This prospective cohort feasibility study was conducted on patients admitted to the 33-bed intensive care unit at a tertiary care academic/teaching hospital. Our local Health Sciences Research Ethics Board approved the study protocol.
Inclusion and exclusion criteria
Following informed consent from the substitute decision maker, we prospectively enrolled 11 consecutive patients who met the inclusion and exclusion criteria, which were similar to previous studies [8, 9]. Briefly, eligible patients were 18 years and older whom spontaneous circulation was restored <60 min following witnessed cardiac arrest due to ventricular fibrillation (VF) or pulseless ventricular tachycardia (VT). We also included patients who had shocks delivered by an automatic external defibrillator, as presumably, these patients had VT or VF. We chose to focus on this cohort of cardiac arrest patients, as the underlying etiology tends to be myocardial ischemia. In contrast, patients who have asystole or pulseless electrical activity may be more heterogeneous in nature. For example, we did not wish to include patients who had a hypoxemic cardiac arrest due to pneumonia and sepsis, as the underlying sepsis syndrome would likely alter their proteomic profile. Patients needed to be enrolled within 6 h of the cardiac arrest. Exclusion criteria included pregnancy, hypothermia on admission (tympanic temperature <30 °C), response to verbal commands following restoration of spontaneous circulation (i.e., the patient was not comatose), persistent hypotension despite fluids/pressors (mean arterial pressure (MAP) <60 mmHg) or hypoxia (O2 saturation <85 %) for more than 30 min after return of spontaneous circulation, a terminal illness that preceded the cardiac arrest, and poor baseline functional status (defined as modified Cerebral Performance Category (CPC) ≥3) prior to the cardiac arrest.
Eligible patients were treated with targeted temperature management using surface cooling as soon as possible. This included ice packs and a commercial cooling blanket. Target core temperature (32–34 °C) was maintained for 24 h, followed by passive rewarming. The hemodynamic parameters of each patient were managed at the discretion of the treating intensivist. Coronary revascularization procedures were also performed at the discretion of the treating attending intensivist and consultant interventional cardiologist.
Clinical data collection
After an informed consent was obtained from the substitute decision maker, each enrolled patient was assigned a case response form. Basic demographic and health history was collected. During the patients’ ICU stay, we recorded clinical data, outcomes, and physiological parameters, including heart rate, mean arterial pressure, and temperature (measured rectally while the patient was undergoing therapeutic hypothermia, orally if patient was awake). To determine the patients’ 3-month outcome, the CPC was used, in line with other investigations of neurologic recovery after cardiac arrest . The CPC is a 5-point scale, which ranges from 1 = none, or mild cerebral disability, 2 = moderate cerebral disability, 3 = severe cerebral disability, 4 = comatose, and 5 = dead. The CPC was dichotomized into good (CPC 1–2) or poor (CPC 3–5) outcomes as done previously [8, 9]. The CPC for each patient was determined either by telephone interview or chart review by the one of the study investigators (J.G.B.).
Sample collection and proteomic separation with 2D-GE
Whole blood was collected at the time of admission to the ICU (within 6 h of cardiac arrest) and at 24 h. It was immediately transported to the core laboratory for centrifugation and isolation of serum using standard protocols. Serum was stored at −80 °C until proteomic assessment. To create a unique protein fingerprint, proteins were separated using 2D-GE as previously described [10, 11]. Briefly, equal quantities of serum proteins were loaded onto a pH (3–11) gradient strip overnight and then ran in an isoelectric focusing machine to separate the serum proteins as per their isoelectric focusing point. The immobilized pH gradient (IPG) strips containing protein were separated on a 12 % acrylamide gel at 4 °C overnight, washed, and then silver stained. A second analysis was performed on the serum after high abundance proteins were depleted using a commercially available high-performance liquid chromatography-based column according to the manufacturer’s instructions (Multiple Affinity Removal Column, Human 14; Agilent Technologies, Canada). This column depletes serum of the following high abundance proteins: albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin.
For non-depleted serum samples, MALDI-TOF MS was used to identify proteins of interest from the 2D gels as previously described . To increase the ability to detect smaller quantities of protein in the depleted samples, triple quadrupole/linear ion trap MS was performed using the QTRAP 5500 System (Sciex, Canada) according to standard protocols. The data files were submitted to Mascot for the protein library search.
To determine if there were significant differences between hemodynamic parameters between patients with good vs. poor neurological outcome, t tests were used. Results were considered significant if p < 0.05. Due to the feasibility nature of this project, no correction was performed for multiple comparisons, as all results are considered exploratory.