Name: Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models
Uploaded: 2024-04-19T13:40:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In-Vitro Models at JoVE.com

Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL167877. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All methods mentioned below are in accordance with institutional review board (IRB) protocols and the institutional human research ethics committee. All healthy volunteers provided written and informed consent prior to blood donation. Of note, all materials referenced within the protocol can be found in the Table of Materials. While human WB and plasma are discussed throughout this protocol, the use of research animal blood and factor-depleted blood products can be purchased and substituted.
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Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

<ol>
	<li>Ali, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspect+of+thrombolytic+therapy:+a+review.">Aspect of thrombolytic therapy: a review.</a> Scientific World Journal. 2014, 586510 (2014).</li><li>Bhogal, P., Andersson, T., Maus, V., Mpotsaris, A., Yeo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+thrombectomy-A+brief+review+of+a+revolutionary+new+treatment+for+thromboembolic+stroke.">Mechanical thrombectomy-A brief review of a revolutionary new treatment for thromboembolic stroke.</a> Clin Neuroradiol. 28 (3), 313-326 (2018).</li><li>Fluri, F., Schuhmann, M. K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Des Devel Ther. 9, 3445-3454 (2015).</li><li>Kaiser, E. E., West, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+animal+ischemic+stroke+models:+replicating+human+stroke+pathophysiology.">Large animal ischemic stroke models: replicating human stroke pathophysiology.</a> Neural Regen Res. 15 (8), 1377-1387 (2020).</li><li>Elnager, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+whole+blood+clot+lysis+for+fibrinolytic+activity+study+using+d-dimer+and+confocal+microscopy.">In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy.</a> Adv Hematol. 2014, 814684 (2014).</li><li>Prasad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+model+to+study+clot+lysis+activity+of+thrombolytic+drugs.">Development of an in vitro model to study clot lysis activity of thrombolytic drugs.</a> Thromb J. 4, 14 (2006).</li><li>Robbie, L. A., Young, S. P., Bennett, B., Booth, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombi+formed+in+a+Chandler+loop+mimic+human+arterial+thrombi+in+structure+and+RAI-1+content+and+distribution.">Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and RAI-1 content and distribution.</a> Thromb Haemost. 77 (3), 510-515 (1997).</li><li>Mutch, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+thrombi+formed+under+flow+reveal+the+role+of+factor+XIII-mediated+cross-linking+in+resistance+to+fibrinolysis.">Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis.</a> J Thromb Haemost. 8 (9), 2017-2024 (2010).</li><li>Blinc, A., Kennedy, S. D., Bryant, R. G., Marder, V. J., Francis, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+through+clots+determines+the+rate+and+pattern+of+fibrinolysis.">Flow through clots determines the rate and pattern of fibrinolysis.</a> Thromb Haemost. 71 (2), 230-235 (1994).</li><li>Mutch, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Chandler+loop+to+examine+the+interaction+potential+of+NXY-059+on+the+thrombolytic+properties+of+rtPA+on+human+thrombi+in+vitro.">The use of the Chandler loop to examine the interaction potential of NXY-059 on the thrombolytic properties of rtPA on human thrombi in vitro.</a> Br J Pharmacol. 153 (1), 124-131 (2008).</li><li>Herbig, B. A., Yu, X., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidic+devices+to+study+thrombosis+in+pathological+blood+flows.">Using microfluidic devices to study thrombosis in pathological blood flows.</a> Biomicrofluidics. 12 (4), 042201 (2018).</li><li>Jigar Panchal, H., Kent, N. J., Knox, A. J. S., Harris, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+haemostasis:+A+review.">Microfluidics in haemostasis: A review.</a> Molecules. 25 (4), 833 (2020).</li><li>Zeng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+conjugated+annular+fibrin+clot+for+multiplexed+real-time+digestion+analysis.">Fluorescently conjugated annular fibrin clot for multiplexed real-time digestion analysis.</a> J Mater Chem B. 9 (45), 9295-9307 (2021).</li><li>Zeng, Z., Christodoulides, A., Alves, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+tracking+of+fibrinolysis+under+constant+wall+shear+and+various+pulsatile+flows+in+an+in-vitro+thrombolysis+model.">Real-time tracking of fibrinolysis under constant wall shear and various pulsatile flows in an in-vitro thrombolysis model.</a> Bioeng Transl Med. 8 (3), e10511 (2023).</li><li>Christodoulides, A., Zeng, Z., Alves, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+thromboelastographic+characterization+of+reconstituted+whole+blood+utilizing+cryopreserved+platelets.">In-vitro thromboelastographic characterization of reconstituted whole blood utilizing cryopreserved platelets.</a> Blood Coagul Fibrinolysis. 32 (8), 556-563 (2021).</li><li>Zeng, Z., Nallan Chakravarthula, T., Christodoulides, A., Hall, A., Alves, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Chandler+loop+shear+and+tubing+size+on+thrombus+architecture.">Effect of Chandler loop shear and tubing size on thrombus architecture.</a> J Mater Sci Mater Med. 34 (5), 24 (2023).</li><li>Touma, H., Sahin, I., Gaamangwe, T., Gorbet, M. B., Peterson, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+investigation+of+fluid+flow+in+a+chandler+loop.">Numerical investigation of fluid flow in a chandler loop.</a> J Biomech Eng. 136 (7), (2014).</li><li>Wojdyla, M., Raj, S., Petrov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption+spectroscopy+of+single+red+blood+cells+in+the+presence+of+mechanical+deformations+induced+by+optical+traps.">Absorption spectroscopy of single red blood cells in the presence of mechanical deformations induced by optical traps.</a> J Biomed Opt. 17 (9), (2012).</li><li>Wu, J. H., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+quench+and+dequench+assay+of+fibrinogen+polymerization,+fibrinogenolysis,+or+fibrinolysis.">A fluorescence quench and dequench assay of fibrinogen polymerization, fibrinogenolysis, or fibrinolysis.</a> Anal Biochem. 224 (1), 83-91 (1995).</li></ol>

Clot formation and labeling 
The Chandler loop has been demonstrated to provide an easy and effective means of reproducibly generating clots that mimic in-vivo thrombi16. Fine-tuning parameters such as tubing size, rotational speeds, drum diameter, and clotting time allow for the rapid generation of clots under differing shear conditions that can capture architectural features appreciated in a range of thrombi mimicking both arterial and venous sources. The additiona...

Diseases fundamentally stemming from thrombo-embolic etiologies present a major source of morbidity and mortality in present-day society. Manifestations of thrombo-embolic pathogenesis include, but are not limited to, myocardial infarctions, ischemic strokes, deep venous thromboses, and pulmonary emboli1. A tremendous amount of ongoing research, spanning multiple disciplines, revolves around the development of safe and effective methods for dealing with pathogenic thrombosis. Variations in arterial and venous manifestations of thrombosis and varying anatomic locations have resulted in the development of different treatment approaches. However, acute treatment generally relies on the use of pharmacologic thrombolysis via plasminogen activators with the potential for mechanical thrombectomy under certain clinical circumstances2.
The development of novel pharmacologic treatment strategies fundamentally relies on both in-vivo animal models and in-vitro digestion models for preclinical testing3,4. In-vivo models naturally benefit from their ability to capture the complex interaction of various physiologic parameters on treatment efficacy that include clearance of pharmaceutical agents as well as cellular interactions with drugs. However, this same complexity often makes such models quite costly and introduces additional issues when attempting to isolate underlying pharmaco-dynamics/kinetics in animals that significantly differ from human physiology. The development of in-vitro models has helped by facilitating a distilled testing setting in which drug development and screening can be performed but often lacks the fidelity necessary to recapitulate the disease state being studied. 
Commonly found in-vitro protocols for testing novel thrombolytics rely on the utilization of clots formed and lysed under static conditions whereby the residual clot mass serves as the primary endpoint5,6. Unfortunately, such techniques fail to account for the mechanical aspects of clot lysis such as turbulent flow and trans-thrombus pressure drops that can significantly alter the pharmacodynamics of test drugs. Additionally, clots formed under static conditions contain microarchitecture that differs from physiologic clots. The presence of shear during clot formation has reproducibly been shown to impact the resulting clot characteristics such as platelet activation and fibrin-crosslinking. Clots being produced under shear flow exhibit complex heterogeneity from tip-to-tail that is absent in statically formed clots7,8. Such departures from physiologic clot architecture may impact important drug development characterization that includes drug penetration within a thrombus and subsequent lysis efficiency9. 
To address some of these limitations associated with the use of static clotting/clot-lysis models, the adoption of the Chandler loop for both clot formation and clot lysis in the presence of shear has seen a resurgence10. Although such systems allow for a better representation of flow dynamics and generate clots with more physiologically relevant architecture compared to relatively static assays, their simplified flow conditions still represent a deviation from physiologic conditions. Lastly, microfluidic approaches have also been undertaken due to their ease of imaging and uniform flow patterns; however, they remain a significant removal from the physiologic conditions expected within the larger vessels primarily affected in most clinically relevant thrombo-embolic disorders11,12. 
With the above discussion in mind, we developed a high-fidelity, in-vitro thrombolysis model for preclinical thrombolytic drug screening. The model aims at addressing some of the current pitfalls detailed above in the realm of novel thrombolytic therapy screening and was validated for reproducibility and sensitivity at varying concentrations of tissue plasminogen activator (tPA). The system described herein offers physiological shear flows utilizing a peristaltic pump, a pressure dampener, a heated reservoir, two pressure sensors, an in-line fluorometer, and a fluorescently labeled Chandler loop shear-formed clot analog to facilitate real-time tracking of fibrinolysis13. Taken together, the overall system is called the Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay)14 and this manuscript will discuss the intricacies of successfully setting up and running assays in this high-fidelity in-vitro thrombolysis model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G&#160;Disposable Hypodermic Needles</td>
			<td>Exel International&#160;</td>
			<td>26439</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>6 mm HSS Lathe Bar Stock Tool 150 mm Long</td>
			<td>uxcell</td>
			<td>B07SXGSQ82</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>96-Well Clear Flat Bottom UV-Transparent Microplate</td>
			<td>Corning</td>
			<td>3635</td>
			<td>Other Consumables, Non-treated acrylic copolymer, non-sterile</td>
		</tr>
		<tr>
			<td>Air-Tite Luer-lock Unsterile 60 mL Syringes</td>
			<td>Air-Tite</td>
			<td>MLB3</td>
			<td>RT-FluFF Apparatus , dampeners</td>
		</tr>
		<tr>
			<td>Arium Mini Plus Ultrapure Water System</td>
			<td>Sartorius</td>
			<td>NA</td>
			<td>DI water source</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Millipore Sigma</td>
			<td>C5670</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Disposable BP Transducers</td>
			<td>AD Instruments</td>
			<td>MLT0670</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drager Siemans HemoMed Pod</td>
			<td>Drager</td>
			<td>5588822</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drager Siemans Patient Monitor</td>
			<td>Drager</td>
			<td>SC 7000</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drum (cylinder, diameter 120 mm, width 85 mm)</td>
			<td></td>
			<td></td>
			<td>Chandler loop,</td>
		</tr>
		<tr>
			<td>Face Shield</td>
			<td>Moxe</td>
			<td>SHIELDS10</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Scientific</td>
			<td>F13191</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile</td>
			<td>Masterflex</td>
			<td>30600-04</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Fluorescein (FITC)</td>
			<td>Thermo Scientific</td>
			<td>119245000</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>General-Purpose Water Bath</td>
			<td>Thermo Scientific</td>
			<td>2839</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Hotplate 4 &#215; 4</td>
			<td>Fisher Scientific</td>
			<td>1152016H</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Human Source Plasma Fresh-Frozen</td>
			<td>Zen-Bio</td>
			<td>SER-SPL</td>
			<td>Other Consumables, CPDA-1 anticoagulant</td>
		</tr>
		<tr>
			<td>Human Whole Blood&#160;</td>
			<td>Zen-Bio</td>
			<td>SER-WB-SDS&#160;</td>
			<td>Other Consumables, CPDA-1 anticoagulant</td>
		</tr>
		<tr>
			<td>L/S Easy-Load II Pump Head for High-Performance Precision Tubing, PPS Housing, SS Rotor</td>
			<td>Masterflex</td>
			<td>77200-62</td>
			<td>RT-FluFF Apparatus, Pump Head</td>
		</tr>
		<tr>
			<td>L/S Variable-Speed Digital Drive Pump with Remote I/O, 6 to 600 rpm; 90 to 260 VAC</td>
			<td>Masterflex</td>
			<td>7528-10</td>
			<td>RT-FluFF Apparatus, Pump</td>
		</tr>
		<tr>
			<td>Motor Speed Controller</td>
			<td>CoCocina</td>
			<td>ZK-MG</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Nalgene Tubing T-Type Connectors</td>
			<td>Thermo Scientific</td>
			<td>6151-0312</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Peristaltic pump tubing&#160;</td>
			<td>Masterflex</td>
			<td>06424-15&#160;</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Millipore Sigma</td>
			<td>P3813</td>
			<td>Other Consumables, Powder, pH 7.4, for preparing 1 L solutions</td>
		</tr>
		<tr>
			<td>SpectraMax M5 multi-detection microplate reader system (or other fluorescence detection)</td>
			<td>Molecular Devices</td>
			<td>M5</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Switching Power Supply</td>
			<td>SoulBay</td>
			<td>UC03U</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Thermo Scientific National Target All-Plastic Disposable Syringes 10 mL</td>
			<td>Thermo Scientific</td>
			<td>S751010</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Tissue plasminogen activator, human</td>
			<td>Millipore Sigma</td>
			<td>T0831</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Tubing ID 1/4&#39;&#39;, OD 3/8&#39;&#39;</td>
			<td>Fisher Scientific</td>
			<td>AGL00017</td>
			<td>Other Consumables, cut into 1.5cm sections use to connect tubing to T-type connectors</td>
		</tr>
		<tr>
			<td>Tubing ID 5/32&#34;, OD 7/32&#34;</td>
			<td>Tygon</td>
			<td>ND-100-65, ADF 00009&#160;</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>V3 365 nm Mini - Black Light UV Flashlight</td>
			<td>uvBeast</td>
			<td>uvB-V3-365-MINI</td>
			<td>Chandler loop, used to check completed clots</td>
		</tr>
		<tr>
			<td>ZGA37RG ZYTD520 DC Motor, 12 V, 100 rpm</td>
			<td>Pangyoo</td>
			<td>ZGA37RG</td>
			<td>Chandler loop,&#160;</td>
		</tr>
	</tbody>
</table>

tracking fibrinolysis of chandler loop-formed whole blood clots under shear flow in an in-vitro thrombolysis model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

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

Thrombosis is associated with numerous medical conditions and current treatments risk severe bleeding. Circulatory system flow significantly impacts blood clot structure. Incorporating flow into in-vitro models may improve their physiological relevance, enhancing the selection of better candidates for in-vivo testing.The assays for the study of fibrinolytics predominantly utilize static clot or microfluidic systems to assess properties such as lysis time, clot structure, or viscoelasticity. Static clot lysis assays typically rely on spectrophotometric measurements or clot mass determination as endpoints to evaluate fibrinolysis efficiency. Current thrombolytic testing uses statically-formed clots, which differ structurally from those formed under flow conditions.Microfluidic systems consider flow dynamics, but their smaller scales create unique hemodynamics and clot structures. The currently developed RT-FluFF assay provides ease in monitoring fibrinolysis in real time under physiological flow conditions. This means we now have a platform that allows control over multiple parameters to mimic conditions closer to in-vivo study models for better translation when screening new thrombolytics.

Chandler loop clot formation 
In forming clots, we generally aimed for quadruplicates to ensure that if any clot outliers (based on gross morphology and mass) existed, we still had the ability to run triplicate thrombolysis assays. Assuming optimal loading conditions, clots should all be quite uniform in length (~3.3 cm), weight (~100 mg), and appearance as is represented in Figure 3. When employing FITC-Fg, we also aimed to examine clots under UV light to ensure relati...

Read this Scientific Article Protocol about Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In-Vitro Models at JoVE.com

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.