Table of Contents  
Year : 2021  |  Volume : 8  |  Issue : 5  |  Page : 18-24

Our approach and review of current concepts of catheter directed procedures in acute limb ischemia

Department of Vascular and Endovascular Services, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra, India

Date of Submission02-Jul-2021
Date of Decision22-Jul-2021
Date of Acceptance28-Jul-2021
Date of Web Publication30-Aug-2021

Correspondence Address:
R Sekhar
Department of Vascular and Endovascular Services, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijves.ijves_72_21

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Acute lower limb ischemia (ALI) is defined as sudden reduction of limb perfusion, leading to a threat to viability of the extremity. With better access to technology and better training in endovascular management in peripheral arterial disease, catheter-directed procedures are an attractive and feasible option in ALI patients. This article deals with our approach to ALI, the basic guidelines and clinical pathway adopted for its management, the hardware and drugs used, and also intraoperative and postprocedural care. It also briefly delves into our experience in using this protocol as also the recent modifications to standard techniques that have been employed, keeping in mind drawbacks such as bleeding and failure of recanalization when employing standard procedures. All patients who presented with ALI from the period of June 2016 to May 2021 with a short history of symptoms <14 days, Class 1, 2a, 2b ALI, and those that were fit and adequately consented for the procedure were studied. Those with age over 80 years, history of gastrointestinal bleed, and history of central nervous system bleed in the last 30 days were automatically excluded from undergoing these procedures. During this period, we saw 112 ALIs in our department, of which 74 cases were primarily deemed suitable for catheter-directed procedures, 60 for catheter-directed thrombolysis, 6 for AngioJet, and 8 using the CAT6 Penumbra catheter. 6 (8.1%) cases subsequently failed the procedure and were converted to open surgery, of which only 2 of 6 had limb/s salvaged, meaning that 4 of 6 converted cases had major amputations. The high amputation rates (25 out of 112, i.e. 22.32%) were clearly attributable on audit, to a delayed referral to a tertiary care center, leading to a large number of Class 3 cases. In our part of the world, open surgical embolectomy is still the mainstay of treatment for ALI. However, an endovascular approach is an option that may be used in selected cases. The structural, personal, and technical conditions of each department must be considered before advocating this modality for therapy in patients.

Keywords: Acute limb ischemia, catheter-directed procedures, thrombolysis

How to cite this article:
Patkar A, Gadhavi R, Kalwadia N, Sekhar R. Our approach and review of current concepts of catheter directed procedures in acute limb ischemia. Indian J Vasc Endovasc Surg 2021;8, Suppl S1:18-24

How to cite this URL:
Patkar A, Gadhavi R, Kalwadia N, Sekhar R. Our approach and review of current concepts of catheter directed procedures in acute limb ischemia. Indian J Vasc Endovasc Surg [serial online] 2021 [cited 2022 Sep 25];8, Suppl S1:18-24. Available from:

  Introduction Top

Acute lower limb ischemia (ALI) is defined as sudden reduction of limb perfusion, leading to a threat to viability of the extremity.

Of all ALI patients, 80%–85% suffer from arterial thrombosis, 15%–20% from embolic occlusions.[1]

In India, until the early 2000s, surgical intervention was considered the only available option for ALI treatment, especially with the Surgery versus Thrombolysis for Ischemia of the Lower Extremity (STILE), Thrombolysis or Peripheral Arterial Surgery (TOPAS), and Rochester studies not showing added advantage of catheter-assisted procedures with increased bleeding risks.[2],[3],[4] However, with better access to technology and better training in endovascular management, catheter-directed procedures have become an attractive option in ALI patients.[5],[6],[7]

Although no strict recommendations exist for using either surgical or endovascular approaches, endoluminal treatment is gaining increased acceptance.[8] Current endovascular approaches to ALI have high technical success rates, and survival, limb salvage, perioperative complications, and length of stay are similar to reported historical open cohorts.

  Basic Approach Top

  1. A proper history often suggests the etiology and should include past/present COVID-19 status and vaccination history
  2. Clinical examination to stratify into class of ischemia
  3. Doppler ultrasound (USG) for locating site and thrombus load and roughly estimating age of the thrombus based on echogenicity
  4. Routine hematological and biochemical tests, an electrocardiogram for rhythm disturbances, and an echocardiogram to exclude a left ventricular or left atrial clot that might fragment during a pharmacological thrombolysis with disastrous consequences
  5. A computed tomography angiogram if thrombus is suspected predominantly in the aortoiliac vessels rather than infrainguinal vessels as these cases may be triaged to the operation theater for quick restoration of circulation since delay may cause profound neurological impact
  6. Consenting for the procedure adequately and appropriately which should include:

    • Details of procedure
    • Need of intensive care unit (ICU) admission
    • Reperfusion, its sequelae including need for a fasciotomy; transient renal functional derangement occasionally needing dialysis
    • Puncture site complications: early and late (pseudo aneurysms)
    • Procedure-related vessel injury, distal embolization, and its consequences
    • Bleeding and need for transfusion/s, including gastrointestinal (GI) and intracranial bleeds
    • The possibility of a bypass and/or stenting at some stage
    • Rethrombosis
    • Reintervention
    • Rarely, acute pancreatitis after rheolytic therapy
    • Failure to revascularize and conversion to open surgical thrombectomy
    • Persistence of neurological deficit despite successful revascularization
    • Projected costs of procedure
    • Occasional limb loss.

  Case Selection Top

  • Short history of symptoms < 14 days
  • Rutherford Class 1, 2a, 2b ALI[9]
  • Thrombus in common femoral artery (CFA), profunda femoris, superficial femoral artery (SFA), popliteal and/or tibial vessels [Figure 1] and [Figure 2].
  • Thrombus in lower brachial and forearm arteries
  • Patient fit and adequately consented for the procedure
  • Exclusion criteria: Age more than 80 years, history of GI bleed, history of central nervous system bleed in the last 30 days.[10],[11]
Figure 1: Midsuperficial femoral artery thrombosis

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Figure 2: Distal superficial femoral artery occlusion

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Four consecutive steps for an endovascular approach to acute limb ischemia

  • Diagnostic angiography
  • Lesion crossing
  • Management of thrombus
  • Approach to underlying lesion if any, typically in acute on chronic thrombosis and graft thrombosis cases.

Catheter-based therapy can be classified as:

  1. Catheter-directed thrombolysis (CDT): Pharmacological thrombolysis where a thrombolytic agent is delivered into the clot via a catheter
  2. Mechanical thrombectomy/percutaneous thromboaspiration: Performed either through an end-hole catheter system or a mechanical device such as Penumbra catheter (aspiration) or Rheolytic device (AngioJet). Currently, only these two devices are available in India. This technique is especially useful when use of thrombolytics is contraindicated[10],[11]
  3. Pharmacomechanical thrombectomy, such as AngioJet.

  Access Top

  • Groin CFA, unilateral or bilateral
  • Brachial access: unilateral (preferably left, to avoid manipulation across an atherosclerotic arch and risk debris dislodgement to cerebral circulation), occasionally bilateral
  • USG guidance for an accurate single-wall puncture
  • Percutaneous and periadventitial local lignocaine infiltration is the preferred method. A standby anesthetist for suitable pain relief and management of any adverse event necessitating higher levels of anesthesia and airway management
  • A contralateral retrograde femoral access on most occasions
  • Sheath size: In general, 6F, frequently upgraded to a 6F/7F 45 cm/60 cm destination long sheath (Terumo Corp™).

  Hardware Top

  • Wires: 0.035” Amplatz type stiff wire for crossover, thereafter 260 cm 0.035” Terumo hydrophilic wire. The “guide wire traversal test” gives a rough estimation of feasibility of catheter-related procedures
  • Catheters: A variety of catheters are available and use depends on the therapeutic modality:

    • Suction catheters: Commonly used are the end-hole guiding catheters 100 cm attached to a 50 cc syringe. For a more distal thrombus, a 0.014 wire compatible 6F/7F/8F Eliminate (Terumo) catheter (rapid exchange) gives a length of 140 cm. The CAT6 Indigo catheter has a 135 cm length and can be used for distal thrombi along with the Penumbra system. The Thrombuster II (Kaneka Corp. Japan) is another less used option for 6F system with a 140 cm length and can be used for acute tibial vessel thrombus aspiration [Figure 3]a, [Figure 3]b, [Figure 3]c
    • Infusion catheters for thrombolysis: We use the 4F/5F Cragg McNamara (EV3) valved infusion catheter with a 135 cm length and 20 cm infusion segment. Alternative is the Cook (USA) Multi sideport infusion catheter with a 130 cm length and 15 cm infusion segment [Figure 4]
    • Rheolytic techniques: The AngioJet (Boston Scientific) system works on the Venturi effect and the catheters - The Solent Omni with a working length of 120 cm for distal SFA and Solent Proxi with a 90 cm length for proximal SFA thrombus are both 6F sheath and 0.035” wire compatible. The Solent™ Dista designed for distal thrombosuction of tibial thrombus did not find favor with us due to poor thrombus clearance [Figure 5].
Figure 3: (a) Length of Indigo catheter permits thromboaspiration till ankle even through retrograde access; (b) good two-vessel recanalization; (c) final result

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Figure 4: Lysis of infragenicular vessels in progress

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Figure 5: AngioJet™ (pharmacomechanical thromboaspiration) in progress

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Distal embolization may occur which gets subsequently lysed by the thrombolytic infused. Hence, it is not suitable in cases where thrombolytics are contraindicated. There might be a case for using a distal protection device; however due to significant increase in cost, we have not used the same. The total period of active thrombosuction/lysis should not exceed 400 s as significant hemoglobinuria occurs postprocedure due to hemolysis caused by the system [Figure 6].[6]
Figure 6: Hemoglobinuria postpharmacomechanical thombectomy

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  Drugs Top

  • Intravenous (IV) unfractionated heparin (UFH) is given in a bolus dose as per body weight, on an average, 7500 international units (IU)
  • Thrombolytics: There are different generations of thrombolytic agents with each generation achieving increased specificity for fibrin-bound plasminogen. While we have stopped using first-generation streptokinase and urokinase due to reports of increased hemorrhagic complications, with no data clearly suggesting effectiveness of one over another second or third generation agent, we use Alteplase (Actilyse, Boehringer Ingelheim, Germany), with a half-life of <5 min, as a thrombolytic agent. In Class 1 and 2a, we prefer CDT with infusion rate of 1 mg/h up to a maximum of 40 mg. There are different available protocols in literature varying from 0.12 mg to 2 mg/h. for alteplase.[6],[12]

There are different techniques for infusion, from pulse-spray (Bookstein technique) to continuous infusion, to initial pulse-spray followed by continuous infusion. We employ continuous infusion for Class 1 and 2a and an initial pulse-spray bolus of 10 mg Actilyse followed by continuous infusion for Class 2b patients.

There are reports of increased hemorrhagic complications with low-dose prolonged infusion and increased risk of distal embolization with pulse-spray rapid infusion with most studies reporting mild bleeding and our experience of both is similar.

We do not have experience with the use of other thrombolytics such as tenecteplase and reteplase.

  • We infuse 400–500 IU/h of UFH simultaneously through the side arm of the sheath to keep it clot free. Activated partial thromboplastin clotting time is not routinely monitored
  • Adequate IV hydration is mandatory to dilute the contrast used in the procedure and renoprotective for the hemolysis and hemoglobinuria seen during and postprocedure

  Monitoring Top

  • All patients are kept in the ICU, with puncture site, blood pressure, return of distal pulses, pain management including compartment syndrome, urine color, and output monitoring
  • Hemoglobin, platelets, fibrinogen levels, renal parameters monitored 12 hourly.

Our protocol for lysis is for a minimum of 12 h with a maximum dose of 40 mg alteplase.

Although fibrinogen levels fall and fibrin degradation product (FDP) levels consequently rise, there are no specific values suggesting a high bleeding risk. Roughly a fibrinogen level <150 suggests a 4 times higher risk of bleeding and an FDP level >400 suggests a 2.5 times higher bleeding risk.[13]

Success of the procedure is gauged as per the classification in [Table 1].[14] Thrombolysis degree 2 and 3 indicate a successful procedure.
Table 1: Angiographic results of catheter directed thrombolysis classification

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  • Postprocedure angiography, if suggestive of an underlying lesion, may be corrected at the same sitting, endovascularly where possible.

  Discharge Protocol (Figure 7) Top

Figure 7: Algorithm for management of acute limb ischaemia

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All patients are discharged on Factor Xa inhibitors (rivaroxaban) 15 mg twice daily for 3 weeks, reduced to 20 mg once daily for 3 months.

Vitamin K analogs (warfarin) are prescribed only if patients cannot afford medications and are compliant with regular monitoring and food restrictions. Antiplatelets are thereafter prescribed long term, specifically if an underlying lesion is treated concurrently.

Follow-ups are monthly for 3 months, then 6 monthly long term.

Our experiences with acute limb ischemia (June 2016–May 2021)

During this period, we saw 112 ALIs in our department, of which 24 were upper limb and 88 were lower limb. 19 (16.96%) of these cases underwent a primary amputation, being Class 3 ALI.

Nineteen (16.96%) cases underwent open surgery after primary evaluation resulting in limb salvage in 17 of 19 (89.47%) cases and 2 (10.52%) major amputations. 74 (66.07%) cases were primarily deemed suitable for catheter-directed procedures, 60 for CDT, 6 for AngioJet, and 8 using the CAT6 Penumbra catheter. Six cases (8.1%) failed the procedure and were converted to open surgery, of which only 2 of 6 had limb/s salvaged and 4 of 6 had major amputations. The high amputation rates (25 out of 112 i.e. 22.32%) were attributable on audit, to delayed referral to a tertiary care center, leading to a large number of Class 3 cases.

Complications of endovascular therapy [Table 2]
Table 2: Complications of endovascular therapy

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Other than conversion, all other complications were seen in the early years of performing the procedures, indicating a learning curve.

In our experience, catheter-directed procedures for ALI in COVID-19-related cases were successful when limb salvage was attempted during the acute pulmonary phase of the disease. In cases appearing in the delayed phase as seen in the second wave of the pandemic, these procedures are primarily unsuitable judging from the nature of the clots seen during open surgery [Figure 8]a and [Figure 8]b.
Figure 8: (a) Acute distal brachial artery thrombus in a COVID-19 patient; (b) postlysis

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Newer thoughts on catheter-directed procedures for ALI

Newer thoughts in the management of ALI are directed to increase limb salvage rates, reduce hospital/ICU stay, and minimize bleeding as a complication.

Fast-track thrombolysis protocol for acute limb ischemia

Fast-track thrombolysis protocol for ALI advocated by Ascher et al.[15] includes USG-guided periadventitial lidocaine injection at arterial puncture site, contrast arteriography of targeted segment, pharmacomechanical rheolytic thrombectomy of occluded arterial segment, infusion of tissue plasminogen activator (tPA) along the occluded segment, balloon maceration of thrombus, and (if necessary) stenting of an area of significant (more than or equal to 30%) stenosis that is refractory to balloon angioplasty and thrombolysis. After the stenosis or thrombus is cleared, patients are prescribed an oral anticoagulant agent.

This approach has exhibited numerous patient benefits including reduced hospital and ICU stay and decreased costs and radiation exposure. Compared with standard CDT, this protocol uses a lower dosage of lytic agent (mean, 9.7 mg) for a limited amount of time (mean, 148.9 min) under hemodynamic monitoring and direct visualization, resulting in a lower complication rate. In this study, only 1 patient out of 42, i.e. 2.4%, experienced complications in the form of bleeding (hematuria) as compared to bleeding complications reported in thrombolysis patients – 11 of 57 (20%) in the Rochester study, 5.2% in STILES, and 12.5% in TOPAS.[2],[3],[4],[14] While this approach looks attractive, cost issues restrict following it in all cases. Balloon maceration of thrombus is also fraught with dangers of distal trashing leading to caution in recommending it strongly.

EKOS ultrasound-enhanced catheter-directed thrombolysis for acute limb ischemia

USG-based devices utilize high-frequency low-power USG to enable delivery of therapeutic agents in peripheral vasculature. This consists of a reusable control system that powers the unit and a single-use infusion catheter system including a drug delivery catheter containing an USG core. It separates clot fibrin for better drug delivery into the clot without fragmentation of emboli and the principle of acoustic streaming hastens penetration of the lytic agent into the clot.[16]

Using ultrasound to enhance the effect of CDT is predicated on the idea that generating acoustic vibration augments Tissue Plasminogen Activator (tPA) delivery throughout the thrombus. This could theoretically lead to lower tPA dosage with a similar clinical efficacy.

A study by George et al., regarding real-world outcome of EKOS (EkoSonic Endovascular System-Boston Scientific) USG-enhanced CDT (UET) for ALI, showed that UET is generally safe and effective at reestablishing in-line flow, yielding high limb salvage rates; however, it is associated with a high rate of reintervention.[17]

In this procedure, after a clinical diagnosis of ALI, patients are systemically heparinized followed by angiography (contralateral CFA access) to evaluate the ischemic limb and EKOS catheter placement for overnight thrombolysis. tPA is infused at 0.5–1 mg/h for pharmacologic thrombolysis and 500 units/h of UFH is given through the sheath to prevent thrombus formation. Once UET is initiated, systemic heparinization is stopped. Patients are kept in the ICU with 6th hourly fibrinogen and coagulation parameter monitoring. UET is continued overnight followed by a lysis check and completion angiography.

Major bleeding occurred in three patients (9.4%) including one intracranial hemorrhage (3.1%), while ipsilateral reintervention was required in 15.6% of patients in the first 30 days and 37.5% within the 1st year.[17]

When comparing standard thrombolysis with USG-accelerated thrombolysis (UST), a multicenter Dutch trial showed a significant reduction in therapy time with UST, but a substantial number of bleeding complications.[14] Large cohort studies or systematic meta-analyses to show clinical benefits are also missing.[17],[18],[19]

Newer thrombolytics

Alfimeprase: Alfimeprase is a recombinantly produced, truncated form of fibrolase, a known fibrinolytic zinc metalloproteinase.

Both fibrolase and alfimeprase have shown direct proteolytic activity against the fibrinogen A α chain. In vivo pharmacology studies have shown that thrombolysis with alfimeprase is up to six times more rapid than with plasminogen activators.

Alfimeprase can be bound and neutralized by serum α 2-macroglobulin, a prevalent mammalian protease inhibitor capable of forming a macromolecular complex with it. Thus, systemic bleeding complications have been greatly reduced due to the inhibitory effects of α 2-macroglobulin.[20]

Alfimeprase first showed promising results in preclinical and pilot studies regarding duration of therapy and bleeding risk. However, in a multicenter study with two blinded, placebo-controlled, randomized trials, it neither showed life-threatening bleeding complications, nor did it have greater effectiveness than placebo.[21]

  Conclusion Top

ALI is a clinical emergency, with a variable presentation that may be life-threatening and lead to limb loss. Immediate diagnosis and treatment are critical to prevent irreversible damage. In our part of the world, open surgical embolectomy is still the mainstay of treatment for ALI; however, an endovascular approach is an attractive and feasible alternative in selected cases. It is recommended that the structural, personal, and technical conditions of each department must be considered before advocating this modality for therapy.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Walker TG. Acute limb ischemia. Tech Vasc Interv Radiol 2009;12:117-29.  Back to cited text no. 1
Weaver FA, Comerota AJ, Youngblood M, Froehlich J, Hosking JD, Papanicolaou G. Surgical revascularization versus thrombolysis for nonembolic lower extremity native artery occlusions: Results of a prospective randomized trial. The STILE Investigators. Surgery versus Thrombolysis for Ischemia of the Lower Extremity. J Vasc Surg 1996;24:513-21.  Back to cited text no. 2
Ouriel K, Veith FJ, Sasahara AA. A comparison of recombinant urokinase with vascular surgery as initial treatment for acute arterial occlusion of the legs. Thrombolysis or Peripheral Arterial Surgery (TOPAS) Investigators. N Engl J Med 1998;338:1105-11.  Back to cited text no. 3
Ouriel K, Shortell CK, DeWeese JA, Green RM, Francis CW, Azodo MV, et al. A comparison of thrombolytic therapy with operative revascularization in the initial treatment of acute peripheral arterial ischemia. J Vasc Surg 1994;19:1021-30.  Back to cited text no. 4
Lichtenberg M, Käunicke M, Hailer B. Percutaneous mechanical thrombectomy for treatment of acute femoropopliteal bypass occlusion. Vasc Health Risk Manag 2012;8:283-9.  Back to cited text no. 5
Lukasiewicz A. Treatment of acute lower limb ischaemia. Vasa 2016;45:213-21.  Back to cited text no. 6
Gupta R, Hennebry TA. Percutaneous isolated pharmaco-mechanical thrombolysis-thrombectomy system for the management of acute arterial limb ischemia: 30-day results from a single-center experience. Catheter Cardiovasc Interv 2012;80:636-43.  Back to cited text no. 7
Björck M, Earnshaw JJ, Acosta S, Bastos Gonçalves F, Cochennec F, Debus ES, et al. Editor's Choice-European Society for Vascular Surgery (ESVS) 2020 Clinical Practice Guidelines on the Management of Acute Limb Ischaemia. Eur J Vasc Endovasc Surg 2020;59:173-218.  Back to cited text no. 8
Rutherford RB, Baker JD, Ernst C, Johnston KW, Porter JM, Ahn S, et al. Recommended standards for reports dealing with lower extremity ischemia: Revised version. J Vasc Surg 1997;26:517-38.  Back to cited text no. 9
Working Party on Thrombolysis in the Management of Limb Ischemia. Thrombolysis in the management of lower limb peripheral arterial occlusion – A consensus document. J Vasc Interv Radiol 2003;14:S337-49.  Back to cited text no. 10
Kearon C, Akl EA, Comerota AJ, Prandoni P, Bounameaux H, Goldhaber SZ, et al. Antithrombotic therapy for VTE disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012;141:S419-96.  Back to cited text no. 11
Hage AN, McDevitt JL, Chick JF, Vadlamudi V. Acute limb ischemia therapies: When and how to treat endovascularly. Semin Intervent Radiol 2018;35:453-60.  Back to cited text no. 12
Giannakakis S, Galyfos G, Sachmpazidis I, Kapasas K, Kerasidis S, Stamatatos I, et al. Thrombolysis in peripheral artery disease. Ther Adv Cardiovasc Dis 2017;11:125-32.  Back to cited text no. 13
Lukasiewicz A. Contemporary management of acute lower limb ischemia: Determinants of treatment choice. J Clin Med 2020;9:1501.  Back to cited text no. 14
Ascher E, Kibrik P, Rizvi SA, Alsheekh A, Marks N, Hingorani A. Fast-track thrombolysis protocol for acute limb ischemia. J Vasc Surg 2021;73:950-9.  Back to cited text no. 15
Fluck F, Augustin AM, Bley T, Kickuth R. Current treatment options in acute limb ischemia. Rofo 2020;192:319-26.  Back to cited text no. 16
George EL, Colvard B, Ho VT, Rothenberg KA, Lee JT, Stern JR. Real-world outcomes of EKOS ultrasound-enhanced catheter-directed thrombolysis for acute limb ischemia. Ann Vasc Surg 2020;66:479-85.  Back to cited text no. 17
Schrijver AM, van Leersum M, Fioole B, Reijnen MM, Hoksbergen AW, Vahl AC, et al. Dutch randomized trial comparing standard catheter-directed thrombolysis and ultrasound-accelerated thrombolysis for arterial thromboembolic infrainguinal disease (DUET). J Endovasc Ther 2015;22:87-95.  Back to cited text no. 18
Schrijver AM, Reijnen MM, van Oostayen JA, Nolthenius RP, van der Valk PH, Hoksbergen AW, et al. Dutch randomized trial comparing standard catheter-directed thrombolysis versus ultrasound-accelerated thrombolysis for thromboembolic infrainguinal disease (DUET): Design and rationale. Trials 2011;12:20.  Back to cited text no. 19
Deitcher SR, Funk WD, Buchanan J, Liu S, Levy MD, Toombs CF. Alfimeprase: A novel recombinant direct-acting fibrinolytic. Expert Opin Biol Ther 2006;6:1361-9.  Back to cited text no. 20
Han SM, Weaver FA, Comerota AJ, Perler BA, Joing M. Efficacy and safety of alfimeprase in patients with acute peripheral arterial occlusion (PAO). J Vasc Surg 2010;51:600-9.  Back to cited text no. 21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2]


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