Name: Forfatter Spotlight: Fremme af trombolytisk testning ved at integrere flowdynamik i in vitro-modeller
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

Forskning rapporteret i denne publikation blev støttet af National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL167877. Indholdet er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institutes of Health.

Alle metoder nævnt nedenfor er i overensstemmelse med Institutional Review Board (IRB) protokoller og den institutionelle humane forskningsetiske komité. Alle raske frivillige gav skriftligt og informeret samtykke forud for bloddonation. Bemærk, at alt materiale, der henvises til i protokollen, kan findes i materialefortegnelsen. Mens human WB og plasma diskuteres i hele denne protokol, kan brugen af dyreblod fra forskning og faktorudtømte blodprodukter købes og erstattes.
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Kombination af Chandler Loop og RT-FluFF til ex vivo overvågning af koagulationslysering

<ol>
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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. 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Dannelse og mærkning af blodpropper Chandler-løkken har vist sig at give et nemt og effektivt middel til reproducerbart at generere blodpropper, der efterligner in vivo tromber16. Finjusteringsparametre såsom rørstørrelse, rotationshastigheder, tromlediameter og koagulationstid giver mulighed for hurtig generering af blodpropper under forskellige forskydningsforhold, der kan fange arkitektoniske træk, der værdsættes i en række tromber, der efterligner både ar...

Sygdomme, der grundlæggende stammer fra trombo-emboliske ætiologier, udgør en væsentlig kilde til sygelighed og dødelighed i nutidens samfund. Manifestationer af tromboembolisk patogenese omfatter, men er ikke begrænset til, myokardieinfarkt, iskæmiske slagtilfælde, dybe venøse tromboser og lungeemboli1. En enorm mængde igangværende forskning, der spænder over flere discipliner, drejer sig om udvikling af sikre og effektive metoder til håndtering af patogen trombose. Variationer i arterielle og venøse manifestationer af trombose og varierende anatomiske placeringer har resulteret i udviklingen af forskellige behandlingstilgange. Akut behandling er dog generelt afhængig af brugen af farmakologisk trombolyse via plasminogenaktivatorer med potentiale for mekanisk trombektomi under visse kliniske omstændigheder2.
Udviklingen af nye farmakologiske behandlingsstrategier er grundlæggende afhængig af både in vivo dyremodeller og in vitro fordøjelsesmodeller til prækliniske test 3,4. In-vivo-modeller drager naturligvis fordel af deres evne til at fange den komplekse interaktion mellem forskellige fysiologiske parametre på behandlingseffektivitet, der inkluderer clearance af farmaceutiske midler såvel som cellulære interaktioner med lægemidler. Men den samme kompleksitet gør ofte sådanne modeller ret dyre og introducerer yderligere problemer, når man forsøger at isolere underliggende farmakodynamik/kinetik hos dyr, der adskiller sig væsentligt fra menneskelig fysiologi. Udviklingen af in vitro-modeller har hjulpet ved at facilitere et destilleret testmiljø, hvor lægemiddeludvikling og screening kan udføres, men ofte mangler den nøjagtighed, der er nødvendig for at rekapitulere den sygdomstilstand, der undersøges.
Almindeligt forekommende in vitro-protokoller til testning af nye trombolytika er afhængige af udnyttelsen af blodpropper dannet og lyseret under statiske forhold, hvor den resterende koagulationsmasse fungerer som det primære endepunkt 5,6. Desværre tager sådanne teknikker ikke højde for de mekaniske aspekter af koagulationslyse, såsom turbulent flow og transtrombetrykfald, der kan ændre farmakodynamikken af testlægemidler betydeligt. Derudover indeholder blodpropper dannet under statiske forhold mikroarkitektur, der adskiller sig fra fysiologiske blodpropper. Tilstedeværelsen af forskydning under koagulationsdannelse har reproducerbart vist sig at påvirke de resulterende koagulationskarakteristika såsom blodpladeaktivering og fibrin-tværbinding. Blodpropper, der produceres under forskydningsstrøm, udviser kompleks heterogenitet fra spids-til-hale, der er fraværende i statisk dannede blodpropper 7,8. Sådanne afvigelser fra fysiologisk koagulationsarkitektur kan påvirke vigtig karakterisering af lægemiddeludvikling, der inkluderer lægemiddelpenetration i en trombe og efterfølgende lyseringseffektivitet9.
For at løse nogle af disse begrænsninger forbundet med brugen af statiske koagulations-/koagulationslysemodeller har vedtagelsen af Chandler-løkken til både koagulationsdannelse og koagulationslyse i nærvær af forskydning set en genopblussen10. Selvom sådanne systemer giver mulighed for en bedre repræsentation af strømningsdynamik og genererer blodpropper med mere fysiologisk relevant arkitektur sammenlignet med relativt statiske analyser, repræsenterer deres forenklede strømningsbetingelser stadig en afvigelse fra fysiologiske forhold. Endelig er der også foretaget mikrofluidiske tilgange på grund af deres lette billeddannelse og ensartede strømningsmønstre; de forbliver dog en betydelig fjernelse fra de fysiologiske tilstande, der forventes i de større kar, der primært påvirkes ved de fleste klinisk relevante tromboemboliske lidelser11,12.
Med ovenstående diskussion i tankerne udviklede vi en high-fidelity, in vitro trombolysemodel til præklinisk trombolytisk lægemiddelscreening. Modellen sigter mod at adressere nogle af de nuværende faldgruber, der er beskrevet ovenfor inden for screening af ny trombolytisk terapi og blev valideret for reproducerbarhed og følsomhed ved varierende koncentrationer af vævsplasminogenaktivator (tPA). Systemet, der er beskrevet heri, tilbyder fysiologiske forskydningsstrømme ved hjælp af en peristaltisk pumpe, en trykdæmper, et opvarmet reservoir, to tryksensorer, et in-line fluorometer og en fluorescerende mærket Chandler loop forskydningsformet koagulationsanalog for at lette realtidssporing af fibrinolyse13. Samlet set kaldes det overordnede system Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay)14 , og dette manuskript vil diskutere forviklingerne ved succesfuld opsætning og kørsel af assays i denne high-fidelity in vitro trombolysemodel.

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			<td>NA</td>
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			<td>MLT0670</td>
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			<td>Drager Siemans HemoMed Pod</td>
			<td>Drager</td>
			<td>5588822</td>
			<td>RT-FluFF Apparatus</td>
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			<td>Drager Siemans Patient Monitor</td>
			<td>Drager</td>
			<td>SC 7000</td>
			<td>RT-FluFF Apparatus</td>
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			<td>Drum (cylinder, diameter 120 mm, width 85 mm)</td>
			<td></td>
			<td></td>
			<td>Chandler loop,</td>
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			<td>Face Shield</td>
			<td>Moxe</td>
			<td>SHIELDS10</td>
			<td>Chandler loop,&#160;</td>
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			<td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Scientific</td>
			<td>F13191</td>
			<td>Other Consumables</td>
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			<td>Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile</td>
			<td>Masterflex</td>
			<td>30600-04</td>
			<td>RT-FluFF Apparatus</td>
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			<td>Fluorescein (FITC)</td>
			<td>Thermo Scientific</td>
			<td>119245000</td>
			<td>Other Consumables</td>
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			<td>General-Purpose Water Bath</td>
			<td>Thermo Scientific</td>
			<td>2839</td>
			<td>Chandler loop,&#160;</td>
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			<td>Hotplate 4 &#215; 4</td>
			<td>Fisher Scientific</td>
			<td>1152016H</td>
			<td>RT-FluFF Apparatus</td>
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			<td>Human Source Plasma Fresh-Frozen</td>
			<td>Zen-Bio</td>
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			<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>
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			<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>
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			<td>Motor Speed Controller</td>
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			<td>ZK-MG</td>
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			<td>Thermo Scientific</td>
			<td>6151-0312</td>
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			<td>Thermo Scientific</td>
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			<td>Tissue plasminogen activator, human</td>
			<td>Millipore Sigma</td>
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			<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>
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tracking fibrinolysis of chandler loop-formed whole blood clots under shear flow in an in-vitro thrombolysis model

Tromboemboli og relaterede komplikationer er en førende årsag til sygelighed og dødelighed på verdensplan, og forskellige analyser er blevet udviklet til at teste trombolytisk lægemiddeleffektivitet både in vitro og in vivo. Der er stigende efterspørgsel efter mere fysiologisk relevante in vitro-koagulationsmodeller til lægemiddeludvikling på grund af kompleksiteten og omkostningerne forbundet med dyremodeller ud over deres ofte manglende oversættelighed til human fysiologi. Flow, tryk og forskydningshastighed er vigtige egenskaber ved kredsløbssystemet, hvor blodpropper, der dannes under flow, viser en anden morfologi og fordøjelseskarakteristika end statisk dannede blodpropper. Disse faktorer er ofte ikke repræsenteret i konventionelle in vitro-blodpropper fordøjelsesassays, hvilket kan have farmakologiske implikationer, der påvirker lægemiddeltranslationelle succesrater.
Real-T ime Fluorometrisk Flowing Fibrinolyse (RT-FluFF) assay blev udviklet som en high-fidelity trombolysetestplatform, der bruger fluorescerende mærkede blodpropper dannet under forskydningsstrøm, som derefter fordøjes ved hjælp af cirkulerende plasma i nærvær eller fravær af fibrinolytiske farmaceutiske midler. Ændring af strømningshastighederne for både koagulationsdannelse og blodpropfordøjelsestrin gør det muligt for systemet at efterligne arterielle, lunge- og venøse forhold på tværs af meget forskellige eksperimentelle opsætninger. Målinger kan foretages kontinuerligt ved hjælp af et in-line fluorometer eller ved at tage diskrete tidspunkter samt en konventionel måling af endepunktsmasse. RT-FluFF-analysen er et fleksibelt system, der giver mulighed for realtidssporing af blodpropper under strømningsforhold, der mere nøjagtigt repræsenterer in vivo fysiologiske forhold, samtidig med at kontrollen og reproducerbarheden af et in vitro-testsystem bevares.

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.

Forfatter Spotlight: Fremme af trombolytisk testning ved at integrere flowdynamik i in vitro-modeller

Sporing af fibrinolyse af Chandler loop-dannede fuldblodspropper under forskydningsflow i en in vitro trombolysemodel

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.

Dannelse af sløjfepropper Ved dannelsen af blodpropper sigtede vi generelt mod firdoble for at sikre, at hvis der fandtes nogen blodpropafvigelser (baseret på grov morfologi og masse), havde vi stadig mulighed for at køre tredobbelte trombolyseanalyser. Under forudsætning af optimale belastningsforhold bør blodpropper alle være ret ensartede i længde (~3,3 cm), vægt (~100 mg) og udseende som vist i figur 3. Ved anvendelse af FITC-Fg sigtede vi også mod at unders?...

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

In vitro-trombolyseassays har ofte kæmpet for at replikere in vivo-tilstande , hvad enten det er i modeltromben, der fordøjes, eller i det miljø, hvor trombolyse forekommer. Heri undersøger vi, hvordan kobling af Chandler-løkken og Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) bruges til high-fidelity, ex-vivo, koagulationslysovervågning.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

Trombose er forbundet med adskillige medicinske tilstande, og nuværende behandlinger risikerer alvorlig blødning. Kredsløbssystemets strømning påvirker blodproppens struktur betydeligt. Inkorporering af flow i in vitro-modeller kan forbedre deres fysiologiske relevans og forbedre udvælgelsen af bedre kandidater til in vivo-testning.Assays til undersøgelse af fibrinolytika bruger overvejende statiske koagulations- eller mikrofluidiske systemer til at vurdere egenskaber såsom lysistid, koagulationsstruktur eller viskoelasticitet. Statiske koagulationslysassays er typisk afhængige af spektrofotometriske målinger eller bestemmelse af koagulationsmasse som endepunkter for at evaluere fibrinolyseeffektiviteten. Nuværende trombolytisk testning bruger statisk dannede blodpropper, som adskiller sig strukturelt fra dem, der dannes under strømningsforhold.Mikrofluidiske systemer overvejer strømningsdynamik, men deres mindre skalaer skaber unik hæmodynamik og koagulationsstrukturer. Det aktuelt udviklede RT-FluFF-assay gør det nemt at overvåge fibrinolyse i realtid under fysiologiske flowforhold. Det betyder, at vi nu har en platform, der tillader kontrol over flere parametre for at efterligne forhold tættere på in vivo-undersøgelsesmodeller for bedre oversættelse ved screening af nye trombolytika.

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JoVE Journal

Bioengineering

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.

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.