Study Design
Xe-HYPOTHECA trial (ClinicalTrials.gov NCT00879892) was a randomized 2-group single-blinded phase 2 clinical drug trial at two multipurpose intensive care units (ICU) in Finland. The study was approved by the ethics committee of the Hospital District of Southwest Finland and the institutional review boards of the Helsinki University Hospital and the Finnish Medicines Agency. All patients’ next of kin or legal representative gave written informed assent within 4 hours after hospital arrival. Consent was sought from patients when they regained consciousness. As described earlier, an independent data and safety monitoring committee reviewed data after enrolment of every 4 patients and after each 6-month interval [9]. The study was conducted according to good clinical practice and the latest revision of the Declaration of Helsinki. Study design and methodology was consistent with the STARD guidelines for reporting diagnostic accuracy studies [13].
Participants
Consecutive comatose survivors of witnessed OHCA from an initial shockable rhythm admitted to the Turku and Helsinki University hospitals between August 2009 and September 2014 were screened for eligibility. Detailed inclusion and exclusion criteria are listed in eAppendix in the Supplement.
We have previously reported the primary and secondary clinical end points of the Xe-HYPOTHECA trial [9, 14]. The protocol of the Xe-HYPOTHECA trial has also been published [9].
Randomization and Blinding
The patients were allocated in a 1:1 ratio with random block sizes of 4, 6, and 8 to receive either therapeutic hypothermia treatment alone for 24 hours or inhaled xenon (LENOXe, Air Liquide Medical GmbH, Düsseldorf, Germany) in combination with hypothermia for 24 hours as described earlier [9]. The neurological end-point evaluators as well as the patients were blinded to the treatment.
Procedures
MRI imaging was scheduled to be performed within 16 hours of rewarming i.e. 36-52 hours after OHCA. Patients were kept intubated and sedated (with sedation interruptions after completion of rewarming) until brain imaging was performed, regardless of neurological status. A predetermined prognostication protocol (eAppendix in the Supplement) was used to preclude premature decisions to withdraw life-sustaining therapy. DTI and 1H-MRS results were not available at the time of prognostication or at any time during the intensive care. The clinical outcome was evaluated at six months after OHCA with modified Rankin Scale (mRS) by experienced neurologists.
After rewarming was completed sedation interruptions were initiated and performed every 6 to 12 hours throughout intensive care stay. Motor score of the Glasgow Coma Scale was assessed during each sedation interruption by either trained intensive care nurse or on-duty intensive care physicians. NSE serum concentration (Immuno-Electro-Chemi-Luminescent assay, Roche Diagnostics GmbH, Mannheim, Germany) was determined at hospital arrival, and at 24 hours, 48 hours and 72 hours after OHCA. An electroencephalogram was recorded only if it was clinically indicated.
Siemens Magnetom Verio 3T scanner (Siemens Medical Solutions, Erlangen, Germany) with 12-element Head Matrix coil was used in both MRI centers. DTI data were acquired using diffusion weighted spin-echo echo planar imaging (SE-EPI) sequence with 20 diffusion encoding directions (see eTable1 in the Supplement for details).
FSL software library (version 6.0, Analysis Group, FMRIB, Oxford, United Kingdom) was used for processing the DTI images, following the tract-based spatial statistics (TBSS) processing [15, 16]. This observer-independent and hypothesis-free method has the ability to spatially locate group differences in the DTI data. Mean fractional anisotropy value of white matter was calculated as a mean value of all the voxels in the skeleton. (see eMethods in Supplement for details).
1H-MRS data were acquired from the region of basal ganglia by utilizing Chemical Shift Imaging (CSI) technique (see eTable 2 for details). Acquired data were analyzed using the LCModel software (version 6.3-0C) [17]. An average of all analyzed voxels, except the ones containing cerebrospinal fluid (CSF) were selected for the final analysis (see Figure 1). The metabolite concentration values were corrected for relaxation effects (eMethods) but absolute concentration values were not feasible to use. Therefore, the amount of tNAA (total N-acetyl aspartate) and total choline were expressed as ratios over total creatine, i.e. tNAA/tCr and tCho/tCr, as it is expected to remain stable. In addition, apart from the tNAA/tCho ratio, the amount of tNAA and tCho was assessed as these individual parameters are related to neuronal density, activity and integrity [18].
Statistical analysis
The sample size of 110 patients was based on a power analysis of the fractional anisotropy values from brain magnetic resonance imaging, i.e. the primary end-point of the Xe-HYPOTHECA trial [9]. The categorical demographic data and baseline clinical characteristics between groups of mRS 0-2 and mRS 3-6 were compared with chi-square or Fisher’s exact test. Two-sample t-test or Mann-Whitney U-test was used to test the differences in continuous demographic data and baseline clinical characteristics between the groups mRS 0-2 and mRS 3-6. The normality of continuous variables was evaluated visually using histograms. The mean differences in mean fractional anisotropy, 1H-MRS data and NSE at 48 and 72 hours after OHCA between the groups were tested with two-sample t-test. Age-, sex-, treatment-, and site-adjusted mean differences between the groups were compared with analysis of covariance. NSE values were log-transformed for statistical analysis due to positively skewed distribution. The associations of the mean fractional anisotropy, 1H-MRS and NSE values with 6-month mortality were analyzed by using Cox regression analysis after adjustment for age, sex, treatment, and site. The follow-up time for survival analysis was calculated from the time of cardiac arrest until death or 6 months. The observation was censored in the survival analysis if the patient was withdrawn from the study or was still alive at the end of the 6-month follow-up. Permutation-based voxel-wise statistical analysis with tract-based spatial statistics in conjunction with family-wise error correction was used for multiple comparisons across space to obtain group differences in the white matter tracts [9, 15, 16].
The results are expressed using adjusted hazard ratios (HR) with 95% confidence intervals (CI). The prognostic values of fractional anisotropy, tNAA/tCr and NSE 72 hours after OHCA and logistic regression derived combined models including selected clinical variables were evaluated post-hoc by calculating the area under the curve (AUC) of receiver operating characteristic (ROC) curve using a nonparametric method. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each prognostic variable was calculated. Optimal cut-off values were chosen by using the Youden Index (sensitivity+specificity-1).
A 2-sided p-value less than 0.05 was considered statistically significant. Statistical analyses were performed with SAS System for Windows, version 9.4 (SAS Institute Inc., Cary, NC) and SPSS Statistics for Macintosh, version 24 (IBM Corp., Armonk, NY). The hazard ratio plot was created using DistillerSR Forest Plot Generator (Evidence Partners, Ottawa, Canada).