Thrombin generation was measured in PPP using Calibrated Automated Thrombography, as described,25 with modifications. Briefly, 40μL of PPP samples was incubated with 10μL of (a) thrombin calibrator; (b) phospholipid vesicles (4μM final concentration), (c) phospholipid vesicles and TF (1pM) (the standard assay condition), or (d) phospholipid vesicles, TF and thrombomodulin (20nM). Samples were incubated for 10 min at 37°C, and thrombin generation was initiated with the addition of 10μL of a mixture of calcium and fluorogenic thrombin substrate. Thrombin activity was measured using a Fluoroskan Ascent Microplate Reader (Thermo Scientific) and quantified using Thrombinoscope software (Diagnostica Stago).
Thrombinoscope software
Manufactured by Diagnostica Stago
Sourced in France
The Thrombinoscope software is a tool designed for the assessment of thrombin generation in plasma samples. It provides quantitative measurement of thrombin activity over time, allowing for the analysis of various parameters related to the coagulation process.
Automatically generated - may contain errors
Lab products found in correlation
Fluoroskan ascent microplate reader, by Thermo Fisher Scientific (1 mentions)
Fluoroskan ascent, by Thermo Fisher Scientific (1 mentions)
Round bottom microtiter plate, by Thermo Fisher Scientific (1 mentions)
Calibrated automated thrombogram, by Diagnostica Stago (1 mentions)
Fluorogenic substrate, by Diagnostica Stago (1 mentions)
Fluorogenic thrombin substrate, by Diagnostica Stago (1 mentions)
Ppp reagent, by Diagnostica Stago (1 mentions)
Fluoroskan ascent fluorometer, by Thermo Fisher Scientific (1 mentions)
10 protocols using thrombinoscope software
1
Calibrated Automated Thrombography for Thrombin Generation
2
Thrombin Generation Assay Protocol
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This was done according to the manufacturer’s instructions using reagents and procedures provided by Diagnostica Stago. Suspensions of cells (50,000/ml) or microparticles (suspended in an equivalent volume as the cells) were mixed with normal plasma and fluorescence development measured with a Fluoroskan Ascent (Thermo electron corporation) microplate fluorimeter and analyzed using Thrombinoscope™ software (Diagnostica Stago).
3
Calibrated Automated Thrombography for Thrombin Generation
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The thrombin generation assay was performed using the Calibrated Automated Thrombography (CAT, Diagnostica Stago, Asnières-sur-Seine, France) according to the manufacturer’s standard procedure. Briefly, 20 µL of starting reagent (containing tissue factor at final concentration of 5 pM) or thrombin calibrator was added to each test well of a 96-well round-bottom microtiter plate (Nunc, Denmark). 80 µL of PPP was added to the test wells and the microtiter plate was then warmed up at 37 °C for 5 min in the CAT instrument. The reaction is initiated when 20 µL of fluorescence substrate/CaCl2 mixture was dispensed into each well. During the 60 min of reaction time, the fluorescence was measured (excitation filter at 390 nm and emission filter at 460 nm) at 20-s intervals. Each assay was performed as triplicate and ETP was calculated by the thrombinoscope software (Diagnostica Stago, Asnières-sur-Seine, France). Numerical values and traces of thrombography were retained for result analysis. ETP represents the total amount of thrombin a plasma sample could generate under pro- and anti-coagulant factors operating simultaneously in the plasma [28 (link)].
4
Calibrated Automated Thrombogram Analysis
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TF‐TG was measured using a Calibrated Automated Thrombogram (Diagnostica Stago, Inc, Parsippany, NJ, USA) per the manufacturer’s recommendations using the PPP‐reagent (5 pM TF/4 µM phospholipids). TF‐TG profiles were analyzed using Thrombinoscope software (Diagnostica Stago) with five parameters: ETP, Peak, lag time, time‐to‐peak, and velocity index. The results were reported as mean ± standard deviation.
5
Thrombin Generation Assay Protocol
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A thrombin generation assay (TGA) was carried out at 37 °C by measuring the fluorescence intensity upon cleavage of the fluorogenic thrombin substrate, Z-Gly-Gly-Arg-AMC. Commercially available pooled normal platelet poor plasma (PNP, 30 donors) from George King Bio-Medical, USA was mixed 1:1 with HBS (20 mM HEPES with 100 mM NaCl at pH 7.4). Phosphatidylcholine (80): phosphatidylserine (20) (PCPS) liposomes were added to obtain a final concentration of 20 μM. Serial dilutions of MPI candidates and UHRA were prepared fresh for these experiments. Experiments were repeated twice with two technical replicates each.
Thrombin calibrator was added to each well following the manufacturer’s instructions and the thrombin generation assay was initiated by the addition of fluorogenic substrate (both from Diagnostica Stago). Substrate hydrolysis was monitored on a Thrombinograph™ plate reader from Diagnostica Stago. The fluorescence intensity was recorded at 37 °C every 30 s over a period of 1.5 h and analyzed using Thrombinoscope™ software from Diagnostica Stago.
Thrombin calibrator was added to each well following the manufacturer’s instructions and the thrombin generation assay was initiated by the addition of fluorogenic substrate (both from Diagnostica Stago). Substrate hydrolysis was monitored on a Thrombinograph™ plate reader from Diagnostica Stago. The fluorescence intensity was recorded at 37 °C every 30 s over a period of 1.5 h and analyzed using Thrombinoscope™ software from Diagnostica Stago.
6
Calibrated Automated Thrombography Assay
7
Comprehensive Hemostasis Evaluation
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Von Willebrand factor antigen (VWF:Ag) and activity (VWF:GPIbR) were measured by chemiluminescense on AcuStar (Werfen Instrumentation Laboratory, Bedford, USA) using corresponding HemosIL reagents. Thrombin generation (TG) was performed by calibrated automated thrombinography using a fluorometer (Fluoroskan Ascent; Thermolab, Massachusetts, USA) with Thrombinoscope software (Diagnostica Stago) with PPP reagent (5 pM tissue factor on PPP. Free tissue factor pathway inhibitor (TFPI) antigen was measured by an ELISA using Asserchrom Free TFPI (Diagnostica Stago) and performed according to the manufacturer’s recommendations.
8
Thrombin Generation Measurement Protocol
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Thrombin generation was measured in a Fluoroskan Ascent Fluorometer (Thermo Lab systems, Helsinki, Finland) as described by Hemker et al. [15 (link)]. Thrombin generation was stimulated by two concentrations of TF (1 and 5 pmol/L). Briefly, 80 µL plasma was mixed with 20 µL of reagents containing TF and phospholipid to a final concentration of 1 or 5 pmol/L TF. Then, 20 µL of a fluorogenic substrate mixed with CaCl2 was added. The thrombin generation amount was measured by using Thrombinoscope software (Diagnostica Stago, Asnieres, France). Four parameters including lag time, time-to-peak, peak thrombin, and endogenous thrombin potential (ETP) can be calculated from the thrombin generation curves (Fig. 1 ). Lag time is the starting point of thrombin generation, time-to-peak is the point to reach the peak thrombin height, peak thrombin is the maximum thrombin height, and ETP is the area under the thrombin generation curve.
9
Thrombin Generation Assay for Anticoagulant Pathway
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Thrombin generation was measured in PPP using the CAT assay (Diagnostica Stago, Asnière-sur-Seine, France), as described in more detail previously (17 (link)). Thrombin generation was measured after a 5 pM tissue factor and 4 µM phospholipid trigger was added in the presence and absence of thrombomodulin to examine the function of the anticoagulant activated protein C pathway. TG parameters lag time, time-to-peak, peak, ETP and velocity index were calculated using the dedicated Thrombinoscope software (Diagnostica Stago, Asnière-sur-Seine, France). The lag time is defined as the time point at which the burst of TG starts, which is defined as 1/6th of the peak height. The peak height represents the highest active thrombin concentration detectable. The time-to-peak is the time until the peak height is reached. The ETP is defined as the area under the curve and represents the total thrombin potential that a plasma sample can generate. The velocity index was calculated as peak height/(time-to-peak – lag time). The generated TG curves were used in thrombin dynamics analysis, as described below.
10
Thrombin Generation Assay Protocol
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Thrombin generation assay was performed using a Thrombinoscope (Thrombinoscope BV, Maastricht, The Netherlands). TF with a final concentration of 5 pmol/L (PPP Reagent High, Thrombinoscope BV) or 1 pmol/L (PPP Reagent Low, Thrombinoscope BV) was used to stimulate thrombin generation. A total of 20 µL of reagent containing TF and phospholipid was mixed with 80 μL of platelet-poor plasma. Then, 20 µL of fluorogenic substrate in buffer containing Hepes and calcium chloride (FluCa-Kit, Thrombinoscope BV) was added to the mixture. The fluorescent signals were detected in a Fluoroskan Ascent fluorometer (Thermo Labsystems OY, Helsinki, Finland). Finally, thrombin generation curves were analyzed using Thrombinoscope software (Diagnostica Stago, Asnières-sur-Seine, France). The endogenous thrombin potential (ETP), peak thrombin, lag time and time to peak thrombin were measured.
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