The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well

My research focuses on measuring secondary hemostasis and fibrinolysis together to measure overall hemostatic balance. Most current assays assess isolated components of coagulation or fibrinolysis. This assay measures both processes simultaneously in one assay.

To begin, turn on the fluorescence plate reader and allow it to equilibrate to 37 degrees Celsius. Configure the filter settings in the software by setting the Number of wavelength pairs to two. Set the excitation wavelength to 355 nanometers for thrombin and 485 nanometers for plasmin.

Then set the emission wavelength to 460 nanometers for thrombin and 525 nanometers for plasmin. Next, set the Plate Type to a 96 Well Greiner Flat Bottom Black Microplate. Navigate to Read Area and define the plate layout using a maximum of 20 wells per plate.

Configure the PMT and Optic settings by setting the PMT Gain to 200 volts and the number of Flashes per read to six. Then navigate to Timing to confirm the Timing Settings with a total runtime of one hour nine minutes and 30 seconds, an Interval of 30 seconds. Navigate to Shake to confirm a shake of three seconds before the first read.

Navigate to More Settings to ensure that the Read Order is set to Column. Configure the Compound Transfer before Read settings by setting the No.of Compound Transfers to one and Initial Volume to 120 microliters. Check to ensure that the Pipette Height is 130 microliters, Volume is 20 microliters Rate is one microliter per second, and Time Point is 14 seconds.

Under Compound Source, confirm the 96 Well Compound Plate format, and specifically choose the Beckman 96 2.3 milliliter option. Confirm the Pipette Tips Layout settings by selecting the correct columns. Now navigate to Compound Tips Columns and select the correct columns.

Set the microplate shaker temperature to 37 degrees Celsius and verify the temperature before use. Then set the shaking intensity to 1, 100 revolutions per minute. Next thaw patient plasma and normal pooled plasma in a 37 degrees Celsius water bath for up to 10 minutes.

Mix the plasma samples thoroughly and verify complete thawing with no visible cryoprecipitate. Using a pipette, add 80 microliters of patient plasma or normal pooled plasma to the preselected wells, using a fresh pipette tip for each sample. Then prepare the substrate mixture by mixing 48 microliters of cephalin with 48 microliters of tissue factor.

To this, add 240 microliters trisodium chloride buffer, then pipette 96 microliters thrombin substrate, and 48 microliters plasmin substrate. Pipette out 20 microliters of the substrate mixture into each well. Shake afterwards.

Now insert the microplate into the fluorescence plate reader and allow it to equilibrate at 37 degrees Celsius for several minutes. Combine trisodium chloride buffer, trisodium chloride calcium chloride buffer, and tissue plasminogen activator to prepare the starting reagent. Vortex thoroughly to mix.

Pipette the starting reagent mixture in all rows of the compound column. Insert the plate containing the starting reagent mixture to the source drawer and start the fluorescence measurement. Record fluorescence signals every 30 seconds for 70 minutes.

Maintain the plate at 37 degrees Celsius throughout the measurement. For data analysis, export the kinetic fluorescence data as a table containing time points and fluorescence values per well. Calculate the first derivative and plot the enzyme generation curves for each well to visualize thrombin or plasmin generation over time.

The hemostasis assay enabled simultaneous, time resolved measurement of thrombin and plasmin generation to provide an integrated assessment of coagulation and fibrinolysis. Several parameters can be derived from the thrombin generation curve, including lag time, peak height, and thrombin potential. Longer lag times, lower peak heights, and reduced thrombin potentials indicate impaired thrombin generation.

The plasmin generation curve also provides multiple parameters, including fibrin lysis time, and plasmin peak height. Shorter fibrin lysis times, and higher peak heights reflect enhanced plasma generation. Thrombin and plasma generation curves from three healthy controls and normal pooled plasma showed reproducible peak height end timing, serving as reference profiles for normal hemostatic function.

In a patient with Factor V activity of 3%thrombin generation was completely absent. However, plasma generation increased marginally. The patient with Factor V activity of 44%showed reduced thrombin generation with a prolonged lag time and decreased peak height while plasma generation remained within the normal range.

In Alpha-2-antiplasmin deficiency, plasma generation increased with decreasing Alpha-2-antiplasmin activity. The homozygous patient with 23%activity displayed an early onset of plasmin generation, a shorter fibrin lysis time, and a higher plasma peak. These alterations were also observed in the heterozygous patient with 73%activity, although they were not as pronounced.

The hemostasis assay is well suited for research into rare coagulation factor deficiencies, fibrinolytic disorders, and hemostatic drug effects. The main challenges are standardization of the technique and normalization of results to improve comparability across rooms and laboratories. Future work may focus on automating the assay and developing adaptations for compatibility with whole blood or plated-rich plasma.