The SpectraMax® i3x Multi-Mode Microplate Reader from Molecular Devices can be used to successfully perform the Technothrombin® thrombin generation assay (TGA).
Assessing thrombin generation in plasma samples enables an improved understanding of coagulation mechanisms and the abnormalities in these mechanisms linked to chronic diseases, such as hemophilia or thrombophilia.
Technothrombin TGA facilitates monitoring of the impact of coagulation-directed drugs designed for the treatment of hemophilia or thrombophilia. This enables evaluation of the thrombogenic activity of immunoglobulin concentrates.
Technoclone GmbH has developed a plate-reader-compatible assay format to accommodate researchers’ need to determine time-dependent changes in thrombin concentrations via a flexible platform.
The Technothrombin TGA assay works by monitoring the fluorescence generated by the fluorogenic substrate’s cleavage by thrombin over time.
Cleavage occurs as the coagulation cascade is activated by various triggers (tissue factor and phospholipids) at different concentrations. The nanomolar concentration of thrombin in the sample can be calculated from changes in fluorescence over time, thereby generating a thrombin calibration curve.
Monitoring thrombin concentration over time allows calculation of the amount of thrombin generated in the sample. Thrombin values versus time can also be plotted for the entire coagulation process, allowing the different phases of thrombin generation to be visualized.
This approach offers a range of benefits, including:
- Consistent results with standardized reagents
- Time savings by running a single calibration curve per substrate lot
- The ability to easily run assays with a fluorescence microplate reader
The Technothrombin TGA assay can be run with different coagulation cascade triggers. Choosing the correct trigger reagent is important because it enables the study of different coagulation mechanisms.
Triggers differ in their phospholipid and/or tissue factor composition, resulting in varying activating capacities that provide insight into distinct components of the coagulation system.
Table 1 shows the different TGA trigger reagents, along with the corresponding tissue factor and phospholipid concentrations, as well as the areas of investigation for which they may be employed.
Each of the three triggers was tested on the SpectraMax i3x reader, with the best performance observed when using a fluorescence detection cartridge. It is important to note that most of the data shown is for Technothrombin TGA RB Trigger, because this is a useful representative value for data processing and assay functionality.
Table 1. Characteristics of and uses for the different TGA trigger reagents. Source: Molecular Devices UK Ltd
| TGA Trigger reagent |
Tissue factor/phospholipid
concentration |
Areas of investigation |
| Technothrombin TGA RB |
Low/low |
- Detecting hypercoagulability and bleeding tendency
- Monitoring FVIII inhibitor bypass therapy with rFVIIa and FEIBA hF VII, hF Xa, hF XIa
|
| Technothrombin TGA RC Low |
High/low |
- Finding correlation of thrombin generation results with thrombotic events
- Measuring the thrombophilic capacity
- Monitoring the thrombogenicity of microparticles
|
| Technothrombin TGA RC High |
High/high |
- Monitoring the anticoagulant therapy with heparin, heparinoids, or direct oral anticoagulant (DOACS)
|
Materials
- SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices) with FI-COFL detection cartridge (Molecular Devices, P/N 0200-7002)
- Immuno Standard Modules, black MaxiSorp (Thermo Fisher Scientific, P/N 475515)
- Technothrombin TGA reagents from Technoclone GmbH:
- 5006010 Technothrombin TGA Kit, including three different triggers, a calibrator, and a control sample
Other individual reagents can also be ordered separately:
- 5006209 Technothrombin TGA RB 5 x 0.5 mL
- 5006210 Technothrombin TGA RB 50 x 0.5 mL
- 5006212 Technothrombin TGA RC Low 5 x 0.5 mL
- 5006213 Technothrombin TGA RC Low 50 x 0.5 mL
- 5006214 Technothrombin TGA RC High 5 x 0.5 mL
- 5006216 Technothrombin TGA RC High 50 x 0.5 mL
- 5006230 Technothrombin TGA SUB 50 x 1.5 mL
- 5006235 Technothrombin TGA SUB 5 x 1.5 mL
- 5006320 Technothrombin TGA Control high 5 x 1 mL
- 5006330 Technothrombin TGA Control low 5 x 1 mL
- 5006345 Technothrombin TGA CAL Set
Methods
Data acquisition
A SoftMax® Pro Software data acquisition protocol was configured using guidelines provided by Technoclone GmbH. A preconfigured protocol is also available on Molecular Devices’ Spectranet customer care portal.
Table 2 summarizes the instrument settings for the SpectraMax i3x reader.
The fluorescence intensity (FI) detection cartridge (coumarin-fluorescein) was used for this assay to ensure optimal sensitivity. The onboard monochromator-based fluorescence detection can also be used, however.
Other instruments suitable for this assay include SpectraMax iD3s, SpectraMax Mini, and SpectraMax iD5e readers.
The SpectraMax i3x reader’s temperature was set to 37 °C 20 minutes before the read. Two independent plates were prepared for this assay: the calibration curve plate and the sample plate. The thrombin calibration curve was initially prepared using calibrator concentrations ranging from 3.6 nM to 361 nM.
The batch-dependent concentration range from the thrombin calibration curve is indicated on the vial’s label. A total of 40 μL of each calibration dilution was pipetted into duplicate assay wells, immediately followed by 50 μL of the fluorogenic substrate.
The plate was then transferred to the plate reader before performing a kinetic read, and data was collected at 30-second intervals for 10 minutes.
A total of 40 µL of sample was initially added to the wells for the sample plate, followed by 10 µL of the corresponding trigger and 50 µL of the fluorogenic substrate. A kinetic read was performed, with data acquired at one-minute intervals for a total of one hour.
Data analysis
Raw data was exported from SoftMax Pro to Excel after being generated using the SpectraMax i3x reader. Data evaluation was performed in the Technothrombin TGA evaluation file (Figure 1), using an optimized algorithm to correct for the inner filter effect.
The raw data from the thrombin calibration curve measurement was used from 30 seconds onward. The same calibration curve can be employed in all further sample measurements using the same Technothrombin TGA substrate lot. The raw data acquired from the sample measurements was copied into the same evaluation file for analysis.
Table 2. Plate Reader settings for thrombin calibration curve and sample plate. Source: Molecular Devices UK Ltd
| Parameter |
SpectraMax i3x |
| Optical Configuration |
FI-COFL Cartridge |
| Read Mode |
Fluorescence |
| Read Type |
Kinetic |
| Wavelengths |
EX 360 nm (35 nm bandpass)
EM 465 nm (35 nm bandpass) |
| PMT and Optics |
Integration Time: 400 ms
Read from Top
Read Height: 0.8 mm* |
| Shake |
Before first read: five seconds, linear, medium |
* Read height should be optimized for each assay volume used.
Figure 1. Layout with all information used for sample measurement: reagent lots, calibrator, operator, instrument, and plate layout. Image Credit: Molecular Devices UK Ltd
Results
Data visualization was performed by plotting the raw data of the thrombin calibrators in the SoftMax Pro software (Figure 2). The Technothrombin TGA evaluation file was used for further data analysis.
The increase in the cleaved fluorogenic substrate’s signal is linked to the thrombin concentration. The signal was calculated as RFU/min for each calibrator and used to generate the calibration curve via the Technothrombin TGA evaluation file (Figure 3).
This calibration curve was used to convert the sample measurement from ∆RFU to thrombin concentration (nM).
Figure 4 features the raw kinetic profiles of the sample measurement in the SoftMax Pro software. This data was transferred to the Technothrombin TGA evaluation file for analysis and plotting of a thrombin generation curve.
An optimized algorithm was initially used to calculate the first derivative of sample raw data. The calibration curve was then used to convert the ∆RFU of sample measurement to thrombin concentration (nM).
The thrombin generation curve (Figure 5) was used to calculate all thrombin generation parameters, including nM peak thrombin, lag time, time to peak, velocity index, and area under the curve (AUC).
The TGA curve generally highlights the variation of the thrombin concentration during the activation of the coagulation cascade.
Figure 6 features the acquired thrombin generation curve for samples activated with the Technothrombin TGA RB trigger. The thrombogenic potential of each sample is reflected by the thrombin generation parameters and the thrombin generation curves.
Table 3 shows results from a sample from a patient with thrombophilia, a sample from a patient with hemophilia A, and a normal plasma sample.
It was noted that parameters such as AUC, peak of thrombin, and velocity index were increased in the thrombophilia patient sample in comparison with those of the normal sample, indicating its high thrombogenic potential.
The same parameters of the hemophilia A patient sample were found to be reduced in comparison to those of the normal sample, however, reflecting the clotting deficiency of these patient groups. This deficiency is caused by a missing or defective factor VIII.
A combination of standardized Technothrombin TGA reagents and a validated SoftMax Pro software protocol on a SpectraMax i3x reader enables a precise measurement with CVs <10% (Table 4 and Table 5). Technothrombin TGA RC low was selected as an example in this instance because this trigger is primarily used for thrombophilic patient samples.
Figure 2. Thrombin calibrators, raw data shown using SoftMax Pro software. Image Credit: Molecular Devices UK Ltd
Figure 3. Thrombin calibration curve plotted in the Technothrombin TGA evaluation file. Image Credit: Molecular Devices UK Ltd
Figure 4. Kinetic traces in SoftMax Pro software for samples triggered with Technothrombin TGA RB. Image Credit: Molecular Devices UK Ltd
Figure 5. TGA curve and phases of clot formation. Lag Phase: phase from the time point when the TGA trigger reagent, including CaCl2, is added until the first burst in thrombin formation. Peak Height: two different parameters are calculated. Peak Thrombin: maximal concentration of thrombin generated. Time to Peak: time at the highest concentration of generated thrombin (peak of Thrombin). Slope: steepest rate of thrombin formation per minute, calculated by the evaluation file as velocity index. AUC: area under the curve representing the endogenous thrombin potential. Image Credit: Molecular Devices UK Ltd
Figure 6. Transformed thrombin generation curves of a normal plasma sample, a sample from a thrombophilia patient, and a sample from a hemophilia A patient, all samples triggered with Technothrombin TGA RB. Curves were plotted using the Technothrombin TGA evaluation file. Image Credit: Molecular Devices UK Ltd
Table 3. Results of a normal plasma sample, a thrombophilia patient sample, and a hemophilia A patient sample; data were analyzed using the Technothrombin TGA evaluation file. Source: Molecular Devices UK Ltd
| Sample |
Trigger
Reagent |
Lag Phase |
Peak Height |
Slope
(Velocity-Index) |
AUC |
| Time [min] |
Thrombin
[nM] |
Time
[min] |
| Thrombophilia patient |
RB |
8 |
269.7 |
15 |
38.53 |
3331 |
| Normal patient |
RB |
14 |
172.6 |
25 |
15.69 |
2570 |
| Hemophilia A patient |
RB |
29 |
33.6 |
45 |
2.10 |
662 |
Table 4. Intra-assay %CVs for TGA parameters. Source: Molecular Devices UK Ltd
|
|
Lag phase |
Peak height |
AUC |
|
|
Time [min] |
Thrombin [nM] |
Time [min] |
Reagent
RC Low |
Target value |
7.3 |
311.4 |
14.2 |
3609 |
| Intra-assay CV % |
6.2 |
6.0 |
5.3 |
1.7 |
Table 5. Inter-assay %CVs for TGA parameters. Source: Molecular Devices UK Ltd
|
|
Lag phase |
Peak height |
AUC |
|
|
Time [min] |
Thrombin [nM] |
Time [min] |
Reagent
RC Low |
Target value |
7.3 |
274.6 |
14.3 |
3312 |
| Intra-assay CV % |
6.5 |
9.8 |
3.3 |
5.1 |
Conclusion
The robust combination of the SpectraMax i3x reader, SoftMax Pro software, and Technothrombin TGA evaluation file is ideally suited to performing thrombin generation assays with excellent precision when using the Technothrombin TGA reagents kit or modular TGA reagents as required.
This platform meets the needs of researchers in the fields of hemophilia, thrombophilia, anticoagulation, microparticle thrombogenicity, and related drug development thanks to its standardized reagents, validated reader settings, and tools for complete assay analysis.
Researchers can now perform accurate thrombin generation measurements that are critical to the success of their work.
Acknowledgments
Produced from materials originally authored by Lieselotte Wagner, PhD, and Richa Amiya, PhD, from Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, and Cathy Olsen, PhD, Cathleen Salomo, PhD, Teresa Castano Martinez, PhD, from Molecular Devices.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance bioanalytical measurement systems, software and consumables for life science research, pharmaceutical and biotherapeutic development. Included within a broad product portfolio are platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. These leading-edge products enable scientists to improve productivity and effectiveness, ultimately accelerating research and the discovery of new therapeutics. Molecular Devices is committed to the continual development of innovative solutions for life science applications. The company is headquartered in Silicon Valley, California, with offices around the globe. For more information, please visit www.moleculardevices.com.
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