This study was a cross-sectional, observational study. In this study, three hundred-three patients [mean age: 53 ± 9, 199 (66%) men) who attended the Kangnam Sacred heart Hospital, Hallym University from the April, 2016 to May, 2018. All patients were referred to division of cardiology for evaluation of cardiac embolic source. Patients with age < 65 and with acute stroke, transient ischemic attack (TIA), or retinal vessel occlusion were included. All patients had undergone TTE, carotid ultrasound, ankle-brachial index (ABI), pulse wave velocity (PWV), 24-h-Holter monitoring, and ASE. We also collected participant data on demographic, anthropometric, and laboratory parameters including cardiac biomarkers. Patients with significant valvular diseases, myocardial infarction, atrial fibrillation (AF), intra-cardiac mass (such as thrombi, myoxoma, papillary fibroelastoma, vegetation, et al.), presence of intra-cardiac shunt except PFO, peripheral arterial disease (PAD), significant obstruction (diameter stenosis ≥ 75%) of carotid arteries, or brain hemorrhage were excluded from this study. Study population were sorted into two groups – positive ASE (n = 92) vs. negative ASE (n = 211). In addition, TEE was performed to 72 consenting patients to confirm PFO (51 patients with positive ASE and 21 patients with negative ASE agreed to be performed TEE). We observed whether there was a recurrence of stroke or suspicion of stroke in both groups for up to 2 years.
Echocardiography
TTE was performed using standard techniques with a 2.5-MHz transducer. The standard 2-D and Doppler echocardiography was performed using a commercially available echocardiographic machine (Vivid 7R GE Medical System, Horten, Norway) with the same setup interfaced with a 2.5-MHz phased-array probe. All measurements were performed according to the guideline [11]. With the study participant in the partial left decubitus position and breathing normally, the observer obtained images from the parasternal long and short axes and from the apical four chamber and two-chamber and long-axis views. Depth setting was optimized to display the LV on the screen as large as possible and the same field depth was kept for both four and two-chamber apical views. Sector width was reduced to increase spatial and temporal resolution. Left ventricular end-diastolic dimensions (LV EDD), end-diastolic interventricular septal thickness (IVSTd), and end-diastolic LV posterior wall thickness (PWTd) were measured at end-diastole according to the standards established by the American Society of Echocardiography. LV ejection fraction (EF) was determined by the biplane Simpson’s method. Maximal left atrial (LA) volume was calculated using the Simpson method and indexed to the body surface area. LV mass was calculated using the Devereux formula = 1.04[(LVEDD + IVSTd + PWTd)3 − (LVEDD)3] − 13.6. Thereafter, the LV mass index (LVMI) was calculated and indexed to body surface area.
Mitral flow velocities were recorded in the apical four-chamber view. Mitral inflow measurements included the peak early (E) and peak late (A) flow velocities and the E/A ratio. The tissue Doppler of the mitral annulus movement was also obtained from the apical four-chamber view. A 1.5-mm sample volume was placed sequentially at the septal annular sites. The analysis was performed for early diastolic (E’), late diastolic (A’), and systolic (S’) peak tissue velocities. As a noninvasive parameter for LV stiffness, the LV filling index (E/E’) was calculated by the ratio of transmitral flow velocity to annular velocity. Adequate mitral and tissue Doppler image (TDI) signals were recorded in all patients.
The mean longitudinal global strain (GS) of LV was calculated from the apical 4,3,2-chamber views by speckle-tracking 2D-strain imaging [12].
Agitated Saline Echo (ASE)
Nine millilitre of normal saline agitated with one millilitre of room air, agitated back and forth between two sterile syringes just before intra-venous bolus injection through a forearm vein to detect right to left shunt (RLS) [13]. The injections performed at rest and provocative maneuver (Valsalva maneuver in this study) to increase the right atrial (RA) pressure. The presence of PFO is presumed 3 to 5 cardiac cycles after complete opacification of the RA. We modified the protocol used for shunt grading incorporated 4 grades [8]: grade 1: < 5 bubbles; grade 2: 5 to 25 bubbles; grade 3: > 25 bubbles; grade 4: opacification of chamber to 3 grades: grade 1: < 5 bubbles only after Valsalva maneuver; grade 2: 5 to 25 bubbles both resting and after Valsalva maneuver; grade 3: > 25 bubbles or opacification (Fig. 1).
Carotid ultrasound
A high-resolution B-mode ultrasound (Vivid 7R GE Medical Systems, Horten, Norway) equipped with a 7.5-MHz linear array transducer was used for carotid ultrasonography. In the longitudinal view, carotid intima-media thickness (IMT) was determined as the distance from the media adventitia interface to the intima lumen interface on the far wall in a region free of plaque [14]. The examiner assessed the presence of carotid plaques, which were defined as focal structures that encroached into the lumen by at least 100% of the surrounding IMT value. Common carotid artery IMT (CCA-IMT) was measured between the origin of the carotid bulb and a point 10 mm proximal to the CCA, and the carotid bulb IMT (CB-IMT) was measured in the carotid bulb region. CCA-IMT and CB-IMT values were determined as the average of the maximum IMT of the left and right CCA and CB.
Pulse Wave Velocity (PWV)
We measured PWV to estimate arterial stiffness, which is generally accepted as the most simple, non-invasive, and validated indicator of arterial stiffness [15]. PWV was measured using a VP-2000 automated device (Colin Co., Komaki, Japan). The right and left brachial-ankle PWV (baPWV) were simultaneously measured. The patients were placed in a supine position about 15 min prior to the test. The pressure waveforms of the brachial and tibial arteries were obtained from the occlusion and monitoring cuffs wrapped around the upper arm and lower leg. All measurements were performed in a quiet, temperature-controlled room (22 ± 1 °C), with the patients having fasted overnight. The baseline brachial systolic and diastolic blood pressure (BP), heart rate (HR), and PWV were simultaneously measured.
Statistical analysis
All continuous data are expressed as mean ± SD, and all categorical data are presented as percentage or absolute numbers. Continuous variables were analyzed using Student’s t-test and dichotomous variables were analyzed using the chi square test. Data was statistically analyzed using SPSS version 20, and p-value < 0.05 was considered statistically significant for all the analyses. Significant factors were tested in a univariate binary logistic regression analysis, and then only significant variables were entered in a stepwise multivariate logistic regression analysis to identify the independent predictors for the positive ASE. In addition, multivariate analysis (logistic regression) was performed.
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The local institutional review board approved this study (IRB file No.: HKS 2018–01-014). Informed consent was not obtained from all individual participants included in the study, because this study was a cross-sectional, observational study.