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.
- Substrate: 3 x 1.5 TGA substrate (SUB) Fluorogenic substrate 1 mM Z-G-G-RAMC, 15 mM CaCl2
- Buffer: 1 x 3 TGA buffer (BUF) Hepes-NaCl-buffer containing 0.5 % bovine serum albumin
- Calibrator: 1 x 0.5 TGA thrombin calibrator ~1.000 nM thrombin in buffer with BSA
- Reagent C Low: 1 x 0.5 TGA reagent C (RC) Low RC Low conc. of phospholipid micelles containing rhTF in Tris-Hepes-NaCl buffer
- Reagent C High: 1 x 0.5 TGA reagent C (RC) High RC High conc. of phospholipid micelles containing rhTF in Tris-Hepes-NaCl buffer
- Reagent D: 1 x 1.5 TGA reagent D (RD) RD conc. of phospholipid micelles in TrisHepes-NaCl buffer
- Control High: 1 x 1 TGA control high (CH) Human plasma with increased thrombin generation, lyophilized.
- Control Low: 1 x 1 TGA control low (CL) Human plasma with decreased thrombin generation, lyophilized.
Each reagent is available separately.
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.
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.
|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.|
|If using a BioTek® fluorescent reader, can I keep the machine on all day?|
|Unfortunately, no, Technoclone and BioTek® recommend running only 2 consecutive runs and to switch the reader off for ~2-3 hours before starting the next run. The intensity of the lamp begins to diminish after too long. In addition, the bulb should be changed once a year (lifespan is ~500 hours anytime the instrument is on).|
|I received a BioTek® “Run-time error ‘1004’ Cannot insert object.” Now what?|
|This can occur with the Gen5 software associated with the BioTek® reader. Some newer versions of Microsoft Excel may be incompatible with older versions of Gen5 Software. Gen5 v1.11 or higher is needed. There is a cost associated with an upgrade to version 2.0, and BioTek® Customer Service must be contacted for this software upgrade. However, Gen5 v1.11 will work and can be downloaded here: https://customer.biotek.com/login/|
(Your Gen5 Software serial number will be needed).
|I received another BioTek® error when trying to power export. What should I do?|
|While some issues may require your IT support, in some situations simply saving the data, and then restarting the computer, opening the data, and selecting power export again will fix the error (eg, this may resolve errors related to ‘cannot find path’). In addition, be sure that screen savers are turned off as this can interrupt the experiment.|
|I receive an error after BioTek® power export, what happened?|
|It’s likely that the data was not correctly copied into the excel evaluation template (Note: Technoclone.com even provides document instructions on this). Cells must be copied to corresponding cell for data analysis of the calibration curve or samples/controls within the ‘Raw Data’ excel sheet tab. Raw data can then be analyzed with Excel protected formulas.|
|The BioTek® instrument reading is completed, how do I analyze my data?|
|You need to select ‘Power Export’. This will automatically open the data in an Excel file. However, as noted above, do not analyze data using standard Microsoft Excel. Your data must be copied and pasted into the Excel Evaluation Template/Software that corresponds to the instrument used.|
|I have created the protocols for the calibration curve and samples/controls, now what?|
|Prepare for running the experiment by opening the fluorescent reader software, creating a new experiment, and selecting the appropriate protocol. For some instruments, pre-heating should begin right away. To run the actual experiment, follow the short instructions provided with the Technothrombin® TGA for the calibration curve and samples/protocol. At some point, the plate layout within the Excel Evaluation Software/Template should be completed in order to help with subsequent data analysis.|
Note: Only one calibration curve has to be done for each lot of substrate.
|How do I set-up the protocol on the BioTek® instrument?|
|Protocol set-up on the instrument software only needs to be completed once. Two protocols are required: one for the calibration curve and one for running the samples and controls. Instrument read settings are critical! Technoclone maintains instrument application documents for the available instruments. The instrument application documents contain screenshots to help with set-up but the overall basic parameters settings will remain similar if the instrument software is ever updated.|
|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 firstname.lastname@example.org.|
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|