Streamlining Thrombin Generation Analysis

This article outlines the successful application of the Technothrombin^® thrombin generation assay (TGA) using Molecular Devices’ SpectraMax^® i3x Multi-Mode Microplate Reader.

Evaluating thrombin generation in plasma samples enhances understanding of coagulation mechanisms and provides insight into how abnormalities in these mechanisms affect chronic diseases such as hemophilia or thrombophilia.

Technothrombin TGA can be used to monitor the effects of coagulation-directed drugs used to treat thrombophilia or hemophilia. This enables the evaluation of the thrombogenic activity of immunoglobulin concentrates.

Technoclone GmbH has developed a plate-reader-compatible assay format that meets researchers’ need for a flexible platform to determine time-dependent changes in thrombin concentrations. The Technothrombin TGA assay is designed to monitor the fluorescence generated by the cleavage of a fluorogenic substrate by thrombin over time.

Cleavage occurs upon activation of the coagulation cascade by various triggers (phospholipids and tissue factor) at varying concentrations. It is possible to calculate the sample’s nanomolar concentration of thrombin from the changes in fluorescence over time, resulting in a thrombin calibration curve.

The amount of thrombin generated in the sample can be calculated by monitoring the increase in thrombin concentration with time. It is also possible to plot thrombin values versus time for the whole coagulation process, offering a useful visualization of the various phases of thrombin generation.

This approach offers a number of advantages, including:

• Running only one calibration curve per substrate lot saves time.
• Standardized reagents ensure consistent results.
• Assays can be run easily with a fluorescence microplate reader.

The Technothrombin TGA assay can be run with various coagulation cascade triggers; it is important to select the appropriate trigger reagent to facilitate the study of different coagulation mechanisms.

These triggers differ in terms of their phospholipid and/or tissue factor composition, resulting in different activating capacities able to inform on a range of coagulation system components.

Table 1 details various TGA trigger reagents, alongside the corresponding tissue factor and phospholipid concentrations and the areas of investigation for which they can be employed.

All three triggers were tested on the SpectraMax i3x reader, with optimal performance observed when using a fluorescence detection cartridge. It should be noted that the majority of the data shown in this article is for the Technothrombin TGA RB trigger, because this is representative of assay functionality and data processing.

Table 1. Characteristics of and uses for the different TGA trigger reagents. Source: Molecular Devices UK Ltd

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

It is also possible to order a range of individual reagents 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

Guidelines provided by Technoclone GmbH were used to configure a SoftMax^® Pro Software data acquisition protocol. Molecular Devices also offers a preconfigured protocol via its customer care portal, Spectranet.

Table 2 summarizes the SpectraMax i3x reader’s instrument settings.

The fluorescence intensity (FI) (coumarin-fluorescein) detection cartridge was employed for this assay to ensure maximum sensitivity, but it is also possible to use the onboard monochromator-based fluorescence detection.

Other instruments suitable for this assay include the SpectraMax iD5e, SpectraMax Mini, and SpectraMax iD3s readers.

Twenty minutes prior to the read, the temperature of the SpectraMax i3x reader was set to 37 °C. Two independent plates were prepared to measure this assay: the sample plate and the calibration curve plate.

The thrombin calibration curve was first prepared using a range of concentrations from 3.6 nM to 361 nM. The curve’s concentration range is batch-dependent and indicated on the vial’s label. A total of 40 μL of each calibration dilution was pipetted into duplicate assay wells. Next, 50 μL of the fluorogenic substrate was immediately added, and the plate was transferred to the plate reader.

A kinetic read was then performed, with data collected at 30-second intervals for a total of 10 minutes.

For the sample plate, 40 µL of sample was added to the wells, followed by 10 µL of the corresponding trigger and 50 µL of the fluorogenic substrate. A kinetic read was performed next, with data collected at one-minute intervals for one hour.

Data Analysis

After generating data with the SpectraMax i3x reader, raw data was exported from SoftMax Pro to Excel. The Technothrombin TGA evaluation file was used to perform data evaluation (Figure 1), with an optimized algorithm employed in correcting for the inner filter effect.

Raw data collected from the thrombin calibration curve measurement was used from 30 seconds. It is possible to use the same calibration curve for all subsequent sample measurements from the same lot of Technothrombin TGA substrate.

Raw data collected 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

^* 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

The raw data of the thrombin calibrators was plotted in SoftMax Pro software (Figure 2) to facilitate data visualization. The Technothrombin TGA evaluation file was used in additional data analysis.

The increased signal of the cleaved fluorogenic substrate relates to thrombin concentration. The signal was calculated as RFU/min for each calibrator to generate the calibration curve using the Technothrombin TGA evaluation file (Figure 3).

This calibration curve was used to convert the sample measurement ∆RFU to thrombin concentration (nM).

Raw kinetic profiles of the sample measurement in the SoftMax Pro software are displayed in Figure 4. This data was transferred to the Technothrombin TGA evaluation file for analysis and to allow a thrombin generation curve to be plotted.

An optimized algorithm was employed to calculate the first derivative of the raw sample data. Next, the calibration curve was leveraged to convert ∆RFU of sample measurement to thrombin concentration (nM).

The thrombin generation curve (Figure 5) was used to calculate all thrombin generation parameters: velocity index, lag time, time to peak, area under the curve (AUC), and nM peak thrombin.

The TGA curve shows thrombin concentration variation during coagulation cascade activation.

The thrombin generation curve for samples activated with the Technothrombin TGA RB trigger is shown in Figure 6. Each sample’s thrombogenic potential is reflected by the thrombin generation parameters and the thrombin generation curves.

Table 3 features results from a normal plasma sample, a sample from a patient with hemophilia A, and a sample from a patient with thrombophilia.

Parameters such as peak thrombin, AUC, and velocity index were found to be higher in the thrombophilia patient sample versus the normal sample, highlighting its high thrombogenic potential.

However, the same parameters for the hemophilia A patient sample were lower than those of the normal sample. This reflects the clotting deficiency of these patient groups, caused by a defective or missing factor VIII.

The use of a validated SoftMax Pro software protocol on a SpectraMax i3x reader and standardized Technothrombin TGA reagents facilitates precise measurements with CVs <10% (Tables 4 and 5). Technothrombin TGA RC Low was chosen as an example in this case, with this trigger mainly employed when working with 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

Table 4. Intra-assay %CVs for TGA parameters. Source: Molecular Devices UK Ltd

Table 5. Inter-assay %CVs for TGA parameters. Source: Molecular Devices UK Ltd

Conclusion

Used together, the SpectraMax i3x reader, SoftMax Pro software, and Technothrombin TGA evaluation file are an ideal platform for conducting thrombin generation assays. This platform offers excellent precision when employing the Technothrombin TGA reagents kit or modular TGA reagents in line with user requirements.

This platform’s powerful combination of validated reader settings, standardized reagents, and tools for complete assay analysis meets the needs of researchers in the fields of thrombophilia, microparticle thrombogenicity, hemophilia, anticoagulation, and related drug development.

This is key to researchers’ success, offering the ability to perform accurate thrombin generation measurements.

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, and 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 life science technology. We make advanced scientific discovery possible for academia, pharma, and biotech customers with platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. From cancer to COVID-19, we've contributed to scientific breakthroughs described in over 230,000 peer-reviewed publications.

Over 160,000 of our innovative solutions are incorporated into laboratories worldwide, enabling scientists to improve productivity and effectiveness – ultimately accelerating research and the development of new therapeutics. Molecular Devices is headquartered in Silicon Valley, Calif., with best-in-class teams around the globe. Over 1,000 associates are guided by our diverse leadership team and female president that prioritize a culture of collaboration, engagement, diversity, and inclusion.

To learn more about how Molecular Devices helps fast-track scientific discovery, visit www.moleculardevices.com.

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