Thromboelastography and turbidity assays are two simple and distinctive methods for clot characterization. These techniques together offer a more comprehensive understanding of how clotting variables impact clot features. Both of this assays, not only offer endpoint clot analysis but also attract fibrin clot formation over time, unlike many other clot characterization tools These techniques can help develop a physiologically relevant synthetic clot platform that can provide reliable diagnosis of the fibrinolytic state of thrombosis patients To monitor clot turbidity over time, use any commercially available spectrometer that has an absorbance range of 350 to 700 nanometers.
Turn on the spectrometer and open the corresponding analysis software. Then select plate one and open the plate settings tab. Click ABS and kinetic to monitor a dynamic absorbance over time.
Select 550 nanometers in the wavelength tab, then switch to the timing tab and adjust the total runtime to 60 minutes with an interval of 30 seconds, select the wells of interest by highlighting them. Pipette 140 microliters of PBS into one well of a UV transparent 96 well plate. Then add 10 microliters of thrombin and mix, immediately initiate clotting by adding 50 microliters of fibrinogen to the well and pipette up and down five times using only the first stop of the pipette taking care to avoid creating bubbles.
Place the plate in the plate holder and click read in the software to start the turbidity reading. When the read has finished retrieve the turbidity data and obtain a turbidity tracing curve by plotting the absorbents change over time, derive maximum turbidity by taking the max absorbance value of the curve over time, then calculate 90%maximum turbidity by multiplying maximum turbidity by 0.9. Derive the time to maximum turbidity by computing the time from clot initiation to 90%maximum turbidity.
Turn on the thromboelastography or TEG analyzer, and wait for the temperature to stabilize at 37 degrees Celsius, then open the TEG software and create an experiment name under the ID section. Click the TEG tab and follow the onscreen prompts to conduct an eTest test for all channels. Then place the lever back to the load position once all checks are complete.
Click done in the information page and input sample information for channels that will be used. Place a clear uncoated TEG cup in its corresponding channel. Slide the carrier up to the top and press the cup bottom five times to a fix the pin to the torsion rod then lower the carrier and press the cup downward into the base until it clicks.
Pipette 20 microliters of thrombin solution into the TEG cup, then initiate clotting by adding 340 microliters of fibrinogen into the cup to obtain a 360 microliter clotting solution. Mix the contents of the cup by pipetting up and down five times. Slide the cup loaded carrier up move the lever to the read position and click start in the software to initiate the TEG reading.
Once the reading is completed retrieve the parameters and obtain a TEG tracing curve by plotting amplitude over time. Elect MA as maximum amplitude, which is indicative of clot strain and TMA as time to maximum amplitude from the software. Representative turbidity and a TEG tracing curves of human and bovine fibrin clots at different fibrinogen levels are shown here.
The tracing curves demonstrate that after a lag period following clot initiation, clot turbidity or clot amplitude increases over time and levels off at the end of clot formation. Maximum turbidity and time to maximum turbidity are the two parameters derived from turbidity, while maximum amplitude and time to maximum amplitude are derived from TEG. At a higher level of fibrinogen in the clotting solution all four values increase.
Maximum turbidity is an optical measure of clot structure, which is indicative of fibrin fiber thickness and fibrin network density. While maximum amplitude is a mechanical measure that reflects absolute clot strain, taken together the two values provide complimentary insight about the clot micro structures. This procedure demonstrates a simplified clotting model to examine variables that mainly affect fibrin polymerization.
This model can be customized by inclusion of additional clotting factors to study other parameters. The clotting model can be easily modified using clinical plasma samples to monitor clot formation and disillusion in the presence of antithrombotic drugs. Results could guide therapeutic management in patients.
