Name: The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well (Video) | JoVE Journal
Uploaded: 2026-02-27T13:15:00.000+00:00
Duration: 8 min 1 s
Description: Learn the Nijmegen Hemostasis Assay for simultaneous fluorogenic measurement of thrombin and plasmin generation in plasma. This integrated method enables comprehensive assessment of coagulation and fibrinolysis balance.

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5 minutes

This study was conducted in accordance with the objectives of EuroBloodNet.

All procedures involving human plasma samples were conducted in accordance with institutional guidelines and approved by the medical ethics committee of Arnhem-Nijmegen. Written informed consent was obtained from all donors prior to sample collection.
1. Preparation of buffers, reagents and plasma samples
<ol>
	<li>Prepare buffer solutions
	<ol>
		<li>Weigh 1.2 g Tris (p.a.) and dissolve in 180 mL of 0.9% NaCl.</li>
		<li>Adjust the pH to 7.4 using HCl.</li>
		<li>Bring the final volume to 200 mL with 0.9% NaCl to obtain 50 mM Tris-150 mM NaCl buffer (TBS).</li>
		<li>Aliquot the TBS into two 50 mL tubes.</li>
		<li>Use the remaining 100 mL TBS to prepare the calcium-containing buffer. Dissolve 7.34 g CaCl2·2H2O in 100 mL of TBS to obtain Tris-NaCl-CaCl2 buffer (TBS-Ca, pH 7.4).</li>
		<li>Aliquot the TBS-Ca into two 50 mL tubes.</li>
		<li>Store both buffers at 4 °C and use within one week.</li>
	</ol>
	</li>
	<li>Prepare fluorogenic substrates
	<ol>
		<li>Dissolve 53.2 mg of the thrombin substrate (Bz-beta-Ala-Gly-Arg-AMC) in 4 mL of Tris-NaCl buffer. 
		NOTE: The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human thrombin in 120 µL reagent yields a final concentration of 76 nmol/L.</li>
		<li>Aliquot 100 µL in Eppendorf tubes and store at −80 °C.</li>
		<li>Thaw an aliquot of thrombin-specific fluorogenic substrate for 10 min at room temperature before use. Vortex and keep on ice during the experiment.</li>
		<li>Dissolve the contents of one vial of plasmin substrate (CBZ-L-Phe-Arg-rhodamine) in 1,955 µL of DMSO. 
		NOTE: The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 µL of added human plasmin in 120 µL reagent yields a final concentration of 200 nmol/L.</li>
		<li>Aliquot 60 µL in Eppendorf tubes and store at −80 °C.</li>
		<li>Thaw an aliquot of plasmin-specific fluorogenic substrate for 10 min at room temperature before use. Vortex and keep at room temperature during the experiment. 
		NOTE This substrate is dissolved in dimethylsulfoxide (DMSO) and must not be cooled to avoid precipitation.</li>
	</ol>
	</li>
	<li>Prepare trigger reagents
	<ol>
		<li>Reconstitute the lyophilized cephalin by adding 1 mL of distilled water. Vortex thoroughly until fully dissolved. 
		NOTE: Cephalin is reconstituted to provide phospholipids, which are essential to catalyze multiple coagulation reactions enabling physiologically relevant coagulation in the assay.</li>
		<li>Aliquot 60 µL in Eppendorf tubes (sufficient for 20 wells) and store at −20 °C.</li>
		<li>Thaw an aliquot before use and keep it on ice. Vortex briefly before pipetting.</li>
		<li>Reconstitute the contents of one vial of recombinant human tissue factor (TF) in 4 mL of distilled water.</li>
		<li>Aliquot 60 µL in Eppendorf tubes and store at −80 °C.</li>
		<li>Thaw an aliquot of TF and vortex briefly.</li>
		<li>Prepare a 1:500 dilution of TF by sequential dilution:
		<ol>
			<li>Add 6 µL of TF to 54 µL of TBS buffer. Vortex.</li>
			<li>Take 6 µL of this dilution and add to 54 µL of TBS. Vortex.</li>
			<li>Take 20 µL of this second dilution and add to 80 µL of TBS to reach a final concentration of 0.28 pM. Vortex.</li>
		</ol>
		</li>
		<li>Reconstitute the contents of one vial (20 mg) of tissue-type plasminogen activator (tPA) in 20 mL of distilled water to obtain a final concentration of 580,000 IU/mL (verify batch-specific concentration).</li>
		<li>Aliquot 50 µL in Eppendorf tubes and store at −80 °C.</li>
		<li>Thaw an aliquot of tPA and vortex briefly.</li>
		<li>Prepare a 1:50 dilution of tPA by adding 20 µL of tPA to 980 µL of Tris-NaCl buffer, reaching a final concentration of 11,600 IU/mL. Vortex.</li>
	</ol>
	</li>
	<li>Prepare plasma samples
	<ol>
		<li>Collect venous blood into 3 mL citrate tubes containing 3.2% citrate, ensuring at least 2.7 mL (90% fill) is collected.</li>
		<li>Do not use the first tube after venipuncture; discard it to avoid activation by tissue factor.</li>
		<li>Invert the tube at least 10 times immediately after collection.</li>
		<li>Centrifuge within 2 h at room temperature, first at 2,500 × g for 5 min, followed by 10,000 × g for 10 min.</li>
		<li>Use a disposable pipette to collect the plasma just above the cell layer.</li>
		<li>Aliquot plasma into labeled cryovials, snap-freeze in liquid nitrogen, and store at −80 °C. 
		NOTE: Ensure the sample is free of clots. Use only fresh or once-thawed plasma. One CTAD tube is sufficient for duplicate analysis.</li>
	</ol>
	</li>
</ol>
2. Instrument preparation and software setup
<ol>
	<li>Prepare the fluorescence plate reader
	<ol>
		<li>Turn on the plate reader and allow it to equilibrate to 37 °C.</li>
		<li>Set the following basic parameters:
		<ol>
			<li>Greiner 96-well, flat-bottom, black.</li>
			<li>General settings: Positioning delay: 0.1 s; Number of kinetic windows: 1.</li>
			<li>Kinetic window: Number of cycles: 140; Measurement start time: 0.0 s; Number of flashes per well per cycle: 10; Cycle time: 30 s.</li>
			<li>Optic: Top optic</li>
			<li>Filter settings: Number of monochromatics: 2. Excitation filter: 355 nm for thrombin; 485 nm for plasmin. Emission filter: 460 nm for thrombin, 520 nm for plasmin. Gain: 600.</li>
		</ol>
		</li>
		<li>Define the layout with a maximum of 20 wells per plate.</li>
		<li>Navigate to Concentrations/Volumes/Shaking and confirm the following settings. Pump 1 = 20 µL; Pump 2 = 0 µL; Speed = 420 µL/s.</li>
	</ol>
	</li>
	<li>Prepare the injection system
	<ol>
		<li>Remove and rinse the injection needles in a container with buffer.</li>
		<li>Set the rinse volume to 2,500 µL and rinse the pumps with 37 °C Tris/NaCl buffer.</li>
		<li>Reset the rinse volume to 500 µL.</li>
	</ol>
	</li>
</ol>
3. Assay setup in microplate format
<ol>
	<li>Microplate shaker setup
	<ol>
		<li>Set the microplate shaker to 37 °C and verify the temperature before use.</li>
		<li>Set the shaking intensity to 1100 rpm.</li>
		<li>Use this shaker for all mixing steps in the assay to ensure homogeneous reagent distribution and consistent enzymatic activity.</li>
	</ol>
	</li>
	<li>Add reagents to the wells
	<ol>
		<li>Thaw patient plasma and normal pooled plasma (NPP) in a 37 °C water bath for up to 10 min.</li>
		<li>Mix thoroughly and verify complete thawing (no cryoprecipitate).</li>
		<li>Add 80 µL of patient plasma or NPP to the preselected wells. Use a fresh pipette tip for each sample. 
		NOTE: Include a NPP sample in every assay run to account for between-run variability (see further in step 5.6).</li>
		<li>Add 2 µL of cephalin.</li>
		<li>Add 2 µL of tissue factor (1:500 dilution). Mix briefly using a plate shaker.</li>
		<li>Add 10 µL of Tris-NaCl buffer. Mix briefly using a plate shaker.</li>
		<li>Add 4 µL of thrombin substrate.</li>
		<li>Add 2 µL of plasmin substrate. Mix briefly using a plate shaker.</li>
	</ol>
	</li>
	<li>Pre-incubation
	<ol>
		<li>Insert the microplate into the plate reader.</li>
		<li>Allow the plate to equilibrate at 37 °C for several min.</li>
	</ol>
	</li>
</ol>
4. Preparation and injection of the starting reagent
<ol>
	<li>Prepare the reagent for Pump 1
	<ol>
		<li>Combine 672 µL of Tris/NaCl buffer, 192 µL of Tris/NaCl/CaCl₂ buffer, and 96 µL of tPA (from step 1.2.5). Vortex thoroughly.</li>
		<li>Pierce two holes in the cap of the reagent tube.</li>
		<li>Insert the pump tubing through one hole to the bottom of the tube.</li>
	</ol>
	</li>
	<li>Prime and start the assay
	<ol>
		<li>Use the software to prime the pump with 500 µL of the reagent.</li>
		<li>Reinsert the injection needles into their designated positions.</li>
		<li>Immediately start the fluorescence measurement.</li>
	</ol>
	</li>
	<li>Manual initiation of the assay 
	NOTE: This manual initiation procedure provides an alternative to the automated injection workflow described in steps 2.2, 4.1, and 4.2.
	<ol>
		<li>Prepare the starting reagent as described in 4.1.1 immediately before use.</li>
		<li>Remove the microplate from the plate reader and quickly add 20 µL of the starting reagent manually to each well using a calibrated pipette or multichannel pipette for faster handling.</li>
		<li>Immediately return the plate to the reader and start the fluorescence measurement without delay. 
		NOTE: Minimize the time between reagent addition and measurement start (&lt;10 s per row) to reduce variability.</li>
	</ol>
	</li>
	<li>Kinetic readout
	<ol>
		<li>Record fluorescence every 30 s for 70 min.</li>
		<li>Maintain the plate at 37 °C during measurement.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Prepare the data for analysis
	<ol>
		<li>Export the kinetic fluorescence data from the plate reader as a table with timepoints and fluorescence values per well.</li>
		<li>Convert the raw fluorescence data into enzyme generation curves by calculating the first derivative using Excel or another program.</li>
		<li>Plot the enzyme generation curves for each well to visualize the enzymatic reaction. 
		NOTE: These curves represent the rate of thrombin or plasmin generation over time and correspond to the graphical output shown in the figures.</li>
	</ol>
	</li>
	<li>Analyze thrombin generation curves
	<ol>
		<li>Identify the following parameters from the thrombin generation curve (see Figure 1):
		<ol>
			<li>Lag time: The time point where the thrombin generation curve rises consistently and reaches 8% of the thrombin peak height.</li>
			<li>Thrombin peak height: The maximum signal value on the y-axis (i.e., maximal thrombin generation rate).</li>
			<li>Thrombin Peak time: The time from the assay start to the thrombin peak height.</li>
			<li>Thrombin potential (area under the thrombin generation curve): The area under the curve from lag time until the thrombin signal returns to baseline. 
			NOTE: The thrombin potential reflects the total thrombin-generating capacity of the sample under assay conditions.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analyze plasmin generation curves
	<ol>
		<li>Identify the following parameters from the plasmin generation curve (see Figure 2):
		<ol>
			<li>Fibrin lysis time: The time from onset of plasmin generation (i.e., the time point where the plasmin generation curve rises consistently and reaches 8% of the plasmin peak height) to the point at which the plasmin peak is reached.</li>
			<li>Plasmin peak height: The first distinct maximum in the curve, corresponding to the initial burst in plasmin generation, is determined at the point where the second derivative of the plasmin generation curve (based on a three-minute moving average) reaches zero after the onset of plasmin generation.</li>
			<li>Plasmin potential (area under the plasmin generation curve): The area under the curve from the onset of plasmin generation to the time of the plasmin peak. 
			NOTE: Signal fluctuations do occur after the initial plasmin peak, and are not included in the analysis of the plasmin generation parameters.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extract parameters manually or using custom software
	<ol>
		<li>Use Microsoft Excel, GraphPad Prism, or a custom script (e.g., in Python or R) to extract kinetic parameters.</li>
		<li>Save the raw data, plotted curves, and calculated parameters in a structured format for archiving and future reference. 
		NOTE: If using macros, verify the correctness of all parameters visually and manually before relying on automated output. Our macro for data conversion is available upon request.</li>
	</ol>
	</li>
	<li>Standardization and normalization
	<ol>
		<li>Establish a physiological reference by measuring 40 individual samples from healthy donors in parallel with the NPP sample used for normalization. Perform this step once per NPP batch.</li>
		<li>Calculate the mean thrombin and plasmin generation parameter values of the healthy donors, and determine their ratio to the corresponding NPP values obtained in the same run.</li>
		<li>For each patient sample in a given run, first normalize the thrombin or plasmin generation parameter to the NPP value measured in that same run.</li>
		<li>Divide this result by the healthy donor-to-NPP ratio previously established for the same NPP batch (see step 5.6.2). This yields a relative value normalized to the average healthy individual, with standardization for inter-run variation using NPP.</li>
		<li>Express parameter values as percentages relative to the average value of healthy individuals (defined as 100%). Present patient sample results accordingly. 
		NOTE: The relative parameter values for each individual patient sample are calculated using the following formula, where run a refers to the measurement run of the patient sample, and run b refers to the reference run in which healthy donor samples were measured in parallel with the same NPP batch. Our Excel file containing the calculations for data normalization is available upon request. 
		<img alt="Equation for relative value calculation; fraction for patient sample analysis; mathematical formula." src="/files/ftp_upload/69701/69701eq1.jpg" style="margin: auto;" data-altupdatedat="1778558422000" data-alt="Equation 1"></li>
	</ol>
	</li>
</ol>
6. Generation of calibration curves
<ol>
	<li>Prepare the thrombin calibration curve
	<ol>
		<li>Reconstitute the lyophilized human α-thrombin as instructed in the manufacturer's datasheet. 
		NOTE: For example, if the datasheet specifies reconstitution in 100 µL water to yield 3.34 mg/mL, this corresponds to 0.334 mg in 100 µL.</li>
		<li>Dilute the reconstituted thrombin 1:10 in Tris/NaCl buffer. 
		NOTE: This yields a stock concentration of approximately 9.1 nmol/mL (0.0091 nmol/µL), based on a molecular weight of 36,700 g/mol.</li>
		<li>Prepare a dilution series in Tris/NaCl buffer to achieve final thrombin concentrations in the assay ranging from 0 to 152 nmol/L.</li>
		<li>Pipette thrombin into selected wells already containing buffer to reach a total volume of 100 µL per well.</li>
		<li>Prepare the substrate solution for Pump 1 by mixing 300 µL of Tris/NaCl buffer with 200 µL of thrombin substrate from step 1.2.2.</li>
		<li>Further dilute this solution by combining 400 µL of the mixture with 360 µL of Tris/NaCl buffer.</li>
		<li>Add 20 µL of this substrate mix to each calibration well, bringing the total assay volume to 120 µL.</li>
		<li>Gently mix the plate and insert it into the plate reader. Run the kinetic assay for 25 cycles, using the same fluorescence settings described in section 2.</li>
		<li>Export the raw fluorescence data and determine the initial slope (rate of fluorescence increase per cycle) for each thrombin concentration using GraphPad Prism or equivalent software.</li>
		<li>Generate a standard curve by plotting the initial slope versus thrombin concentration. 
		NOTE: Ensure that all buffers are preheated to 37 °C and handle enzymes on ice. Always prepare fresh dilutions and mix thoroughly. The resulting standard curve is used to calculate the relative thrombin activity in unknown samples using the macro described in section 5.</li>
	</ol>
	</li>
	<li>Preparation of the plasmin calibration curve
	<ol>
		<li>Reconstitute lyophilized plasmin in distilled water (e.g., 0.543 mL) according to the manufacturer's instructions. 
		NOTE: Plasmin has a molecular weight of 75,400 g/mol. The resulting stock concentration is approximately 24.3 nmol/mL (0.024 nmol/µL), assuming 1 mg in 0.543 mL.</li>
		<li>Prepare a dilution series in Tris/NaCl buffer to achieve final plasmin concentrations in the assay ranging from 66 to 532 nmol/L.</li>
		<li>Pipette plasmin into wells containing Tris/NaCl buffer to reach a final volume of 100 µL per well.</li>
		<li>Prepare the substrate solution for Pump 1 by mixing 680 µL of Tris/NaCl buffer with 80 µL of plasmin/rhodamine substrate (pre-diluted 1:1 if required) from step 1.2.5.</li>
		<li>Add 20 µL of this substrate solution to each calibration well, bringing the total volume to 120 µL.</li>
		<li>Gently mix the plate and insert it into the plate reader. Run the kinetic assay for 25 cycles using the same fluorescence settings as described in Section 2.</li>
		<li>Export the raw fluorescence data and determine the initial slope of the fluorescence increase for each plasmin concentration by applying linear regression to the linear phase of the kinetic curve using GraphPad Prism or equivalent software.</li>
		<li>Generate a standard curve by plotting the initial slope versus plasmin concentration. 
		NOTE: Due to batch-to-batch variation in plasmin activity, calibration should be repeated with each new lot. If unexpectedly high activity is observed, a 1:2 dilution of the stock is recommended before preparing the calibration series.</li>
	</ol>
	</li>
	<li>Use the calibration curve for quantification
	<ol>
		<li>Use the calibration curves generated in Section 6 to convert fluorescence slope values into relative enzyme activity.</li>
		<li>Use only the linear portion of the calibration curve for interpolating unknown samples.</li>
	</ol>
	</li>
</ol>

<ol><li>Chapin, J. C., Hajjar, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinolysis+and+the+control+of+blood+coagulation%5BTitle%5D)+AND+%22Blood+Rev%22%5BJournal%5D)">Fibrinolysis and the control of blood coagulation.</a> Blood Rev. 29 (1), 17-24 (2015).</li>
<li>Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+thrombosis%5BTitle%5D)+AND+%22Hematology%22%5BJournal%5D)">Pathogenesis of thrombosis.</a> Hematology. 2009 (1), 255-258 (2009).</li>
<li>Teruya, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+normal+aPTT+does+not+guarantee+adequate+coagulation+factor+levels%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">A normal aPTT does not guarantee adequate coagulation factor levels.</a> Anesthesiology. 94 (3), 542(2001).</li>
<li>Puetz, J., Hugge, C., Moser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+aPTT+in+children+with+mild+factor+XI+deficiency%5BTitle%5D)+AND+%22Pediatr+Blood+Cancer%22%5BJournal%5D)">Normal aPTT in children with mild factor XI deficiency.</a> Pediatr Blood Cancer. 65 (4), pbc.26910(2018).</li>
<li>Toulon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+sensitivity+of+different+activated+partial+thromboplastin+time+reagents+to+mild+clotting+factor+deficiencies%5BTitle%5D)+AND+%22Int+J+Lab+Hematol%22%5BJournal%5D)">In vitro sensitivity of different activated partial thromboplastin time reagents to mild clotting factor deficiencies.</a> Int J Lab Hematol. 38 (4), 389-396 (2016).</li>
<li>Massignon, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prothrombin+time+sensitivity+and+specificity+to+mild+clotting+factor+deficiencies+of+the+extrinsic+pathway%3Aevaluation+of+eight+commercial+thromboplastins%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Prothrombin time sensitivity and specificity to mild clotting factor deficiencies of the extrinsic pathway:evaluation of eight commercial thromboplastins.</a> Thromb Haemost. 75 (4), 590-594 (1996).</li>
<li>Burns, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paradoxic+effect+of+multiple+mild+coagulation+factor+deficiencies+on+the+prothrombin+time+and+activated+partial+thromboplastin+time%5BTitle%5D)+AND+%22Am+J+Clin+Pathol%22%5BJournal%5D)">Paradoxic effect of multiple mild coagulation factor deficiencies on the prothrombin time and activated partial thromboplastin time.</a> Am J Clin Pathol. 100 (2), 94-98 (1993).</li>
<li>van Geffen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+hemostasis+assay+for+the+simultaneous+measurement+of+coagulation+and+fibrinolysis%5BTitle%5D)+AND+%22Hematology%22%5BJournal%5D)">A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.</a> Hematology. 16 (6), 327-336 (2011).</li>
<li>van Geffen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacodynamics+of+recombinant+activated+factor+VII+and+plasma-derived+factor+VII+in+a+cohort+of+severe+FVII+deficient+patients%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients.</a> Thromb Res. 132 (1), 116-122 (2013).</li>
<li>Hemker, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+calibrated+automated+thrombogram+%28CAT%29%3Aa+universal+routine+test+for+hyper-+and+hypocoagulability%5BTitle%5D)+AND+%22Pathophysiol+Haemost+Thromb%22%5BJournal%5D)">The calibrated automated thrombogram (CAT):a universal routine test for hyper- and hypocoagulability.</a> Pathophysiol Haemost Thromb. 32 (5-6), 249-253 (2002).</li>
<li>Kristensen, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation+measured+on+ST+Genesia%2C+a+new+platform+in+the+coagulation+routine+lab%3Aassessment+of+analytical+and+between-subject+variation%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">Thrombin generation measured on ST Genesia, a new platform in the coagulation routine lab:assessment of analytical and between-subject variation.</a> Res Pract Thromb Haemost. 6 (1), e12654(2022).</li>
<li>Hemker, H. C., Béguin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotyping+the+clotting+system%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Phenotyping the clotting system.</a> Thromb Haemost. 84 (5), 747-751 (2000).</li>
<li>Tripodi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation+assay+and+its+application+in+the+clinical+laboratory%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">Thrombin generation assay and its application in the clinical laboratory.</a> Clin Chem. 62 (5), 699-707 (2016).</li>
<li>Valke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinolytic+assays+in+bleeding+of+unknown+cause%3Aimprovement+in+diagnostic+yield%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">Fibrinolytic assays in bleeding of unknown cause:improvement in diagnostic yield.</a> Res Pract Thromb Haemost. 6 (2), e12681(2022).</li>
<li>van Geffen, M., van Heerde, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+haemostasis+assays%2C+from+bench+to+bedside%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">Global haemostasis assays, from bench to bedside.</a> Thromb Res. 129 (6), 681-687 (2012).</li>
<li>Luddington, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombelastography%2Fthromboelastometry%5BTitle%5D)+AND+%22Clin+Lab+Haematol%22%5BJournal%5D)">Thrombelastography/thromboelastometry.</a> Clin Lab Haematol. 27 (2), 81-90 (2005).</li>
<li>Hartmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viscoelastic+testing%3Aan+illustrated+review+of+technology+and+clinical+applications%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">Viscoelastic testing:an illustrated review of technology and clinical applications.</a> Res Pract Thromb Haemost. 7 (1), 100031(2023).</li>
<li>Schoeman, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+chamber+and+microfluidic+approaches+for+measuring+thrombus+formation+in+genetic+bleeding+disorders%5BTitle%5D)+AND+%22Platelets%22%5BJournal%5D)">Flow chamber and microfluidic approaches for measuring thrombus formation in genetic bleeding disorders.</a> Platelets. 28 (5), 463-471 (2017).</li>
<li>Bavinck, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viscoelastic+testing+in+patients+with+very+rare+bleeding+disorders+of+secondary+hemostasis%3Aa+scoping+review%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">Viscoelastic testing in patients with very rare bleeding disorders of secondary hemostasis:a scoping review.</a> Thromb Res. 2025, 109449(2025).</li>
<li>Mansouritorghabeh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+utility+of+total+thrombus-formation+analysis+system+%28T-TAS%29+in+the+thrombosis+and+hemostasis+field%3Aa+scoping+review%5BTitle%5D)+AND+%22Int+J+Lab+Hematol%22%5BJournal%5D)">The utility of total thrombus-formation analysis system (T-TAS) in the thrombosis and hemostasis field:a scoping review.</a> Int J Lab Hematol. 47 (2), 201-211 (2025).</li>
<li>Lecchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow-chamber+device+%28T-TAS%29+to+diagnose+patients+suspected+of+platelet+function+defects%5BTitle%5D)+AND+%22Blood+Transfus%22%5BJournal%5D)">Flow-chamber device (T-TAS) to diagnose patients suspected of platelet function defects.</a> Blood Transfus. 22 (1), 55-64 (2024).</li>
<li>Zekavat, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+thrombin+generation+assay+with+conventional+coagulation+tests+in+evaluation+of+bleeding+risk+in+patients+with+rare+bleeding+disorders%5BTitle%5D)+AND+%22Clin+Appl+Thromb+Hemost%22%5BJournal%5D)">Comparison of thrombin generation assay with conventional coagulation tests in evaluation of bleeding risk in patients with rare bleeding disorders.</a> Clin Appl Thromb Hemost. 20 (6), 637-644 (2014).</li>
<li>Brummel-Ziedins, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation+and+bleeding+in+haemophilia+A%5BTitle%5D)+AND+%22Haemophilia%22%5BJournal%5D)">Thrombin generation and bleeding in haemophilia A.</a> Haemophilia. 15 (5), 1118-1125 (2009).</li>
<li>Al Dieri, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation%3Awhat+have+we+learned%5BTitle%5D)+AND+%22Blood+Rev%22%5BJournal%5D)">Thrombin generation:what have we learned.</a> Blood Rev. 26 (5), 197-203 (2012).</li>
<li>Binder, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+use+of+thrombin+generation+assays%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Clinical use of thrombin generation assays.</a> J Thromb Haemost. 19 (12), 2918-2929 (2021).</li>
<li>Verhagen, M. J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+patients+with+hemophilia%2C+a+decreased+thrombin+generation+profile+is+associated+with+a+severe+bleeding+phenotype%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">In patients with hemophilia, a decreased thrombin generation profile is associated with a severe bleeding phenotype.</a> Res Pract Thromb Haemost. 7 (2), 100062(2023).</li>
<li>Haisma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+thrombin+and+plasmin+generation+profiles+in+alpha-2-antiplasmin-deficient+patients%3Adata+from+the+Rare+Bleeding+Disorders+in+the+Netherlands+study%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">Enhanced thrombin and plasmin generation profiles in alpha-2-antiplasmin-deficient patients:data from the Rare Bleeding Disorders in the Netherlands study.</a> Res Pract Thromb Haemost. 8 (7), 102604(2024).</li>
<li>Haisma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+thrombin+generation+platforms+for+patients+with+coagulation+factor+deficiencies%3Aa+comprehensive+assessment%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies:a comprehensive assessment.</a> Thromb Res. 240, 109045(2024).</li>
<li>van Geffen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrospective+evaluation+of+bleeding+tendency+and+simultaneous+thrombin+and+plasmin+generation+in+patients+with+rare+bleeding+disorders%5BTitle%5D)+AND+%22Haemophilia%22%5BJournal%5D)">Retrospective evaluation of bleeding tendency and simultaneous thrombin and plasmin generation in patients with rare bleeding disorders.</a> Haemophilia. 18 (4), 630-638 (2012).</li>
<li>Haisma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((genotype%2C+and+laboratory+assessment+of+congenital+fibrinogen+disorders%3Adata+from+the+Rare+Bleeding+Disorders+in+the+Netherlands+study%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">genotype, and laboratory assessment of congenital fibrinogen disorders:data from the Rare Bleeding Disorders in the Netherlands study.</a> Thromb Res. 249, 109317(2025).</li>
<li>Bouma, B. N., Meijers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin-activatable+fibrinolysis+inhibitor+%28TAFI%2C+plasma+procarboxypeptidase+B%2C+procarboxypeptidase+R%2C+procarboxypeptidase+U%29%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma procarboxypeptidase B, procarboxypeptidase R, procarboxypeptidase U).</a> J Thromb Haemost. 1 (7), 1566-1574 (2003).</li>
<li>Marx, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin-activatable+fibrinolysis+inhibitor%5BTitle%5D)+AND+%22Curr+Med+Chem%22%5BJournal%5D)">Thrombin-activatable fibrinolysis inhibitor.</a> Curr Med Chem. 11 (17), 2335-2348 (2004).</li>
<li>Rijken, D. C., Lijnen, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+insights+into+the+molecular+mechanisms+of+the+fibrinolytic+system%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">New insights into the molecular mechanisms of the fibrinolytic system.</a> J Thromb Haemost. 7 (1), 4-13 (2009).</li>
<li>Depasse, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation+assays+are+versatile+tools+in+blood+coagulation+analysis%3Aa+review+of+technical+features%2C+and+applications+from+research+to+laboratory+routine%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Thrombin generation assays are versatile tools in blood coagulation analysis:a review of technical features, and applications from research to laboratory routine.</a> J Thromb Haemost. 19 (12), 2907-2916 (2021).</li>
<li>Subcommittee on Control of Anticoagulation of the SSC of the ISTH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+a+recommendation+for+the+standardization+of+the+measurement+of+platelet-dependent+thrombin+generation%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Towards a recommendation for the standardization of the measurement of platelet-dependent thrombin generation.</a> J Thromb Haemost. 9 (9), 1859-1861 (2011).</li>
<li>Duarte, R. C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+and+evaluation+of+the+performance+of+the+thrombin+generation+test+under+hypo-+and+hypercoagulability+conditions%5BTitle%5D)+AND+%22Hematol+Transfus+Cell+Ther%22%5BJournal%5D)">Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions.</a> Hematol Transfus Cell Ther. 41 (3), 244-252 (2019).</li>
<li>Dargaud, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+standardization+and+normalization+on+imprecision+of+calibrated+automated+thrombography%3Aan+international+multicentre+study%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">Effect of standardization and normalization on imprecision of calibrated automated thrombography:an international multicentre study.</a> Br J Haematol. 139 (2), 303-309 (2007).</li>
<li>Ninivaggi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recommendations+for+the+measurement+of+thrombin+generation%3Acommunication+from+the+ISTH+SSC+Subcommittee+on+Lupus+Anticoagulant%2FAntiphospholipid+Antibodies%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Recommendations for the measurement of thrombin generation:communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies.</a> J Thromb Haemost. 19 (5), 1372-1378 (2021).</li>
<li>Duchemin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+coagulation+factors+and+tissue+factor+concentration+on+the+thrombin+generation+test+in+plasma%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma.</a> Thromb Haemost. 99 (4), 767-773 (2008).</li>
<li>Miszta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-fat+diet+delays+plasmin+generation+in+a+thrombomodulin-dependent+manner+in+mice%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A high-fat diet delays plasmin generation in a thrombomodulin-dependent manner in mice.</a> Blood. 135 (19), 1704-1717 (2020).</li>
</ol>

W.L. van Heerde is founder and employee of Enzyre BV and co-owner of Van Heerde Holding BV, which is a shareholder of Enzyre. Enzyre has received funding for contract research from Takeda and Novo Nordisk. W.L van Heerde has also received travel grant support from SOBI and CAF-DCF. The remaining authors declare no competing financial interests.

The Nijmegen Hemostasis Assay (NHA) is a dual-enzyme fluorogenic assay that enables the simultaneous, time-resolved measurement of thrombin and plasmin generation in platelet-poor plasma. By capturing both processes in a single well, the method facilitates high-resolution kinetic analysis of secondary hemostasis and fibrinolysis. This dual readout offers distinct methodological advantages over conventional assays and has been successfully applied in the study of coagulation factor deficiencies, hyperfibrinolytic conditio...

Hemostasis is the result of a dynamic balance between coagulation and fibrinolysis. Thrombin and plasmin are the key enzymes driving these processes: Thrombin converts fibrinogen into fibrin, while plasmin degrades fibrin to dissolve clots. Disruption of this balance can lead to bleeding or thrombotic events1,2. However, the laboratory tests used to evaluate hemostasis in clinical practice typically focus on isolated steps of coagulation or fibrinolysis, providing limited insight into the overall hemostatic balance. In patients with a suspected bleeding tendency, routine screening tests such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) are commonly used to screen for coagulation factor deficiencies. However, these assays measure the time to initial fibrin formation and do not capture thrombin generation beyond clotting onset, where most physiologically relevant thrombin activity occurs1,2,3. Consequently, they may yield normal results in patients with mild coagulation factor deficiencies3,4,5,6,7,8. To identify these specific deficiencies, coagulation factor activity assays are used. While informative for diagnosis, these assays assess individual proteins in isolation and do not reflect the overall hemostatic balance. As a result, in patients with mild deficiencies, factor levels often correlate poorly with the bleeding phenotype9,10. This complicates clinical risk assessment and the optimization of patient management, including the timing and dosing of therapy, as well as interventions in response to thrombotic or severe hemorrhagic events. These limitations highlight the need for assays that offer a more comprehensive view of the hemostatic process over time and may help bridge the gap between laboratory results and clinical presentation.
The Nijmegen Hemostasis Assay (NHA) was developed to address these limitations by enabling the simultaneous, time-resolved measurement of thrombin and plasmin generation in platelet-poor plasma8,9. It uses two synthetic fluorogenic substrates with distinct excitation and emission spectra to quantify both enzymes in the same microplate well without cross-reactivity. Enzyme generation curves allow derivation of quantitative parameters that reflect the dynamics of secondary coagulation and fibrinolysis, including lag time, thrombin peak height, fibrin lysis time and plasmin peak height. The NHA provides several advantages over existing thrombin generation assays, such as the Calibrated Automated Thrombogram (CAT), Ceveron s100, and ST Genesia, which provide valuable information on thrombin dynamics but do not assess fibrinolytic activity10,11,12,13. Clot lysis assays, in turn, capture aspects of fibrinolysis but lack information on thrombin generation14. This limits their usefulness in evaluating the complex interactions that determine overall hemostatic balance. While some laboratory-developed approaches have combined thrombin and plasmin generation measurements, these typically require separate wells or parallel assays, limiting temporal alignment and throughput15. Global assays such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM) provide complementary information by capturing primary hemostasis, secondary coagulation, and fibrinolysis16,17. Flow chamber systems represent another emerging yet experimental approach to assess total hemostasis under flow conditions18. While these global assays offer a broader physiological context, they are generally less sensitive to mild bleeding disorders19,20,21. By simultaneously measuring thrombin and plasmin generation in the same reaction well, the NHA combines the sensitivity of conventional TGAs with the integrated view of global assays, enabling direct assessment of secondary hemostasis, fibrinolysis and their interactions.
Thrombin generation assays have already shown considerable promise for translational applications, such as individualized bleeding and thrombotic risk profiling and therapy monitoring22,23,24,25. In clinical research, the NHA has demonstrated reduced thrombin generation in patients with hemophilia A, hemophilia B, and rare coagulation factor deficiencies, as well as elevated plasmin generation in hyperfibrinolytic conditions such as &#945;2-antiplasmin deficiency26,27,28,29. Differences in both pathways have also been observed in patients with hypofibrinogenemia, a disorder associated with both bleeding and an increased risk of thrombosis. In this population, lower fibrinogen levels correlated with reduced plasmin generation, likely due to the lack of plasminogen-binding sites, while patients with a history of thrombosis showed increased thrombin generation30. Additionally, the assay detects the effects of therapeutic agents, including anticoagulants, procoagulants, and antifibrinolytics8. In a previous study, we proposed a standardization and normalization approach to improve reproducibility and enable comparison of results across runs and laboratories, an essential step towards clinical implementation28. This protocol outlines the full procedure and analytical approach of the NHA for implementation in clinical research or translational settings.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcium chloridedihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;.2H2O)</td><td>Merck</td><td>1.02382</td><td></td></tr><tr><td>Cephalin (C.K. Prest 5 APTT reagent kit)</td><td>Stago</td><td>00847</td><td>Stable for 6 months at -20 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Distilled water</td><td></td><td></td><td>Record the date of opening. Use within 1 day after first opening.</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Merck</td><td>1.02950.0500</td><td></td></tr><tr><td>Human alpha-thrombin</td><td>Enzyme Research Laboratories</td><td>HT 1002a</td><td>The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 &amp;micro;L of added human thrombin in 120 &amp;micro;L reagent yields a final concentration of 76 nmol/L.</td></tr><tr><td>Human plasmin</td><td>Enzyme Research Laboratories</td><td>HPlas</td><td>The box contains a leaflet with a certificate of analysis. The volume of buffer to add depends on the specified concentration and should be adjusted so that 1 &amp;micro;L of added human plasmin in 120 &amp;micro;L reagent results in a final concentration of 200 nmol/L.</td></tr><tr><td>Normal pooled plasma</td><td>In-house preparation</td><td>Not applicable</td><td>Stable for 3 hours after thawing if kept on ice.</td></tr><tr><td>Patient plasma</td><td>Not applicable</td><td>Not applicable</td><td></td></tr><tr><td>Plasmin substrate (CBZ-L-phenylalanyl-L-arginine amide)-rhodamine-morpholino urea</td><td>Symeres</td><td></td><td>Stable for 6 months at -80 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Recombinant human tissue factor (TF): Innovin</td><td>Dade Behring</td><td>B4212</td><td>Stable for 6 months at -80 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Recombinant tissue-type plasminogen activator (tPA): Alteplase/Actilyse</td><td>Boehringer Ingelheim</td><td></td><td>Stable for 12 months at -80 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Tris(hydroxylmethyl)aminomethane (H&lt;sub&gt;2&lt;/sub&gt;NC(CH&lt;sub&gt;2&lt;/sub&gt;OH)&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>1.08382.2500</td><td></td></tr><tr><td>Tris-NaCl buffer, pH 7,4 (TBS)</td><td>In-house preparation</td><td>Not applicable</td><td></td></tr><tr><td>Tris-NaCl-CaCl&lt;sub&gt;2&lt;/sub&gt; buffer, pH 7,4 (TBS-Ca)</td><td>In-house preparation</td><td>Not applicable</td><td></td></tr><tr><td>Thrombin substrate Bz-beta-Ala-Gly-Arg-7-amino-4-methylcoumarin</td><td>Symeres</td><td></td><td>Stable for 6 months at -80 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fluostar Optima fluorimeter / microplate reader with temperature control and reagent injectors</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>96-well microtiter plate, polystyrene, flat-bottom, black, medium binding surface (Microlon equivalent)</td><td>Greiner BIO-ONE</td><td>655076</td><td></td></tr><tr><td>Analytical balance (top-loading balance)</td><td></td><td></td><td></td></tr><tr><td>Blue pipette tips (for 100-1000 &amp;micro;L)</td><td></td><td></td><td></td></tr><tr><td>Citrate Blood Collection Tubes 3mL (3.2% Citrate)</td><td>BD Vacutainer</td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td></td></tr><tr><td>Ice machine</td><td></td><td></td><td></td></tr><tr><td>Incubator / microplate shaker</td><td></td><td></td><td></td></tr><tr><td>Magnetic stirrer</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge tube rack</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge tubes, closable</td><td></td><td></td><td></td></tr><tr><td>pH-meter</td><td></td><td></td><td></td></tr><tr><td>Pipette tips 0.1-10 &amp;micro;L, non-sterile</td><td></td><td></td><td></td></tr><tr><td>Pipettes (2, 10, 20, 100, and 1000 &amp;micro;L)</td><td></td><td></td><td></td></tr><tr><td>Screwdriver (used as a probe)</td><td></td><td></td><td></td></tr><tr><td>Tube mixer</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Yellow pipette tips (for 20-200 &amp;micro;L)</td><td></td><td></td><td></td></tr></tbody></table>

the nijmegen hemostasis assay: simultaneous fluorogenic measurement of thrombin and plasmin generation in a single well

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

Thrombin and plasmin both play a central role in hemostasis, regulating coagulation and fibrinolysis, respectively. Conventional hemostatic assays often focus on isolated components or limited phases of hemostasis; for example, activated partial thromboplastin time and prothrombin time evaluate only the initiation of fibrin formation, while factor activity assays assess the function of individual coagulation factors. Although valuable, these assays lack a comprehensive view of the overall process of secondary hemostasis and fibrinolysis, which unfolds dynamically over time. The Nijmegen Hemostasis Assay (NHA) addresses this limitation by enabling simultaneous, time-resolved measurement of thrombin and plasmin generation in a single microplate well. Using two synthetic fluorogenic substrates with distinct excitation and emission spectra and no cross-reactivity, the assay allows accurate quantification of both enzymes in parallel. Hemostasis is initiated in platelet-poor plasma using tissue factor, calcium and tissue-type plasminogen activator, and fluorescence is recorded every 30 s over 70 min at 37 &#176;C. Enzyme activity curves are generated using calibration standards, from which parameters such as lag time, thrombin potential, fibrin lysis time, and plasmin peak height are derived.&#160;The assay reliably captures simultaneous thrombin and plasmin generation with good intra- and inter-assay precision, and detects modulation of coagulation and fibrinolysis by effectors such as heparin, activated protein C, and epsilon-aminocaproic acid. In clinical samples, significantly reduced thrombin generation was observed in patients with coagulation factor deficiencies, while patients with fibrinolytic disorders showed elevated plasmin generation profiles. By combining thrombin and plasmin generation measurements, the NHA provides a comprehensive insight into the hemostatic balance beyond conventional assays. This integrated approach holds promise for research and clinical applications, such as the characterization of hemostatic phenotypes, assessment of bleeding and thrombotic risk, and monitoring of targeted therapeutic interventions. This protocol outlines the NHA procedure that aims to enhance the understanding and assessment of hemostatic balance.

Representative thrombin and plasmin generation curves obtained with the Nijmegen Hemostasis Assay (NHA) are shown in Figure 3. The signal intensity of each curve is expressed as a percentage relative to the concurrently measured normal pooled plasma (NPP) sample. A sample with NPP is included in every run as an internal control to verify assay validity and ensure inter-run comparability.
Figure 3A,B display results fr...

This protocol describes the Nijmegen Hemostasis Assay, which enables simultaneous, time-resolved measurement of thrombin and plasmin generation to provide an integrated assessment of coagulation and fibrinolysis. The assay aims to improve characterization of hemostatic balance in research and clinical settings beyond the scope of conventional hemostatic tests.

Thrombin and plasmin both play a central role in hemostasis, regulating coagulation and fibrinolysis, respectively. Conventional hemostatic assays often ...

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.

the-nijmegen-hemostasis-assay-simultaneous-fluorogenic-measurement

Research

JoVE Journal

Medicine

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin.  The contribution of  enoxaparin is assessed by removing its influence through digestion with heparinase.  A fuller description of the assay is detailed in our published paper.1   The assay still requires clinical confirmation.

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

This video demonstrates the use of a UV-visible spectrophotometer for monitoring insoluble fibrin clot formation as a function of the change in the solution turbidity. The UV-visible spectrophotometer measures the solution’s absorbance over time, which represents the turbidity change due to the insoluble fibrin clot formation.

UV-Visible Spectroscopy for Monitoring Fibrin Clot Formation

This video demonstrates an assay to study the plasma membrane resealing efficiency in mammalian cells. The cells are exposed to a bacterial pore-forming toxin, which forms pores in the cell membrane. In the presence of extracellular calcium ions, the pore causes calcium influx, inducing membrane-resealing events. The resealing efficiency is assessed using DNA-binding propidium iodide dye, which only enters cells without a resealed membrane.

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics in Assessing Platelet Function

The present protocol provides a comprehensive set of procedures necessary to analyze procoagulant platelets, which exhibit overlapping features of necrosis, apoptosis, and platelet activation.

Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

This work describes a microfluidic assay that uses shear stress to trigger thrombus formation in blood and characterizes the thrombus by multiple dimensions of readout. The assay can measure the prothrombotic tendency of blood samples, and is thus useful in disease diagnosis, drug discovery, as well as basic mechanistic studies related to thrombosis.

Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

Learn the Nijmegen Hemostasis Assay for simultaneous fluorogenic measurement of thrombin and plasmin generation in plasma. This integrated method enables comprehensive assessment of coagulation and fibrinolysis balance.

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

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