Comparison of longitudinal myocardial tissue velocity, strain, and strain rate measured by two-dimensional speckle tracking and by color tissue Doppler imaging in healthy dogs

Journal of Veterinary Cardiology (2011) 13, 31e43

www.elsevier.com/locate/jvc

Comparison of longitudinal myocardial tissue velocity, strain, and strain rate measured by two-dimensional speckle tracking and by color tissue Doppler imaging in healthy dogs* Gerhard Wess, DVM*, Lisa J.M. Keller, DVM , Michael Klausnitzer, BSc, Markus Killich, DVM , Katrin Hartmann, DVM, PhD Clinic of Small Animal Internal Medicine, LMU-University, Veterinaerstr. 13, 80539 Munich, Germany Received 4 January 2010; received in revised form 6 August 2010; accepted 7 August 2010

KEYWORDS TDI; Canine; 2D-ST; Single point analysis; Average analysis

Abstract Objectives: Two-dimensional speckle tracking (2D-ST) is a new method to measure tissue velocity (TV), strain and strain rate (SR), but it is unclear if results are comparable to color tissue Doppler imaging (TDI). The objective was therefore to compare the two modalities 2D-ST and TDI. Animals: 100 healthy dogs Methods: TDI images were acquired from the interventricular septal wall (IVS) and the left ventricular free wall (LVFW) to measure longitudinal TV, strain, and SR, and grayscale images were collected for 2D-ST analysis. A software program was developed, that allowed extraction of single points (SP) from the 2D-ST data set to compare SP with averages of segments (AOS) results, which are usually displayed by the 2D-ST software. Results: A good agreement was found between AOS and SP measurements using 2D-ST. Although most data were within limits of agreement, significant differences were found between TDI and 2D-ST measurements for selected parameters. The differences were small in the IVS, but higher and of clinical relevance in the LVFW. 2D-ST was feasible and reproducible in the IVS, but less reliable in the LVFW. Conclusions: 2D-ST and color TDI can be used interchangeably in the IVS, but the methods reveal different results in the LVFW. ª 2010 Elsevier B.V. All rights reserved.

*

Preliminary results of this study have been presented at the Human World Congress of Cardiology 2006, Barcelona, Spain. * Corresponding author. E-mail address: [email protected] (G. Wess).

1760-2734/$ - see front matter ª 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jvc.2010.08.001

32

Introduction Myocardial tissue imaging using echocardiography is the method of choice for noninvasive assessment of regional myocardial function in humans and is increasingly used in veterinary medicine.1e14 Different modalities, such as pulsed wave tissue Doppler imaging (PW TDI), color tissue Doppler imaging (color TDI) and most recently two-dimensional speckle tracking (2D-ST) are available to measure myocardial tissue velocity (TV). Myocardial strain and strain rate (SR) can only be measured using color TDI or 2D-ST. However, it is currently unknown if these modalities can be used interchangeably, or if separate reference values are necessary. The first Doppler method used for the assessment of myocardial function was the measurement of TV using PW TDI. Color TDI has been first introduced as an alternative method to measure TV and has been evaluated in healthy dogs.2,15,16 A recent study compared TV results obtained by PW TDI and color TDI and found that TV measurement using PW TDI is significantly higher and that both methods should not be used interchangeably.17 Using color TDI, strain and SR measurements were later introduced to clinical practice to evaluate regional myocardial deformation magnitude and rate, respectively.18,19 Doppler-based strain and SR were validated both experimentally using ultrasonic crystals and clinically with tagged magnetic resonance imaging, and are considered to be less affected by tethering, translational artifacts, and traction than Doppler measurements of myocardial velocities.20,21 Currently, color tissue Doppler analysis is the most commonly used method in veterinary medicine for the measurement of TV, strain and SR.4,13 Using this technique, measurements are performed by placing a small region of interest (ROI) into the myocardial wall and therefore, measurements of single myocardial points are obtained. Tissue tracking has to be done manually, which is often time-consuming, and as all Doppler methods, TDI is angle-dependent. Recently, 2D-ST has been introduced which determines myocardial deformation from continuous frame-by-frame tracking of a small image block of “natural acoustic markers”.22 The appearance of these acoustic markers is considered to be relatively stable between subsequent image frames, whereas a change in their position is assumed to follow tissue motion. Tracking is performed semi-automatically and is based on searching the new location of the marker in the subsequent frame using a block-matching algorithm.23,24 Tissue velocity,

G. Wess et al. strain, and SR are then calculated from the displacement and rate of displacement of each marker. Two-dimensional speckle tracking has been compared to Doppler-based methods in humans, where good correlation between both methods was demonstrated.25,26 In veterinary medicine only systolic radial strain, and SR were compared to TDI-based measurements.27 Twodimensional speckle tracking results are calculated and displayed as averages over whole myocardial segments (basal, middle and apical segment), in contrast to the single point (SP) measurements of color TDI. So far, there are no investigations in human or veterinary medicine evaluating if the averaged measurements actually agree with the conventionally obtained SP measurements using the 2D-ST method. Furthermore, the assessment of systolic and diastolic longitudinal myocardial function using 2D-ST has never been investigated in a large group of healthy dogs and 2D-ST used for longitudinal TV-, strain-, and SR-measurements has not been compared to TDI-based methods. The hypothesis of this study is that 2D-ST and conventional color-coded TDI may reveal different results due to technical differences. The aims of this study are (1) to test the feasibility, as well as reproducibility of the new 2D-ST software in the interventricular septal wall (IVS) and in the left ventricular free wall (LVFW), (2) to analyze the differences between averaged measurements and the measurement of single myocardial points and (3) to compare the results of systolic and diastolic longitudinal myocardial TV, strain, and SR obtained by the traditionally used color Doppler method to those obtained using the new 2D-ST method.

Animals, material and methods Dogs The study population consisted of 100 healthy dogs of different breeds and origins (companion animals presenting for vaccinations or cardiac screening programs as well as student- or faculty-owned dogs). Owner consent was obtained before enrollment into the study. The study protocol was in compliance with the institutional Animal Care and Use Committee and the German law on laboratory animals and animal care. Dogs receiving medication or that had a history of cardiac or systemic diseases were excluded. Dogs were considered to be healthy based on the following examinations: unremarkable physical examination, systolic blood pressure < 160 mmHg using noninvasive arterial

Comparison of TDI software programs blood pressure measurement,a normal sinus rhythm or respiratory sinus arrhythmia using a 12-lead ECG,b and normal complete 2D, M-mode, and Doppler echocardiography. In Doberman Pinschers, a 24-h ambulatory ECGc was performed and the number of ventricular premature contractions per 24-h had to be less than 50 to be included into the study. Dogs were excluded from the investigation if any cardiovascular or systemic disease was identified.

33 acquisition of the images with TDI mode, the same image was stored as grayscale image for 2D-ST analysis. The heart rate of the TDI images and the 2D images had to be within 10 bpm. Grayscale receive gain was set to allow a good detection of endocardial boundaries. Three to 5 consecutive heart cycles using a simultaneous one-channel ECG were stored in digital format for subsequent off-line analysis.

Off-line Analysis Conventional echocardiography and Doppler examination All ultrasound examinations were performed by one investigator (GW) without sedation in right and left lateral recumbency in accordance to the recommendations of the Echocardiography Committee of the Specialty of Cardiology of the American College of Veterinary Internal Medicine.28 The ultrasound machined was equipped with 2.2- to 3.5-MHz and 5.5to 7.5-MHz phased-array transducers. All data were stored digitally for further off-line analysis. Echocardiographic M-mode measurements were performed using the right parasternal long-axis and short-axis views and had to be within reported reference ranges, before the dog was included into the study.29 All valves were examined using color Doppler and only trivial valve insufficiencies were allowed as inclusion criteria. Velocities over the aortic and pulmonary valves were measured using continuous wave Doppler examinations and had to be below 2.2 m/sec.30 Left atrial dimensions were measured in the right parasternal short axis at the level of the heart base.31

Tissue Doppler echocardiography and 2D-ST images The left ventricle was visualized using the left apical view. Images of the interventricular septum (IVS) and left ventricular free wall (LVFW) were obtained using a narrow ultrasonic sector with high frame rates. The ultrasound beam was placed as parallel as possible to the longitudinal motion of the myocardium. Real-time color Doppler images were superimposed on the grayscale with high frame rates and stored for off-line color TDI analysis. The Doppler receive gain was adjusted to maintain optimal coloring of the myocardium, and Doppler velocity range was set as low as possible to avoid aliasing artifact. Immediately after Flow Detector, Parks Medical Electronics, Oregon, USA. Cardiovit AT-10, Schiller, Baar, Switzerland. c Custo tera, Arcon Systems GmbH, Starnberg, Germany. d Vivid 7 Dimension, General Electric Medical Systems, Horten, Norway.

Off-line analysis of tissue Doppler data was performed using a commercially available software.e TV, SR, and strain were measured in the IVS and LVFW using both methods. The mean of peak systolic (TV-S, SR-S and strain), early diastolic (TV-E, SR-E) and late diastolic (TV-A, SR-A) TDI was calculated from 3 cycles. For the conventional TDI analysis, the ROI was placed in the middle of the basal, middle, and apical segment of each wall. To ensure, that the ROI remained within the myocardium manual tracking was performed. In each of the 3 to 5 cardiac cycles, the ROI was placed at end-systole and enddiastole in the middle of the myocardial wall. The software then followed the myocardial movement. The ROI size was chosen to fit the diastolic myocardial wall size. According to the dog’s weight in most dogs the following settings were used: 3
3 mm for < 10 kg, 4
4 mm for 11e20 kg, 5
5 mm for 21e30 kg, 6
6 mm for 31e40 kg and 7
7 mm for dogs > 40 kg. Temporal filter settings for the TDI mode were 30 ms for TV, and 40 ms for strain and strain rate. In the TDI mode, several time markers were set within the cardiac cycle to mark systole (where TV-S, SR-S, and strain occur), isovolumic relaxation, early diastole (where TV-E and SR-E occur), late diastole (where TV-A and SR-A occur), and isovolumic contraction.32 One additional time marker was set at the beginning of the P-wave of the simultaneously recorded ECG indicating the beginning of late diastole (Fig. 1). Setting time markers was done to enable easier allocation of peak systolic and diastolic TDI values and for the automatic extraction of peak SP measurements in the 2D-ST using a specially developed software (see below). For the 2D-ST analysis, only one cardiac cycle can be analyzed at a time. Peak values of 2D-ST variables were measured from three different cardiac cycles and then averaged. Semi-automatic tissue tracking and analysis of 2D grayscale images (for 2D-ST) was performed using a commercially available software.f

a

b

e EchoPac BT06, Q Analysis, General Electric Medical Systems, Horten, Norway. f EchoPac BT 06, 2D Strain, General Electric Medical Systems, Horten, Norway.

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G. Wess et al.

Figure 1 Time markers set in a tissue velocity curve of the septum. AVO ¼ Aortic Valve Opening: set at the zero crossing point for the ascending limb of the systolic Swave (S); AVC ¼ Aortic Valve Closure: set at the zero crossing point for the descending limb of S; MVO ¼ Mitral Valve Opening: set at the zero crossing point for the descending limb of early diastolic E-wave (E); MVC ¼ Mitral Valve Closure: set at the zero crossing point for the ascending limb of the A wave (A); P: time marker set at the beginning of the p-wave in the simultaneous recorded ECG; IVR ¼ isovolumic relaxation time, IVC ¼ isovolumic contraction time.

For semi-automatic tracking, the endocardial border had to be marked manually. Tracking of the myocardial wall was performed automatically by the software by following 2D speckles. The software automatically divided the myocardial wall into a basal, a midventricular, and an apical myocardial segment (Fig. 2). Manual adjustment of the myocardial wall borders was performed when necessary. In each of the myocardial segments, TV-S, TV-E, TVA, SR-S, SR-E, SR-A, and peak systolic strain were measured and displayed as averages over the selected segment (AOS) automatically by the software. Myocardial TDI profiles were displayed by the software as curves. Peak values were marked automatically, and AOS results were displayed in a table. Peak values were corrected manually if necessary.

Extraction of SP measurements using 2D-ST Results of the 2D-ST analysis are usually displayed as averages over segments (basal, mid and apical segment), whereas SP measurements are not displayed using this software. Results obtained by the traditionally used TDI method are SP measurements. Fig. 3 shows the difference between both methods using a 4-chamber view (for analysis of the parameters IVS and LVFW were recorded and analyzed as single wall images to ensure a better alignment with the ultrasound

Figure 2 Tissue velocity, strain rate and strain measured in the basal, middle and apical segment of the interventricular septum using 2D speckle tracking. The curves represent averages over each segment and are calculated automatically by the software. S ¼ peak systolic wave, E ¼ early diastolic wave, A ¼ late diastolic wave, IVR ¼ isovolumic relaxation time, IVC ¼ isovolumic contraction time.

Comparison of TDI software programs

35

Measurement reliability

Figure 3 Difference between 2D-ST and color TDI measurements. Using 2D-ST analysis, the software creates three segments in each myocardial wall and averages the results of multiple measurements for each of the segments, whereas a region of interest (ROI) is placed manually in the middle of each segment using the color TDI method (bullets). For demonstration purposes a 4-chamber view is shown, whereas for analysis single wall views were used.

beam and to acquire higher frame rates). However, it is unknown, if single point measurements and averages over segments would result in the same values. In order to compare results of both methods from the same myocardial area and to extract SP results using 2D-ST, the same region of interest was chosen manually using the 2DSTST software in the basal, mid, and apical segment of IVS and LVFW. A special software was developed, that extracts the raw data results from these single point measurements displayed as a column of numbers in relation to the time at which they are occurring. Each time point of the curve is associated with the numerical value of the curve at that time. Time correlation was achieved using simultaneous ECG recordings and the use of time markers (Fig. 1). The time markers, placed using the TDI mode (as mentioned above), were then exported into the special software to tell the software at what time point systole, isovolumic relaxation, early and late diastole, and isovolumic contraction starts and finishes. The software program was developed especially for the purpose to read out peak values in these different phases of the cardiac cycle from the column of numbers in relation to the time they are occurring. Peak values in systole corresponded to TV-S, SR-S, and strain; peak values in early diastole corresponded to TVE and SR-E, and late diastolic values represent TV-A and SR-A measurements.

Measurement variability was determined for systolic and diastolic TV, SR, and strain variables for both methods used in this study (color TDI and 2D-ST). Ten echocardiograms were randomly selected to be subjected to 3 repeated analyses at 3 different time points on a given day by the same investigator (LK) to determine intra-observer within-day variability and on 3 different days to determine intra-observer between-day measurement variability for all parameters (systolic and diastolic TV and SR, and peak systolic strain), for both, the IVS and LVFW. Each variable was measured 3 times on 3 consecutive cardiac cycles using the same recorded loop, and the mean value was used to determine the intra-observer variabilities. The same 10 echocardiograms were subjected to independent repeated analyses by a second investigator (GW) to determine interobserver measurement variability using the same protocol as stated above. Both investigators were unaware of the results of the prior echocardiographic analyses. The effect of image acquisition was tested by acquiring 2D and color TDI ultrasound images of the IVS and LVFW on three consecutive days of 6 dogs (1 Doberman Pinscher, 1 German Shepherd, 1 Beagle, 1 Jack Russell terrier, 1 Golden Retriever and 1 Australian Shepherd). The ultrasound examinations were performed by one investigator (GW). The 2D-ST and color TDI examinations were performed by one investigator (LK) by measuring each variable 3 times on 3 consecutive cardiac cycles using the recorded loops, and the mean value was used to determine the effect of image acquisition variability.

Statistical analysis All data were visually inspected and tested for normality by the Kolmogorov Smirnov test. Statistical analyses were performed by computer software.g All data are presented as mean values with standard deviation (SD). Data were normally distributed and therefore a paired t-test was used to compare the methods. Limits of agreement were analyzed using Bland-Altman analysis.33 A P-value < 0.05 was considered statistically significant. The intra- and inter-observer coefficients of variations (CV) were calculated using a variance component analysis.

g SPSS; Version 13.0 Statistical Package for the Social Science.

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G. Wess et al.

Results An attempt to measure TV, SR, and strain was made in 100 dogs of various breeds: 15 Doberman Pinschers, 10 Golden Retrievers, 8 Jack Russel terriers, 8 German shepherds, 32 of various other breeds (each breed less than 5 dogs) and 27 mixed breed dogs. The 100 dogs (46 females and 54 males) had a mean weight of 25.4 kg (14.1 kg; range 2e58 kg) and a mean age of 5.2 years (3.2 years; range 1e14 years). For the comparison of SP and AOS results, using the 2D-ST method, measurements were possible in 885 segments (95 dogs) of the IVS and in 765 segments of the LVFW (85 dogs). In the remaining dogs, a semi-automatic tracking of the myocardial border was not possible, despite several attempts (especially in the LVFW due to breathing artifacts). Measurements were possible in all 900 segments of the IVS (100 dogs) and in 882 segments of the LVFW (94 dogs) using the color TDI method. Coefficients of variation (CV) of within and between day intra-, as well as inter-observer variability of TV, SR, and strain were lower in the IVS compared to LVFW (Table 1). Results of the 2D-ST SP versus 2D-ST AOS method showed a good agreement between the methods, with most data sets lying within the limits of agreement (Fig. 4). The Bland-Altman scattergrams display the differences of SP and AOS plotted against average values (of SP and AOS), separately displayed for the IVS and LVPW. TV (combined S, E and A waves), SR (combined S, E, and A waves) and strain are displayed separately. The limits of agreement are shown as separate lines (1.96 SD of the mean difference). The graphs show that the limits of agreement were lower for

the LVFW, but more values were outside the limits of agreement compared to the measurements obtained from the IVS. There was no systematic over- or underestimation by either of the two methods, and the mean difference for TV, SR or strain between the two methods was very small. The paired t-test showed no significant differences between the SP and AOS results for TV, SR, and strain, in the IVS and in the LVFW (all P-values > 0.05). Therefore, SP and AOS can be used interchangeably. As the 2D-ST method only displays AOS results, only the AOS results of the 2D-ST analysis were compared to the results of the color TDI analysis, as those results are used and obtained under clinical conditions. The results of the color TDI analysis, and the 2D-ST analysis showed a good agreement between both methods. Most data sets were within the limits of agreements as shown by the Bland-Altman scattergrams, separately displayed for the septal and lateral wall and as separate figures for TV, SR and strain (Fig. 5). The limits of agreements were smaller for the IVS, compared to the LVFW. The mean (SD) of each method and the mean difference between the two methods and P-values for the IVS are shown in Table 2, and for the LVFW in Table 3. All TV, SR, and strain results in the IVS were significantly different between color TDI and 2D-ST analysis (except SR-S). However, differences were very small and clinically not relevant, as most of the measurements were within the limits of agreement as shown in the Bland-Altman difference plots (Table 2 and Fig. 5). In the LVFW, TV-S, and TV-E were significantly higher using color TDI compared to 2D-ST, and these differences may be of clinical relevance. SR-S, SR-A, and strain were

Table 1 Intra- (within and between day) and inter-observer repeatability and the effect of image acquisition of two different methods (color TDI and 2D-ST) to measure systolic and diastolic TV (tissue velocity), systolic and diastolic strain rate and systolic strain in the IVS and the LVFW. IVS Intra-observer repeatability (within-day) CV in % Intra-observer repeatability (between-day) CV in % Inter-observer repeatability CV in % Effect of image acquisition CV in %

TV Strain Strain TV Strain Strain TV Strain Strain TV Strain Strain

Rate

Rate

Rate

Rate

LVFW

color TDI

2D-ST

color TDI

2D-ST

2.9 7.7 2.7 6.4 11.8 3.7 8.8 14.8 9.3 7.3 12.8 6.3

3.9 7.1 3.1 5.1 11.2 4.1 8.1 14.4 9.5 8.8 13.3 7.2

4.2 12.2 7.2 7.5 17.2 10.2 11.0 17.3 14.9 9.1 16.3 12.9

5.9 13.1 8.6 6.9 19.1 9.5 11.4 19.7 15.1 9.4 18.7 13.1

Comparison of TDI software programs

37

Figure 4 Bland-Altman difference plots showing the differences of single points (SP) and averages over segments (AOS) plotted against average values (of SP and AOS) using the 2D-ST method. Results for the interventricular septal wall (IVS) and left ventricular free wall (LVFW) are displayed separately. Results are displayed for tissue velocity imaging (TV; combined S, E and A waves), strain rate (SR; combined S, E, and A waves) and strain. The limits of agreement are shown as separate lines (1.96 SD of the mean difference).

also significantly different between the two methods, but the differences were small and considered not to be of clinical relevance (1) because most of the measurements were within the limits of agreement as shown in the Bland-Altman difference plots and (2) the mean differences are in the range or below of the measurement variability (Table 3 and Fig. 5). Average values for TV measured using 2D-ST and color TDI are shown in Table 4, SR average values in Table 5 and strain average values in Table 6. TV showed in both walls, and with both methods a significant (P < 0.001) velocity gradient from the basal to the apical segment for all velocities measured (systolic and diastolic velocities). SR was not different (P ¼ 1.0) between the segments using 2D-ST in the LVFW and

IVS. Using the TDI method, SR in the apical segment was significantly (P ¼ 0.08) lower than the middle segment in the IVS. All other SR values were not different (P ¼ 1.0) between segments (Table 5). There were no significant differences (P ¼ 0.83) between the strain values in the basal, middle, and apical segment of the IVS and of the LVFW for both methods.

Discussion Recently, improved hardware and software have allowed angle-independent quantification of myocardial deformation based on 2D-ST methods in B-mode.34e36 Currently, color TDI is the most

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G. Wess et al.

Figure 5 Bland-Altman difference plots showing the differences of the color tissue Doppler imaging (TDI) software and the 2D-ST software plotted against average values (of color TDI and 2D-ST results). Results for the interventricular septal wall (IVS) and left ventricular posterior wall (LVFW) are displayed separately. Results are displayed for tissue velocity imaging (TV; combined S, E and A waves), strain rate (SR; combined S, E, and A waves) and strain. The limits of agreement are shown as separate lines (1.96 SD of the mean difference).

commonly used technique in veterinary medicine to measure TV, SR and strain. However, it has been argued, that the clinical use of SR measured by

tissue Doppler is limited to experienced users due to the low signal-to-noise ratio.37 Additionally, it requires manual tracking of the myocardial wall,

Table 2 Results of systolic (S), early diastolic (E) and late diastolic (A) tissue velocity imaging (TV) and strain rate (SR) as well as systolic strain measured in the interventricular septum (IVS) by two different methods: color TDI and 2D-ST. Results are shown as mean (SD). TV-S

TV-E

TV-A

SR-S

SR-E

SR-A

Strain

n (segments) 285 283 286 285 287 284 283 TDI 6.46 (2.93) 4.20 (1.59) 3.32 (1.57) 2.16 (0.80) 1.98 (0.83) 1.46 (0.70) 17.81 (4.97) 2D-ST 6.27 (2.77) 4.37 (1.60) 3.89 (1.47) 2.09 (0.71) 1.75 (0.75) 1.34 (0.68) 16.89 (4.26) Mean difference 0.20 (1.31) 0.17 (1.19) 0.57 (1.17) 0.07 (0.75) 0.23 (0.77) 0.12 (0.70) 0.92 (4.32) TDI e 2D-ST P-value 0.011 0.018 <0.001 0.092 <0.001 0.006 <0.001

Comparison of TDI software programs

39

Table 3 Results of systolic (S), early diastolic (E) and late diastolic (A) tissue velocity imaging (TV) and strain rate (SR) as well as systolic strain measured in the left ventricular free wall (LVFW) by two different methods: color TDI (TDI) and 2D-ST. Results are shown as mean (SD). TV-S n (segments) TDI 2D-ST Mean difference TDI e 2D-ST P-value

TV-E

TV-A

SR-S

SR-E

SR-A

Strain

210 210 207 210 210 206 209 6.86 (2.93) 8.21 (3.53) 4.88 (2.44) 1.70 (0.95) 2.46 (1.07) 1.70 (0.81) 14.15 (4.44) 5.26 (2.80) 6.77 (3.34) 4.75 (2.71) 1.88 (1.18) 2.36 (1.33) 1.54 (0.94) 15.18 (5.86) 1.60 (2.38) 1.45 (2.59) 0.13 (2.48) 0.17 (0.88) 0.10 (0.92) 0.16 (0.82) 1.03 (5.82) <0.001

<0.001

0.442

0.005

which is time-consuming. For those cardiologists with limited experience or limited time to do manual analyses, a more automated approach seems to be advantageous.26 This study shows that the new 2D-ST method is feasible in dogs and that the results obtained by this method can be used interchangeably with the results of the currently most commonly used color TDI method. In humans, 2D-ST shows better agreement with magnetic resonance imaging tagging for regional myocardial strain and strain rate than measurements based solely on TDI.38 Additionally, as the 2D-ST method is angle-independent, uses automatic tracking, and is therefore faster for data analysis, it has several advantages over TDI. In veterinary medicine, 2D-ST have been used to measure radial strain,27 but longitudinal systolic SR and strain have not yet been assessed and feasibility of 2D-ST has not yet been compared between the IVS and the LVFW. However, before a new technique can be used, it has to show a good repeatability and reproducibility. Intra- and inter-observer reproducibility appears to be good for TV and strain in both myocardial walls. Reproducibility is acceptable for SR in the IVS, but not adequate in the LVFW (between day intra-observer CV 19.1; interobserver CV 19.7%). The reproducibility of 2D-ST is

0.128

0.006

0.011

similar to the results of color TVI. The effect of image acquisition was comparable to the effect of inter-observer variability for all parameters and therefore, the variability is adequate for all values except SR in the LVFW. One study reported a comparatively better reproducibility for radial systolic SR that was similar to systolic strain measurements using 2D-ST in dogs,27 but the poorer reproducibility of SR compared to TV and strain (as shown in this study) is in agreement with studies reporting reproducibility in humans.38 The present study found a better reproducibility of the IVS, compared to the LVFW, independent of which software was used. The IVS was also more feasible than the LVFW using 2D-ST in this study. Whereas the software was able to measure TV, SR, and strain in 95% of the cases in the IVS (885 of 900 segments), the software could achieve measurements in the LVFW in only 69.7% (627 of 900 segments). Therefore, the use of 2D-ST in the LVFW is limited. An explanation for the lower feasibility in the LVFW may be that it is more difficult to align the LVFW with the ultrasound beam (compared to the IVS) and that there is a higher rate of breathing artifacts with lung tissue overlying the image (reverberation artifacts) and dropouts. This reduces the ability of the software to follow the speckles throughout the cardiac cycle in

Table 4 Average values for myocardial tissue velocity (mean SD) for 2D-ST and color TDI from 95 healthy dogs in the interventricular septal wall (IVS) and 85 dogs in the left ventricular free wall (LVFW). There was a significant velocity gradient from the basal to the apical segment for all velocities, in walls and for both methods. Tissue Velocity in cm/s IVS segments basal 2D-ST

color TDI

S E A S E A

7.5 5.0 4.5 7.8 5.1 4.1

(2.7) (1.4) (1.4) (2.7) (1.3) (1.5)

middle 6.1 4.4 4.0 6.3 4.4 3.3

(2.5) (1.5) (1.3) (2.7) (1.4) (1.5)

S ¼ systolic, E ¼ early diastolic and A ¼ late diastolic wave.

LVFW segments apical 4.9 3.6 3.0 5.2 3.2 2.4

(2.5) (1.5) (1.3) (2.7) (1.4) (1.3)

basal 6.2 7.4 5.7 7.6 8.4 5.9

(3.0) (3.4) (2.8) (2.9) (3.2) (2.%)

middle 5.1 6.9 4.8 6.5 8.0 4.7

(2.8) (3.4) (2.6) (2.9) (3.7) (2.3)

apical 4.5 5.8 3.6 5.8 7.2 3.5

(2.6) (3.3) (2.5) (2.8) (3.7) (2.5)

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G. Wess et al.

Table 5 Average values for strain rate (mean SD) for 2D-ST and color TDI from 95 healthy dogs in the interventricular septal wall (IVS) and 85 dogs in the left ventricular free wall (LVFW). Strain Rate in s1 IVS segments basal 2D-ST

S E A S E A

color TDI

1.9 1.6 1.3 2.3 2.1 1.4

(0.6) (0.8) (0.6) (0.9) (1.0) (0.8)

LVFW segments

middle 2.1 1.6 1.3 2.4 1.9 1.6

apical 2.1 1.8 1.4 2.0 2.1 1.5

(0.8) (0.6) (0.7) (1.0) (0.7) (0.9)

(0.7) (0.8) (0.7) (0.9)a (0.9) (0.7)

basal 2.1 2.3 1.6 1.8 2.4 1.8

(1.2) (1.3) (1.2) (1.0) (1.1) (1.1)

middle 1.8 2.2 1.5 1.5 2.2 1.6

(1.1) (1.2) (1.0) (0.8) (1.0) (0.7)

apical 1.9 2.4 1.6 1.7 2.5 1.7

(1.3) (1.6) (1.0) (1.0) (1.1) (0.9)

S ¼ systolic, E ¼ early diastolic and A ¼ late diastolic wave. a apical segment significantly lower than middle segment.

the LVFW, compared to the IVS. Similar findings have been reported in human studies.34,39 The software programs used for 2D-ST and color TDI analysis measure and report the results in different ways. Whereas the software used for color TDI analysis uses a ROI, which is comparable to a SP measurement, the software used for 2D-ST averages the results obtained by speckle tracking over the segment, in which the TV, SR, and strain are measured. However, no study in human or veterinary medicine has tested if the results of SP and AOS reveal the same results. At least in theory, SR and strain results are equal, independent in which myocardial segment they are measured, and therefore there should be no difference between SP and AOS. It is different for TV, as a basal to apical velocity gradient has been reported in humans and animals, with higher velocities measured in the basal segments.3,4,40e45 Thus, especially for TV, results between SP and AOS may be different, dependent on where the SP is measured. It was necessary to develop a special software for this study in order to compare SP and AOS results, as only AOS results are displayed by the commercial software used for 2D-ST analysis.f The special software enabled automatic SP extractions from the 2D-ST raw data, based upon time markers. The region for SP extraction and the

ROI using color TDI were placed in the middle of the segment and not, as in some other studies, in the lower part of the segment.46,47 This may explain, why this study revealed a good agreement between results of SP and AOS. Slightly higher myocardial velocities below the ROI (closer to the heart base), and lower myocardial velocities above the ROI (closer to the heart apex) may equal out, and are then comparable to the results of the AOS measurements. If a clinician would place the ROI in the lower part of the segment in a large breed dog (with a large segment), then higher velocities may be measured compared to the AOS method. The agreement between SP and AOS was better in the LVFW compared to the IVS, but fewer segments were available for analysis using the LVFW, which may explain the differences. AOS results were used for the comparison between color TDI results and 2D-ST results, as only AOS results are displayed by the software and those results are therefore used under clinical circumstances. The software programs used for 2D-ST and color TDI analysis use not only different ways to display the results (AOS versus SP), but also different techniques to obtain the measurements. Whereas color tissue Doppler images are used for the color TDI technique, 2D-ST uses 2D speckle tracking to obtain the measurements. Frame rates are higher

Table 6 Average values for systolic strain (mean SD) for 2D-ST and Q-analysis from 95 healthy dogs in the IVS and from 85 dogs in the left ventricular free wall (LVFW). There was no significant difference between the basal, middle and apical segment. Strain in % IVS segments 2D-ST Doppler

S S

LVFW segments

basal

middle

apical

basal

middle

apical

16.4 (4.5) 17.8 (5.0)

16.4 (4.5) 18.2 (5.3)

17.1 (4.1) 17.4 (4.7)

14.5 (6.2) 14.8 (5.3)

15.3 (5.7) 13.4 (4.4)

15.7 (6.1) 14.2 (4.6)

Comparison of TDI software programs using color TDI techniques, and were in this study above 200 frames per segment in all cases. For 2D-ST the recommended frame rates are between 70 and 120. However, this difference in frame rates did not seem to influence the result of the two methods, as the results of TV, SR, and strain, obtained by color TDI and by 2D-ST, were comparable and can be used interchangeably in the IVS. There were some statistically significant differences between the two methods in the IVS, but differences were so small that they are not clinically relevant, because most of the measurements were within the limits of agreement as shown in the Bland-Altman difference plots and the mean differences are in the range or below of the measurement variability. However, in the LVFW, TV results were significantly higher using color TDI compared to 2D-ST and these differences may be of clinical relevance. An explanation may be that the LVFW was not always well aligned with the ultrasound beam and that angular errors may have influenced the results. The Doppler-based software used for TDI analysis may actually measure a combination of longitudinal, as well as radial movements of the myocardial wall resulting in higher measurements results compared to 2D-ST measurements that uses angle-independent quantification of myocardial motion.48 The comparison between the basal, middle, and apical segment showed significant differences between the segments for TV in both walls, with a basal to apical velocity gradient. This is in accordance with other human and animal studies.1,3,9,11,16,43,49 This velocity gradient is another disadvantage of TV compared to SR and strain, as separate reference ranges are necessary for each myocardial segment. SR and strain however did not show differences between the segments in this study. Therefore, separate SR and strain reference values for different segments seem not to be necessary, according to the present study, which makes interpretations easier. Other studies found some differences between segments, which might be explained by the use of Doppler-based analysis software and possibly due to angular errors.38,50 Special caution was used in this study to ensure a proper manual tracking using the color TDI software. This may explain why no segmental differences occurred, but manual tracking is time-consuming. Additionally, due to the low signal-to-noise ratio analysis of color TDI methods, there is a steep learning curve, and experience is required for a correct interpretation of the signals. This may be a reason, why the technique is not yet more commonly used in clinical practice.26,38 The new 2D-ST method in contrary uses a semi-automatic tracking system

41 and has fewer problems with the signal-to-noise ratio, as it averages the results over the segment and uses grayscale pixel tracking. A limitation of this study is that the influence of breed could not be assessed, as there were many different breeds with only few dogs per breed available for analysis. It has been recommended to establish separate reference values for different breeds16 and this might be necessary, at least for certain breeds in which cardiomyopathy has a high prevalence. In these breeds TV, SR and strain may be helpful in the early diagnosis of these diseases. However, it was not the intention of this study to establish breed specific reference ranges, but to compare different methods of obtaining TV, SR and strain data. Other potential physiologic influence factors such as heart rate, weight and gender were also not tested in this study, as these factors have been assessed already in other studies. Additionally, these factors are not relevant for the comparison between two methods of analysis. Another limitation of this study is that the time necessary to perform an analysis was not recorded. Analysis using 2D-ST is usually possible within 30 s per myocardial wall. Using color TDI, first manual tracking of each ROI in each segment has to be done, and then manual measurements of the peak TV, SR and strain curves is performed, which will take several minutes. Therefore, there is no doubt that 2D-ST is much faster than Q-analysis. In conclusion, this study shows that AOS results displayed by the new 2D-ST method are comparable with SP measurements, which are usually obtained by TDI methods. Furthermore, reproducibility data were comparable between the color TDI software using manual tracking, and the 2D-ST software using automatic tracking, 2D-ST worked well in the IVS, but could not perform measurements in the LVFW in about 30% of the cases. Semi-automatically tracking of the myocardial wall and automatic measurements of TV, SR and strain using 2D-ST is potentially time saving and convenient for the user. Whereas TV and strain have a good reproducibility in both myocardial walls, reproducibility is acceptable for SR in the IVS, but not adequate in the LVFW. The reproducibility of 2D-ST is similar to the results of color TVI. Therefore, as this study shows a good agreement between the methods, the new 2D-ST method is a valuable alternative with several advantages over the color TDI methods to measure TV, SR, and strain in dogs.

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