Recruitment of participants
The aim was to include 15 to 20 males and females, all employed (past and present) at our hospital, per age decade from 30 to 80 years. An invitation was sent to 250 potential participants. All had to give informed written consent in order to be given an appointment for a screening visit. A total of 195 persons came to this visit, which included a 2DE examination to exclude subclinical cardiac abnormalities such as significant valvular disease (stenosis or aortic-/mitral regurgitation >1/3) or apparent abnormalities of cardiac chambers. 29 individuals were excluded for various reasons, including; antihypertensive use (n = 14), new diagnosis of hypertension (repeated blood pressure > 160/90 mmHg, n = 3), malignant disease (n = 2), a history of coronary heart disease (n = 1), treatment for arrhythmia (n = 1), congenital heart disease (n = 1), diabetes mellitus (n = 1), poor image quality (n = 1), left bundle branch block (n = 1), large atrial septal aneurysm (n = 1), chronic pericardial effusion (n = 1). Two subjects who had given their written consent did not attend the screening. BSA was calculated according to the DuBois et DuBois formula [7].
Ethics
The study was approved by both the Regional Ethics Committee of the South-Eastern Norway Regional Health Authority and the Norwegian Social Science Data Service.
Echocardiographic examinations
All examinations were performed using a Philips IE 33® with 3D QLab advanced software (version 6) installed. According to the manufacturer this software was modified to include both left ventricular and atrial volumes as well as RV volume calculations.
2DE measurements of RV areas (A) in end-diastole (ED) and end-systole (ES) were established from the apical 4-chamber view, with simple planimetry allowing the calculation of RV area fraction (AF). Harmonic RT3DE imaging was performed using a ×3-matrix array transducer with the participant in the left lateral decubitus position. A wide-angled "full-volume" acquisition mode, in which wedge-shaped subvolumes are obtained over 4–5 consecutive cardiac cycles, was used during held respiration in end-expirium. Both an apical 4-chamber view of the RV and an orthogonal view were extracted from the pyramidal dataset in the same manner as applied for LV and atrial recordings [8]. Five anatomic landmarks were then manually initialized, including two points to identify the tricuspid valve annulus in each of the two apical views and one point to identify the apex in either view. Following manual identification of these points, the program automatically identified the 3D endocardial surface using a deformable shell model.
The orthogonal view, however, was not obtainable in all participants. In addition, the automatic border detection occasionally included various parts of the right atrium as well. The criteria for a successful recording were both a clear delineation of the RV cavity in both apical views and the exclusion of the right atrium. If that could not be obtained within three attempts then the participant was excluded from the study. Adjustments of the automatic surface detection, in order to include all trabeculae in the RV cavity that could be visualized, were done without exception in the two apical views. End-diastolic volume (EDV) was automatically computed from voxel counts. Thereafter, end-systole (ES) was selected by identifying the frame with the smallest RV dimension. Surface detection, including initialization and editing, was repeated on this frame to obtain the ESV. RVEF was calculated from these volumes.
Figure 1 presents RV in ED and figure 2 RV in ES, and demonstrate the two apical views, the short axis view, the resulting 3D model of the RV cavity obtained, and the-time volume curve. The performance of these measurements took approximately five to six minutes per participant.
Reproducibility studies
A: Blinded study
Two to four weeks after the initial study a repeated 2DE and RT3DE examination was performed on 20 participants (7 males and 13 females) selected at random. One examiner did all 2DE recordings (EA) and another all RT3DE recordings (JEO). Since we wanted to accurately reflect the scenario as in daily clinical practice, the repeated study was done blindly and without access to previous images or results. With both methods care was taken to include trabeculae in the RV cavity. This study only allowed us to evaluate intraobserver variability for the two investigators. In order to replicate clinical practice the presented 2DE data are taken from only one heartbeat during both examinations.
B. Unblinded study
This study was performed in order to optimize the reproducibility with both methods. 22 participants were picked at random (9 females and 13 males). All had given their informed consent to undergo two new separate echocardiographic examinations which were performed within a mean interval of five days. None of these persons had participated in the blinded study.
RT3DE
The same investigator (JEO) did all examinations and measurements. RV endocardial tracings, after manual editing from the first examination, were available during the second examination. Since only one investigator had been properly trained to carry out this method, interobserver variability data could not be obtained.
2DE
These examinations and measurements were performed by the same investigator of the RT3DE recordings (JEO). Data were transferred to an EchoPAC® analysis system (GE Healthcare). Offline RV tracings were performed by both investigators (JEO and EA) in order to obtain both intra- and interobserver variability. Loops and tracings from the first examination were available during the second, and all values presented are the mean of three heartbeats. To obtain the single plane measurements with three manual tracings per variable took approximately 5–10 minutes.
Statistical analysis
Paired and unpaired t tests were used for comparison of continuous data between groups of subjects. Two-tailed p-values below 0.05 were considered statistically significant. Upper normal limit was calculated as mean +2 standard deviations (SD) and lower normal limit as mean -2SD. Pearson's correlation was used for analyses on relationship between volumes and age.
The intra- and interobserver variability for repeated RT3DE and 2DE measurements has been expressed as coefficient of variability (CV). The CV was calculated as the SD of the differences divided by the mean of the parameter under consideration [9]. All analyses were implemented using SPSS® 16.0 (SPSS Inc, Chicago, IL).