The present study was designed to internally validate the analysis of FMD within the same tester and between testers. The major findings in this study are two-fold: first, the FMD analysis protocol is reproducible within same tester and second, the FMD analysis protocol is reproducible between testers.
In general, FMD protocols follow international guidelines [5, 10, 11]. Our laboratory is not an exception. All images are obtained with an isonation angle of 60° and images are obtained with the help of an ECG-gated system to avoid changes of vessel diameter observed within one cardiac cycle. These technical details provide more accurate and clear images of the studied vessel. The recording duration of 180 s, 30 s prior to deflation accompanied by 150 s post-deflation, was determined to be the most adequate method in which FMD could be determined [5, 10]. The 30 s prior to deflation allows to record images of the vessel that reflect differences with baseline diameters [7, 21]. After the cuff deflation, the hyperemic blood flow will rapidly increase eliciting the vascular response, which will peak anywhere between 30 and 90 s in an average 18–35 years old adult. The additional 60 s help to account for outliers in the vasodilatory response as well as return to baseline measurements.
The present study showed that the intratester CVs from two independent testers (tester 1 and 2) were very similar in all studied variables (range 2.62–4.95%, Table 3) and there no significant difference between the CVs from both testers. Brachial baseline and peak CVs are all below 3.75%, while FMD CV was slightly higher in one of the testers (4.95%). The CVs obtained in the present study are lower than the CVs from previous reports, which are already considered as low methodological error [16, 17, 22]. Therefore, the intratester validation presented here provides data for brachial baseline and peak artery dilation, making this data an acceptable reproducible measurement of brachial artery recording. Similar results were observed for the intertester analysis (Table 4). CVs between both testers were lower or at 5.50% for all studied variables. All the studied diameters, brachial baseline and peak and femoral baseline and peak, and femoral FMD showed no significant difference between testers. Only brachial FMD showed a significant difference between testers. This difference might be the results of slightly larger peak brachial diameter observed on tester 1, which could produce a larger difference when the FMD ratio is applied. Interestingly, and according to our best knowledge, this is the first study designed to determine reproducibility between two independent testers. The results of the present study show that the FMD protocol use in our laboratory is reproducible within a ~ 5% of variation between testers.
Other studies using similar approaches have shown comparable results than the present study. For example, Avery et al. [16] used the same automated edge detection system [15] as the present study. Their CVs were slightly higher than 5% (4–10%). This difference might be explained by the difference on their acquisition system, a 10.5 MHz ultrasound probe, which could decrease the image definition [5, 15]. Woodman et al. [17], using a tailored automated edge detection system, also found slightly higher CVs than the ones presented here (6.7%). Interestingly, their image acquisition was also performed with a 10 MHz ultrasound probe, which can produce less defined images [5]. Finally, Ghiadoni et al. [12, 22] used a new automated edge detection system that does not use ECG-gated image selection, using the average vessel diameter within a cardiac cycle. In addition, they recommend using 7.5 to 10 MHz ultrasound probes, which decreases images definition [5]. These two major differences might be responsible of higher CVs (7.6–11.6%) than the ones observed in the present study [22].
The final impact that this study had was the reproducibility of the observed results. A major factor of clinical utilization of FMD as a diagnostic technique is the accurate replicability between analyzers [1, 5, 11]. In this laboratory, two individuals with different backgrounds in research performed this validation study. One tester was a research assistant for nearly 2 years in this lab, while the other had been working in the lab for only 1 month prior to this analysis. The same techniques were provided by the principal investigator to both analyzers, with the same allotted practice time and techniques given from previous studies [17, 23]. The correlation between testers provided by this data proves the reproducibility of this laboratory procedure and advances the application of FMD as a future clinical diagnostic tool.
In essence, the present study was designed to provide a basis of support and assess the capabilities of our laboratory to provide accurate analysis and a specific, reproducible protocol for FMD testing. One of the main problems with FMD becoming a clinical assessment tool is the wide variety of results between laboratories and the dependency upon experimenter knowledge and background [23]. To solve the problem, our laboratory has created a structured methodological approach, accompanied by several sessions of background information about FMD that discussed background knowledge, methods, and common misconceptions, including image acquisition, recordings storage, and analysis software training. The present approach provides a basis of support for the protocol being easily reproducible and helps laboratories performing FMD to use it as a potential biomarker.
This study was not without limitations. First, the present study used the calculation of FMD proposed by Atkinson et al. [19, 20, 24]. This method deviated from previous studies, which used %FMD = [(Peak Diameter – Baseline Diameter)/(Baseline Diameter)] × 100 and implements a mathematical correction to account for the confounding influence of allometric scaling between individuals [19, 20, 23, 24]. Atkinson et al. approach allows constructing a mathematically efficient and more accurate representation of FMD ratio, which in the present study are different from data presented from other laboratories [4,5,6, 11, 16, 17, 22]. Secondly, the ECG-gated trigger system produced some incorrect readings on the QRS complexes, sometimes choosing a frame picture that was not at the correct time line. However, this limitation was non-significant as an occurrence in less than 1 in 1000 frames was observed. Finally, one individual, as highlighted in the results section, had to be removed from the intratester analysis due to technical difficulties that could not be amended, limiting our number of participants (n = 9).