Thrombin generation test

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Correction of microplate location effects improves performance of the thrombin generation test

Correction of microplate location effects improves performance of the thrombin generation test

coagulation assays to pre-analytical conditions has been known for decades yet the causes of TGT variability have only recently been investigated [19], but location artifacts were not discussed in the literature. Location ef- fect can be introduced in various ways. We found that if a coagulation enzyme, e.g., FXIa or FVIIa, is added manually to multiple wells on a microtiter plate, a well- to-well drift appears due to the different durations of contact between the enzyme and plasma inhibitors. A similar effect may be observed if TF or contact activator is added manually before recalicification. In our experi- ence, long exposure of plasma to fluorogenic substrate prior to recalcification also decreases the TGT response. Edge effects can be caused by uneven heating of microplate inside the reader and artifacts of liquid dis- penser. It should be noted that internal normalization on the thrombin-α2macroglobulin activity calibrator (e.g., as proposed by Hemker et al. [21]) is not helpful here because the thrombin calibrator only corrects for fluorogenic signal-related inaccuracies and fails to cor- rect for biological and pipetting variables [22]. All lo- cation effects may be corrected with the use of the strip-plot sample design. Specifically, when edge arti- facts are minor, our symmetrical strip-plot design
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A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis

A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis

In general thrombin generation tests (TGT) uses some trigger to imitate vessel wall damage (e.g. tissue factor). In platelet poor plasma (PPP) procoagulant phospholipids (in general about 4 μM) amplify the effects of tissue factor [29]. Depending on the question of the test the amount of added TF may reflect different kinds of factor composi- tions. That means with large amounts (>10pM) TF factor VIII, IX and XI are bypassed, but between 2 and 5 pM TG depends on factor VIII and IX and at even lower con- centrations factor XI might become more important [30]. In contrast when using platelet rich plasma (PRP) the platelets take the role of phospholipids over as amp- lifying surface. By this the reaction reflects the interplay of platelet activation and plasmatic coagulation. The most relevant parameters deriving from the thrombin generation tests are the lag-time (time to start), the time to peak, the peak height, and the endogenous thrombin potential (ETP) [28].
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New considerations on pathways involved in acute traumatic coagulopathy: the thrombin generation paradox

New considerations on pathways involved in acute traumatic coagulopathy: the thrombin generation paradox

automated analyzer (STA-R Evolution, Stago, Asnieres sur Seine, France). PT, aPTT times, and fibrinogen con- centrations were measured with neoplastin Cl + 10, tri- niclot aPTTb, and STA liquid fib, respectively. Specific factor-depleted plasmas (Stago, Asnieres sur Seine, France) were used to determine coagulation factor activ- ities. The thrombin generation test was performed using the Thrombinoscope CAT (Calibrated Automated Thrombogram, Maastricht, The Netherlands) assay ac- cording to the manufacturer’s instructions (Diagnostica Stago, Asnières, France) [7, 8]. Twenty microliters of plasma was incubated with 20 μL PPP-ReagentTM (con- taining 5 pM recombinant tissue factor and 4 μM phos- pholipids) for 10 min in round-bottomed 96-well black microplates. For each sample, a calibrator (Thrombin CalibratorTM) was run in parallel in order to correct the fluorescence signal for substrate consumption and plasma color variability. Thrombin generation was initi- ated by the addition of 20 μL of FluCa-KitTM). Fluores- cence was detected by a Fluoroskan Ascent1 fluorimeter (Thermo Fischer Scientific, Waltham, MA), and the thrombin generation curves were analyzed by the throm- binoscope software (Thrombino- scope BV, Maastricht, The Netherlands). Thrombin generation curves was characterized by 5 parameters: “endogenous thrombin potential” (ETP), the area under the curve expressed in nM/min; “lagtime,” the length of time required before thrombin generation starts; “peak,” the highest thrombin concentration; “time to peak,” the length of time until peak; and “ start tail, ” the duration to end-point of thrombin generation. Platelet aggregometry was per- formed with a Multiplate analyzer (Verum Diagnostica GmbH, Munich, Germany) in a whole blood sample, as described by the manufacturer. Three platelet agonists specific to three pathways were tested: “PAR-4 test” (70 mmol/L, PAR-4 receptor, SIGMA, St. Louis, USA); “ADP test” (10 mmol/L, ADP receptor, Roche Diagnos- tics GmbH, Sandhofer Mannheim, Germany); and
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PubMedCentral-PMC5383133.pdf

PubMedCentral-PMC5383133.pdf

Globally different treatment protocols are used for treating haemophilia[5], where some regimens aim at obtaining a certain factor trough level, and others on tailoring the treatment according to individual bleeding phenotype. It is however difficult, to predict the exact dose of FVIII or FIX needed to stop or prevent bleeding episodes in individual patients. This makes optimizing haemophilia treatment based on individual needs challenging[6], and improved methodologies are needed to address these issues. Evidence is accumulating to support the hypothesis that the thrombin generation test (TGT) may reflect the bleeding risk of patients with coagulation disorders[7]. By utilizing the TGT researchers have reported correlations between TGT parameters and the clinical bleeding phenotype as well as response to treatment in human HA patients [8, 9].
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Impact of Thrombomodulin and Thrombin-Activatable Fibrinolysis Inhibitor on the Anticoagulant and Profibrinolytic Effects of Rivaroxaban

Impact of Thrombomodulin and Thrombin-Activatable Fibrinolysis Inhibitor on the Anticoagulant and Profibrinolytic Effects of Rivaroxaban

of TAFIa at 0 o C supports this notion, as this movement would be minimized at such a low temperature. Additionally, the stabilizing effects of the reversible inhibitors previously mentioned can be attributed to their ability to reduce the flexibility of the dynamic flap (234, 236-238). Several unnatural mutations within this area have been found to result in a stabilizing effect, thereby increasing the overall stability of TAFIa. Ceresa et al. (239) combined each of these known mutations and constructed TAFI Ser305Cys/Thr325Ile/Thr329Ile/His333Tyr/His335Gln (TAFI-CIIYQ). This combination of substitutions results in a 180-fold increase in stability, which is the greatest observed for any construct to date (239). After TAFIa has been thermally inactivated to TAFIai, proteolytic cleavage by thrombin can occur at Arg302 and cleavage by plasmin can occur at Arg302, Lys327 or Arg330 (240, 241). Moreover, at high concentrations of plasmin, TAFIa may be cleaved by plasmin at Arg302, Lys327 or Arg330 prior to any
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Implication of free fatty acids in thrombin generation and fibrinolysis in vascular inflammation in Zucker rats and evolution with aging

Implication of free fatty acids in thrombin generation and fibrinolysis in vascular inflammation in Zucker rats and evolution with aging

FIGURE 1 | Platelet aggregation, thrombin generation, and fibrinolysis in LZR and MSZR rats. (A) Mean maximum aggregation in washed platelets in response to collagen (5 µg/ml) and in (B) platelet-rich plasma (PRP) in response to ADP (5 µM), with the platelet count adjusted to 200 × 10 9 platelets/l. (C) Calibrated automated thrombinography (CAT) in rat plasma. Mean thrombin generation curves in platelet free plasma (PFP) triggered by 5 pM tissue factor in LZR and MSZR at 25 and 80 weeks of age. (D) Endogenous thrombin potential (ETP) in PFP and PRP of 25 and 80 week-old LZR and MSZR, expressed as ratios of values for 25 week-old LZR. (E) Ultrastructure of fibrin fibers was visualized by scanning electron microscopy. Pictures were made at 10,000 × magnification. (F,G) Fiber thickness and fiber density of fibrin clot in LZR and MSZR. (H) ELISA results of PAI-1 measured in PFP (n = 17–19). (I) Representative curves of fibrinolytic tests in PFP in LZR and MSZR. (J,K) Half-lysis time and maximal lysis speed of fibrinolytic tests in LZR and MSZR. Results are mean ± standard error of the mean (n = 7–11). *p < 0.05 vs. LZR at the same age; # p
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Inhibition of endotoxin induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti tissue factor antibody in chimpanzees

Inhibition of endotoxin induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti tissue factor antibody in chimpanzees

reproducible and dose-dependent elevations in serum TNF and IL-6, as well as marked increases in thrombin generation in vivo as measured by immunoassays for prothrombin activation fragment F1 + 2, thrombin-antithrombin III complexes, and fibrinopeptide A. Activation of the fibrinolytic mechanism was monitored with assays for plasminogen

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Eosinophil cationic granule proteins impair thrombomodulin function  A potential mechanism for thromboembolism in hypereosinophilic heart disease

Eosinophil cationic granule proteins impair thrombomodulin function A potential mechanism for thromboembolism in hypereosinophilic heart disease

This complete abrogation by MBP of the ability of endothelial cells to support thrombin-dependent APC generation is not attributable to a direct cytotoxic effect of MBP on endothelial ce[r]

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Endogenous thrombin potential following hemostatic therapy with 4 factor prothrombin complex concentrate: a 7 day observational study of trauma patients

Endogenous thrombin potential following hemostatic therapy with 4 factor prothrombin complex concentrate: a 7 day observational study of trauma patients

Coagulation factor concentrates such as purified human fibrinogen concentrate and prothrombin complex concen- trate (PCC) are considered as potential alternatives to FFP [10,11]. These substances are immediately available and contain well-defined quantities of coagulation proteins. Coagulation management based on infusion of concen- trates under guidance from point-of-care coagulation monitoring (thrombelastography or thromboelastometry) has been proposed [12]. Fibrinogen concentrate is adminis- tered first line to correct low levels of fibrinogen, which are rapidly detectable by impaired fibrin-based clot formation (FIBTEM assay or functional fibrinogen assay) [13-16]. Among patients with adequate fibrinogen levels but persistent bleeding and prolonged initiation of coagula- tion (rapidly detectable by prolonged clotting time), PCC may be administered to increase thrombin generation (TG) [15]. However, there is little evidence to support PCC use in trauma-induced coagulopathy (TIC) [17,18].
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Studies on a family with combined functional deficiencies of vitamin K dependent coagulation factors

Studies on a family with combined functional deficiencies of vitamin K dependent coagulation factors

Two siblings with m ild hemorrhagic symptoms had combined functional deficiencies of vitamin K-dependent clotting factors. Prothrombin (0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) and Stuart factor (Factor X, 0.18-0.20 U/ml) were most severely affected. Antigenic amounts of affected coagulation factors were normal and normal generation of thrombin activity occurred in the patients' plasmas after treatment with nonophysiologic activators that do not require calcium for prothrombin activation. Hepatobilary disease, malabsorptive disorders, and plasma warfarin were not present. Both parents had normal levels of all coagulation factors. The patients' plasmas contained prothrombin that reacted both with antibody directed against des-gamma-carboxyprothrombin and native
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Thrombin use in surgery: an evidence-based review of its clinical use

Thrombin use in surgery: an evidence-based review of its clinical use

For any given surgical procedure, it is mainly dependent on surgeon preference which thrombin or combination prod- uct is used. In general, the stand-alone thrombin spray is used to assist in hemostasis of large, raw surface areas such as in the case of solid viscera injury and resection of retroperitoneal tumors. Gelatin/thrombin combinations are very helpful in achieving hemostasis in vascular and spinal procedures and the fibrin glues are most commonly used in reoperative cardiac procedures which are complicated by persistent and difficult to control mediastinal bleeding. The effectiveness of the gelatin/ thrombin combination to achieve hemostasis has been exten- sively studied and they have been found to be quite effective. The different fibrin glues have also been studied in numerous surgical settings including liver resections, 48 vascular PTFE
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Local hemostasis, immunothrombosis, and systemic disseminated intravascular coagulation in trauma and traumatic shock

Local hemostasis, immunothrombosis, and systemic disseminated intravascular coagulation in trauma and traumatic shock

A in trauma patients with DIC immediately after the arrival to the emergency department have been described [61]. In addition, higher levels of these molecular markers of thrombin generation at an early stage of trauma have been repeatedly confirmed [5-7,47,48,53]. Dunbar and Chandler demonstrated excessive non-wound-related thrombin generation in trauma patients with both DIC and ‘ ACOTS’ immediately after arrival to the hospital [63,64]. Their first study showed marked systemic throm- bin generation due to circulating procoagulants that initi- ate thrombin generation systemically as well as a reduced ability to localize hemostasis at the wound site due to the loss of antithrombin. Their second study found that tissue factor activity made up approximately 80% of the total procoagulant activity. Reports showing a signifi- cant correlation between tissue factor and the markers of thrombin generation and microparticle formation by activated platelets support these results [66,67]. Importantly, the term ‘ ACOTS’ in the first study was changed to ‘DIC’ in their second study [63,64].
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Test Pattern Generation Using Lfsr With Reseeding Scheme for Bist Designs

Test Pattern Generation Using Lfsr With Reseeding Scheme for Bist Designs

able to achieve high transition fault coverage using functional broadside tests based on A. The hardware used in this paper for generating the primary input sequence A consists of a reseeding scheme with linear- feedback shift-register (LFSR) as a random source [15], and of a small number of gates (at most six gates are needed for every one of the benchmark circuits considered). The gates are used for modifying the random sequence in order to avoid cases where the sequence takes the circuit into the same or similar reachable states repeatedly. This is referred to as repeated synchronization [13]. In addition, the on-chip test generation hardware consists of a single gate that is used for determining which tests based on will be applied to the circuit. The result is a simple and fixed hardware structure, which is tailored to a given circuit only through the following parameters.
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The control of thrombin generation in haemoglobinopathies and other haemolytic red cell disorders in children

The control of thrombin generation in haemoglobinopathies and other haemolytic red cell disorders in children

The polymerisation and gelation of HbS upon deoxygenation is believed to play a central role in microvascular occlusion. The rate and extent of this is dependent on a number of physiological factors such as the intracellular concentration and composition of Hb, the percentage of oxygen saturation, temperature, pH and 2,3-DPG. Occlusion is likely to occur whenever intra- and extracellular conditions promote the generation of sufficient numbers of rigid sickle cells with polymerised Hb capable of blocking capillaries and initially it was postulated that this occurred when the transit time through the microcirculation was delayed beyond the lag time required for initiation of HbS polymerisation. However it is now evident that some SSRBCs, especially ‘dense cells’ (see below), contain polymerised HbS even at arterial oxygen saturation thus reducing the lag time to gelation during microvascular transit (Eaton and Hofrichter, 1987; Noguchi and Schechter, 1981). There is also evidence that rapid deoxygenation can occur, resulting in the formation of extensive HbS polymers without classic morphological sickling but resulting in selective trapping of dense cells (Kaul et al. 1989). Situations that slow microvascular transit time such as altered vascular reactivity and increased adhesion of SSRBC to the endothelium may be as important as HbS gelation in modulating microvascular occlusion in vivo (see below) (Eaton and Hofrichter, 1987).
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Modelling of thrombin generation under flow in realistic left anterior descending geometries

Modelling of thrombin generation under flow in realistic left anterior descending geometries

For the simulations under flow we calculated the parameters of TGA: the lag 266 time Tlag, the time until thrombin reaches its maximum concentration ttP, the maximum 267 concentration of[r]

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A test generation for pairwise testing

A test generation for pairwise testing

Another approach to generating a pairwise test set is to use orthogonal arrays. The original method of orthogonal arrays requires that all parameters have the same number of values and that each pair of values be covered the same number of times [8]. The rst requirement can be relaxed by adding don't care values for missing values. But the use of don't care values creates extra tests [9]. The second requirement is considered unnecessary for software testing and also creates extra tests for pairwise testing [1].

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Automatic Test Packet Generation

Automatic Test Packet Generation

In ATPG, test packets are create algorithmically from the configuration files and FIB, with minimum number of packets required completing test. Test packets are provide into the network so that every rule is checked directly from the data plane. Since ATPG treats links just like normal forwarding rules, it’s full testing of every link in the network [2].

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Automatic Test Packet Generation

Automatic Test Packet Generation

Network managers today use primitive tools such as and. Our survey results indicate that they are eager For more sophisticated tools. Other fields of engineering indicate that these desires are not unreasonable: For example, both the ASIC and software design industries are buttressed by billion- Dollar tool businesses that supply techniques for both static (e.g., design rule) and dynamic (e.g., timing) verification. In fact, many months after we built and named our system, we discovered to our surprise that ATPG was a well-known acronym in hardware chip testing, where it stands for Automatic Test Pattern Generation [2]. We hope network ATPG will be equally useful for automated dynamic testing of production networks.
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Packages Automatic Test Generation

Packages Automatic Test Generation

Testing liveness of a network is a fundamental problem for ISPs and large data center operators. Sending probes between every pair of edge ports is neither exhaustive nor scalable . It suffices to find a minimal set of end-to-end packets that traverse each link. However, doing this requires a way of abstracting across device specific configuration files, generating headers and the links they reach, and finally determining a minimum set of test packets (Min-Set- Cover).

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Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies

Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies

In FVIII-deficient plasma, 5-, 12-, or 15-HETE-PL liposomes supported a significant dose-dependent increase in thrombin generation (Figure 1). For 12- or 15-HETE-PLs, thrombin generation was restored to levels that were higher than HETE-PL–free control values obtained using healthy plasma (Figure 1, H, I, K, and L). This demonstrates that the lipids can overcome a clinically relevant factor deficiency to promote hemoastasis. Also, it shows that the procoagulant action of HETE-PLs that we recently demonstrated in healthy plasma is at least partially dependent on FVIII (25). Although HETE-PLs enhanced thrombin generation in FVIII-deficient plasma, they were considerably more active in healthy plasma (seen as steeper slopes in concentration dose–response curves in Figure 1, G–L, for healthy vs. FVIII-deficient plasma). We note that different pooled plasma preparations contain different amounts of coagulation factors and inhibitors; thus, HETE-PE–free controls are not identical between separate experiments. Within single experiments, the same platelet-poor plasma (PPP) preparation was always used for controls and samples containing HETE-PLs. HETE-PL also increases thrombin generation in the absence of FIX (a model for hemophilia B) and FXI (Figure 2, A–D).
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