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Submitted on 22 Oct 2019
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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�
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
Manuscript version: Accepted Manuscript
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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
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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
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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
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8-200401000-0001729
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Dworkin H J, Smith J R and Bull F E 1966 A reaction following administration of1
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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
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PrandoniP2015ThePostthromboticSyndrome:Evidence-BasedPrevention,Diagnosis16
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JoyalF,KassisJ,SolymossS,DesjardinsL,JohriMandGinsbergJS2008Determinants19
of health-related quality of life during the 2 years following deep vein thrombosis. J.20
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7836.2008.03002.x22
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&rendertype=abstract30
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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
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clots quantitativelymeasured in vivowith shear-wave ultrasound imaging in a rabbit9
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deep vein thrombosis. Arterioscler. Thromb. Vasc. Biol. 31 479–84 Online:16
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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
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during isotopeperfusion scansusinghumanmacroaggregatesof albumin.Nucl.Med.22
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aheterodyneinterferometerAppl.Phys.Lett.61153–15525
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databaseSyst.Rev.CD002783Online:http://www.ncbi.nlm.nih.gov/pubmed/1549503427
WrightC,HynynenKandGoertzD2012Invitroandinvivohighintensityfocusedultrasound28
thrombolysisInvest.Radiol.4721729
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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
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Figure31
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Figure41
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Figure51
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Figure61
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