Technothrombin® Thrombin Generation Assay (TGA)
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Technothrombin® TGA is a thrombin generation assay (TGA) based on monitoring the formation of thrombin by means of a fluorogenic substrate upon activation of the coagulation cascade by tissue factor.
The use of Technothrombin® TGA for such diverse applications is made possible because Technothrombin® TGA allows to measure the whole kinetics of thrombin generation not only during the initiation phase of thrombin formation with the end point fibrin formation, but also during the phase of down regulation of thrombin formation and inactivation of the formed thrombin. Technothrombin® TGA is therefore a universal tool for analysis and research of the hemostatic system on an individual basis.
| Reagent | Size | Description |
| SUB | 3×1.5mL | TGA substrate (SUB) fluorogenic substrate 1 mM Z-G-G-RAMC, 15 mM CaCl2 |
| BUF | 1x3mL | TGA buffer (BUF) Hepes-NaCl-buffer containing 0.5 % bovine serum albumin |
| CAL | 1×0.5mL | TGA thrombin calibrator (CAL) ~1.000 nM thrombin in buffer with BSA |
| RC Low | 1×0.5mL | TGA reagent C (RC) Low RC Low conc. of phospholipid micelles containing rhTF in Tris-Hepes-NaCl buffer |
| RC High | 1×0.5mL | TGA reagent C (RC) High RC High conc. of phospholipid micelles containing rhTF in Tris-Hepes-NaCl buffer |
| RD | 1×1.5mL | TGA reagent D (RD) RD conc. of phospholipid micelles in TrisHepes-NaCl buffer |
| CH | 1x1mL | TGA control high (CH) human plasma with increased thrombin generation, lyophilized |
| CL | 1x1mL | TGA control low (CL) human plasma with decreased thrombin generation, lyophilized |
Each reagent is available separately.
Downloads
- Package Insert (PDF)
- Safety Data Sheet (PDF)
- Brochure (PDF)
- Technothrombin® TGA RA/MFC Package Insert (PDF)
- TGA Mouse Protocol (PDF)
- Thrombin Generation Triggers Presentation (PowerPoint)
- Ceveron® Alpha Brochure (PDF)
- Technothrombin® TGA on Ceveron (PDF)
- Technothrombin® TGA Brochure 2 (PDF)
- Immunoglobulin Testing Assays Flyer (PDF)
- "Count on Diapharma" Anticoagulation Measurement Flyer (PDF)
- Thrombin Generation Assays Comparison Sheet (PDF)
- Technoclone Haemophilia A Testing Brochure (PDF)
- Global Hemostasis and The Thrombin Generation Assay Slideshow (PowerPoint)
- We Deliver Solutions (PDF)
- Gene Therapy Research Flyer (PDF)
Images
Videos
Are the tissue factor (TF) and phospholipid (PL) compositions available?
Exact TF concentration is not available. The manufacturing process for TF/PLs is proprietary. A mixture of PL and TF is used, which form micelles of varying PL/TF concentrations and thrombin generation is then tested for release (not concentrations). Generally, TF is in the low pM range, and PLs consist of a mixture of phosphatidylcholine and phosphatidylserine.
Can I prepare my own controls?
Yes. However, Technoclone TGA controls are preselected for TGA use; care is taken to not induce microparticle formation. Using one’s own controls or another source might have differences in microparticles between plasmas.
Can thrombin generation of microparticles be determined?
Yes. This again will vary depending on the experimental goals as well as the source of microparticles. Trial and error may be needed in this situation. For example, cell-derived microparticles are not the same as plasma-derived microparticles. To measure thrombin generation from microparticles, isolation of the microparticles is required first, which can be completed by research protocols in the literature or using a Microparticle Filtration Unit such as the Ceveron® MFU 500.
Can Technothrombin® TGA measure thrombin generation of cells?
Yes. However, the experiment protocol will vary depending on the experimental goals, as thrombin generation depends on any trigger that initiates thrombin generation. In some cases, cells are used as the trigger in plasma and tested for their capacity to generate thrombin.
Can Technothrombin® TGA be used with other species samples besides human?
Yes. However, researchers develop their own protocols and often vary the initial plasma sample dilution. For example, the dilution factor may vary depending on the species, but some researchers have had success diluting mouse plasma 1:2 in TGA buffer and dog plasma 1:3. This requires validation in the researcher’s laboratory.
Where is the TGA evaluation software?
The manufacturer refers to the Microsoft Excel data analysis templates as evaluation software. These are templates created by Technoclone that can be used for data analysis with a number of fluoresecent readers. The excel templates are available at www.technoclone.com or you can request one at info@diapharma.com.
Do not analyze your data with standard Microsoft Excel.
What type of Fluorescent Reader do I need?
While the manufacturer currently recommends using the Ceveron® alpha TGA analyzer, alternative fluorescent readers can be used. Excel data evaluation templates have been created for the BioTek® FLx800™, Spectramax M5, BMG FLUOstar, Perkin Elmer Wallac Viktor, TECAN, and Thermo Fluoroskan.
Protein concentrations in plasma
| Component | Molecular Weight kDa | Plasma Concentration mg/l | Plasma Concentration μmol/l |
|---|---|---|---|
| Fibrinogen | 330 | 3000 | 9 |
| Prothrombin | 72 | 150 | 2 |
| Factor V | 330 | 20 | 0.05 |
| Factor VII | 50 | 0.5 | 0.01 |
| Factor VIII | 330 | 0.1 | 0.0003 |
| Factor IX | 56 | 5 | 0.09 |
| Factor X | 59 | 8 | 0.13 |
| Factor XI | 160 | 5 | 0.03 |
| Factor XII | 80 | 30 | 0.4 |
| Factor XIII | 320 | 10 | 0.03 |
| Protein C | 62 | 4 | 0.06 |
| Protein S | 70 | 10 (free) | 0.14 |
| Protein Z | 62 | 2 | 0.03 |
| Prekallikrein | 86 | 50 | 0.6 |
| HMW kininogen | 120 | 70 | 0.6 |
| Fibronectin | 450 | 300 | 0.7 |
| Plasminogen | 92 | 200 | 2 |
| t-PA | 60 | 0.005 | 0.0001 |
| Urokinase | 53 | 0.004 | 0.0001 |
| Antithrombin | 58 | 145 | 2.5 |
| Heparin Cofactor II | 66 | 80 | 1.2 |
| Plasmin Inhibitor | 63 | 60 | 1 |
| Protein C Inhibitor | 57 | 4 | 0.07 |
| α2-Macroglobulin | 725 | 2000 | 3 |
Measurement Principle
Technothrombin® TGA is based on monitoring the fluorescence generated by thrombin cleavage of a fluorogenic substrate over time upon activation of the coagulation cascade by different concentrations of tissue factor (RA, RB, RC Low and RC High) and negatively charged phospholipids in whole blood or plasma. From the changes in fluorescence over time, the concentration of thrombin (nM) in the sample can be calculated using the respective thrombin calibration curve. The rate of increase in thrombin concentration over time then allows to calculate generation of thrombin in the sample per minute and to plot this value over time for the whole coagulation process. This then results in the visualization of the different phases of clot formation.
Background
The Technothrombin® TGA kit is an assay system for determination of thrombin generation over time in platelet poor or platelet rich plasma (PPP or PRP) upon activation of the clotting cascade by micelles of negatively charged phospholipids containing different amounts of human tissue factor and CaCl2. The kit can be used to monitor hemophiliacs during inhibitor bypassing therapy, to monitor anticoagulation therapy, to calculate INR values for patients and to determine states of bleeding disorders or thrombophilia as well as the activity of circulating micro particles. This broad range of applications is possible by providing different tissue factor concentrations and by monitoring the whole kinetic of thrombin generation during initiation, amplification and down regulation of thrombin formation. Technothrombin® TGA is therefore a universal assay kit for analyzing and researching the function of the hemostatic system.
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