pulsed cavitational therapy using high-frequency

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Pulsed cavitational therapy using high-frequencyultrasound for the treatment of deep vein thrombosis in

an in vitro model of human blood clotG Goudot, T Mirault, Benoît Arnal, C Boisson-Vidal, B Le Bonniec, P.

Gaussem, A Galloula, M. Tanter, E. Messas, M Pernot

To cite this version:G Goudot, T Mirault, Benoît Arnal, C Boisson-Vidal, B Le Bonniec, et al.. Pulsed cavitationaltherapy using high-frequency ultrasound for the treatment of deep vein thrombosis in an in vitromodel of human blood clot. Physics in Medicine and Biology, IOP Publishing, 2017, 62 (24), pp.9282-9294. �10.1088/1361-6560/aa9506�. �hal-02327100�

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Physics in Medicine and Biology

ACCEPTED MANUSCRIPT

Pulsed cavitational therapy using high-frequency ultrasound for thetreatment of deep vein thrombosis in an in vitro model of human bloodclotTo cite this article before publication: Guillaume Goudot et al 2017 Phys. Med. Biol. in press https://doi.org/10.1088/1361-6560/aa9506

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Pulsed cavitational therapy using high-frequency 1

ultrasound for the treatment of deep vein thrombosis in 2

an in vitro model of human blood clot3

G. Goudot1, T. Mirault2, 3, B. Arnal1, C. Boisson-Vidal4, B. Le Bonniec4, P.4

Gaussem2,4,A.Galloula2,3,M.Tanter1,E.Messas2,3,M.Pernot1*5

(1) Institut Langevin, INSERM U979, ESPCI Paris, CNRS UMR 7587, PSL Research6

University,Paris,France7

(2) Georges–Pompidou European Hospital, APHP, Paris Descartes University – USPC8

SorbonneParisCité,Paris,France9

(3) INSERM U970 PARCC, Paris Descartes University – USPC Sorbonne Paris Cité10

University,Paris,France11

(4)INSERMUMRS1140,ParisDescartesUniversity–USPCSorbonneParisCitéUniversity,12

Paris,France13

*:Correspondingauthor14

MPandEMsharetheseniorco-authorship15

Abstract16

Post-thromboticsyndrome,a frequentcomplicationofdeepvenousthrombosis,canbe17

reducedwithearlyveinrecanalization.Pulsedcavitationaltherapy(PCT)usingultrasoundis18

a recent non-invasive approach. We propose to test the efficacy and safety of high-19

frequency focused PCT for drug-free thrombolysis (thrombotripsy) in a realistic in vitro20

modelofvenousthrombosis.21

Toreproducevenousthrombosisconditions,humanwholebloodwasallowedtoclotby22

stasisinsiliconetubes(6mminternaldiameter)ata30cmH20pressure,maintainedduring23

thewhole experiment.We engineered an ultrasound device composed of dual 2.25MHz24

Page 1 of 23 AUTHOR SUBMITTED MANUSCRIPT - PMB-106169.R1

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transducerscenteredarounda6MHzimagingprobe.Atherapeuticfocuswasgeneratedat1

a3.2cmdepthfromtheprobe.Thrombotripsywasperformedbylongitudinallyscanningthe2

thrombusat3differentspeeds:1mm.s-1(n=6);2mm.s-1(n=6);3mm.s-1(n=12).Restored3

outflowwasmeasuredevery3passages.Filterswereplacedtoevaluatethedebrissize.4

24occlusive thrombi,of2.5cmmean lengthand4.4kPameanstiffness,werestudied.5

Flowrestorationwassystematicallyobtainedby9subsequentpassages(4.5minmaximum).6

By varying the device’s speed,we found an optimal speed of 1mm.s-1 to be efficient for7

effectiverecanalizationwith90s (3passages).Within90s, flowrestorationwasof80,628

and74%atrespectively1,2and3mm.s-1.Forallgroups,cavitationclouddrilleda1.7mm9

meandiameterchannelthroughouttheclot.Debrisanalysisshowed92%ofdebris<10μm,10

withnofragment>200µm.11

12

CONCLUSION:High-frequencythrombotripsyallowedfastandeffectiverecanalizationof13

whole-bloodthrombusinvitro,withoutanyparietalalterationorbulkydebrisformation.14

15

Keywords:thrombolysis–histotripsy–venousthrombosis–therapeuticultrasound16

Wordcount:abstract:252words,manuscript2860words. 17

Page 2 of 23AUTHOR SUBMITTED MANUSCRIPT - PMB-106169.R1

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I. Purpose1

Deepvenousthrombosisofthelowerlimbsisafrequentdisease,affectingapproximately2

0.1% to 0.2% of people per year [1, 2, 3]. The main initial risk is the migration of3

fibrinocruoricemboliresponsibleforpulmonaryembolisminabout30%ofcases(Heitetal4

2016). In the long term, themain risk forproximal thrombosis is theappearanceofpost-5

thrombotic syndrome in about 20 to 50% of cases (Kahn et al 2015), with an increased6

incidenceincasesofiliofemoralpersistentocclusion(Delisetal2004).Itischaracterizedby7

functional impotence,pain,pruritusanddistaltrophicdisorders,withamajoralterationof8

thequality of life (Kahnet al 2008). Current treatment of venous thrombosis is basedon9

effectiveanticoagulation,expectedtoavoidembolicmigrationandtoreducemorbidityand10

mortality. On the other hand, anticoagulation is frequently inefficient for recanalizing the11

occluded vessel [6, 7], with a consequently low influence on the incidence of post-12

thromboticsyndrome.Severalstudiessuggestthattheflowrestorationintheoccludedvein13

allows for long-term venous recanalization and thus limits the risk of post-thrombotic14

syndrome(WatsonandArmon2004,Endenetal2012).Theuseofplasminogenactivators15

or other thrombolytic agents has been shown to significantly improve the16

repermeabilization of the occluded vein. It was however associated with a 10% rate of17

severe haemorrhagic events (Watson and Armon 2004). Similarly, effective endovascular18

invasive recanalization procedures were associated with a decrease in post-thrombotic19

syndromeat6weeks(Endenetal2012)whereasthepersistenceofaresidualthrombuswas20

associated with an increased risk of post-thrombotic syndrome (Comerota et al 2012).21

Howeverthisinterventionisassociatedwithasubstantialriskofhematoma,falseaneurysm,22

reocclusionorstentrupture.23

Therapeuticultrasoundhasbeeninvestigatedbyseveralgroupsasapromisingdrug-free24

approach for non-invasive recanalization in deep venous thrombosis settings. Various25

techniques such as histotripsy based on short high intensity ultrasound pulses or High26

IntensityFocusedUltrasound(HIFU)basedonlongerexcitationpulseshavebeenproposed27

as a way to fragment thrombi by acoustic cavitation without the need of injecting28

microbubbles.Vascularwalldamagecanhoweverbeobservedwhencavitationoccursnear29

thevesselwalls(Maxwelletal2009).Toovercomethisissue,microtripsyhasbeenrecently30

introduced togenerateacavitationcloudcontained ina small volume, thereforeavoiding31


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anydamagetothevesselwalls(Zhangetal2015b).Microtripsyrequiresaveryhighnegative1

pressuretoreachtheintrinsiccavitationthreshold(–30MPa),whichremainschallengingto2

achieve in vivo.Wepropose hereby an alternative approach by increasing the ultrasound3

frequency(afrequencylowerthan1.5MHzisusedinmosthistotripsyapplications)inorder4

todecreasethefocussizeandachieveveryaccuratefragmentationofthethrombuswithout5

damaging the vesselwalls. The goal of this studywas to1) evaluate the feasibilityof this6

approachonaninvitromodelofhumanbloodclot,2)assesstherecanalizationefficacyof7

pulsedcavitationalultrasoundand3)quantifythesizeofthedebris.8

II. Materialsandmethods9

1) Obtaininganocclusivethrombus10

Human citrated (3.2%) whole blood was obtained from healthy volunteers from the11

FrenchBloodBankInstitute(EtablissementFrançaisduSang,Paris,France,agreementref.C12

CPSL UNT n°13/EFS/064). The subjects had normal complete blood count, denied having13

taken drugs interfering with haemostasis in the past 10 days and gave written informed14

consent. Coagulation was induced by adding 20 mM calcium chloride (CaCl2, number C15

5080; Sigma Chemical©, St. Louis, MO, USA). Aprotinin (100 kIU/mL final concentration;16

Trasylol500000kIU/50mL,Bayer©)wasaddedtoblocktheendogenousfibrinolysisduring17

therecanalizationprocedure.Clotsof2.5cminlengthwereobtainedbycoagulationunder18

stasis at 37 °C in sealed roughened silicone tubes (6mmwide, 1mm thick, close to the19

characteristicsofthehumanfemoralvein)andheldinverticalpositionfor1hourafterthe20

depositionof0.8mlofcitratedwholeblood.Aftercoagulationandthrombusretraction,the21

capwasremovedandthetubeplacedhorizontallyandchargedwithasalinesolution(0.9%22

NaCl)viaapressurecolumnat30cmH2O.Non-obstructivethrombiwerenotretainedfor23

the experiments. The silicon tube was positioned in order to approach a realistic depth24

basedonanexampleofhumanfemoralvein(Figure1).25

2) Thrombistiffnessevaluation26

Shear wave elastography was performed using a clinical ultrafast scanner (Aixplorer©,27

Supersonics Imagine©, Aix-en-Provence, France) and a linear ultrasound probe (SL10-2,28

centralfrequency6MHz,192elements).Thrombusstiffnesswasobtainedbymeasuringthe29


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mean elasticity of three circular regions of interest at a distance from thewalls to avoid1

interferencewiththesiliconetube’swallstiffness.2

3) Thrombotripsydevice3

Usinganin-househeterodyneinterferometer(Royeretal1992),two2.25MHzfocused4

transducers (central frequency2.25MHz, focaldistance38mm,F/D=1, Imasonic©,Voray-5

sur-l’Ognon,France)werepositionedconfocallyoneithersideofaSL10-2probe.Thewhole6

systemwasassembledthanksto3Dprintedparts,immersedinadegassedwater-bath,and7

movedbyamotoralongthesiliconetube.Thefocalpointwaslocated3.2cmawayfromthe8

imagingprobe(Figure2).Thesizeofthefocalspot(-6dB)intheplaneofthe2transducers9

was0.45x1.25mmat lowpressure.Thetransducersalonehadatheoreticalfocalspotof10

0.67mmx4.667mm.Usingthesetwoapertureconfocaltransducersallowedreachinghigh11

enough peak negative pressure for cavitation inception even though their aperture was12

small (38mmdiameter). The use of two confocal transducers allowed reducing the non-13

linearpropagationeffectsasshowninsimulationbyFowleretal.andLafondetal.(Fowler14

et al 2013, Lafond et al 2017). As a consequence, such a setup allowed a better spatial15

localizationof the focalpointandahighernegativepressurecompared towhatwouldbe16

generatedbyasinglelargeaperture.Thetwotransducersweredrivenbyasignalgenerator17

(Tektronix©)amplifiedbyagainof60dBbya2.5kWpoweramplifier (GA-2500,RITEC©,18

USA).Thesignalswerecomposedofburstsof8-cyclesat2.25MHztransmittedwithaPulse19

RepetitionFrequency(PRF)of100Hz.Thenegativepressure’speakgeneratedatthefocusof20

thetwoconfocaltransducerswasmeasuredwithanopticalinterferometerat–15MPa.21

4) Recanalization:22

The SL10-2probewasused to appropriately align thedevicewith the tube inorder to23

placethefocalspotinthecenterofthetube.Eachgroupreceived3sequencesofcavitation24

(1sequence=3passages)alongthethrombus,withassessmentoftherestoredflowafter25

eachsequence.Onepassage isthedisplacementofthedevicealongthethrombus length.26

Wefixedthreedifferentpassagespeedsalongthethrombus:1mm.s-1(n=6;1passage=3027

s), 2 mm.s-1 (n=6: 1 passage=15 s) and 3mm.s-1 (n=12; 1 passage=10 s). Thus, the total28

cavitationtime(3sequences)rangedbetween1.5min(at3mm.s-1)to4.5min(at1mm.s-1).29

Thedrilledchannelwaslongitudinallyscannedbyplane-by-planevolumetricacquisitionwith30


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adedicatedprobe(SuperLinear™VolumetricSLV16-5,Supersonics,AixenProvence,France)1

toassessthecontinuityofthethrombusrecanalization.2

5) Outflowevaluation3

Aftereachsequence,outflowwas investigatedbymeasuringthevolumeoftheoutflow4

solution for a definedperiod of 1minute. Initial flowmeasurementwas carried out after5

mounting the tube with the pressure column (30 cmH2O i.e. 22 mmHg). Results are6

expressedasapercentageofthemaximumflowrate(flowratemeasuredwiththesilicone7

tubewithoutthrombusunderthesameconditions).8

6) Debrisanalysis9

The outflow was filtered through 2 consecutive filters with 100 μm and 40 μm nylon10

mesh filters (Cell Stainer, BD Biosciences©). Each filter was rinsed with a saline solution,11

dried, and analysed under an optical microscope. Debris were counted by scanning the12

whole filter under amicroscope, using x100 and x200opticalmagnifications. Small debris13

(<40µm)werecollectedandthenanalysedbysamplingusingacomputerisedcounter(Cell14

CounterBioRadLaboratory®).Thediameterdistributionofthesmalldebrisisrepresentedby15

thenumberofdebrisnormalizedtotheremovedthrombusvolume.16

7) Statisticalanalysis17

Continuous variables, presentedas theirmean± standarddeviation,were comparedwith18

theMann–WhitneyU-test, or Kruskal–Wallis testwhen comparingmore than2 groups (319

differentspeedgroups).VariancesbetweengroupswerecomparedusingLevene’stestwith20

aBonferronicorrectionformultiplecomparisons.Two-sidedpvalues<0.05wereconsidered21

significant.AllstatisticalcomputationsusedtheRsoftware.22

III. Results23

Twenty-four consecutive adherent thrombi were evaluated. After 1 hour of clot24

formationandretraction,thethrombusoccupiedthetubevolume(6mminternaldiameter)25

and2.51cmin lengthonaverage.Onlyocclusivethrombiwereretained,withamicroflow26

through the thrombus lower than0.5mL.min-1. Shearwaveelastographyof the thrombus27

showed an average stiffness of 4.4 ± 1.8 kPa, corresponding to a recent deep venous28

thrombosis(Mfoumouetal2014).29


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1) Recanalizationefficiency:1

Histotripsy formed a circular channel of 1.7 ± 0.4 mmmean cross sectional diameter2

throughouttheclot(theaveragevolumeofthrombusremovedwas5.70mm3)(amovieof3

the recanalization procedure is presented in the supplementary materials). Transverse4

alignmentof thedevicemade itpossibletocenterthecavitationcloudso itdidnotreach5

the tube’s walls. End experiment examination revealed a residual thrombus coating the6

tube’sinnerwallsof2.1mmmeanthickness.Thisensuredthatthecavitationcloudwaswell7

centeredandfarfromthetube’swalls,whilerespectingthematerial’sintegrity(Figure3).8

2) Speedandtimeforthrombusrecanalization9

A speed of 1mm.s-1 allowed efficient recanalization (80±7%) after only one sequence (310

passages),correspondingtoatreatmentdurationof90s(Figure4).Forthesameduration11

time,weobtainedonly61±32%at2mm.s-1and74±22%at3mm.s-1(p=NSforallgroups).12

When comparing the time required for effective recanalization, regardless of the13

transducer’sspeed,anaveragetimeof76±17swasrequiredtoobtainaflowrecoveryat14

70%and101±45sforaflowat80%oftheinitialflow(Figure5).However,flowrestoration15

variance between thrombi recanalizationswas significantly lower at a speed of 1mm.s-1,16

7.4%,comparedtootherspeeds(32.5%for2mm.s-1:p=0.003;22.4%for3mm.s-1:p=0.027).17

3) Debrisanalysis18

Gross examination of the 100 µm filter at the end of each experiment revealed no19

macroscopic debris, whatever the speed group. Microscopic analysis of the nylon mesh20

filters (Figure6)foundraredebris(1.6±1.7perthrombus)butnonebiggerthan200µm,21

whichisconsistentwiththegrossexaminationfindings(Figure7).Thesmalldebrisdiameter22

distribution(lessthan40μm)isrepresentedinFigure8,accordingtothedevice’sspeedand23

reported by the number of debris, normalized to the removed thrombus volume. No24

significant difference was noticed between the three groups of speed: p=0.30 with the25

Kruskal-Wallis test. For theabsolutenumberof small debris, anon-significant trend toan26

increaseofsmalldebriswasobservedataspeedof1mm.s-1,whichcorrespondstoaslightly27

larger volume of recanalized channel (89±29mm3 for 1mm.s-1 vs. 50±14mm3 for other28

speeds,p=0.008)withalowerresidualthrombus(1.93±0.20vs.2.20±0.11mm,p=0.01).29


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IV. Discussion1

We aimed at demonstrating the effectiveness of thrombotripsy to recanalize a silicon2

tube containing an occlusive thrombus obtained with human whole blood stasis,3

reproducingas closelyaspossible theconditionsofa recent femoral vein thrombosis. For4

thisreason,weperformedthrombus instasisat37°C, inwholebloodobtainedfromfresh5

humanblood.Obtaining a thrombus adheringwell to thewallswas an important step, in6

order to prevent themigration of the entire thrombuswhen loadedwith the pressurized7

salinesolution.Moreover, itavoidedtheuseofstenosis toblocktheclotasused inother8

studies (Zhang et al 2015a, 2015b, 2016).We abandoned this procedure, first because it9

deviatesfromphysiologicalconditions,secondbecausewhenrecanalizationisperformed,it10

turnsoutthatthethrombusmigratesinsidethestenosis,thereforereducingthedebristobe11

drained by the restored outflow.Our initial use of a 600 kHz transducer failed to form a12

cavitation cloudwithin the channel without creating cavitation on the outer walls of the13

tube.Thisshieldingreducedtheacousticpoweratthefocusdepthandthusthecavitation14

activity in the channel.Moreover, the formationof a large cavitation cloudcoulddamage15

the vessel walls. Depending on the emitted acoustic power, the cavitation cloud position16

variedunpredictably.17

In our study, the use of higher frequency confocal transducers solved these issues. It18

allowedformingacloudoflimitedsizewithisotropicdimensions(2x2x2mm).Comparedto19

large phased array systems powered with multi-channel electronics, this system was20

designedandbuiltatamuchlowercost.Itinducedacompleterepeatabilityofthecavitation21

cloudposition,whichwillenableahigherinnocuousnessofthetreatmentforfutureinvivo22

trials.23

Sonothrombolysis usually corresponds to multiple techniques whose only common24

feature is the use of ultrasound during the thrombolysis procedure. Themain techniques25

developedareHigh Intensity FocusedUltrasound (HIFU), forwhich themain advantage is26

theuseofthethermalpropertiesofultrasoundwavestofacilitatetheactionofachemical27

thrombolysis (Bader et al 2016, Wright et al 2012). Thrombus fragmentation techniques28

using microbubbles underlined the mechanical thrombolytic effects of gas microbubbles,29

whichcanbe injectedordirectly formedbycavitationalone. Inordertodevelopastrictly30

non-invasive technique, our work stands on a model of cavitation alone, according to31


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previous work from the University ofMichigan [11, 18, 19, 20]. In order to improve this1

method, we used a higher frequency 2-channel device, developed a model close to the2

humanvenousthrombosisanddefinedtheoptimaltherapeutictimeforarecentthrombus3

lengthof2.5cmasstudiedherein.Theadvantageofourtechnique,eventhoughithasnot4

beentestedyetinvivo,istheuseofasmallerdevice,easiertohandle.Cavitationwithour5

devicecanbeachievedwithcommerciallyavailablegeneratorsandamplifiers.6

Flowrestorationwasinitiatedassoonasalittlechannelwasformedalongthethrombus,7

witharapidincreaseduringtheprocedurerelatedtothechannelenlargement.Longitudinal8

alignmentof thethrombus,withcontrolof thechanneldrillingthroughouttheprocedure,9

allowed rapid recanalization by performing continuous cavitation with a fixed transducer10

speed.11

Wedefinedthe1mm.s-1speedastheoptimalspeedoftherapyfromour3experimental12

conditions.Indeed,itallowedaminimalnumberofpassages(3)toachievetheoptimalflow13

rate(>80%)withashorttimeoftherapy(90s).Thetimerequiredtorecanalizeathrombus14

didnotdependsignificantlyon the speed.Athighervelocities (2and3mm.s-1), complete15

recanalization requiredmorepassages,which ended to the same recanalization than at 116

mm.s-1. We can note, however, that a higher variability of the recanalization time was17

observed at higher speed. We can anticipate that a lower speed would also provide a18

completerecanalization,butitwouldrequirelongertreatmenttime,whichwouldincrease19

the exposure risk of the vessel wall. Our objective being to allow a fast and safe20

recanalization,we did not choose to explore lower speeds than 1mm.s-1. Thismethod of21

longitudinal thrombolysis greatly shortened the procedure’s duration. However, this22

requires adapting the displacement of the transducer in order to follow the venous23

thrombus.Suchadevicecanbedevelopedusingimageguidancecombinedtoaroboticarm.24

The purpose of the thrombus recanalization procedure was not to remove the entire25

thrombus clotted inside the tube but to create a small flowing channel in the clot.26

Recanalizationwasconductedatadistancefromthetube’swalls,inordertolimitasmuch27

aspossibletheriskofparietal injury.Moreover,evenwithasmalldiameterchannel,good28

outflow (more than 80% of the maximal flow) was obtained. Although the presence of29

residual thrombusmayexpose to the riskofpost-procedurevenousocclusion,weplan in30

further pre-clinical studies to perform the procedure under effective anticoagulation.We31


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expect the recanalization to allow the heparin treatment to act locally andmaintain the1

vessel’spermeability.2

Thrombus elastography was achieved to verify our thrombosis model. An average3

thrombuselasticity of 4.5 kPawas consistentwith thematuration timeof 1 hour after in4

vitrocoagulationinduction.AccordingtothestudycarriedoutbyMfoumouetal[16],with5

thesameAixplorer©apparatusoninvivothrombosisintherabbit,5kPathrombusactually6

corresponded to a thrombus triggered in 1 to 2 hours. We did not test our device on7

thrombuswith ahigher stiffness, due to the conditionsneeded for thrombusmaturation,8

withaprogressiveenrichmentinfibrinaswellasthelossoftheplatelets,whichcouldnotbe9

achievedinourinvitroconditions.10

Debrisformationisinherenttoanyrecanalizationprocedure.Thereleaseofsmalldebris11

isnecessarytoallowsecurevenousrecanalization.Inourexperiment,wedidnotnoticeany12

macroscopicdebrisoranydebriswithadiameterover200µm.Occlusionofthepulmonary13

artery or its proximal branches, responsible for proximal pulmonary embolism is only14

triggered by bulky macroscopic thrombi. However, overall innocuousness of the debris15

obtainedisdifficulttoassessinvitro.Duringaperfusionlungscintigraphy,between20000016

and700000particlesofalbuminaggregatesareinjected,withadiameterbetween10and17

90µmandwithamaximumdiameterof150µm.Eventhoughalbuminaggregateswillblock18

somelungcapillaries(between1/200and1/1000(Dworkinetal1966)),thisexaminationis19

consideredassafe,withoutsignificanthypoxemiaduetoarteriolarblockade(Renowdenet20

al 1991) and is performed in daily practice to diagnose pulmonary embolism.Wemainly21

obtaineddebriscorrespondingtofragmentsofredbloodcells,size<10µmnotthreatening22

tothepulmonaryvasculartree.Debrislikelytoobstructapulmonaryarteriole,i.e.over10023

µmdiameterintheory,wereveryscarce(morethan99%ofthedebriswereunder40µm).24

Theriskofpoortoleranceduetotheembolizationofthesedebrisisthereforeverylowwith25

asmallnumberofaffectedarterioles.26

Thelimitationofthisinvitromodelofdeepvenousthrombosisistherelativelyshortsize27

of the thrombus.This limits theprocedure to suspendedproximal thrombosis. Finally, the28

potentialdangerofthedebrisrequiresaspecificevaluationinanimals.Inordertoobtaina29

sufficientvenousdiameterclose to thehumanfemoralvein, thepigmodel is thesmallest30

animalmodelappropriateforthispurpose,asalreadytested(Maxwelletal2009,Zhanget31


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al2017).Inaninvivoapplication,weneedtoconsidertissueattenuation.Withanaverage1

depthof3cmofthefemoralveininpig,andanaveragetissueattenuationof0.6dB.MHz-21.cm-1, we expect a total attenuation of 4.05 dB with a 2.25 MHz transducer. This3

attenuation factor can be easily compensated by increasing the amplification gain by 1.64

fold. On the other hand with histotripsy, tissue heating remains limited due to the low5

energy transmitted.With our setup at PRF of 100Hz, the total temporal average acoustic6

powertransmittedintissueisevaluatedtobelessthan1Wwiththereforenoriskofheating7

thetissueonthebeampath.8

Our thrombotripsy device has demonstrated its efficacy and safety in a recanalization9

model close to the conditions of human deep venous thrombosis. We carried out10

recanalization through recent thrombi, at a distance from the vessel’s walls, without11

generating largedebris.This firstexperiment isencouraging in thedevelopmentofanon-12

invasivetoolforvenousrecanalization.13

14

Acknowledgements:15

Wewould liketothankCardiawave©forthefundingsupport.WealsothankMrs.Beatrice16

Walkerforhercarefulproofreadingofthemanuscript.17

V. References18

BaderKB,BouchouxGandHollandCK2016Sonothrombolysis.Adv. Exp.Med.Biol.88019

339–62Online:http://www.ncbi.nlm.nih.gov/pubmed/2648634720

ComerotaAJ,GrewalN,MartinezJT,ChenJT,DisalleR,AndrewsL,SepanskiDandAssiZ21

2012Postthromboticmorbidity correlateswith residual thrombus following catheter-22

directed thrombolysis for iliofemoral deep vein thrombosis J. Vasc. Surg. 55 768–7323

Online:http://dx.doi.org/10.1016/j.jvs.2011.10.03224

Delis K T, Bountouroglou D and Mansfield A O 2004 Venous claudication in iliofemoral25

thrombosis: long-termeffectsonvenoushemodynamics,clinicalstatus,andqualityof26

life. Ann. Surg. 239 118–26 Online:27

http://content.wkhealth.com/linkback/openurl?sid=WKPTLP:landingpage&an=000006528

8-200401000-0001729


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Dworkin H J, Smith J R and Bull F E 1966 A reaction following administration of1

macroaggregatedalbumin(maa)foralungscan.Am.J.Roentgenol.RadiumTher.Nucl.2

Med.98427–33Online:http://www.ncbi.nlm.nih.gov/pubmed/59251133

Enden T, Haig Y, Kløw N-E, Slagsvold C-E, Sandvik L, GhanimaW, Hafsahl G, Holme P A,4

HolmenLO,NjaastadAM,SandbækGandSandsetPM2012Long-termoutcomeafter5

additional catheter-directed thrombolysis versus standard treatment for acute6

iliofemoral deep vein thrombosis (the CaVenT study): a randomised controlled trial.7

Lancet (London, England) 379 31–8 Online:8

http://www.ncbi.nlm.nih.gov/pubmed/221722449

FowlerR-A, LafondM,PoizatA,Mestas J-L,Chavrier F,Béra J-CandLafonC2013 Inertial10

cavitationenhancementusingconfocalultrasoundJ.Acoust.Soc.Am.134423411

HeitJA,SpencerFAandWhiteRH2016TheepidemiologyofvenousthromboembolismJ.12

Thromb. Thrombolysis 41 3–14 Online:13


KahnSR,ComerotaAJ,CushmanM,EvansNS,GinsbergJS,GoldenbergNA,GuptaDKand15

PrandoniP2015ThePostthromboticSyndrome:Evidence-BasedPrevention,Diagnosis16

,andTreatmentStrategiesCirculation1301636–6117

KahnSR,ShbakloH,LampingDL,HolcroftCA,ShrierI,MironMJ,RoussinA,DesmaraisS,18

JoyalF,KassisJ,SolymossS,DesjardinsL,JohriMandGinsbergJS2008Determinants19

of health-related quality of life during the 2 years following deep vein thrombosis. J.20

Thromb. Haemost. 6 1105–12 Online: http://doi.wiley.com/10.1111/j.1538-21

7836.2008.03002.x22

LafondM,PrieurF,ChavrierF,MestasJ-LandLafonC2017Numericalstudyofaconfocal23

ultrasonic setup for cavitation creation. J. Acoust. Soc. Am. 141 1953 Online:24

http://aip.scitation.org/doi/10.1121/1.497806125

Maxwell A D, Cain C A, Duryea A P, Yuan L, Gurm H S and Xu Z 2009 Noninvasive26

thrombolysisusingpulsedultrasoundcavitationtherapy-histotripsy.UltrasoundMed.27

Biol. 35 1982–94 Online:28

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2796469&tool=pmcentrez29

&rendertype=abstract30

MaxwellAD,OwensG,GurmHS,IvesK,MyersDDandXuZ2011Noninvasivetreatmentof31


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deep venous thrombosis using pulsed ultrasound cavitation therapy (histotripsy) in a1

porcine model. J. Vasc. Interv. Radiol. 22 369–77 Online:2

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3053086&tool=pmcentrez3

&rendertype=abstract4

Messas E, Wahl D and Pernod G 2016 [Management of deep-vein thrombosis: A 20155

update]. J. Mal. Vasc. 41 42–50 Online:6


MfoumouE,TripetteJ,BlosteinMandCloutierG2014Time-dependenthardeningofblood8

clots quantitativelymeasured in vivowith shear-wave ultrasound imaging in a rabbit9

model of venous thrombosis Thromb. Res. 133 265–71 Online:10

http://dx.doi.org/10.1016/j.thromres.2013.11.00111

Patel K, Fasanya A, Yadam S, Joshi A A, Singh A C andDuMont T 2017 Pathogenesis and12

Epidemiology of Venous Thromboembolic Disease Crit. Care Nurs. Q. 40 191–20013

Online:http://www.ncbi.nlm.nih.gov/pubmed/2855789014

PopuriRKandVedanthamS2011Theroleof thrombolysis in theclinicalmanagementof15

deep vein thrombosis. Arterioscler. Thromb. Vasc. Biol. 31 479–84 Online:16

http://atvb.ahajournals.org/cgi/doi/10.1161/ATVBAHA.110.21341317

PuurunenMK,GonaPN,LarsonMG,MurabitoJM,MagnaniJWandO’DonnellCJ201618

Epidemiology of venous thromboembolism in the Framingham Heart Study Thromb.19

Res.14527–33Online:http://dx.doi.org/10.1016/j.thromres.2016.06.03320

Renowden SA,Dunne J A andHaywardMW1991Changes in arterial oxygen saturation21

during isotopeperfusion scansusinghumanmacroaggregatesof albumin.Nucl.Med.22

Commun.12959–63Online:http://www.ncbi.nlm.nih.gov/pubmed/175415623

RoyerD,DuboisNandFinkM1992Opticalprobingofpulsed,focusedultrasonicfieldsusing24

aheterodyneinterferometerAppl.Phys.Lett.61153–15525

Watson L I andArmonMP2004 Thrombolysis for acutedeep vein thrombosis.Cochrane26

databaseSyst.Rev.CD002783Online:http://www.ncbi.nlm.nih.gov/pubmed/1549503427

WrightC,HynynenKandGoertzD2012Invitroandinvivohighintensityfocusedultrasound28

thrombolysisInvest.Radiol.4721729

ZhangX,MacoskeyJJ,IvesK,OwensGE,GurmHS,ShiJ,PizzutoM,CainCAandXuZ201730


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Non-invasiveThrombolysisUsingMicrotripsyinaPorcineDeepVeinThrombosisModel1

Ultrasound Med. Biol. Online:2

http://www.sciencedirect.com/science/article/pii/S03015629173005953

ZhangX,MillerRM,LinK-W,LevinAM,OwensGE,GurmHS,CainCAandXuZ2015aReal-4

time feedback of histotripsy thrombolysis using bubble-induced color Doppler.5

Ultrasound Med. Biol. 41 1386–401 Online:6

http://www.umbjournal.org/article/S030156291400800X/fulltext7

ZhangX,OwensGE,CainCA,GurmHS,MacoskeyJandXuZ2016HistotripsyThrombolysis8

on Retracted Clots. Ultrasound Med. Biol. 42 1903–18 Online:9

http://linkinghub.elsevier.com/retrieve/pii/S030156291630003510

ZhangX,OwensGE,GurmHS,DingY,CainCA,ArborA,DiseasesC,ArborAandArborA11

2015b Non-invasive Thrombolysis using Histotripsy beyond the ‘Intrinsic’ Threshold12

(Microtripsy)IEEETransUltrasonFerroelectrFreqControl6213

14

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VI. FiguresLegends1

Fig. 1. Comparisonof thedepth anddiameterof the femoral vein in a patientwith an2

occlusivethrombus(A)versustheparametersusedinoursetup(B).3

Fig.2. Illustrationof thethrombotripsyexperimentationsetup(A).Thetwotransducers4

arecoupledandcenteredbyalinearprobeof7MHz(B).Itisthenimmergedandcentered5

ontheocclusivethrombusinthesilicontube(C).6

Fig.3.Imagingofarecanalyzedthrombus.Ultrasonicacquisitionbeforethrombolysis(A).7

Complete recanalization after 3 series of 3 passages of thrombotripsy (B). Volumetric8

acquisitionwithadedicatedprobeconfirms the recanalization’scontinuity, remotely from9

thewalls(C).10

Fig.4.Rateofrecanalizationaccordingtothrombotripsypassagesanddevice’sspeed:111

mm.s-1 (red), 2 mm.s-1 (purple), 3 mm.s-1 (blue). The restored outflow is presented as a12

percentageofthemaximumflow(initialflowwithoutthrombus).13

Fig. 5. Recanalization rate according to the cavitation time. The restored outflow is14

presentedasapercentageofthemaximumflow(initialflowwithoutthrombus).15

Fig. 6. Setup for debris collection (A). The100µmand40μm filters allowed to collect16

debris>40µm.PicturesBandCshowdebris>100µmandpicturesDandEshowdebris>4017

µm.18

Fig.7.Debriscount>40μmaccordingtothedevice’sspeed.Debrisonfiltersof40µmand19

100μmwerecountedunderlightmicroscopy.20

Fig.8.Smalldebris(<40μm)diameterdistributionrepresentedbythenumberofdebris21

standardizedtotheremovedthrombusvolume.Debriscountwasobtainedbytheautomatic22

Counterandpresentedaccordingtodevice’sspeed. 23


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Figure11

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Figure21

2 3


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Figure31

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Figure41

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Figure51

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Figure61

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Figure71

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Figure81

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pulsed cavitational therapy using high-frequency

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