Journal of Stroke 2020;22(1):130-140 https://doi.org/10.5853/jos.2019.01648 Original Article Timing of Recanalization and Functional Recovery in Acute Ischemic Stroke Georgios Tsivgoulis,a,b,c,* Maher Saqqur,d,e,* Vijay K. Sharma,f Alejandro Brunser,g,h Jürgen Eggers,i,j Robert Mikulik,k,l Aristeidis H. Katsanos,b,m Theodore N. Sergentanis,n Konstantinos Vadikolias,c Fabienne Perren,o Marta Rubiera,p Reza Bavarsad Shahripour,a,q Huy Thang Nguyen,r Patricia Martínez-Sánchez,s,t Apostolos Safouris,b,u Ioannis Heliopoulos,c Ashfaq Shuaib,d Carol Derksen,d Konstantinos Voumvourakis,b Theodora Psaltopoulou,n Anne W. Alexandrov,a Andrei V. Alexandrov,a,q CLOTBUST-PRO investigators a Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA b Second Department of Neurology, Attikon University Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece c Department of Neurology, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis, Greece d Division of Neurology, Department of Medicine, University of Alberta, Edmonton, AB, Canada e Department of Neuroscience, Hamad General Hospital, Doha, Qatar f Division of Neurology, National University Hospital, Yong Loo Lin School of Medicine, National University of Singapore, Singapore g Vascular Neurology Program, Neurology Service, Department of Medicine, Clinica Alemana of Santiago, University of Desarrollo, Santiago, Chile h Department of Emergency Medicine, Clinica Alemana of Santiago, University of Desarrollo, Santiago, Chile i Department of Neurology, University Hospital Schleswig-Holstein, Campus Lubeck, Lübeck, Germany j Department of Neurology, Sana Hospital Lubeck, Lübeck, Germany k Neurology Department and International Clinical Research Center, St. Anne’s Hospital, Brno, Czech Republic l Medical Faculty, Masaryk University, Brno, Czech Republic m Department of Neurology, University of Ioannina School of Medicine, Ioannina, Greece n Department of Hygiene, Epidemiology, and Medical Statistics, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece o Stroke Unit, Department of Neurological Sciences, LUNIC Laboratory, HUG, University Hospital and Medical Faculty of Geneva, Geneva, Switzerland p Stroke Unit, Department of Neurology, Vall d’Hebron University Hospital, Vall d’Hebron Research Institute, Barcelona, Spain q Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, USA r Department of Cerebrovascular Disease, 115 The People Hospital, Ho Chi Minh, Vietnam s Department of Neurology and Stroke Center, IdiPAZ Health Research Institute, La Paz University Hospital, Autonomous University of Madrid, Madrid, Spain t Torrecardenas Hospital, University of Almeria School of Health Sciences, Almeria, Spain u Stroke Unit, Department of Neurology, Brugmann University Hospital, Brussels, Belgium Background and Purpose Although onset-to-treatment time is associated with early clinical Correspondence: Andrei V. Alexandrov Department of Neurology, University of recovery in acute ischemic stroke (AIS) patients treated with intravenous tissue plasminogen Tennessee Health Science Center, 855 activator (tPA), the effect of the timing of tPA-induced recanalization on functional outcomes Monroe Avenue, Suite 415, Memphis, TN 38163, USA remains debatable. Tel: +1-901-4486199 Methods We conducted a multicenter, prospective observational cohort study to determine Fax: +1-901-4487440 E-mail: avalexandrov@att.net whether early (within 1-hour from tPA-bolus) complete or partial recanalization assessed during 2-hour real-time transcranial Doppler monitoring is associated with improved outcomes in patients Received: June 28, 2019 with proximal occlusions. Outcome events included dramatic clinical recovery (DCR) within 2 and Revised: September 23, 2019 Accepted: September 23, 2019 24-hours from tPA-bolus, 3-month mortality, favorable functional outcome (FFO) and functional independence (FI) defined as modified Rankin Scale (mRS) scores of 0–1 and 0–2 respectively. *These authors contributed equally to the manuscript as first author. Results We enrolled 480 AIS patients (mean age 66±15 years, 60% men, baseline National Copyright © 2020 Korean Stroke Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 130 http://j-stroke.org pISSN: 2287-6391 • eISSN: 2287-6405 Vol. 22 / No. 1 / January 2020 Institutes of Health Stroke Scale score 15). Patients with early recanalization (53%) had significantly (P<0.001) higher rates of DCR at 2-hour (54% vs. 10%) and 24-hour (63% vs. 22%), 3-month FFO (67% vs. 28%) and FI (81% vs. 39%). Three-month mortality rates (6% vs. 17%) and distribution of 3-month mRS scores were significantly lower in the early recanalization group. After adjusting for potential confounders, early recanalization was independently associated with higher odds of 3-month FFO (odds ratio [OR], 6.19; 95% confidence interval [CI], 3.88 to 9.88) and lower likelihood of 3-month mortality (OR, 0.34; 95% CI, 0.17 to 0.67). Onset to treatment time correlated to the elapsed time between tPA-bolus and recanalization (unstandardized linear regression coefficient, 0.13; 95% CI, 0.06 to 0.19). Conclusions Earlier tPA treatment after stroke onset is associated with faster tPA-induced recanalization. Earlier onset-to-recanalization time results in improved functional recovery and survival in AIS patients with proximal intracranial occlusions. Keywords Thrombolysis; Stroke; Reperfusion; Outcomes Introduction Methods Intravenous thrombolysis (IVT) with tissue plasminogen activa- Study design tor (tPA) remains the only approved systemic therapy for acute We conducted this prospective, open-label, observational co- ischemic stroke (AIS), with onset-to-treatment time being di- hort study in 12 participating tertiary care stroke centers in rectly associated with improved short-term and long-term North America, South America, Europe, and Asia. The rationale clinical outcomes.1-3 Since recanalization following IVT is di- for the study, design details, and eligibility criteria have been rectly associated with early clinical recovery,4 it is logical to as- previously published.9 Exclusion criteria together with other sume that faster recanalization is the mechanism why earlier detail on the methodology of our trial are available in the Sup- treatment with tPA can lead to improved 3-month functional plementary Material. outcomes.5-8 However, the potential correlation between onset- to-tPA treatment time and elapsed time between tPA-bolus Study population and beginning of recanalization has not been established yet. We included patients with AIS attributable to a proximal intra- Furthermore, the influence of timing of tPA-induced recanali- cranial occlusion eligible for IVT, who presented within 4.5 zation on functional outcomes and risk of symptomatic intra- hours to the participating sites.9 The study was approved by the cerebral hemorrhage (sICH) in AIS remains debatable. Ethics Committees and the Institutional Review Boards of par- In view of these considerations, we designed the ‘PROspec- ticipating Institutions as previously described. All patients or tive multi-national Combined Lysis of Thrombus in Brain Isch- their substitute decision-makers provided written consent prior emia Using Transcranial Ultrasound and Systemic tPA (CLOT- to participation.9 BUST) collaboration on reperfusion therapies for stroke’ (CLOT- BUST-PRO)9 that sought to determine whether early recanali- Insonation protocol zation (within 1-hour from tPA-bolus), assessed with real-time All eligible patients received continuous 2-MHz TCD or tran- transcranial Doppler (TCD) monitoring, is independently associ- scranial color-coded duplex (TCCD) assessment of recanaliza- ated with improved 3-month outcomes in AIS patients with tion up to 2 hours following tPA-bolus by experienced sonog- proximal intracranial arterial occlusions. We also investigated raphers. The duration of continuous TCD monitoring varied be- the association between the elapsed time between symptom tween 60 and 120 minutes, because if there was no ultrasono- onset and tPA-bolus with the elapsed time between tPA-bolus graphic evidence of recanalization within 60 minutes from and beginning of recanalization. We hypothesized the early tPA tPA-bolus and the patient was considered by the treating phy- delivery may result to swift recanalization which in turn may sician as a candidate for additional revascularization therapies translate into better clinical outcomes. (intra-arterial thrombolysis or mechanical thrombectomy), TCD monitoring was discontinued and an additional TCD evaluation was performed at the end of endovascular reperfusion thera- https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 131 Tsivgoulis et al. CLOTBUST-PRO pies. This therapeutic approach towards endovascular reperfu- was evaluated at 3-month by the on-site investigators (certified sion therapies was based on the lack of available randomized- in the assessment of mRS) who were unaware of TCD monitoring controlled clinical trial data supporting the clinical efficacy of findings. All radiological examinations were independently eval- mechanical thrombectomy at the time of study design and pa- uated by institutional radiologists/neuroradiologists who were tient enrollment (2008 to 2014). blinded to both TCD findings and patient clinical data. In patients with clinical deterioration following initial im- provement within the first 24 hours from symptom onset, an Statistical analyses additional TCD recording was performed to assess the presence We enrolled 480 patients to identify at least 120 cases of early of delayed re-occlusion. complete recanalization and have power of 82.4% to detect a TCD findings were interpreted using previously validated cri- statistically significant difference of at least 15% in FI rates at teria, including the thrombolysis in brain ischemia (TIBI) flow 3-month between patients with early recanalization and per- grade definitions.10,11 sisting occlusion/delayed recanalization.5 Dichotomous vari- ables were presented with their absolute values and corre- Outcomes sponding percentages, while continuous variables were pre- The main hypothesis and primary efficacy outcome of the sented as mean values with their corresponding standard devi- study was to determine whether early tPA-induced partial or ations for normally distributed data, or median values with complete recanalization, achieved during the first hour of TCD their corresponding interquartile ranges (IQRs) when distribu- monitoring following tPA-bolus, was independently associated tions were skewed. We compared baseline characteristics and with more favorable early and long term clinical outcomes. The outcomes between patients with complete or partial recanali- primary outcome of interest was the difference in the favor- zation and those with persisting occlusion with the Pearson’s able functional outcome (FFO; defined modified Rankin Scale chi-square or Mann-Whitney-Wilcoxon test where appropri- [mRS] score of 0–1 at 3-month) rates in patients with early ate. The distributions on the mRS-score between groups at complete or partial recanalization compared to those with per- 3-month follow-up were compared with the Cochran-Mantel- sisting occlusion at 1-hour TCD monitoring. The exact time of Haenszel test, as previously described.13 the beginning, duration, timing and amount of recanalization Univariable and multivariable logistic regression analyses were and re-occlusion were recorded for all patients. Beginning of performed to evaluate the associations between baseline char- recanalization was marked as the time point in which at least acteristics and outcomes (sICH, 3-month FFO and mortality). We 1 point improvement in TIBI score was noticed during the TCD also performed additional sensitivity analyses after dichotomiz- monitoring.12 A table summarizing the definitions of the afore- ing time from symptom onset to beginning of recanalization us- mentioned outcomes is provided in the Supplementary Table 1. ing the variable’s observed median value as threshold (>178 and Early clinical outcome was assessed with the rates of dra- ≤178 minutes) and persisting occlusion set as reference category. matic clinical recovery (DCR), defined by a change of equal or Only the factors proven significant (P<0.05) at the univariable more than 10 points in the National Institutes of Health Stroke analyses were entered into the multivariable models. We also Scale (NIHSS) or total NIHSS score of less or equal to 3 within performed a post hoc subgroup analysis by including only pa- 2 and 24 hours after tPA-bolus.11 Finally, we prospectively fol- tients with middle cerebral artery (MCA) occlusions. lowed all patients up to 3 months to record also functional in- Apart from the analysis in the total sample, a nested analysis dependence (FI; defined as 3-month mRS of 0–2) and deaths (univariable and multivariable logistic regression analysis) was from any cause during the 3-month follow-up period. performed in the subgroup of patients with sustained complete sICH with clinical worsening (≥4 NIHSS points) that in the or partial early recanalization at the end of 2-hour TCD-moni- opinion of the treating physician was causatively and tempo- toring. The same approach was followed as in the total sample, rally related to neurological deterioration during the first 24 with the exception of time from symptom onset to the begin- hours following tPA infusion,9 was considered as the primary ning of recanalization, which was treated in increments of 30 safety endpoint. minutes. In this subgroup of patients, the potential correlations For all patients, serial assessments on NIHSS scores were ob- between onset-to-treatment time (elapsed time between tained at baseline (pre-tPA-bolus), within 10 minutes of the be- symptom onset and tPA-bolus) and baseline characteristics, in- ginning of recanalization or re-occlusion, at the end of TCD cluding elapsed time between tPA-bolus and beginning of re- monitoring and at 24-hour after symptom onset by vascular canalization, were assessed with simple and multiple regres- neurologists certified in the NIHSS scoring.9 Functional outcome sion analyses, adjusting for potential confounders. Statistical 132 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 analyses were performed with STATA/SE version 13 (Stata Corp, Table 1. Baseline characteristics and outcomes of study population (n=480) according to early recanalization status achieved during the first hour of College Station, TX, USA). TCD-monitoring Delayed Data availability statement Early complete recanalization or partial De-identified patient dataset will be made available upon rea- Variable or Persisting P recanalization occlusion sonable request from the corresponding author. (n=251) (n=229) Baseline characteristic Results Age (yr) 63±14 69±15 <0.001* Male sex 162 (65) 127 (55) 0.048† The predetermined sample size of 480 AIS patients (mean age NIHSS-score (point) 14 (9–19) 15 (10–20) 0.112* 66±15 years, 60% men) with proximal intracranial occlusions, Hypertension 164 (65) 161 (71) 0.217† who underwent continuous 2-hour TCD-monitoring following Diabetes mellitus 60 (24) 56 (25) 0.867† tPA-bolus, was enrolled during a 7-year period (January 2008 Atrial fibrillation 69 (27) 76 (33) 0.174† to December 2014) from 12 participating tertiary care stroke Hypercholesterolemia 108 (43) 78 (35) 0.073† centers from North America (n=2), South America (n=1), Eu- Coronary artery disease 64 (25) 55 (24) 0.707† rope (n=7), and Asia (n=2). The complete list of participating Baseline TIBI score 0–1 138 (55) 113 (49) 0.217† centers is available in the Supplementary Material. The median Isolated ICA or tandem 25 (10) 43 (19) 0.006† admission NIHSS-score was 15 points (IQR, 10 to 19) and the ICA/MCA occlusion median onset to treatment time was 140-minute (IQR, 116 to Systolic blood pressure 160±26 154±26 0.022* 170). Baseline characteristics of the study population are pre- before tPA-bolus (mm Hg) sented in Supplementary Table 2. The distribution of proximal intracranial occlusions was as follows: M1-MCA 48% (n=231), Diastolic blood pressure 90±15 85±16 <0.001* before tPA-bolus M2-MCA 29% (n=141), terminal internal carotid artery (ICA) (mm Hg) 9% (n=44), tandem ICA/MCA 5% (n=24), basilar artery 7% Serum glucose before 130±53 135±47 0.140* (n=61), intracranial vertebral artery 0.2% (n=1), and posterior tPA-bolus (mg/dL) cerebral artery 1% (n=6). No patient was lost during the Time from symptom on- 135 (110–165) 145 (120–180) 0.002* set to tPA-bolus (min) 3-month follow-up. Endovascular reperfusion 4 (2) 8 (3) 0.183‡ Complete or partial recanalization was detected in 180 therapies (38%) and 71 (15%) patients at 1-hour TCD monitoring, re- Outcome spectively, and in 211 (44%) and 90 (19%) patients at the Symptomatic intracere- 6 (2) 13 (6) 0.065 † 2-hour TCD-monitoring point, respectively. Sustained complete bral hemorrhage or partial recanalization was achieved in 201 (42%) and 85 Dramatic clinical recovery 129 (54) 21 (10) <0.001† (18%) patients, respectively at the end of 2-hour TCD-monitor- at 2-hr§ ing, while re-occlusion was documented in 18 cases (4%). The Dramatic clinical recovery 155 (63) 46 (22) <0.001† at 24-hr∥ median onset to recanalization and the median bolus to reca- 3-mo favorable function- 169 (67) 63 (28) <0.001† nalization times were 178-minute (IQR, 145 to 214) and al outcome mRS 0–1 24-minute (IQR, 22 to 53), respectively. Comparisons of the 3-mo functional inde- 203 (81) 89 (39) <0.001† baseline characteristics between patients with persisting occlu- pendence mRS 0–2 sion or any (partial or complete) recanalization within the first 3-mo mortality 16 (6) 39 (17) <0.001† hour of TCD monitoring are presented in Table 1. Patients with 3-mo mRS score 1 (0–2) 3 (1–5) <0.001¶ complete or partial early recanalization during the first hour of Values are presented as mean±standard deviation, number (%), or median TCD-monitoring as compared to patients with persisting occlu- (interquartile range). TCD, transcranial Doppler; NIHSS, National Institutes of Health Stroke sion/delayed recanalization were younger (63±14 years vs. Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; 69±15 years, P<0.001), more likely to be men (65% vs. 55%, MCA, middle cerebral artery; tPA, tissue plasminogen activator; mRS, mod- P=0.048) and had lower prevalence of isolated ICA or tandem ified Rankin Scale. *P-value derived from Mann-Whitney-Wilcoxon test for independent sam- ICA/MCA occlusion (10% vs. 19%, P=0.006). Patients with early ples; †P-value derived from Pearson’s chi-square; ‡P-value derived from Fish- recanalization had shorter median onset-to-treatment times er’s exact test; §Missing data on 2-hour NIHSS for 27 patients; ∥Missing (135-minute vs. 145-minute, P=0.002), higher pre-treatment data on 24-hour NIHSS for 24 patients; ¶P-value derived from Cochran– Mantel–Haenszel test. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 133 Tsivgoulis et al. CLOTBUST-PRO systolic (160±26 mm Hg vs. 154±26 mm Hg, P=0.022), and di- onset to recanalization time (178 minutes). More specifically, a astolic blood pressure levels (90±15 mm Hg vs. 85±16 mm Hg, more pronounced beneficial effect on 3-month FFO was docu- P<0.001). sICH rates were not different between patients with mented in patients with initiation of recanalization within the early complete or partial recanalization and patients with per- first 178 minutes after symptom onset (OR [vs. the reference sisting occlusion (2% vs. 6%, P=0.065). Patients with early re- group of persisting occlusion], 14.70; 95% CI, 7.90 to 27.33; canalization had significantly (P<0.001) higher rates of DCR at P<0.001) (Table 4) compared to patients with a more delayed re- 2-hour (54% vs. 10%) and 24-hour (63% vs. 22%), 3-month canalization after 178 minutes from symptom onset (OR [vs. the FFO (67% vs. 28%) and 3-month FI (81% vs. 39%). Three- reference group of persisting occlusion], 6.72; 95% CI, 3.78 to month mortality rates (6% vs. 17%) and distribution of 11.92; P<0.001) (Table 4). An independent association with lower 3-month mRS scores (1 [0 to 2] vs. 3 [1 to 5]) were significant- likelihood of 3-month mortality was detected for both groups of ly lower in the early recanalization subgroup. The respective early (≤178-minute from symptom onset; OR, 0.37; 95% CI, 0.16 comparisons of the baseline characteristics and outcomes be- to 0.85; P=0.018) or delayed (>178-minute from symptom on- tween patients with persisting occlusion and those with any set; OR, 0.22; 95% CI, 0.10 to 0.51; P<0.001) recanalization ver- (partial or complete) recanalization within 2 hours of TCD mon- sus persisting occlusions (Table 5). In the subgroup analysis of itoring are available in Supplementary Table 3. patients with MCA occlusions (n=374) similar differences in the In multivariable analyses adjusting for potential confounders, outcomes of interest were documented between patients with early complete or partial recanalization within the first hour early complete or partial recanalization compared to patients after tPA-bolus was independently associated with higher odds with delayed recanalization or persisting occlusion (Supplemen- of 3-month FFO (odds ratio [OR], 6.19; 95% confidence inter- tary Table 4). This subgroup analysis confirmed the associations val [CI], 3.88 to 9.88; P<0.001) (Table 2) and lower likelihood of documented in our primary analysis including all patients. 3-month mortality (OR, 0.34; 95% CI, 0.17 to 0.67; P=0.002) In the nested subgroup analysis (Supplementary Table 5) of (Table 3) compared to delayed recanalization or persisting oc- patients with sustained complete or partial recanalization at clusion. the end of 2-hour TCD-monitoring (n=301), an independent These associations were reproduced in our sensitivity analyses association was detected between increasing elapsed time stratifying patients with complete or partial recanalization from symptom onset to the beginning of recanalization and during the 2-hour TCD monitoring by the median of symptom reduced odds of 3-month FFO (OR per 30-minute increase, Table 2. Association of early (within 1-hour from tPA-bolus) recanalization with 3-month favorable functional outcome in the study population (n=480) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 0.96 (0.95–0.97) <0.001 0.97 (0.95–0.99) 0.001 Male sex 1.64 (1.13–2.37) 0.009 1.70 (1.06–2.71) 0.027 NIHSS-score (/1 point increase) 0.86 (0.83–0.89) <0.001 0.85 (0.82–0.89) <0.001 Hypertension 0.64 (0.44–0.95) 0.026 1.18 (0.71–1.99) 0.518 Diabetes mellitus 0.83 (0.54–1.26) 0.372 - Atrial fibrillation 0.73 (0.49–1.07) 0.108 - Hypercholesterolemia 1.08 (0.75–1.56) 0.687 - Coronary artery disease 0.98 (0.65–1.48) 0.913 - Baseline TIBI score 0–1 0.64 (0.45–0.92) 0.015 1.05 (0.64–1.72) 0.842 Isolate ICA or tandem ICA/MCA occlusion 0.30 (0.17–0.55) <0.001 0.53 (0.26–1.09) 0.083 Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.96 (0.90–1.03) 0.275 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.98 (0.87–1.11) 0.749 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 0.96 (0.93–1.00) 0.050 - Time from symptom onset to tPA-bolus (/10 min increase) 0.97 (0.93–1.01) 0.128 - Early recanalization 5.43 (3.67–8.04) <0.001 6.19 (3.88–9.88) <0.001 Endovascular reperfusion therapies 2.18 (0.65–7.33) 0.209 - - tPA, tissue plasminogen activator; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery. 134 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Table 3. Association of early (within 1-hour from tPA-bolus) recanalization with 3-month mortality in the study population (n=480) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 1.05 (1.03–1.08) <0.001 1.04 (1.01–1.06) 0.003 Male sex 0.65 (0.37–1.14) 0.131 - NIHSS-score (/1 point increase) 1.16 (1.11–1.22) <0.001 1.16 (1.10–1.22) <0.001 Hypertension 1.07 (0.58–1.96) 0.834 - Diabetes mellitus 1.47 (0.79–2.72) 0.220 - Atrial fibrillation 1.79 (1.00–3.17) 0.048 1.16 (0.60–2.24) 0.649 Hypercholesterolemia 0.85 (0.47–1.54) 0.592 - Coronary artery disease 1.72 (0.94–3.12) 0.078 - Baseline TIBI score 0–1 1.20 (0.68–2.12) 0.521 - Isolated ICA or tandem ICA/MCA occlusion 2.35 (1.20–4.59) 0.013 1.32 (0.63–2.77) 0.462 Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.93 (0.84–1.04) 0.195 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.89 (0.74–1.07) 0.221 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 1.02 (0.96–1.07) 0.536 - Time from symptom onset to tPA-bolus (/10 min increase) 1.00 (0.94–1.06) 0.923 - Early recanalization 0.33 (0.18–0.61) <0.001 0.34 (0.17–0.67) 0.002 Endovascular reperfusion therapies Not estimable* >0.999 † - tPA, tissue plasminogen activator; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery. *Not estimable due to lack of events (deaths) in the subgroup of patients receiving endovascular treatment; †P-value derived from Fisher’s exact test. Table 4. Association of elapsed time between symptom onset and beginning of recanalization with 3-month favorable functional outcome in the study popu- lation (n=480) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 0.96 (0.95–0.97) <0.001 0.97 (0.95–0.99) 0.002 Male sex 1.64 (1.13–2.37) 0.009 1.55 (0.96–2.52) 0.076 NIHSS-score (/1 point increase) 0.86 (0.83–0.89) <0.001 0.85 (0.81–0.89) <0.001 Hypertension 0.64 (0.44–0.95) 0.026 1.33 (0.77–2.31) 0.303 Diabetes mellitus 0.83 (0.54–1.26) 0.372 - Atrial fibrillation 0.73 (0.49–1.07) 0.108 - Hypercholesterolemia 1.08 (0.75–1.56) 0.687 - Coronary artery disease 0.98 (0.65–1.48) 0.913 - Baseline TIBI score 0–1 0.64 (0.45–0.92) 0.015 1.21 (0.73–2.01) 0.460 Isolate ICA or tandem ICA/MCA occlusion 0.30 (0.17–0.55) <0.001 0.57 (0.27–1.21) 0.143 Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.96 (0.90–1.03) 0.275 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.98 (0.87–1.11) 0.749 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 0.96 (0.93–1.00) 0.050 - Time from symptom onset to tPA-bolus (/10 min increase) 0.97 (0.93–1.01) 0.128 - Endovascular reperfusion therapy 2.18 (0.65, 7.33) 0.209 - - Time from symptom onset to beginning of recanalization Persisting occlusion (reference category) ≤178 min 13.23 (7.82–22.39) <0.001 14.70 (7.90–27.33) <0.001 Persisting occlusion (reference category) >178 min 5.55 (3.39–9.10) <0.001 6.72 (3.78–11.92) <0.001 CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cere- bral artery; tPA, tissue plasminogen activator. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 135 Tsivgoulis et al. CLOTBUST-PRO Table 5. Association of elapsed time between symptom onset and beginning of recanalization with 3-month mortality in the study population (n=480) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 1.05 (1.03–1.08) <0.001 1.04 (1.01–1.06) 0.002 Male sex 0.65 (0.37–1.14) 0.131 - NIHSS-score (/1 point increase) 1.16 (1.11–1.22) <0.001 1.16 (1.10–1.23) <0.001 Hypertension 1.07 (0.58–1.96) 0.834 - Diabetes mellitus 1.47 (0.79–2.72) 0.220 - Atrial fibrillation 1.79 (1.00–3.17) 0.048 1.21 (0.62–2.34) 0.574 Hypercholesterolemia 0.85 (0.47–1.54) 0.592 - Coronary artery disease 1.72 (0.94–3.12) 0.078 - Baseline TIBI score 0–1 1.20 (0.68–2.12) 0.521 - Isolate ICA or tandem ICA/MCA occlusion 2.35 (1.20–4.59) 0.013 1.26 (0.59–2.70) 0.546 Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.93 (0.84–1.04) 0.195 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.89 (0.74–1.07) 0.221 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 1.02 (0.96–1.07) 0.536 - Time from symptom onset to tPA-bolus (/10 min increase) 1.00 (0.94–1.06) 0.923 - Endovascular reperfusion therapy Not estimable* >0.999† - - Time from symptom onset to beginning of recanalization Persisting occlusion (reference category) ≤178 min 0.28 (0.14–0.59) 0.001 0.37 (0.16–0.85) 0.018 Persisting occlusion (reference category) >178 min 0.26 (0.12–0.55) 0.001 0.22 (0.10–0.51) <0.001 CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cere- bral artery; tPA, tissue plasminogen activator. *Not estimable due to lack of events (deaths) in the subgroup of patients receiving endovascular treatment; †P-value derived from Fisher’s exact test. 1 0.84; 95% CI, 0.73 to 0.97; P=0.015) (Figure 1). Moreover, among all baseline characteristics only onset-to-treatment Probability of favourable functional outcome time correlated positively to the elapsed time between tPA-bo- 0.8 lus and beginning of recanalization (unstandardized linear re- gression coefficient, 0.13; 95% CI, 0.06 to 0.19; P<0.001) (Fig- 0.6 ure 2) in the subgroup of patients with complete or partial re- canalization at the end of TCD-monitoring (Supplementary Ta- 0.4 ble 6). In a post hoc sensitivity analysis after adjusting for pre- defined potential confounders (baseline age, NIHSS score, presence of tandem occlusion, and endovascular treatment), a 0.2 similar correlation of elapsed time from symptom onset to 0 100 200 300 400 Time between symptom onset and the beginning of recanalization (min) tPA-bolus with elapsed time from tPA-bolus to the beginning of recanalization documented (unstandardized linear regres- Figure 1. Frequency of favorable functional outcome by elapsed time be- sion coefficient, 0.13; 95% CI, 0.06 to 0.20; P<0.001). tween symptom onset and the beginning of recanalization, as predicted by unadjusted analysis in patients with sustained complete or partial recanali- We detected no association between elapsed time from symp- zation at the end of transcranial Doppler-monitoring (n=301). The recana- tom onset and beginning of recanalization with sICH in multi- lization cohort (including patients with complete or partial sustained re- variable analyses of the study population (Supplementary Table canalization) was divided into groups of about 30 on the basis of deciles in the time to recanalization. The red circles show the proportion with favor- 7) and in the subgroup of patients with sustained complete or able functional outcome and mean time to recanalization in each group, partial recanalization (n=301) at the end of TCD-monitoring and the red horizontal lines depict the range of time included in that group. The solid curve shows the model results from the logistic regression analy- (Supplementary Table 8). Three-month mortality was not associ- sis, with 95% confidence interval prediction bands shown in dashed curves. ated with the timing of tPA-induced recanalization in the group Favorable functional outcome was defined as a modified Rankin Scale of patients with sustained complete or partial recanalization at score of ≤1. the end of 2-hour TCD-monitoring (Supplementary Table 9). 136 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 150 vessel recanalization, in the era of mechanical thrombectomy it could only be useful in case of other imaging unavailability, Time from tPA bolus to recanalization onset (min) and should not delay any systemic or endovascular therapies 100 when employed for clinical or research purposes. We detected sustained complete early recanalization in 42% of patients within 2 hours after tPA-bolus and DCR in 50% at 50 the end of TCD-monitoring. Although the rates of tPA-induced recanalization are higher in our cohort than those reported for 0 patients with large vessel occlusions (LVOs) following treat- ment with systemic thrombolysis,14,15 it should be highlighted that they are in accordance with the results from the CLOT- 0 100 200 300 Time from stroke onset to tPA bolus (min) BUST trial,11 reporting sustained complete recanalization in 46% and DCR in 44% of the patients receiving tPA administra- Figure 2. Correlation of elapsed time between symptom onset and tissue tion coupled with continuous TCD-monitoring.11 Our findings plasminogen activator (tPA)-bolus (onset to treatment time) with elapsed time between tPA-bolus to the beginning of recanalization in patients with are also in line with a longitudinal cohort of consecutive AIS sustained complete or partial recanalization at the end of transcranial patients treated with IVT that reported sustained recanaliza- Doppler-monitoring (n=301). The 95% confidence interval (CI) forecast tion in 51% of the study population at the end of the 2-hour bands are also presented. The correlation between the two time intervals was statistically significant (unstandardized linear regression coefficient, TCD-monitoring.16 Finally, the sICH rate in our population (4%) 0.13; 95% CI, 0.06 to 0.19; P<0.001). was comparable to the treatment arm of the recently pub- lished multicenter, double-blind, phase 3, randomized con- Discussion trolled trial on the safety and efficacy of 2 hours continuous transcranial ultrasound insonation coupled with tPA treatment Our prospective observational study showed that faster tPA-in- in AIS patients (3%), providing further evidence of no increase duced recanalization represents the link between shorter on- in the sICH risk with TCD insonation.17 set-to-treatment time and improved 3-month functional out- The timing of tPA-induced recanalization after stroke onset comes in AIS patients with proximal intracranial occlusions. was a strong predictor of clinical outcomes in the whole study Shorter onset-to-tPA-bolus time was associated with more population and among patients with sustained recanalization at prompt recanalization because of shorter tPA-bolus-to-recan- the end of TCD-monitoring. This observation is corroborated by alization time. Elapsed time between symptom onset and be- the findings of a retrospective, single-center study of 508 AIS ginning of recanalization was an independent predictor of patients treated with IVT (54% with proximal intracranial occlu- 3-month functional outcome among patients who achieved sions).18 The investigators reported an inverse relationship be- sustained early complete or partial recanalization. Finally, de- tween the probability of early recanalization, evaluated by TCD layed recanalization was not related to the risk of sICH in the at 1-hour following the end of tPA infusion and elapsed time whole study population. Our study supports ultra-fast delivery from symptom onset.18 However, no association between onset- of tPA after patient arrival to hospital. Taking also into account to-treatment and tPA-bolus to initiation of recanalization time that the vast majority of complete or partial vessel recanaliza- was found.18 The inverse relationship between onset-to-treat- tions were found to occur within 1-hour from tPA-bolus and ment time and the rate of tPA-induced recanalization has also that very few patients recanalized after 1-hour from tPA-bolus, been identified in another study of 102 AIS patients with proxi- our results also provide unflinching support the strategy of not mal intracranial occlusions treated with IVT.19 Early recanaliza- delaying endovascular procedures in cases of persisting occlu- tion at the end of alteplase infusion (1-hour after tPA-bolus) was sion after tPA administration. Therefore, our results on the as- determined using magnetic resonance angiography (MRA). The sociation of shorter onset-to-treatment time with faster in- investigators of this study reported that tPA administration duced recanalization and improved 3-month functional out- within 130 minutes from symptom onset was independently as- comes, should not only apply for systemic tPA treatment, but sociated with a 3-fold higher likelihood of complete recanaliza- also for endovascular therapies and align with the continuous tion at the end of tPA infusion.19 Finally, our results are also in efforts for reduction of onset-to-groin puncture and groin accordance with an experimental study protocol supporting that puncture-to recanalization times. Although TCD can be used as early administration of tPA after stroke onset is independently a very reliable tool for continuous, real-time assessment of associated with better thrombus resolution.20 https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 137 Tsivgoulis et al. CLOTBUST-PRO The main study finding, that the beneficial effect of tPA-in- monitoring was discontinued and they were treated with addi- duced recanalization on clinical outcomes of AIS patients with tional revascularization therapies, if available at the participat- proximal intracranial occlusions are time-dependent (16% de- ing centers. Although the number of patients receiving addi- crease in the likelihood of FFO for every 30-minute delay in the tional endovascular reperfusion therapies was limited (2% of elapsed time between symptom onset and initiation of recana- the study population), the duration of monitoring was not lization) parallel the recent experience with mechanical throm- consistent among the whole study population. Finally, determi- bectomy in AIS patients with LVOs.21-27 Both randomized-con- nation of recanalization status using TCD-monitoring is chal- trolled and observational data have highlighted a time depen- lenging and highly operator-dependent. Although no central dency of the beneficial effect of endovascular reperfusion adjudication of TCD recording was available, it should be noted therapies in AIS due to LVO.21-27 More specifically, in a pre- that the centers participating in CLOTBUST-PRO recruited specified analysis of the Interventional Management of Stroke skilled and experienced sonographers implementing predefined III (IMS III) trial, every 30-minute delay in angiographic reper- ultrasound criteria that have been shown to accurately (>80%) fusion of AIS patients with LVO was associated with a 12% predict complete recanalization in real-time compared to digi- decrease in the likelihood of 3-month FI on multivariable anal- tal subtraction angiography.29 Moreover, we do not have data yses adjusting for potential confounders.26 Similarly, the Highly on the imaging modalities used to screen proximal intracranial Effective Reperfusion Evaluated in Multiple Endovascular occlusions and therefore we were not able to assess potential Stroke Trials (HERMES) collaborators have recently reported discrepancies between TCD and computed tomography angiog- that each 1-hour delay to reperfusion was associated with a raphy/MRA findings. 19% reduction in the odds of 3-month FI among LVO patients On the other hand, certain strengths of the present study who achieved reperfusion following mechanical thrombectomy may also be acknowledged. CLOTBUST-PRO represents a pro- and standard therapy.27 spective, international adequately powered observational study To the best of our knowledge, CLOTBUST-PRO is the first that sought to assess and establish the association of the prospective, international multicenter observational study, with speed of tPA-induced recanalization with shorter symptom- adequate statistical power, a predefined protocol and no miss- onset-to-treatment time using a predefined previously pub- ing follow-up evaluation at 3 months, which provides real- lished protocol.9 To the best of our knowledge, we consider time information on arterial recanalization dependency with that this association represents the causative mechanism that shorter symptom-onset-to-treatment time linking it to accounts for the improved functional outcomes documented 3-month functional outcomes. The correlation between earlier with earlier tPA delivery.1,2 The former association has previ- onset to treatment time and shorter elapsed time between ously been established in AIS patients receiving endovascular tPA-bolus and initiation of recanalization has not been previ- reperfusion therapies21-27 but has never been documented in ously reported. Our results provide further support to the asso- AIS patients receiving systemic thrombolysis, since TCD repre- ciation of ultra-early tPA administration with favorable out- sents the only imaging modality that can assess recanalization comes3 and highlight the potential role of mobile stroke units in real-time in patients receiving treatment with IVT.30 in prompt tPA administration in this ultra-early time window.28 Certain limitations of the present study need to be acknowl- Conclusions edged. First, per study protocol, no screening log was provided from the participating centers and thus the percentage of ex- In conclusion, our observational cohort study provides observa- cluded patients, with corresponding reasons for exclusion (e.g., tional evidence that faster tPA delivery is associated with earlier insufficient temporal window or tPA contraindications) is not tPA-induced recanalization, which in turn results in improved available. However, it should be highlighted that included pa- functional recovery and survival in AIS patients with proximal tients from all institutions were consecutive and thus we con- intracranial occlusions. Our study further underscores the need sider the possibility of selection bias negligible. Second, we to arrange acute stroke services in ways that shortest possible evaluated arterial recanalization only within the first 2 hours onset-to-arrival and arrival-to-tPA-bolus time be achieved. of treatment initiation. However, per our study protocol all pa- tients experiencing clinical deterioration during the first 24 Supplementary materials hours from symptom onset received an additional TCD record- ing. Third, in patients with no ultrasonographic evidence of Supplementary materials related to this article can be found successful recanalization at 60-minute after tPA initiation, TCD online at https://doi.org/10.5853/jos.2019.01648. 138 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Disclosure 10. Demchuk AM, Burgin WS, Christou I, Felberg RA, Barber PA, Hill MD, et al. Thrombolysis in brain ischemia (TIBI) transcra- The authors have no financial conflicts of interest. nial Doppler flow grades predict clinical severity, early recov- ery, and mortality in patients treated with intravenous tissue Acknowledgments plasminogen activator. Stroke 2001;32:89-93. 11. Alexandrov AV, Molina CA, Grotta JC, Garami Z, Ford SR, Alva- Robert Mikulik has been supported by the project no. LQ1605 rez-Sabin J, et al. Ultrasound-enhanced systemic thrombolysis from the National Program of Sustainability II (MEYS CR) and by for acute ischemic stroke. N Engl J Med 2004;351:2170-2178. the project FNUSA-ICRC no. CZ.1.05/1.1.00/02.0123 (OP VaVpI). 12. Demchuk AM, Christou I, Wein TH, Felberg RA, Malkoff M, Grot- ta JC, et al. Accuracy and criteria for localizing arterial occlusion References with transcranial Doppler. J Neuroimaging 2000;10:1-12. 13. Saver JL, Gornbein J. Treatment effects for which shift or bi- 1. Hacke W, Donnan G, Fieschi C, Kaste M, von Kummer R, nary analyses are advantageous in acute stroke trials. Neu- Broderick JP, et al. Association of outcome with early stroke rology 2009;72:1310-1315. treatment: pooled analysis of ATLANTIS, ECASS, and NINDS 14. Campbell BCV, Mitchell PJ, Churilov L, Yassi N, Kleinig TJ, rt-PA stroke trials. 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The golden 35 min of stroke intervention with emic stroke in mobile stroke unit and hospital settings. ADAPT: effect of thrombectomy procedural time in acute isch- Stroke 2018;49:1996-1999. emic stroke on outcome. J Neurointerv Surg 2018;10:213-220. 29. Tsivgoulis G, Ribo M, Rubiera M, Vasdekis SN, Barlinn K, 25. Khatri P, Yeatts SD, Mazighi M, Broderick JP, Liebeskind DS, Athanasiadis D, et al. Real-time validation of transcranial Demchuk AM, et al. Time to angiographic reperfusion and Doppler criteria in assessing recanalization during intra-ar- clinical outcome after acute ischaemic stroke: an analysis of terial procedures for acute ischemic stroke: an international, data from the Interventional Management of Stroke (IMS III) multicenter study. Stroke 2013;44:394-400. phase 3 trial. Lancet Neurol 2014;13:567-574. 30. Tsivgoulis G, Alexandrov AV. Ultrasound in neurology. Continu- 26. Khatri P, Abruzzo T, Yeatts SD, Nichols C, Broderick JP, Tom- um (Minneap Minn) 2016;22(5, Neuroimaging):1655-1677. 140 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Supplementary Material travenous thrombolysis (IVT); (8) patient or substitute decision- maker refusal to give informed consent to participate in the study; (9) patients treated directly with endovascular therapies List of participating institutions (without receiving tissue plasminogen activator [tPA] preatrea- 1. Department of Neurology, University of Alabama at Birming- ment); and (10) patients in whom tPA was initiated in other set- ham, Birmingham, AL, USA tings before being transferred to the participating in the current 2. Second Department of Neurology, Attikon University Hospi- study institutions (“drip and ship” patients). Concomitant severe tal, School of Medicine, National and Kapodistrian University stenosis or occlusion of the proximal internal carotid artery (ICA), of Athens, Athens, Greece established by either carotid duplex ultrasonography, magnetic 3. Department of Neurology, University of Alberta, Edmonton, resonance angiography, CT angiography, or digital subtraction AB, Canada angiography of the neck vessels was not an exclusion criterion 4. Division of Neurology, National University Hospital, Yong Loo and did not preclude patient enrollment into the study as long as Lin School of Medicine, National University of Singapore, patient has proximal intracranial occlusion on their baseline di- Singapore agnostic TCD. 5. Neurology Service, Department of Medicine, Clinica Alemana Arterial occlusion at baseline TCD assessment was defined as of Santiago, University of Desarrollo, Santiago, Chile residual thrombolysis in brain ischemia (TIBI) flow grades 0–3, 6. Department of Neurology, Sana Hospital Lubeck, Lübeck, complete recanalization was diagnosed if flow improved to TIBI Germany grades 4–5 and partial recanalization was identified if flow im- 7. Neurology Department and International Clinical Research proved by one grade or more from the baseline but not to Center, St. Anne’s Hospital, Brno, Czech Republic grades 4–5 on the TIBI scale. Patients with re-occlusion follow- 8. Department of Neurology, University Hospital of Alexandrou- ing IVT were diagnosed, if after an improvement by at least one polis, Democritus University of Thrace, Alexandroupolis, TIBI flow grade during TCD monitoring the residual flow wors- Greece ens and an arterial occlusion is diagnosed at the end of moni- 9. Stroke Unit, Department of Neurological Sciences, LUNIC toring with TIBI grades 0–3. In patients with concomitant and Laboratory, HUG, University Hospital and Medical Faculty of persisting severe stenosis or occlusion of the proximal ICA, Geneva, Geneva, Switzerland complete recanalization of the middle cerebral artery (MCA) 10. Stroke Unit, Department of Neurology, Brugmann University was considered if TCD showed low-resistance waveforms (TIBI Hospital, Brussels, Belgium 2 or greater) over both M1 and M2 segments, with an im- 11. Department of Cerebrovascular Disease, 115 The People Hos- provement in mean flow velocity to more than 20 cm/seconds. pital, Ho Chi Minh, Vietnam The following baseline characteristics were included as can- 12. Department of Neurology and Stroke Center, IdiPAZ Health didate variables in logistic regression models: age (per 1-year Research Institute, La Paz University Hospital, Autonomous increase), male sex, National Institutes of Health Stroke Scale University of Madrid, Madrid, Spain score (per 1-point increase), hypertension, diabetes mellitus, atrial fibrillation, hypercholesterolemia, coronary artery disease, Supplementary methods baseline TIBI score 0–1, isolated ICA or tandem ICA/MCA oc- Per study protocol, we excluded from further evaluation patients clusion, systolic blood pressure before tPA-bolus (per 10 mm with: (1) hypodensity on computed tomography (CT) scan with Hg increase), diastolic blood pressure before tPA-bolus (per 10 unknown stroke onset time; (2) acute intracranial hemorrhage mm Hg increase), mean serum glucose before tPA-bolus (per (ICH) on baseline CT examination; (3) history of ICH or subarach- 10 mg/dL increase), endovascular reperfusion therapies (intra- noid hemorrhage; (4) history of intracerebral malignancy; (5) ab- arterial thrombolysis and/or mechanical thrombectomy), time sent temporal windows on pre-bolus transcranial Doppler (TCD) from symptom onset to tPA-bolus (per 10-minute increase), examination; (6) pre-treatment systolic blood pressure equal or and early (within 1-hour from tPA-bolus) recanalization. The more than 185 mm Hg or diastolic blood pressure equal or more variables that were significantly (P<0.05) associated with the than 110 mm Hg not responding to antihypertensive therapy; (7) outcome events in the univariable analyses were selected for other standard institutional or regional contra-indications to in- inclusion in the multivariable models. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 1 Tsivgoulis et al. CLOTBUST-PRO Supplementary Table 1. Outcome definitions from the transcranial Doppler monitoring Outcome variable Definition Early recanalization Complete or partial recanalization within the first 60 minutes following tPA-bolus Delayed recanalization Complete or partial recanalization within the time interval between 60–120 minutes following tPA-bolus Sustained recanalization Complete or partial recanalization at 120 minutes following tPA-bolus Persisting occlusion Lack of any recanalization within 120 minutes following tPA-bolus Timing of recanalization Elapsed time between symptom onset to beginning of recanalization Beginning of recanalization The time point that at least 1 point improvement in TIBI score is noticed during the TCD monitoring tPA, tissue plasminogen activator; TIBI, thrombolysis in brain ischemia; TCD, transcranial Doppler. Supplementary Table 2. Baseline characteristics of the whole study population (n=480) Variable Value Age (yr) 66±15 Male sex 289 (60) NIHSS-score (points) 15 (10–19) Hypertension 325 (70) Diabetes mellitus 116 (24) Atrial fibrillation 145 (30) Hypercholesterolemia 186 (39) Coronary artery disease 119 (25) Baseline TIBI score 0–1 251 (52) Isolated ICA or tandem ICA/MCA occlusion 68 (14) Systolic blood pressure before tPA-bolus (mm Hg) 157±26 Diastolic blood pressure before tPA-bolus (mm Hg) 87±16 Serum glucose before tPA-bolus (mg/dL) 133±51 Time from symptom onset to tPA-bolus (min) 140 (116–170) Endovascular reperfusion therapy 12 (2) Values are presented as mean±standard deviation, number (%), or median (interquartile range). NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery; tPA, tissue plasminogen activator. 2 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Supplementary Table 3. Baseline characteristics and outcomes of study population (n=480) according to recanalization status during the 2-hour TCD-moni- toring Complete or partial recanalization Persisting occlusion Variable P (n=301) (n=179) Baseline characteristic Age (yr) 64±14 69±15 <0.001* Male sex 196 (65) 93 (52) 0.005† NIHSS-score (point) 14 (9–19) 16 (10–20) 0.013* Hypertension 196 (65) 129 (73) 0.096† Diabetes mellitus 68 (23) 48 (27) 0.280† Atrial fibrillation 87 (29) 58 (32) 0.420† Hypercholesterolemia 126 (42) 60 (35) 0.123† Coronary artery disease 78 (26) 41 (23) 0.460† Baseline TIBI score 0–1 155 (52) 96 (54) 0.650† Isolated ICA or tandem ICA/MCA occlusion 30 (10) 38 (21) 0.001† Systolic blood pressure before tPA-bolus (mm Hg) 159±26 154±27 0.085* Diastolic blood pressure before tPA-bolus (mm Hg) 89±15 85±16 0.001* Serum glucose before tPA-bolus (mg/dL) 130±52 136±48 0.104* Time from symptom onset to tPA-bolus (min) 135 (113–168) 145 (120–172) 0.138* Endovascular reperfusion therapy 6 (2) 6 (3) 0.356† Outcome Symptomatic intracerebral hemorrhage 8 (3) 11 (6) 0.058† Dramatic clinical recovery at 2-hr ‡ 143 (50) 7 (4) <0.001† Dramatic clinical recovery at 24-hr§ 181 (61) 20 (12) <0.001∥ 3-mo favorable functional outcome, mRS 0–1 199 (66) 33 (18) <0.001† 3-mo functional independence, mRS 0–2 238 (79) 54 (30) <0.001† 3-mo mortality 19 (6) 36 (20) <0.001† 3-mo mRS score 1 (0–2) 4 (2–5) <0.001¶ Values are presented as mean±standard deviation, number (%), or median (interquartile range). TCD, transcranial Doppler; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia, ICA, internal carotid artery; MCA, middle ce- rebral artery; tPA, tissue plasminogen activator; mRS, modified Rankin Scale. *P-value derived from Mann-Whitney-Wilcoxon test for independent samples; †P-value derived from Pearson’s chi-square test; ‡Missing data on 2-hour NI- HSS for 27 patients; §Missing data on 24-hour NIHSS for 24 patients; ∥P-value derived from Fisher’s exact test; ¶P-value derived from Cochran–Mantel– Haenszel test. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 3 Tsivgoulis et al. CLOTBUST-PRO Supplementary Table 4. Baseline characteristics and outcomes of patients with middle cerebral artery occlusion (n=374) according to early recanalization status achieved during the first hour of TCD-monitoring Early complete or partial recanalization Delayed recanalization or Variable P (n=195) Persisting occlusion (n=179) Baseline characteristic Age (yr) 63±14 69±15 <0.001* Male sex 125 (64) 92 (52) 0.015† NIHSS-score (point) 13 (9–18) 14 (9–18) 0.625* Hypertension 128 (66) 121 (68) 0.632† Diabetes mellitus 43 (22) 39 (22) 0.867† Atrial fibrillation 54 (28) 63 (35) 0.174† Hypercholesterolemia 81 (41) 49 (28) 0.008† Coronary artery disease 51 (26) 42 (24) 0.548† Baseline TIBI score 0–1 103 (53) 74 (41) 0.026† Systolic blood pressure before tPA-bolus (mm Hg) 160±26 150±25 <0.001* Diastolic blood pressure before tPA-bolus (mm Hg) 90±15 83±15 <0.001* Serum glucose before tPA-bolus (mg/dL) 129±55 133±47 0.532* Time from symptom onset to tPA-bolus (min) 135 (110–160) 145 (120–180) 0.008* Endovascular reperfusion therapies 3 (2) 3 (2) 0.916‡ Outcome Symptomatic intracerebral hemorrhage 5 (3) 12 (7) 0.055 † Dramatic clinical recovery at 2-hr§ 98 (53) 19 (11) <0.001† ∥ Dramatic clinical recovery at 24-hr 123 (64) 37 (23) <0.001† 3-mo favorable functional outcome, mRS 0–1 136 (70) 54 (30) <0.001† 3-mo functional independence, mRS 0–2 164 (84) 78 (44) <0.001† 3-mo mortality 10 (5) 27 (15) <0.001† 3-mo mRS score 1 (0–2) 3 (1–5) <0.001¶ Values are presented as mean±standard deviation, number (%), or median (interquartile range). TCD, transcranial Doppler; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia, tPA, tissue plasminogen activator; mRS, modified Rankin Scale. *P-value derived from Mann-Whitney-Wilcoxon test for independent samples; †P-value derived from Pearson’s chi-square; ‡P-value derived from Fisher’s ex- act test; §Missing data on 2-hour NIHSS for 23 patients; ∥Missing data on 24-hour NIHSS for 19 patients; ¶P-value derived from Cochran–Mantel–Haenszel test. 4 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Supplementary Table 5. Association of elapsed time between symptom onset and beginning of recanalization with 3-month favorable functional outcome in patients with sustained complete or partial recanalization at the end of TCD-monitoring (n=301) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 0.96 (0.94–0.98) <0.001 0.97 (0.95–0.99) 0.007 Male sex 1.24 (0.75–2.03) 0.399 - NIHSS-score (/1 point increase) 0.86 (0.82–0.90) <0.001 0.87 (0.82–0.91) <0.001 Hypertension 0.75 (0.45–1.25) 0.265 - Diabetes mellitus 1.01 (0.57–1.79) 0.972 - Atrial fibrillation 0.64 (0.38–1.07) 0.086 - Hypercholesterolemia 0.93 (0.57–1.51) 0.775 - Coronary artery disease 0.83 (0.48–1.42) 0.491 - Baseline TIBI score 0–1 0.61 (0.37–0.99) 0.044 1.27 (0.70–2.31) 0.431 Isolate ICA or tandem ICA/MCA occlusion 0.35 (0.16–0.76) 0.007 0.56 (0.23–1.35) 0.194 Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.92 (0.83–1.01) 0.066 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.84 (0.71–1.00) 0.050 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 0.98 (0.94–1.03) 0.387 - Time from symptom onset to tPA-bolus (/10 min increase) 0.95 (0.90–1.00) 0.052 - Endovascular reperfusion therapies 2.60 (0.30–22.58) 0.385 Time from symptom onset to beginning of recanalization (/30 min 0.83 (0.73–0.94) 0.003 0.84 (0.73–0.97) 0.015 increase) TCD, transcranial Doppler; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery; tPA, tissue plasminogen activator. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 5 Tsivgoulis et al. CLOTBUST-PRO Supplementary Table 6. Association of baseline characteristics with the elapsed time between tPA-bolus and start of recanalization in patients with sus- tained complete or partial recanalization at the end of TCD-monitoring (n=301) Univariable analysis Multivariable analysis Variable Unstandardized linear Unstandardized linear P P regression coefficient (95% CI) regression coefficient (95% CI) Age (/1 yr increase) 0.19 (–0.02 to 0.41) 0.080 - - Male sex –0.78 (–7.32 to 5.77) 0.816 - - NIHSS-score (/1 point increase) –0.02 (–0.50 to 0.45) 0.924 - - Hypertension 1.82 (–4.72 to 8.36) 0.585 - - Diabetes mellitus –3.13 (–10.56 to 4.29) 0.407 - - Atrial fibrillation 3.50 (–3.37 to 10.37) 0.317 - - Hypercholesterolemia –1.54 (–7.86 to 4.78) 0.632 - - Coronary artery disease –0.31 (–7.41 to 6.78) 0.931 - - Baseline TIBI score 0–1 –2.30 (–8.53 to 3.92) 0.467 - - Isolate ICA or tandem ICA/MCA occlusion 7.52 (–2.82 to 17.86) 0.153 - - Systolic blood pressure before tPA-bolus (/10 mm Hg increase) –0.60 (–1.70 to 0.60) 0.351 - - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) –0.40 (–2.50 to 1.70) 0.719 - - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 0.36 (–0.22 to 0.96) 0.225 - - Endovascular reperfusion therapies 7.57 (–10.73 to 25.87) 0.416 - - Elapsed time between stroke onset and tPA-bolus (min) 0.13 (0.06 to 0.19) <0.001 0.13 (0.06 to 0.19) <0.001 tPA, tissue plasminogen activator; TCD, transcranial Doppler; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery. 6 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Supplementary Table 7. Association of elapsed time between symptom onset and beginning of recanalization with symptomatic intracranial hemorrhage in the study population (n=480) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 1.02 (0.99–1.05) 0.219 - Male sex 2.55 (0.83–7.79) 0.102 - NIHSS-score (per 1 point increase) 1.06 (0.99–1.14) 0.084 - Hypertension 0.81 (0.31–2.09) 0.656 - Diabetes mellitus 1.88 (0.72–4.89) 0.196 - Atrial fibrillation 1.07 (0.40–2.87) 0.894 - Hypercholesterolemia 0.18 (0.04–0.81) 0.026 0.11 (0.02–0.62) 0.012 Coronary artery disease 0.80 (0.26–2.47) 0.701 - Baseline TIBI score 0–1 2.03 (0.76–5.43) 0.159 - Isolate ICA or tandem ICA/MCA occlusion 0.33 (0.04–2.49) 0.280 - Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 1.08 (0.90–1.29) 0.400 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 1.01 (0.75–1.37) 0.929 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 1.10 (1.04–1.17) 0.002 1.14 (1.06–1.22) <0.001 Time from symptom onset to tPA-bolus (/10 min increase) 1.08 (0.98–1.19) 0.103 - Endovascular reperfusion therapies 2.27 (0.28–18.57) 0.444 - - Time from symptom onset to beginning of recanalization Persisting occlusion (reference category) ≤178 min 0.42 (0.13–1.34) 0.142 - Persisting occlusion (reference category) >178 min 0.42 (0.13–1.36) 0.149 - CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cere- bral artery; tPA, tissue plasminogen activator. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 7 Tsivgoulis et al. CLOTBUST-PRO Supplementary Table 8. Association of elapsed time between symptom onset and beginning of recanalization with symptomatic intracranial hemorrhage in patients with sustained complete or partial recanalization at the end of TCD-monitoring (n=301)* Univariable analysis Variable Odds ratio (95% CI) P Age (/1 yr increase) 1.03 (0.98–1.09) 0.244 Male sex 3.87 (0.47–31.91) 0.208 NIHSS-score (/1 point increase) 1.09 (0.98–1.20) 0.100 Hypertension 0.88 (0.21–3.76) 0.865 Diabetes mellitus 2.10 (0.49–9.00) 0.320 Atrial fibrillation 0.81 (0.16–4.10) 0.801 Hypercholesterolemia 0.19 (0.02–1.57) 0.124 Coronary artery disease 1.74 (0.41–7.44) 0.458 Baseline TIBI score 0–1 2.88 (0.57–14.50) 0.200 Isolate ICA or tandem ICA/MCA occlusion Not estimable† >0.999‡ Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 1.02 (0.78–1.33) 0.909 Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.96 (0.60–1.54) 0.879 Mean serum glucose before tPA-bolus (/10 mg/dL increase) 1.05 (0.95–1.16) 0.338 Time from symptom onset to tPA-bolus (/10 min increase) 1.09 (0.95–1.26) 0.231 Time from symptom onset to beginning of recanalization (/30 min increase) 1.20 (0.87–1.67) 0.270 Endovascular reperfusion therapies Not estimable † >0.999‡ TCD, transcranial Doppler; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery; tPA, tissue plasminogen activator. *No multivariable analysis was performed as no since no significant (P<0.05) predictors were identified at the univariable analyses; †Not estimable due to lack of events (symptomatic intracranial hemorrhage) in the subgroups of patients with isolate ICA or tandem ICA/MCA occlusion or those receiving endovascular treatment; ‡P-value derived from Fisher’s exact test. 8 http://j-stroke.org https://doi.org/10.5853/jos.2019.01648 Vol. 22 / No. 1 / January 2020 Supplementary Table 9. Association of elapsed time between symptom onset and beginning of recanalization with 3-month mortality in patients with sus- tained complete or partial recanalization at the end of TCD-monitoring (n=301) Univariable analysis Multivariable analysis Variable Odds ratio (95% CI) P Odds ratio (95% CI) P Age (/1 yr increase) 1.11 (1.05–1.16) <0.001 1.08 (1.03–1.14) 0.003 Male sex 0.92 (0.35–2.41) 0.862 - NIHSS-score (/1 point increase) 1.21 (1.12–1.31) <0.001 1.18 (1.08–1.28) <0.001 Hypertension 0.71 (0.28–1.83) 0.483 - Diabetes mellitus 1.63 (0.60–4.47) 0.342 - Atrial fibrillation 1.47 (0.56–3.86) 0.439 - Hypercholesterolemia 0.79 (0.30–2.08) 0.638 - Coronary artery disease 2.77 (1.08–7.08) 0.034 2.35 (0.82–6.75) 0.114 Baseline TIBI score 0–1 1.31 (0.51–3.35) 0.576 - Isolate ICA or tandem ICA/MCA occlusion 2.62 (0.81–8.46) 0.109 - Systolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.92 (0.77–1.10) 0.364 - Diastolic blood pressure before tPA-bolus (/10 mm Hg increase) 0.94 (0.68–1.30) 0.692 - Mean serum glucose before tPA-bolus (/10 mg/dL increase) 1.01 (0.92–1.10) 0.884 - Time from symptom onset to tPA-bolus (/10 min increase) 0.99 (0.90–1.10) 0.871 - Time from symptom onset to beginning of recanalization (/30 min 1.00 (0.79–1.27) 0.994 - increase) Endovascular reperfusion therapies Not estimable* >0.999† - TCD, transcranial Doppler; CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; TIBI, thrombolysis in brain ischemia; ICA, internal carotid artery; MCA, middle cerebral artery; tPA, tissue plasminogen activator. *Not estimable due to lack of events (deaths) in the subgroup of patients receiving endovascular treatment; †P-value derived from Fisher’s exact test. https://doi.org/10.5853/jos.2019.01648 http://j-stroke.org 9