Our investigation highlighted variations in the GSM and area of plaques when measured on a frame-by-frame basis throughout ultrasound image sequences. Image normalisation did not reduce the extent of the GSM variations and in some cases resulted in greater variation. These results demonstrate that frame-by-frame variations in the plaque GSM cannot be offset by applying normalisation factors based on the selection of blood and adventitia regions in one of the frames. Furthermore, selecting separate reference regions in all images introduced an additional source of variability due to the subjective nature of the process. The reference regions were user-selected and not computerized as they are easily identified by the operator and it would have been difficult to ensure the accuracy of a computerized selection. In NORM1 normalisation, the coefficients of variation for GSM changed after normalization for some plaques. This occurred when the blood reference regions had non-zero GSM, causing an intercept to be introduced into the linear relationship between the normalized and un-normalized greyscale values. Furthermore, as GSM values are limited to the range 0 to 255, normalization could result in the clipping of the GSM values outside this range, thus affecting the coefficients of variation. The increased coefficients of variation for GSM, in the case of the NORM2 normalisation, provided evidence that the frame-by-frame variations seen in GSM were unlikely to be, at least significantly, due to a general temporal variability in the overall image brightness.
The extent of the GSM variations seen in the study were similar in magnitude to those reported by Elatrozy et al. [12], who found that, after normalisation, the coefficient of variation among 4 different observers was 4.7% for the GSM of plaques. However, the variations captured by that study did not include variations that may have been observed to the selection of different still images as each of the four observers appeared to have used the same image to assess the GSM. Therefore, the true inter-observer variabilities may have been greater than that suggested by the results of that study.
The findings of our study have two implications. Firstly, in the case of studies which have already considered intra/inter-observer variabilities, the variabilities found may have been under-estimated unless the same image frames were not used in multiple assessments of the GSM. Secondly and conversely, in the case of inter/intra-sonographer or across-study variabilities of GSM measurements, some of these variabilities may have been due to the selection of different image frames corresponding to the differences in the exact cross-sections being imaged.
The variations seen in the plaque GSM and area may be due to a number of different factors. While changes to the acquisition settings during a single acquisition would not be expected, changes in the plaque GSM could occur, for example, if the distance between the plaque and the transducer face changed during an acquisition. Patient or probe motion may also change the location and orientation of the scan plane with respect to the plaque being imaged, affecting both the measured GSM and the observed cross-sectional area. These are likely to be significant contributors to the variations seen in the GSM and area of plaques in this study. Deformation or compression of the plaque under the pulse pressures may also cause changes in the measured plaque GSM and cross-sectional area and the observation of periodical variations with physiologically reasonable frequencies in the plaque GSM and area for several of the plaques provided evidence to support this hypothesis. However, it should be noted that such cyclic variations could also have been caused by periodic variations in the scan plane location and orientation due to out-of-plane plaque, patient, or probe motion. The poor correlation between the inter-frame coefficients of variation of GSM and plaque area suggested that at least some of the variations seen in the plaque GSM were likely to have occurred due to factors other than changes in the observed plaque area. This was also supported by the results of the regression analysis, which did not highlight the parameters based on the plaque area as being statistically significant contributors to the variabilities seen, across plaque samples, in the extent of the inter-frame GSM variations.
Other factors that may have caused apparent changes in the plaque GSM and cross-sectional area included unclear plaque boundaries (e.g. poor quality image or substantial image noise), which may have caused fluctuations in the detected plaque boundaries. However, the images used in this study were of sufficiently good quality that any variations due to such fluctuations were not thought to be a major contributor to the GSM variations observed.
The statistically significant difference found in the mean GSM of plaques in the symptomatic and asymptomatic groups is in accord with previous findings that have shown symptomatic plaques, in general, had lower GSM [3, 4]. The differences between the two groups in the case of the parameters describing the inter-frame variations in the GSM were not statistically significant which was plausible as out-of-plane plaque, patient, and probe motion appeared to be a significant sources of variation for GSM measurements.
Manual delineation of the plaque boundaries separately for each image frame was found to increase the extent of the frame-by-frame variations observed in the plaque GSM and area (7.7% and 8.0%, respectively, compared with 5.4% and 4.0% for our method) due to the greater subjectivity of the manual delineation process.
The main limitations of our study were the use of two dimensional ultrasound and the absence of any attempts to fix the scan plane location and orientation with respect to the plaque being imaged, other than those measures normally taken in the clinics (e.g. holding the probe fixed and asking the patient to remain still). It should be remembered that the method of ultrasound acquisition commonly used in carotid plaque GSM studies, namely two dimensional ultrasound, provides only a cross section of the whole plaque volume. Since the plaque GSM measured using two dimensional techniques reflects only a cross-section, these measurements are susceptible to variations due to out-of-plane plaque, probe and patient motion. Studies incorporating three dimensional techniques may overcome this limitation and enable further investigation of the nature of any intrinsic frame-by-frame variations in the plaque GSM. Such follow-up studies may also identify whether any inter-frame variations seen in the GSM and volume of plaques can provide additional insight into the dynamic behaviour of carotid plaques, thus improving clinical utility.
Another limitation of our assessment of the plaque GSM and cross-sectional area was with regard to anechoic regions of plaques and regions of plaques in areas of acoustic shadowing. These regions were excluded from analysis. The cross-sectional areas of plaques that had such regions were, thus, under-estimated and neither the plaque area nor the GSM reflected the true values. The excluded regions also had an effect on the magnitude of the frame-by-frame variations that were measured for the affected plaques. In the case of anechoic regions of plaques, the inclusion of the anechoic regions would have reduced the magnitude of the variations observed. However, this would have been only because of the absence of echogenicity in these regions. In the case of the regions of acoustic shadowing, these regions need to be excluded from analysis due to the absence or deterioration of plaque texture information resulting from acoustic shadowing. Although Colour Doppler is useful for subjectively defining the plaque-lumen boundary, it is not suitable for quantifying the plaque area throughout the cardiac cycle, since colour filling of the lumen is dependent on blood flow velocity [17]. Nevertheless, our results demonstrated that variations in the plaque GSM and cross-sectional area were observed, in the visible parts of the plaques that were not in regions of acoustic shadowing. Such variations are important as they could lead to an error in a potential diagnostic test that uses the GSM as the selection criterion, particularly for plaques of intermediate echogenicity where a coefficient of variation of 5% may provide enough bias to move the plaque between the high-risk and low-risk groups. As the plaque GSM is not generally used to inform clinical decision making, these variations do not currently have a clinical impact. Nevertheless, the variations should be appreciated for research studies which increasingly utilize the plaque GSM.
Our results did not find the cross-sectional plaque area to affect the extent of the frame-by-frame variations observed in the plaque GSM significantly. A major source of variation in the inter-frame plaque GSM may in fact be the movement of the plaque cross-section with respect to the scan plane and this may be a bigger problem for smaller plaques. However, our results showed that the observed GSM could vary on a frame-by-frame basis substantially for large plaques as well the minor stenoses.
Since previous studies typically quantified GSM in single frames of ultrasound images, the variations found in this study have been previously neglected. Improved attempts to standardise GSM measurements and reduce variability between centres should account for these findings, for example, by performing GSM measurements at peak systolic/diastolic frames or by carrying out an assessment of the average GSM throughout the cardiac cycle. Techniques such as GSM assessment using multiple cross-sectional views of plaques and plaque texture analysis can also be used to improve diagnostic precision compared to a single cross-sectional assessment. The best option would be to carry out these processes in three dimensions, however, three dimensional ultrasound techniques are still under development and are not widely available. It should be noted that we do not propose the technique we have used in our study as a replacement for other methods but we highlight the variations in plaque GSM and cross-sectional area that may be observed on a frame-by-frame basis using single-view, two dimensional ultrasound.