significant differences in their concentrations and anticoagulant effects when studied ex vivo in orthopedic patients.

Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: two observational studies in patients undergoing total hip or total knee replacement.

INTRODUCTION: Dabigatran and rivaroxaban have recently been added to the armamentarium for thromboprophylaxis in orthopedic surgery. Although this is their first licensed indication, others will soon follow. Owing to their claimed predictable anticoagulant response that dispenses with the need for monitoring coagulation, their effects are poorly described in routine cases. However, interpreting blood coagulation results and evaluating whether a treatment is properly targeted in the case of untoward incidents will become a common concern for clinicians. METHODS: Eighty patients undergoing total hip or knee replacement were included in two studies. Forty of them received dabigatran (study 1) and 40 rivaroxaban (study 2). Blood samples (n = 176 and 166) were taken preoperatively and twice a week from the first postoperative day. RESULTS: Dabigatran increased aPTTr about two-fold and PT about 1.2-fold, and it was mostly an initiation-phase modulator of thrombin generation. Mean circulating concentrations as measured by a diluted thrombin time were 105 ± 85 ng/mL at T(max) in samples from patients receiving the full dosing. They depended significantly on renal function, body weight and gender. Rivaroxaban increased aPTTr and PTr around 1.5 fold and modified the initiation and amplification phases of thrombin generation, with a lowered and prolonged thrombin burst. Mean circulating concentrations as measured by an antiXa test were 117 ± 78 ng/mL at T(max). With both drugs, routine coagulation tests, thrombin generation curves and functionally determined concentrations exhibited high interindividual variability. CONCLUSION: Routine coagulation tests are altered in patients receiving dabigatran or rivaroxaban, but their alterations poorly reflect the circulating concentrations as determined by functional approaches.

Comparability of D-dimer assays in clinical samples.

D-dimers (DD) have shown sufficient proof of their efficiency in the last 10 years to play an important role in hemostasis laboratories for excluding thromboembolic events. Numerous reagents are available on the market but their performances differ. This overview takes stock of the methods used to evaluate the performances of DD assays, the results published in the literature, the technical parameters influencing assay performance, the difficulties caused by the lack of harmonization of DD units, and the attempts to tackle this problem. It raises the issue of the potential optimization of their use with regard to better adaptation to multidisciplinary diagnostic strategies and to target patient populations.