Trial Design

WAKE-UP was an investigator-initiated, multicenter, randomized, double-blind, placebo-controlled clinical trial involving patients with an unknown time of onset of stroke. All the patients otherwise met the clinical criteria for intravenous thrombolysis. Patients could undergo randomization if in the judgment of the investigator MRI showed an acute ischemic lesion on diffusion-weighted imaging but no parenchymal hyperintensity with standard window settings on FLAIR (Fig. S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org).

The trial protocol (also available at NEJM.org) was approved by the national regulatory authority in each participating country. The trial was approved by the national or local ethics committee or institutional review board. Patients or their legal representatives provided written informed consent according to national and local regulations. There was an exception from explicit informed consent in emergency circumstances in some countries, as described previously.13

The trial, which was supported by the European Union Seventh Framework Program, was performed at 70 centers in eight European countries. Sites were selected if they were experienced stroke research centers, had a history of routine use of alteplase in standard stroke care, and could perform MRI for emergency stroke imaging. Investigators were certified by Web-based training on image interpretation and were certified in the use of the clinical examination scales for entry criteria and outcome assessment. A central image-reading committee reviewed all images acquired for patient enrollment, evaluated the decisions of local investigators regarding imaging inclusion and exclusion criteria, and provided feedback on disagreements on these matters to trial sites.

The trial was overseen by a steering committee and an independent data and safety monitoring board. The authors vouch for the accuracy and completeness of the data and adverse event reporting and for the fidelity of the trial to the protocol. There was no industry funding or involvement in any aspect of the trial.

Patients

Patients were eligible if they presented with clinical signs of acute stroke, were 18 to 80 years of age, and had been able to carry out usual activities in their daily life without support before the stroke. The patient either recognized stroke symptoms on awakening or could not report the timing of the onset of symptoms (e.g., as a result of aphasia or confusion). The time that had elapsed since the patient was last known to be well had to be more than 4.5 hours (with no upper limit) in order to exclude patients who otherwise would have fulfilled the standard eligibility criteria for the use of alteplase. Patients underwent MRI examination that included diffusion-weighted imaging, FLAIR, a sequence sensitive to hemorrhage, and time-of-flight magnetic resonance angiography of the circle of Willis. Patients underwent randomization if they had a mismatch between the presence of an abnormal signal on MRI diffusion-weighted imaging and no visible signal change on FLAIR in the region of the acute stroke. Patients were excluded if MRI showed intracranial hemorrhage or lesions larger than one third of the territory of the middle cerebral artery. Also excluded from the trial were patients in whom thrombectomy was planned and those with severe stroke, which was defined as a score of more than 25 on the National Institute of Health Stroke Scale (NIHSS), which ranges from 0 to 42, with higher values indicating a greater neurologic deficit. Patients were also excluded if they had generally recognized contraindications to treatment with alteplase (except for the unknown time of symptom onset).14

Randomization and Treatment

Patients underwent randomization by means of a Web-based procedure with a permuted-block design according to trial center. Patients were assigned in a 1:1 ratio to receive 0.9 mg of alteplase per kilogram of body weight (with 10% administered as a bolus and the remainder by infusion during a 60-minute period) or matching placebo. Randomization was stratified according to age (≤60 or >60 years) and severity of symptoms as assessed on the NIHSS (score, ≤10 or >10). Concomitant treatment and procedures were performed according to the standard of care at each center in compliance with European or national guidelines for acute stroke.14

Clinical and Imaging Assessment

Clinical assessments were performed at baseline, at 22 to 36 hours after randomization, at 5 to 9 days (or at hospital discharge, if earlier), and at 90 days. Assessments included a recording of demographic characteristics, taking of a medical history, evaluation of laboratory values and scores on the NIHSS, an assessment of disability on the basis of the modified Rankin scale (which ranges from 0 [no symptoms] to 6 [death]), a review of concomitant medications, and an assessment of adverse events. Brain MRI was performed at baseline and 22 to 36 hours after randomization to detect intracranial hemorrhage and to assess infarct volume.

Outcome Measures

The primary efficacy end point was a favorable clinical outcome, which was defined as a score of 0 or 1 on the modified Rankin scale 90 days after randomization. Secondary efficacy end points were the ordinal score on the modified Rankin scale at 90 days; the proportion of patients with a treatment response at 90 days (defined as a score on the modified Rankin scale of 0 for patients with an NIHSS score of ≤7, a score of 0 or 1 for patients with an NIHSS score of 8 to 14, and a score of 0 to 2 for patients with an NIHSS score of >14); a global outcome score at 90 days, which was defined as a good outcome on four scales (a score of 0 or 1 on the modified Rankin scale and the NIHSS, a score of 95 to 100 on the Barthel Index [which assesses 10 categories of daily function and ranges from 0 to 100, with higher values indicating better independent function], and a score of 5 on the Glasgow Outcome Scale [which ranges from 1 to 5, with higher values indicating better neurologic recovery]); the 90-day score on the Beck Depression Inventory (which ranges from 0 to 63, with higher scores indicating more severe depressive symptoms); 90-day scores on two EuroQol–5 Dimensions (EQ-5D) scales (including a total score that ranges from 0 to 10, with higher values indicating greater problems across five dimensions of self-care and self-assessment of well-being, and a score on a visual analogue scale that ranges from 0 to 100, with higher scores indicating better health); and infarct volume on MRI 22 to 36 hours after randomization.

The primary safety end points were death and a composite outcome of death or dependence, which was defined as a score of 4 to 6 on the modified Rankin scale at 90 days. Secondary safety end points were symptomatic intracranial hemorrhage causing deterioration in neurologic symptoms2,3,15-17 and the incidence of parenchymal hematoma type 2 (as defined by clots exceeding 30% of the infarct area) on MRI 22 to 36 hours after randomization.15 The central image-reading board assessed the outcome on MRI at 22 to 36 hours in a blinded manner. Symptomatic intracranial hemorrhage was evaluated by a central safety-adjudication committee whose members were unaware of trial-group assignments.

Statistical Analysis

Primary and secondary efficacy outcomes were assessed in the intention-to-treat population, which included all the patients who had undergone randomization. The primary efficacy outcome was determined with the use of an unconditional logistic-regression model fitted to estimate the odds ratio and 95% confidence interval.18 The secondary efficacy outcome of the score on the modified Rankin scale was analyzed by fitting a proportional-odds logistic-regression model to calculate the common odds ratio as a measure of the likelihood that alteplase would lead to lower scores on the modified Rankin scale than would placebo (shift analysis). The global outcome score was analyzed with the use of a global estimate of the odds ratio (Wald-type test) from generalized estimation equations based on a linear logistic-regression model. Odds ratios and common odds ratios were adjusted for stratification factors of age and symptom severity at randomization. Analyses of secondary outcomes were not corrected for multiple comparisons, so the results cannot be used for hypothesis testing or inference. Post hoc Bonferroni-corrected results of secondary outcome analyses were calculated for reference with the P values of secondary outcomes. Missing data in the primary outcome analysis were imputed with the use of multiple imputation techniques (five imputed data sets),18 with prediction based on trial group, baseline age, baseline NIHSS score, and NIHSS score measured on day 7. Analyses were performed with a two-sided alpha level of 0.05.

The same analyses were repeated in the per-protocol population after the exclusion of all the patients who had major protocol violations. Safety end points were assessed in the safety population with the use of an unconditional logistic-regression model to estimate the odds ratio and 95% confidence intervals associated with treatment effect, after adjustment for the stratification factors used at randomization. In the safety population, patients who received any amount of alteplase were assigned to the alteplase group; all other patients who received any amount of placebo were assigned to the placebo group. The sample-size calculation was based on pooled data from trials of thrombolysis for the treatment of stroke,19 with an expected absolute between-group difference of 10 percentage points in the proportion of patients with the primary efficacy outcome. We calculated that 370 patients per group would be required to provide a power of 80% to show the expected treatment effect. Taking into account possible protocol violations and dropouts, we intended to enroll 800 patients (400 per trial group).