Name: Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis (Video) | JoVE Journal
Uploaded: 2026-01-09T12:40:00.000+00:00
Duration: 8 min 50 s
Description: Learn a microfluidics-based thrombus profiling assay for comprehensive biomechanical thrombogenesis characterization using human blood. This method enables seven-dimensional analysis of thrombus formation under arterial shear conditions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Research related to this paper and the development of the thrombus profiling assay in the Chen lab were supported by National Heart, Lung, and Blood Institute grant R00HL153678 (Y.C.), National Institute on Aging, the Claude D. Pepper Older Americans Independence Center Award #P30-AG024832 (Y.C.), UTMB Team Science Pilot Research Award (Y.C.), American Heart Association Postdoctoral Fellowship 20POST35080023 (Y.C.), and American Heart Association Transformational Project Award 25TPA1471420 (Y.C.). This work was performed in part at the San Diego Nanotechnology Infrastructure (SDNI) of UCSD, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (Grant ECCS-2025752).

This protocol follows the guidelines of and has been approved by the Human Research Ethics Committee of The University of Texas Medical Branch. The experimental hardware setup consists of a microfluidic device, perfusion components (connectors, tubing, syringe, and syringe pump), and an inverted microscope with optical components enabling bright-field and multi-color fluorescence imaging. A multi-LED light house and a multi-pass filter cube are used in the microscope to split 4 fluorescent channels with minimal mutual bleed-through: excitation: 391/32, 479/33, 554/24, 638/31, and emission: 435/30, 519/25, 594/32, 695/58. Wear personal protective equipment, including gloves, eye protection, and a lab jacket, for all experimental procedures. The reagents and the equipment used are listed in the Table of Materials.
1. Microfluidic device preparation
<ol>
	<li>Design a master mold to contain rectangular channels (200 &#181;m wide &#215; 50 &#181;m high) that incorporate a site of 80% luminal narrowing. Each channel has dedicated inlets and outlets for tubing connections (Figure 1; see Supplementary File 1 for the original design file).</li>
	<li>Based on the design, use a silicon wafer to fabricate an SU-8 photoresist master mold via standard photolithography. Tape the mold at the bottom of a 15-cm Petri dish (Figure 2A).</li>
	<li>Prepare PDMS mixture by mixing the prepolymer base and curing agent in the silicone elastomer kit at a 10:1 weight ratio. Pour the mixture onto the master mold (Figure 2B).</li>
	<li>Place the plate containing the PDMS mixture in a vacuum desiccator to degas and eliminate entrapped air bubbles. 
	NOTE: Maintain the plate under vacuum for a minimum of 2 h until bubbles are completely removed.</li>
	<li>Thermally cure the PDMS mixture by incubating it at 75 &#176;C for 2 h.</li>
	<li>Carefully cut out the cured PDMS from the master mold (Figure 2C,D) and section it into individual chip units.</li>
	<li>Create the inlet and outlet by punching holes at the designated locations using a probe needle (Figure 2E). 
	NOTE: Use a compressed air duster to remove any residual debris from the punched holes. To minimize surface contamination, clean the PDMS devices using adhesive sealing tape prior to bonding.</li>
	<li>In a chemical hood, spray 75% ethanol on a task wipe, and use the wetted task wipe to wipe and clean glass slides. Leave the glass slides to dry.</li>
	<li>In a chemical hood, treat the glass slides and PDMS chips with a high-frequency generator for 30 s and 20 s, respectively, and then align each chip with a glass slide. Gently press the chip onto the glass slide surface so that it can be bound. 
	NOTE: This step needs to be done in a chemical hood because the high-frequency generator produces ozone during usage, which is harmful to health.</li>
	<li>Thermally bond the devices by incubating them at 150 &#176;C for 15 min. After cooling down, the devices will be ready to use (Figure 2F).</li>
	<li>Store the devices at room temperature under dust-free conditions.</li>
	<li>Use a pincer to remove the plastic part of the probe needles, and bend the metal tubes to 90&#176;. These bent tubes will be used as connectors for the microfluidic devices.</li>
</ol>
2. Fluorescent sensor preparation
NOTE: The experiment uses a total of 7 fluorescent sensors: SZ22-FITC, fibrinogen-Alexa Fluor 405, 2.2.9-Alexa Fluor 555, AK4-Alexa Fluor 647, Annexin V-Pacific Blue, MBC 370.2-Alexa Fluor 555, PAC-1-Alexa Fluor 647. Among them, fibrinogen, 2.2.9, and MBC 370.2 are only commercially available in the unconjugated form and need to be fluorescently conjugated in the lab.
<ol>
	<li>To conjugate fibrinogen with Alexa Fluor 405:
	<ol>
		<li>Make a fresh 0.1 M sodium bicarbonate buffer, pH 8.5.</li>
		<li>Dilute fibrinogen stock to 1 mg/mL in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4).</li>
		<li>Add 1/10 volume of sodium bicarbonate buffer to the fibrinogen solution.</li>
		<li>Mix 1 mL fibrinogen with 1 mg Alexa Fluor 405 NHS-ester and incubate for 1 h at room temperature on a rotator. Protect&#160;from light.</li>
		<li>Remove the storage buffer from a purification column by centrifuging the column at 1,100 &#215; g for 2 min, and discard the flow-through. Load the reaction solution onto the column, mount the column onto a compatible collection vial, and centrifuge at 1,100 &#215; g for 5 min to harvest the fibrinogen-Alexa Fluor 405.</li>
		<li>Use a spectrophotometer to measure the absorbance at 280 nm (A280; protein signal) and 405 nm (A405; dye signal) wavelength. Calculate protein concentration and F/P ratio (fluorescence: protein molar ratio). 
		NOTE: The F/P ratio should be in the range of 8-10.</li>
		<li>Store fibrinogen-Alexa Fluor 405 at 4 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>To conjugate 2.2.9 and MBC 370.2 antibodies with Alexa Fluor 555:
	<ol>
		<li>Make a fresh 0.1 M sodium bicarbonate buffer, pH 8.5.</li>
		<li>Dilute antibody stock to 1 mg/mL in amine-free buffer.</li>
		<li>Add 1/10 volume of sodium bicarbonate buffer to the antibody solution.</li>
		<li>Mix 100 &#181;L antibody with 1 vial (100 &#181;g) of Alexa Fluor 555 NHS-ester and incubate for 1 h at room temperature on a rotator. Protect&#160;from light.</li>
		<li>Remove the storage buffer from a purification column by centrifuging the column at 1,100 &#215; g for 2 min, and discard the flow-through. Load the reaction solution onto the column, mount the column onto a compatible collection vial, and centrifuge at 1,100 &#215; g for 5 min to harvest the 2.2.9- or MBC 370.2-Alexa Fluor 555.</li>
		<li>Use a spectrophotometer to measure the absorbance at 280 nm (A280; protein signal) and 555 nm (A555; dye signal) wavelength. Calculate antibody concentration and F/P ratio (fluorescence: antibody molar ratio). 
		NOTE: The F/P ratio should be in the range of 1-1.5.</li>
		<li>Store 2.2.9- and MBC 370.2-Alexa Fluor 555 in 4 &#176;C in the dark. 
		NOTE: Avoid over-labeling - high F/P ratio may impair biological function.</li>
	</ol>
	</li>
</ol>
3. Blood collection from human subjects
NOTE: This procedure must be conducted by qualified medical personnel (e.g., licensed nurses, certified phlebotomists). Also, obtain written informed consent from the individuals participating in the study.
<ol>
	<li>Prepare the syringe by aspirating 500 &#181;L of Tyrode buffer (12 mM NaHCO3, 10 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 5.5 mM D-Glucose, 0.5% bovine serum albumin, pH 7.4) containing 0.32 U/mL heparin per 10 mL of blood to be collected.</li>
	<li>Collect blood via venipuncture. Use an empty syringe to collect the first 2 mL of blood to discard. Then, change to the heparin-containing syringe to collect the blood sample. Pull the syringe slowly to minimize platelet activation.</li>
	<li>Transfer the collected blood into a 15-mL centrifuge tube and keep it under 37 &#176;C. 
	NOTE: Blood samples should be used for experiments within 6 h of collection.</li>
</ol>
4. Thrombus profiling assay
NOTE: It is recommended to perform all procedures involving blood handling in a biosafety cabinet whenever possible to avoid blood spills on the experimentalist. If this is not possible, then use a benchtop splash shield.
<ol>
	<li>Dilute VWF monomer to 2 &#181;g/mL in PBS. Pre-coat the microfluidic devices with VWF monomer and incubate for 1 h at room temperature.</li>
	<li>Incubate the blood sample with fluorescent sensor Set 1 or 2 for 10 min at room temperature:
	<ol>
		<li>Set 1: SZ22-FITC (0.5 &#181;g/mL), fibrinogen-Alexa Fluor 405 (60 &#181;g/mL), 2.2.9-Alexa Fluor 555 (1 &#181;g/mL), AK4-Alexa Fluor 647 (1 &#181;g/mL).</li>
		<li>Set 2: SZ22-FITC (0.5 &#181;g/mL), Annexin V-Pacific Blue (1 &#181;g/mL), MBC 370.2-Alexa Fluor 555 (1 &#181;g/mL), PAC-1-Alexa Fluor 647 (1 &#181;g/mL). 
		NOTE: Because two sensor sets need to be used separately, each blood sample needs to be tested at least twice (once with sensor Set 1 and once with sensor Set 2) to acquire a complete data set.</li>
	</ol>
	</li>
	<li>Connect a microfluidic device with inlet and outlet connectors and tubing. Connect the other end of the inlet connector with a tube containing PBS, and the other end of the outlet connector with a syringe. Mount the syringe onto a syringe pump. Mount the microfluidic device onto an inverted microscope, and manually maneuver the stage to find the site of stenosis under the microscope (Figure 3A).</li>
	<li>Fill the microfluidic channel and tubing with PBS using the syringe pump at a flow rate of 0.5 mL/min. Ensure that no air bubbles are present around the site of stenosis.</li>
	<li>Connect the inlet tubing with the blood sample, and perfuse the blood sample through the microfluidic channel using the syringe pump at a flow rate of 0.018 mL/min (Figure 3A).</li>
	<li>Set the exposure time and gain of each fluorescence channel. Perform real-time multi-color fluorescence imaging by alternating among the 4 channels, exciting one kind of fluorophore at a time. Meanwhile, find the focus plane of the thrombus, and record signals at the site of stenosis, typically spanning 15-30 min (Figure 4). 
	NOTE: The exposure time of each channel needs to be adjusted so that the fluorescent signal is easily distinguishable while avoiding saturation (Figure 4). In the present system, healthy subjects&#39; blood samples typically render the following signal intensity range within the formed thrombus: SZ22-FITC, 70-110; fibrinogen-Alexa Fluor 405, 60-100; 2.2.9-Alexa Fluor 555, 70-110; AK4-Alexa Fluor 647, 45-75; Annexin V-Pacific Blue, 35-65; MBC 370.2-Alexa Fluor 555, 45-85; PAC-1-Alexa Fluor 647, 10-30. Notably, however, a small proportion of healthy subjects would render a non-typical readout. Therefore, it is recommended to test at least 5 healthy subject&#39;s blood samples to make sure that the exposure time for each channel is selected properly.</li>
</ol>
5. Data analysis
<ol>
	<li>Open the data file using ImageJ 1.53 (Fiji, National Institutes of Health). 
	NOTE: Each file should contain a total of 4 videos from 4 different channels. The following procedure is generally applicable to any time point that the user is interested in analyzing.</li>
	<li>Use the SZ22-FITC channel to identify the contour of the thrombus, and use &#39;Polygon selections&#39; to roughly select the area of the thrombus (Figure 5A,B).</li>
	<li>For each channel, use Image -&#62; Adjust -&#62; Threshold to eliminate the background (Figure 5C), and then use Analyze -&#62; Measure to measure the area and average intensity of the signal within the thrombus. 
	NOTE: SZ22-FITC stains platelets and thus reflects thrombus size. In Set 1, fibrinogen-Alexa Fluor 405 reflects fibrinogen enrichment level, 2.2.9-Alexa Fluor 555 reflects von Willebrand factor (VWF) enrichment level, and AK4-Alexa Fluor 647 reflects P-selection expression. In Set 2, Annexin V-Pacific Blue reflects phosphatidylserine (PS) exposure, MBC 370.2-Alexa Fluor 555 reflects integrin &#945;IIb&#946;3 activation to the extended conformation (E+ &#945;IIb&#946;3), and PAC-1-Alexa Fluor 647 reflects integrin &#945;IIb&#946;3 full activation (Act. &#945;IIb&#946;3)21.</li>
</ol>

<ol><li>Lindstrom, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+burden+of+cardiovascular+diseases+and+risks+collaboration%2C+1990-2021%5BTitle%5D)+AND+%22J+Am+Coll+Cardiol%22%5BJournal%5D)">Global burden of cardiovascular diseases and risks collaboration, 1990-2021.</a> J Am Coll Cardiol. 80 (25), 2372-2425 (2022).</li>
<li>Lim, H. Y., O'malley, C., Donnan, G., Nandurkar, H., Ho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+global+coagulation+assays+-+is+there+a+role+in+thrombosis+risk+prediction%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">A review of global coagulation assays - is there a role in thrombosis risk prediction.</a> Thromb Res. 179, 45-55 (2019).</li>
<li>Paniccia, R., Priora, R., Liotta, A. A., Abbate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+function+tests%3A+A+comparative+review%5BTitle%5D)+AND+%22Vasc+Health+Risk+Manag%22%5BJournal%5D)">Platelet function tests: A comparative review.</a> Vasc Health Risk Manag. 11, 133-148 (2015).</li>
<li>Zhang, Y., Jiang, F., Chen, Y., Ju, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+mechanobiology+inspired+microdevices%3A+From+hematological+function+tests+to+disease+and+drug+screening%5BTitle%5D)+AND+%22Front+Pharmacol%22%5BJournal%5D)">Platelet mechanobiology inspired microdevices: From hematological function tests to disease and drug screening.</a> Front Pharmacol. 12, 779753(2021).</li>
<li>Gorog, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+direct+comparison+of+platelet+reactivity+and+thrombolytic+status+between+Japanese+and+Western+volunteers%3A+Possible+relationship+to+the+%26%2334%3BJapanese+paradox%26%2334%5BTitle%5D)+AND+%22Int+J+Cardiol%22%5BJournal%5D)">First direct comparison of platelet reactivity and thrombolytic status between Japanese and Western volunteers: Possible relationship to the &#34;Japanese paradox&#34.</a> Int J Cardiol. 152 (1), 43-48 (2011).</li>
<li>Gorog, D. A., Becker, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Point-of-care+platelet+function+tests%3A+Relevance+to+arterial+thrombosis+and+opportunities+for+improvement%5BTitle%5D)+AND+%22J+Thromb+Thrombolysis%22%5BJournal%5D)">Point-of-care platelet function tests: Relevance to arterial thrombosis and opportunities for improvement.</a> J Thromb Thrombolysis. 51 (1), 1-11 (2021).</li>
<li>Lopez-Jaime, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clot+stiffness+measured+by+seer+sonorheometry+as+a+marker+of+poor+prognosis+in+hospitalized+COVID-19+patients%5BTitle%5D)+AND+%22Clin+Appl+Thromb+Hemost%22%5BJournal%5D)">Clot stiffness measured by seer sonorheometry as a marker of poor prognosis in hospitalized COVID-19 patients.</a> Clin Appl Thromb Hemost. 28, 10760296221112085(2022).</li>
<li>Bochsen, L., Wiinberg, B., Kjelgaard-Hansen, M., Steinbruchel, D. A., Johansson, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+TEG+platelet+mapping+assay+in+blood+donors%5BTitle%5D)+AND+%22Thromb+J%22%5BJournal%5D)">Evaluation of the TEG platelet mapping assay in blood donors.</a> Thromb J. 5, 3(2007).</li>
<li>Lipets, E. N., Ataullakhanov, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+assays+of+hemostasis+in+the+diagnostics+of+hypercoagulation+and+evaluation+of+thrombosis+risk%5BTitle%5D)+AND+%22Thromb+J%22%5BJournal%5D)">Global assays of hemostasis in the diagnostics of hypercoagulation and evaluation of thrombosis risk.</a> Thromb J. 13 (1), 4(2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Thromboelastography+loinc+code+67790-6%22">Thromboelastography loinc code 67790-6</a>. , LOINC Committee. (2022).</li>
<li>Volod, O., Viola, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+quantra+system%3A+System+description+and+protocols+for+measurements%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">The quantra system: System description and protocols for measurements.</a> Methods Mol Biol. 2663, 743-761 (2023).</li>
<li>Nesbitt, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+shear+gradient-dependent+platelet+aggregation+mechanism+drives+thrombus+formation%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.</a> Nature Medicine. 15 (6), 665-673 (2009).</li>
<li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+integrin+alphaiibbeta3+intermediate+affinity+state+mediates+biomechanical+platelet+aggregation%5BTitle%5D)+AND+%22Nat+Mater%22%5BJournal%5D)">An integrin alphaiibbeta3 intermediate affinity state mediates biomechanical platelet aggregation.</a> Nat Mater. 18 (7), 760-769 (2019).</li>
<li>Li, M., Hotaling, N. A., Ku, D. N., Forest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+thrombosis+under+multiple+shear+rates+and+antiplatelet+therapy+doses%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Microfluidic thrombosis under multiple shear rates and antiplatelet therapy doses.</a> PLoS One. 9 (1), e82493(2014).</li>
<li>Colace, T. V., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+observation+of+von+Willebrand+factor+elongation+and+fiber+formation+on+collagen+during+acute+whole+blood+exposure+to+pathological+flow%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">Direct observation of von Willebrand factor elongation and fiber formation on collagen during acute whole blood exposure to pathological flow.</a> Arterioscler Thromb Vasc Biol. 33 (1), 105-113 (2013).</li>
<li>Westein, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atherosclerotic+geometries+exacerbate+pathological+thrombus+formation+poststenosis+in+a+von+Willebrand+factor-dependent+manner%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner.</a> Proc Natl Acad Sci USA. 110 (4), 1357-1362 (2013).</li>
<li>Brazilek, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+strain+rate+gradient+microfluidic+device+to+von+Willebrand%27s+disease+screening%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Application of a strain rate gradient microfluidic device to von Willebrand's disease screening.</a> Lab Chip. 17 (15), 2595-2608 (2017).</li>
<li>Tovar-Lopez, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidics+device+to+monitor+platelet+aggregation+dynamics+in+response+to+strain+rate+micro-gradients+in+flowing+blood%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood.</a> Lab Chip. 10 (3), 291-302 (2010).</li>
<li>Kim, D. A., Ku, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+shear-induced+platelet+aggregated+clot+formed+in+an+in+vitro+arterial+thrombosis+model%5BTitle%5D)+AND+%22Blood+Adv%22%5BJournal%5D)">Structure of shear-induced platelet aggregated clot formed in an in vitro arterial thrombosis model.</a> Blood Adv. 6 (9), 2872-2883 (2022).</li>
<li>Receveur, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shear+rate+gradients+promote+a+bi-phasic+thrombus+formation+on+weak+adhesive+proteins%2C+such+as+fibrinogen%2C+in+a+vwf-dependent+manner%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Shear rate gradients promote a bi-phasic thrombus formation on weak adhesive proteins, such as fibrinogen, in a vwf-dependent manner.</a> Haematologica. 105 (10), 2471-2483 (2020).</li>
<li>Din, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-parametric+thrombus+profiling+microfluidics+detects+intensified+biomechanical+thrombogenesis+associated+with+hypertension+and+aging%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Multi-parametric thrombus profiling microfluidics detects intensified biomechanical thrombogenesis associated with hypertension and aging.</a> Nat Commun. 15, 9067(2024).</li>
<li>Stein, P. D., Sabbah, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measured+turbulence+and+its+effect+on+thrombus+formation%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Measured turbulence and its effect on thrombus formation.</a> Circ Res. 35 (4), 608-614 (1974).</li>
<li>Mahalingam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numerical+analysis+of+the+effect+of+turbulence+transition+on+the+hemodynamic+parameters+in+human+coronary+arteries%5BTitle%5D)+AND+%22Cardiovasc+Diagn+Ther%22%5BJournal%5D)">Numerical analysis of the effect of turbulence transition on the hemodynamic parameters in human coronary arteries.</a> Cardiovasc Diagn Ther. 6 (3), 208-220 (2016).</li>
<li>Thomson, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gigapixel+imaging+with+a+novel+multi-camera+array+microscope%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Gigapixel imaging with a novel multi-camera array microscope.</a> Elife. 11, e74988(2022).</li>
</ol>

The authors have no conflicts of interest to disclose.

By combining the microfluidic stenosis assay with multi-color fluorescence imaging, the thrombus profiling assay provides a convenient and powerful approach to study biomechanical thrombogenesis and platelet mechanobiology. Meanwhile, the assay is useful in a wide range of applications. For instance, it can be used to screen anti-thrombotic agents that specifically inhibit biomechanical platelet aggregation, where the molecular target and mechanism of action can be deduced from the effect barcode21</sup...

Thrombosis is a main cause of cardiovascular diseases, responsible for millions of deaths worldwide every year1. Currently, no bioassay is available in standard clinical settings for evaluating risks of thrombosis. Among the commercialized laboratory and point-of-care hematological function assays, conventional coagulation assays and aggregometry have been proven unreliable in predicting thrombosis or major adverse cardiovascular events2,3,4. Global thrombosis test5, PFA-100/2006, and global coagulation assays7,8,9,10,11 also have limited data supporting their performance.
Based on the current understanding, the process of thrombogenesis is mainly contributed to by three mechanisms. Besides the two conventionally acknowledged mechanisms, namely, biochemical platelet aggregation and coagulation, a third mechanism that is under-studied and often under-estimated is shear-driven platelet aggregation, which was also termed as &#34;biomechanical platelet aggregation&#34;12,13. In biomechanical platelet aggregation, high shear stress and shear gradient serve as the main drive for platelet crosslinking via&#160;GPIb&#945;-von Willebrand factor (VWF), integrin &#945;IIb&#946;3-VWF, and integrin &#945;IIb&#946;3-fibrinogen interactions. In arterial thrombosis, biomechanical platelet aggregation likely serves as the most essential mechanism, considering that it is greatly reinforced by high shear flow caused by arterial stenosis. Therefore, thrombogenesis driven by biomechanical platelet aggregation was termed &#39;biomechanical thrombogenesis&#39;12,14.
In previous works, a common method for experimentally observing biomechanical platelet aggregation is the microfluidic stenosis assay, wherein a site of severe stenosis is embedded into a straight channel. When blood is perfused over the channel under a physiological wall shear stress, pathologically high shear stress is generated around the stenotic site, which drives the accumulation of platelets to form a thrombus. However, previous works only utilized a single (for platelets, reflecting the thrombus size)15,16,17,18,19 or at most two (one for platelets and one for another biomarker) readout13,20, which are thus unable to achieve comprehensive characterization of the thrombus.
A thrombus profiling assay was recently developed, which incorporates multi-color fluorescence imaging in the microfluidic stenosis assay, achieving real-time tracking of 7 biomarkers (platelets, fibrinogen level, von Willebrand factor level, P-selectin expression level, phosphatidylserine exposure level, extended integrin &#945;IIb&#946;3 expression level, fully active integrin &#945;IIb&#946;3 expression level) in a thrombus, which sets the basis for comprehensively characterizing biomechanical thrombogenesis21. In this work, detailed protocols are provided on the preparation and performance of the thrombus profiling assay as well as the related data analysis. The hardware required for the assay includes an inverted multi-color fluorescence microscope and a microfluidic system. The assay uses a relatively small amount of human whole blood (less than 2 mL), has high cost-effectiveness (~$12 per sample), and derives results within 30 min. The assay can accurately detect the multi-dimensional prothrombotic abnormalities of individuals and evaluate the effects of anti-thrombotic agents in changing the size, composition, and platelet activation status of the thrombus, endorsing its wide application for both research and clinical purposes in the future21. It is noteworthy that the assay must use freshly collected heparinized blood. Storing the blood at 4 &#176;C or for over 6 h or using anticoagulants other than heparin will either prevent thrombus from forming or render inaccurate results.

<table><tbody><tr><td>10-mL Syringes</td><td>Henke Sass Wolf</td><td>5100-X00V0</td><td>Blood sample collection</td></tr><tr><td>15-mL falcon tubes</td><td>Genesee Scientific</td><td>28-101</td><td>Blood sample storage</td></tr><tr><td>1-mL gastight glass syringe</td><td>Hamilton</td><td>81331</td><td>Hardware assembly</td></tr><tr><td>2.2.9</td><td>MERU VasImmune</td><td></td><td>Thrombus staining</td></tr><tr><td>20 X1/2 Probe Needles</td><td>McMaster-Carr</td><td>M919</td><td>Making PDMS devices</td></tr><tr><td>20-mL Syringes</td><td>Henke Sass Wolf</td><td>5200-X00V0</td><td>Blood sample collection</td></tr><tr><td>70% ethanol</td><td>Sigma-Aldrich</td><td>EX0281-1</td><td>Disinfection of gastight syringes and cleaning of glass slides</td></tr><tr><td>AK4-Alexa Fluor 647</td><td>BioLegend</td><td>304918</td><td>Thrombus staining</td></tr><tr><td>Alexa Fluor 405 NHS Ester</td><td>Invitrogen</td><td>A30000</td><td>Fibrinogen labeling</td></tr><tr><td>Alexa fluor 555 antibody labeling kit</td><td>Invitrogen</td><td>A88065</td><td>MBC 370.2 and 2.2.9 labeling</td></tr><tr><td>Annexin V-Pacific Blue</td><td>Invitrogen</td><td>501121505</td><td>Thrombus staining</td></tr><tr><td>Cleaning Duster</td><td>Office Depot</td><td>911245</td><td>Making PDMS devices</td></tr><tr><td>Craft Hobby Knife Set with Wooden Box</td><td>Excel Blades</td><td>44282</td><td>Making PDMS devices</td></tr><tr><td>Deionized water&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>751-628</td><td>Washing of gastight syringes</td></tr><tr><td>Desiccator</td><td>Bel Art</td><td>F420270000</td><td>Degasing PDMS</td></tr><tr><td>Disposable Multipurpose Laboratory Spatula</td><td>LevGo</td><td>17211</td><td>Mixing the silicone elastomer base and curing agent&amp;nbsp;</td></tr><tr><td>Dye and Biotin Removal Spin Column</td><td>Zeba</td><td>A44296S</td><td>Fibrinogen labeling</td></tr><tr><td>Fibrinogen</td><td>Innovative Research</td><td>IHUFBG25MG</td><td>Thrombus staining</td></tr><tr><td>Fusion 200-X syringe pump</td><td>Chemyx Inc.</td><td>0720X</td><td>Hardware assembly</td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H3149</td><td>Blood sample anticoagulation</td></tr><tr><td>High Frequency Generator</td><td>Electro-technic product Inc.</td><td>BD-20</td><td>Making PDMS devices</td></tr><tr><td>Human VWF monomer</td><td>Sino Biological Inc.</td><td>10973-H08C</td><td>Microfuidic device coating</td></tr><tr><td>Image J v1.53 software</td><td>Fiji, National Institute of Health</td><td></td><td>Data analysis</td></tr><tr><td>Inverted microscope</td><td>Leica</td><td>DM IL LED; filter cube: Leica DFT51010; lighthouse: LED5</td><td>Hardware assembly</td></tr><tr><td>Kimwipes</td><td>Kimberly- Clark Professional</td><td>34120</td><td>Cleaning of glass slides</td></tr><tr><td>Luer lock caps</td><td>International Medical Industries, Inc.</td><td>57100B</td><td>Blood sample collection</td></tr><tr><td>MBC 370.2</td><td>Kerafast</td><td>EBW104</td><td>Thrombus staining</td></tr><tr><td>Microscope cover glasses</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>ES0107222</td><td>Making PDMS devices</td></tr><tr><td>Mini-Razor Blade Scraper</td><td>Stanley</td><td>28-100</td><td>Making PDMS devices</td></tr><tr><td>NanoDrop 2000 UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td>ND-2000</td><td>Protein concentration and F/P ratio measurement</td></tr><tr><td>Oven</td><td>Lab-Line Instruments</td><td>3512</td><td>Making PDMS devices</td></tr><tr><td>PAC-1-Alexa Fluor 647</td><td>BioLegend</td><td>362806</td><td>Thrombus staining</td></tr><tr><td>Plastic cup</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>S04589</td><td>Mixing the silicone elastomer base and curing agent&amp;nbsp;</td></tr><tr><td>SU-8 photoresist master&amp;nbsp; mold</td><td>UC San Diego Nano3 Nanofabrication Cleanroom Facility</td><td></td><td>Making PDMS devices</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Krayden Dow</td><td>DC4019862</td><td>PDMS</td></tr><tr><td>SZ22-FITC&amp;nbsp;</td><td>Beckman Coulter</td><td>IM 1756U</td><td>Thrombus staining</td></tr><tr><td>Tubing</td><td>Cole -Palmer Instrument Co.</td><td>06422-01</td><td>Hardware assembly</td></tr></tbody></table>

thrombus profiling assay: a microfluidics-based platform for comprehensively characterizing biomechanical thrombogenesis

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

Thrombosis is a pathological condition describing the abnormal accumulation of platelets and clotting factors in a blood vessel. While many works focused on platelet activation by soluble agonists as an underlying mechanism of thrombosis, it has often been overlooked that blood flow also facilitates thrombus formation. Especially, in the arteries, thrombosis is generally associated with arterial stenosis, which elevates the shear stress in the blood flow and facilitates the process of thrombogenesis, a phenomenon termed biomechanical thrombogenesis. For a long time, no bioassay was available to provide all-around and detailed insights into the process of biomechanical thrombogenesis. To address this, a thrombus profiling assay was developed by combining microfluidics with multi-color fluorescence imaging, which allows comprehensive characterization of biomechanical thrombogenesis with seven readouts covering the size and composition of the thrombus as well as platelet activation level. This thrombus profiling assay can be used to evaluate the prothrombotic tendency in humans and the efficacy of anti-thrombotic agents, and it is also useful for further understanding the mechanisms underlying arterial thrombosis.

The thrombus profiling assay perfuses blood through a stenotic channel to allow shear-driven thrombus formation, and performs real-time multi-color fluorescence imaging to collect multi-dimensional information on the formed thrombus. By completing experiments using both Set 1 and Set 2 sensors, one should be able to characterize the thrombus in aspects including size, enrichment of crosslinking proteins (von Willebrand factor, fibrinogen), as well as platelet activation level (reflected by P-selection expression, PS expo...

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.

Thrombosis is a pathological condition describing the abnormal accumulation of platelets and clotting factors in a blood vessel. While many works focused ...

We have developed a new method that can adequately capture the biomechanical attributes of thrombosis and also detect prothrombotic abnormalities in humans. To begin, use a silicon wafer to fabricate an SU8 photoresist master mold via standard photolithography based on the design and secure the master mold at the bottom of a 15 centimeter Petri dish with tape. Prepare the polydimethylsiloxane, or PDMS, mixture by mixing the prepolymer base and curing agent from the silicone elastomer kit at a 10 to 1 weight ratio.Pour the PDMS mixture onto the master mold. Then place the plate containing the PDMS mixture in a vacuum desiccator to degas it. Thermally cure the PDMS mixture by placing it in an incubator set to 75 degrees Celsius for two hours.After removing the sample from the incubator, carefully cut out the cured PDMS from the master mold, and section the cured PDMS into individual chip units. Using a probe needle, punch holes at the designated locations to create the inlet and outlet. In a chemical hood, use a 75%ethanol wetted task wipe to clean the glass slides and dry them.Next, using a high frequency generator, treat the glass slides for 30 seconds and the PDMS chips for 20 seconds. Align each chip with a glass slide and gently press the chip onto the glass slide surface to bind it. Now thermally bond the devices by placing them in an oven set to 150 degrees Celsius for 15 minutes.After bonding, store the devices at room temperature under dust-free conditions. Then, using a pincer, remove the plastic part of the probe needles and bend the metal tubes to 90 degrees to create connectors for the microfluidic devices. After setting up the reactions for conjugating fibrinogen and required antibodies with Alexa Fluor, centrifuge the purification columns at 1, 100 G for two minutes to remove the storage buffer.After discarding the flow through, load the reaction solution onto the purification column mounted in a compatible collection vial and centrifuge at 1, 100 G for five minutes to harvest the required mix. Using a spectrophotometer, measure the absorbance for the protein and the dye signal. Calculate the protein concentration and the fluorescence-to-protein molar ratio.Store the dyes at four degrees Celsius in the dark. Add heparin to the Tyrode buffer to a final concentration of 0.32 units per milliliter per 10 milliliters of blood to be collected, and prepare the syringe by aspirating 500 microliters of Tyrode buffer at pH 7.4. After collecting the blood via venipuncture in the heparin-containing syringe, transfer it into a 15 milliliter centrifuge tube and keep it at 37 degrees Celsius.Dilute Von Willebrand factor monomer to two micrograms per milliliter in PBS. Precoat the microfluidic devices with the diluted von Willebrand factor monomer and incubate them for one hour at room temperature. Now incubate the blood sample with fluorescent sensor set 1 or set 2 for 10 minutes at room temperature.Next, connect a microfluidic device with inlet and outlet connectors and tubing. Connect the other end of the inlet connector to tubing containing PBS, and then connect the other end of the outlet connector to a syringe. Mount the syringe onto a syringe pump and the microfluidic device onto an inverted microscope.Manually maneuver the microscope stage to locate the site of stenosis. Fill the microfluidic channel and tubing with PBS using the syringe pump at a flow rate of 0.5 milliliters per minute. Inspect the device to ensure no air bubbles are present around the site of stenosis.Connect the inlet tubing to the blood sample and perfuse the blood sample through the microfluidic channel using the syringe pump at a flow rate of 0.018 milliliters per minute. In an inverted microscope, set the exposure time and gain for each fluorescence channel in the software. Perform real-time multicolor fluorescence imaging by alternating among the four channels and exciting one fluoro-4 at a time.Adjust the focus plane to the thrombus and record fluorescent signals at the site of stenosis for 15 to 30 minutes. For data analysis, open the data file using ImageJ version 1.53. Use the SZ 22 fit C channel to identify the contour of the thrombus.Then using polygon selections, roughly select the area of the thrombus. For each channel, eliminate background signal by selecting image, choosing Adjust, and then selecting Threshold. Finally, measure the area and average signal intensity within the thrombus by selecting Analyze and choosing Measure.Representative snapshots showed thrombi formed in healthy blood within the stenotic channel with platelets, fibrinogen, von Willebrand factor, and P-selectin visualized using sensor set 1, and platelets, phosphatidylserine, E-positive integrin alpha IIb beta 3, an activated integrin alpha IIb beta three visualized using sensor set 2. A single fluorescence channel image was processed by selecting the thrombus area, removing background signal, and measuring the signal area and average brightness to enable quantitative analysis. Continuous analysis of fluorescent signal intensity over time generated a signal versus time curve for platelet accumulation during thrombus formation.For each time point, total signal intensities were obtained for four biomarkers in sensor set one and four biomarkers in sensor set two. Thrombus size was calculated and a seven-dimensional thrombus profile was generated. Currently, no bioassay is available to evaluate the prothrombotic tendency of human patients, and our work attempts to fill this gap.By detecting the biomechanical activity of blood samples, our assay can comprehensively evaluate the prothrombotic attributes of subjects. Our assay can be developed into a clinical test for detecting prothrombotic tendency in patients, and it can also be used to screen next generation antithrombotic drugs.

thrombus-profiling-assay-microfluidics-based-platform-for

Research

JoVE Journal

Bioengineering

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

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

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

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics 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

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.

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

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly.  Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days.  Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Learn a microfluidics-based thrombus profiling assay for comprehensive biomechanical thrombogenesis characterization using human blood. This method enables seven-dimensional analysis of thrombus formation under arterial shear conditions.

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

Explore More Videos