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Use of Strain Imaging to Predict Myocardial Function Recovery after Percutaneous Revascularization of Infarct Related Artery 


Mohamed SHEHATA1 MD, Walaa ADEL1 MD, Sherief MANSOUR1 MD, Sameh SAMIR1 MD, Nagwa ELMAHALAWY1 MD

1Department of Cardiology, Faculty of Medicine, Ain Shams University, Cairo, Egypt






Corresponding & first author:
Mohamed Abdelsamie Shehata, MD
Department of Cardiology, Faculty of Medicine, Ain Shams University
Abbasia. P.O.: 11381. Cairo, Egypt.
Telephone: +20224535936
Mobile: +201223224282
E-mail:  HYPERLINK "mailto:smarttmann@hotmail.com" smarttmann@hotmail.com
 

Authorship:
- Walaa Adel, MD
  Department of Cardiology, Faculty of Medicine, Ain Shams University
  Abbasia. P.O.: 11381. Cairo, Egypt

-Sherief Mansour, MD
 Department of Cardiology, Faculty of Medicine, Ain Shams University
 Abbasia. P.O.: 11381. Cairo, Egypt

- Sameh Samir, MD
  Department of Cardiology, Faculty of Medicine, Ain Shams University
  Abbasia. P.O.: 11381. Cairo, Egypt

- Nagwa Elmahalawy, MD
  Department of Cardiology, Faculty of Medicine, Ain Shams University
  Abbasia. P.O.: 11381. Cairo, Egypt
ABSTRACT

Background: Speckle tracking echocardiography is integrated into echocardiographic systems for automated evaluation of left ventricular (LV) function by means of Automated Function Imaging (AFI). This study aimed at evaluating role of strain imaging [AFI & Tissue Velocity Imaging (TVI)] in predicting post-percutaneous coronary intervention (PCI) LV function recovery.
Methods: Fifty patients with anterior wall myocardial infarction & impaired LV ejection fraction (LVEFd"45%), were prospectively enrolled. All patients showed positive viability results concerning left anterior descending (LAD) artery territory using low-dose dobutamine stress echocardiography (LDSE) & significant LAD disease for PCI. All patients underwent strain imaging using AFI & TVI; at rest, at peak dose of LDSE and 3 months after PCI. 
Results: Mean age of the study population was 54.17�7.6 years, 32(64%) being males. 23(46%) patients showed post-PCI LV function recovery after 3 months. They showed higher pre-PCI LVEF% & strain values (TVI&AFI-based).Logistic regression analysis presented baseline LVEF% as an independent predictor of LV function recovery (Odds ratio=0.7025, 95%CI:0.53-0.92).Concerning LAD territory at rest, TVI-based strain value of-8.4% (sensitivity: 92% & specificity: 100%) & AFI-based strain value of-4% (sensitivity: 84% & specificity: 75%), predicted LV function recovery. Concerning global LV at rest, TVI-based strain value of-14% (sensitivity: 79% & specificity: 44%) & AFI-based strain value of-9.3% (sensitivity: 50% & specificity: 50%), predicted LV function recovery after PCI to LAD.
Conclusion: Assessment of global & territorial LV strain (AFI&TVI-based) is of added value upon viability assessment using LDSE. Higher baseline LVEF% (independent predictor) & strain values are associated with post-PCI LV function recovery.
.
Keywords: viability, strain imaging, tissue velocity imaging, dobutamine echocardiography.







INTRODUCTION
         
             Dobutamine stress echocardiography (DSE) is widely used for assessing the presence, location, and extent of coronary artery disease (CAD), but it remains limited by its subjective interpretation and dependence on both image quality and experience [1-3]. The need for a more quantitative method for evaluation of stress echocardiography has provoked the introduction of several imaging methods [4-6], but translation and tethering effects between normal and hypocontractile segments may limit the applicability of these techniques [7]. By measuring deformation derived from tissue Doppler velocity, strain imaging had largely overcome these limitations. However, the routine clinical use of these measurements is constrained by problems that are inherent in the use of Doppler, that relate to signal noise and angle dependency [8, 9]. 
          Speckle tracking echocardiography (STE) is a new technique based on pure 2D grayscale ultrasound acquisition allowing calculation of segmental strains [10]. Because of scattering, reflection, and interference of the ultrasound beams in myocardial tissue, speckle formations in gray scale echocardiographic images represent tissue markers that can be tracked from frame to frame throughout the cardiac cycle [11]. To facilitate clinical application, speckle tracking has been integrated into the most recent echocardiographic systems for quick, automated evaluation of left ventricular (LV) function by means of Automated Function Imaging (AFI). AFI is clinically applicable and an effective means of assessing LV function due to its short acquisition time, feasibility and accuracy, whatever the experience of the operator [10]. Similar in concept to MRI tagging, AFI objectively analyzes myocardial motion by tracking features (�natural acoustic tags�) in the ultrasonic image in two dimensions. AFI could potentially be used to differentiate diseased from non-diseased segments and to learn more about the various strain patterns indicative of specific disease types. The computerized assessment presents the data in four different modes: a parametric image, an anatomical m mode, a strain graph and bull�s-eye display. The AFI algorithm non-invasively tracks and analyzes peak systolic strain based on 2D strain. In addition to providing clinical decision support, AFI also decreases LV function assessment variability and streamlines workflow while improving laboratory quality assurance [12-14]. In the current study, the authors sought to explore the feasibility of using AFI based 2D strain & tissue velocity imaging (TVI) based strain in order to predict LV function recovery after coronary revascularization. The study included patients with demonstrated myocardial viability using low-dose dobutamine stress echocardiography (LDSE).

METHODS

 Patient selection
          Fifty patients referred to our stress echocardiography unit were prospectively enrolled between March 2012 & April 2013. Patients were considered eligible for inclusion if they suffered from anterior wall ST segment elevation myocardial infarction (STEMI), managed using thrombolytic therapy, mounting to left ventricle ejection fraction (LVEF%) value of d"45%. Moreover, eligibility criteria included the presence of single vessel disease i.e. left anterior descending (LAD) artery; amenable for percutaneous coronary intervention (PCI) and the presence of demonstrated myocardial viability using LDSE. Patients with significant valvular diseases, myocardial diseases apart from ischemia, significant multi-vessel disease or having any contraindication for dobutamine infusion (e.g. history of complex ventricular arrhythmia), were excluded. Before inclusion, informed written consent was obtained after explanation of study protocol that was approved by our local institutional human research committee as it conforms to the ethical guidelines of the 1975 Declaration of Helsinki, as revised in 2008. Patients were subjected to thorough history taking, clinical examination, 12 lead electrocardiogram (ECG) recording & resting transthoracic echocardiography (TTE), upon enrollment & three months later. Coronary angiography was done within 48 hours after acute anterior wall STEMI. This was then followed (within 48 hours) by LDSE for myocardial viability assessment. PCI to LAD was done within 1 week, after demonstrated viability.  AFI & TVI based strain imaging; were used to assess segmental & global LV function at rest, at peak dose of dobutamine infusion (20�g/kg/min) and 3 months after PCI to LAD.
Baseline echocardiographic assessment 
          Assessment of regional and global LV systolic functions; in addition to recording of LV internal dimensions were performed for all patients by trans-thoracic echocardiography (TTE) using a General Electric Vivid 7 cardiac ultrasound machine (General Electric, Horten, Norway), equipped with harmonic imaging capabilities. A 2.5 MHz phased array probe was used to obtain standard 2D, M-mode and Doppler images. Patients were examined in the left lateral recumbent position using standard parasternal and apical views. Images were digitized in cine-loop format, and saved for subsequent playback and analysis. Views were analyzed by a single echocardiographer, blinded to the study protocol. Regional wall motion was assessed according to the standard 17-segment model, recommended by the American Society of Echocardiography [15], as shown in Fig. 1. LAD territory included seven segments; basal anterior, basal anteroseptal, mid anterior, mid anteroseptal, apical anterior, apical septal & apex (apical cap) [15]. Regional wall motion abnormalities were visually assessed for each segment individually, considering both endocardial excursion and systolic thickening. Each segment was graded according to the semi-quantitative scoring system described by Knudsen et al. [16]. Segments with poorly-defined endocardial borders for 50% or more of their length were considered non-visualized and assigned a score of 0 [17]. Wall thickening was assessed at a distance of at least 1 cm from the adjacent segment, to minimize the effect of tethering [18].
Low-dose dobutamine stress echocardiography (LDSE) for viability assessment
          All included patients showed demonstrated myocardial viability in LAD territory using LDSE, done within 48 hours after coronary angiography. Dobutamine (Dobutamine MYLAN�, MYLAN S.A.S, France) was administered by intravenous infusion starting at a dose of 5 �g/kg/min and raised incrementally every three minutes up to a maximum of 20 �g/kg/min or until a study end-point was reached. LVEF% by modified Simpson's method, LV internal dimensions using m-mode & global LV wall motion score index (WMSI) were recorded for all patients; at rest and at peak dose of dobutamine infusion. WMSI was calculated by the standard formula; sum of the segment scores divided by the number of segments scored [19]. End-points for terminating the test included: attainment of the maximum dose of dobutamine (20 �g/kg/min.); symptoms judged to be unacceptable by the attending cardiologist; serious arrhythmia detected by ECG monitoring, ST-segment elevation >0.1 mV at 80 ms from the J point, systolic blood pressure >200 mmHg, diastolic blood pressure >110 mmHg or a decrease in systolic blood pressure >30 mmHg from the baseline [20]. Standard views were recorded at baseline, at the end of each stage of dobutamine infusion, as well as during recovery. Visual assessment of wall motion and systolic thickening was performed as mentioned before in baseline assessment.
 2D strain measurement using Automated Function Imaging (AFI)
         Global longitudinal 2D strain for LV was assessed using the AFI technique, which provides a new imaging modality based on 2D longitudinal strain imaging [10, 14]. Longitudinal strain (percentage) is defined as the physiological change in length of the region of interest from end-diastole to end-systole. During this period, strain in the longitudinal direction is a negative value as the length of the region of interest decreases. Longitudinal strain can be calculated using the following formula: longitudinal strain (%) = [L(end-systole) - L(end-diastole)] / L(end-diastole) x100%; where L is the length of the region of interest. The AFI algorithm tracks the percent of wall lengthening and shortening in a set of three longitudinal 2D-image planes (apical long, two chambers and four chambers) and displays the results for each plane. It then combines the results of all three planes in a single bull�s-eye summary (agreeing with the standard 17-segment model), which presents the analysis for each segment along with a global peak systolic value for the LV. An example of AFI based strain acquisition is illustrated in Fig. 2. The following steps were followed: (i) Acquiring APLAX, A4-C & A2-C views. (ii) Marking the aortic valve closure timing, and then anchors three points inside the myocardial tissue. (iii)Offline processing using the three-click method that minimizes variability potentially created in a manual tracing process. Two points placed at the base along the mitral valve annulus, and one at the apex, trigger the automated process. The clinicians can override the processed image results at any time [14]. For global LV longitudinal strain analysis, digital cine-loops were off-line processed using commercially available software (Echo Pac 6.1, GE Medical Systems, Horten, Norway). Mean frame rate of the obtained images was 70 (40 �100) frames/second (fps). AFI measurements were acquired at rest, at peak dose of dobutamine infusion & 3 months after PCI to LAD.
TVI based strain measurement
          TVI based strain imaging was done for all patients at rest, at peak dose of dobutamine infusion & 3 months after LAD revascularization. Separate harmonic color TVI images were saved with a color frame rate of 100�140 fps (depending on the sector width), in the three standard apical views. These color TVI images were recorded with digital media using high spatial resolution, at a depth of 16 cm, with pulse repetition frequencies between 500 and 1000 Hz, resulting in aliasing velocities between 16 and 32 cm/s. Three cardiac cycles were saved in digital format onto a magneto-optical disk for off-line analysis using the Echo Pac system. Data were measured from the slope of the regression line of all the velocity estimates between two points in the middle of each myocardial segment, separated by a distance of 12 mm. Measurements were avoided from walls that were poorly visualized, with aliasing on tissue velocity or with insonation angles >30o. The region of interest was tracked manually in each frame, in order to maintain a mid-myocardial position and avoid intra-cavity velocities. Endsystolic strain was measured at end-systole on the TVI curves. End-systole on color tissue Doppler images is marked by a brief color change at the base of the mitral leaflet and septum on the TVI velocity images, attributed to aortic valve closure. That was done in the following steps: (i) obtaining color coded tissue velocity imaging (TVI) in apical views, (ii) cine loop recordings (3 cycles), (iii) samples were defined on each segment, (iv) computer-derived graphs & strain measurements were obtained [21].
          Analysis of intra-observer variability revealed a close correlation between repeated measurements by the single operator, with a correlation coefficient; r = 0.92 for wall motion assessment, r = 0.91 for TVI based strain assessment & r = 0.95 for AFI based strain assessment.
Follow up 

          Three months after percutaneous revascularization, all patients were re-evaluated through conventional TTE data & strain imaging (TVI & AFI) data. Post-PCI LV functional recovery was defined as: improvement of LVEF% by e" 5%. Accordingly, the studied population was divided in to 2 groups; Group (A) included patients who showed post-PCI LV function recovery, while Group (B) included patients who did not show post-PCI LV function recovery.
Statistics
          Data were analyzed using Statistical Package for Special Science (SPSS) software computer program version 15 & described using mean � standard deviation for quantitative (numerical) variables. Comparison of continuous variables was done using Student t-test. P values were used to describe significance. Stepwise forward logistic regression analysis was used to detect the independent predictor of post-PCI LV function recovery. Receiver operating characteristic (ROC) curves were constructed resulting in the sensitivity-specificity plot. The optimal cut-off value was defined by providing the maximal sum of sensitivity and specificity. 

RESULTS

       Baseline clinical characteristics   
          A total of fifty patients suffering from recent anterior wall STEMI were prospectively enrolled in this study after demonstration of significant single vessel disease (LAD) using coronary angiography and sufficient viable myocardium in LAD territory using LDSE. The mean age of the whole study cohort was 54.17 � 7.6 years, 32 (64%) being male patients. Baseline clinical data are summarized in Table 1.
Low-dose DSE
          All included patients showed positive results indicating substantial myocardial viability in LAD territory, using LDSE. Recorded data for each patient included LVEF% using modified Simpson's method, LV internal dimensions using m-mode, WMSI and mean TVI & AFI derived strain values concerning both LAD territory & global LV. Mean strain value was calculated by dividing the sum of segmental strains by number of myocardial segments i.e. seven segments in case of LAD territory & 17 segments in case of global LV. Apical segments were excluded upon calculating mean TVI based strain due to in-applicability of obtaining TVI based strain for apical segments (angle-dependency). Therefore, LAD territory included four segments only & global LV included 12 segments only. Data collected at LDSE are summarized in Table 2.                    
Detecting post-PCI LV function recovery
          Follow-up echocardiography data were collected three months after PCI, showing significantly higher LVEF% and mean territorial strain values. Collected data are summarized in Table 3. Based on detection of post-PCI LV function recovery (improvement of LVEF% by e"5%), the studied population was divided in to 2 groups: Group (A): 23 (46%) patients showing LV function recovery. Group (B): 27 (54%) patients who did not show LV function recovery. Upon comparing the pre-PCI echocardiographic data of both groups at rest, it was found that group A originally showed statistically significant higher LVEF% & mean strain values (AFI & TVI based). Thus, higher pre-PCI resting LVEF% & strain values (more negative) were associated with post-PCI LV function recovery. Comparative data are summarized in Table 4. Stepwise forward logistic regression analysis presented pre-PCI LVEF% as an independent predictor of LV function recovery (Odds ratio=0.7025, 95%CI: 0.53-0.92).
TVI based strain cut-off values
           Receiver Operating Characteristic (ROC) curve analysis was used to determine mean TVI based strain cut-off values capable of predicting post-PCI LV function recovery. Regarding global LV at rest, a cut-off value of -14% was concluded [sensitivity: 79% (95% CI: 49.2-95.3), specificity: 44% (95% CI: 19.8-70.1), positive predictive value (PPV):55% & negative predictive value (NPV):70%.; Area under the curve (AUC) = 0.580 (95% CI: 0.387-0.757)]. However, at peak dose of dobutamine infusion a cut-off value of -19% was concluded [sensitivity: 71% (95% CI: 41.9-91.6), specificity: 56% (95% CI: 29.9-80.2), PPV: 58.8% & NPV: 69.2%; AUC = 0.638 (95% CI: 0.444-0.805)]. Regarding LAD territory at rest, a cut-off value of -8.4% was concluded [sensitivity: 92% (95% CI: 74.9-99.1), specificity: 100% (95% CI: 39.8-100), PPV: 100% & NPV: 66.7%; AUC = 0.923 (95% CI: 0.765-0.988)]. However, at peak dose of dobutamine infusion, a cut-off value of -13% was concluded [sensitivity: 100% (95% CI: 86.8-100) & specificity of 100% (95% CI: 39.8-100); AUC = 1 (95% CI: 0.884-1.000)]. 
Longitudinal 2D strain (AFI based) cut-off values
          Receiver Operating Characteristic (ROC) curve analysis revealed the following mean 2D strain cut-off values predicting post-PCI LV function recovery. Regarding global LV at rest, a cut-off value of -9.3 % was concluded [sensitivity: 50% (95% CI: 23-77), specificity: 50% (95% CI: 24.7-75.3), PPV: 46.7% & NPV: 53.3%; AUC = 0.500 (95% CI: 0.313-0.687)]. However, at peak dose of dobutamine infusion, a cut-off value of -10.5% was concluded [sensitivity: 57 % (95% CI: 36.4-79.3), specificity: 50 % (95% CI: 15.7-84.3), PPV: 50% & NPV: 57%; AUC = 0.458 (95% CI: 0.244-0.671)]. Regarding LAD territory at rest, a cut-off value of -4% was concluded [sensitivity: 84 % (95% CI: 65.1-95.6), specificity: 75% (95% CI: 19.4-99.4), PPV: 95.7% & NPV: 42.9%; AUC = 0.913 (95% CI: 0.752-0.985)]. However, at peak dose of dobutamine infusion, a cut-off value of -6% was concluded [sensitivity: 80% (95% CI: 60.6-93.4), specificity: 100% (95% CI: 39.8-100), PPV: 100% & NPV: 44.4; AUC = 0.865 (95% CI: 0.691-0.962)]. Mean strain cut-off values are summarized in Table 5.
 
DISCUSSION

          In the current study, we evaluated using AFI based 2D strain & TVI based strain in conjunction with LDSE test, in order to predict post-PCI LV function recovery after percutaneous LAD revascularization. TVI based strain is sensitive to angulation issues like other Doppler techniques. During acquisition, every effort should be taken to align the tissue direction parallel with the beam direction, although this is technically challenging. The second limitation of TVI based strain acquisition is signal noise. In the current study, we attempted to optimize the approach to acquisition and processing, including high frame-rate and lateral resolution acquisition, use of an offset distance (strain length) of 12 mm and tracking of the sample on the gray scale image during post-processing. However, these measures make this technique rather time consuming [22]. Besides its angle-independence, the benefit of speckle tracking analysis is its ability to differentiate between active and passive motion by quantification of myocardial deformation. High-quality images are needed for assessment of AFI based global longitudinal LV strain because the technique is frame rate-dependent. Assessment of global LV longitudinal strain using the AFI technique is easily obtained and takes few minutes. The software uses a step-by-step approach and provides continuous support during the process. In the current study, fifty patients with single vessel (LAD) disease were enrolled after an acute anterior wall STEMI and positive LDSE test for viability. Selecting patients with single vessel disease (infarct-related artery) for inclusion was done aiming at getting more precise & discrete results. Moreover, territorial strain assessment was included in the study methodology in order to achieve the same goal. LAD territory was chosen as a major representative of LV, supplied by the main LV blood supplier. Follow-up echocardiography was done three months after successful PCI to LAD. Data recording showed statistically significant improvement of LVEF%, WMSI, territorial AFI based & TVI based strain values. Post-PCI LV function recovery was defined as improved LVEF% by e"5%. Accordingly, patients were divided in to two groups. Interestingly, the group of patients who showed post-PCI LV function recovery [23 (46%) patients]; originally showed pre-PCI favorable echocardiographic parameters. This group of patients showed significantly higher baseline LVEF%, strain values at both global LV & territorial levels using both strain measuring modalities. The authors hypothesized that higher strain values recorded basically before PCI could help predict post-PCI LV function recovery, although higher baseline LVEF%, is still the most powerful predictor. The relatively small number of patients studied did not hinder obtaining significant differences concerning strain values. This seemed more pronounced for LVEF%, indicating the powerful impact of favorable baseline LVEF% on prediction of LV function recovery (independent predictor). Clinical practitioners still call for standardized cut-off values for segmental, territorial & global LV strain. However, prior studies defined a normal range for global longitudinal LV strain (20.3%- 24.1%) [14,  23]. Analyzed data from the current study aided extraction of informative cut-off values of both strain measuring modalities, in order to predict post-PCI LV function recovery. 
Comparison with previous studies
         The current study showed that improved WMSI and LVEF% in a group of patients at the end of LDSE test following acute myocardial infarction (MI), was associated significantly with improvement of these parameters after three months. A prior study reported similar results obtained after a shorter follow-up period of time (25 +/- 11 days) [24]. Longer follow-up period in the current study aimed at achieving more tissue recovery after possible stunning or hibernation for proper assessment of LV function recovery. Similar to the present study, a prior study pointed out the value of strain imaging in predicting LV function recovery after acute MI [25]. The current study showed that higher baseline LVEF% and global 2D strain was associated with post-PCI LV function recovery. Similarly, Mollema et al. reported that baseline global longitudinal LV strain after acute MI, was significantly associated with LV function recovery after one year follow-up period [26]. Another study including Non STEMI patients reported concordant results [27]. On the other side, a prior trial reported weak correlation between LVEF% (by Simpson's method) & global LV strain obtained by AFI in STEMI patients [28]. A follow-up period was not included in this study protocol. The current study reported that a cut-off value of -9.3% for global LV AFI based strain predicted LV function recovery. Mollema et al. reported a higher cutoff value (-13.7%) for the same parameter with higher sensitivity and specificity values among a larger number of STEMI patients and after a longer follow-up period. It is worth mentioning that primary PCI was the adopted reperfusion strategy in their study. Interestingly, patients having the LAD as their infarct-related artery had the lowest (less negative) baseline AFI based global LV strain and demonstrated the smallest increase in LVEF% at one-year follow-up, probably due to the relatively larger infarct size [26]. This suggests that infarct location might influence the relation between baseline AFI based global LV strain and recovery of LV function during follow-up. The current study included only patients with defective anterior coronary circulation. A prior study reported that TVI based strain could predict viability in both anterior and posterior circulations, while speckle tracking based 2D strain measurements could predict viability only in the anterior circulation; assuming that TVI based strain is more accurate [29].
Clinical implications
         Speckle tracking based strain has been associated with the extent of viable myocardial tissue in patients with chronic ischemic heart disease and was recently validated against MRI and sonomicrometry [30, 31]. The great value of myocardial viability detection before coronary revascularization is always brought by later achievement of post-revascularization LV function recovery. Strain imaging can be integrated with low dose dobutamine echocardiography test as an approach to predict LV function recovery, thus augmenting the value of LDSE in detection of viable myocardium in patients considered for PCI. This can be of special importance in patients showing border line or equivocal LDSE results when the operator depends on visual assessment as an only way to assess wall motion changes. Favorable segmental, territorial & global strain values votes for positive viability result in this situation. This is also true in case of conflicting results obtained by different modalities of myocardial viability assessment in addition to LDSE e.g. using nuclear imaging. TVI based strain assessment requires a relatively well experienced echocardiographer. However, AFI based 2D strain assessment requires a lower level of operator experience. This automated process was invented to allow a rather operator in-dependent 2D longitudinal strain assessment.
Limitations of the study
         The data presented in our study only apply for patients defined by inclusion & exclusion criteria. This is a single-centre study with a relatively small sample size of the cohort, a fact that makes it difficult to generalize the results to all patients surviving an acute STEMI. This study included only patients with anterior wall STEMI. Other studies are needed to evaluate patients with more than one territory affection, for the same purpose. Another limitation is lack of long-term follow-up of functional recovery after LAD revascularization on both levels; territorial & global. The current study protocol did not directly compare between the two modalities of myocardial strain assessment (AFI based & TVI based). This was outside the scope of the study.  
Conclusion
          Assessment of global longitudinal LV strain (using AFI) & TVI based strain could be of added value during myocardial viability assessment using LDSE. Higher baseline LVEF% (modified Simpson�s method) & strain values (territorial & global) are significantly associated with post-PCI LV function recovery. Baseline LVEF% is considered an independent predictor of LV function recovery.
Acknowledgment
          The authors would like to express their gratitude for medical, nursing & technical staff of stress echocardiography & cardiac catheterization laboratories in thecardiology department for their sincere co-operation to accomplish this work.
Conflicts of interest     Authors declare that they have no conflicts of interest regarding publication of this original research work.
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