As A Bioassay, Should Flow-mediated Dilation Be Measured In Triplicate?

Abstract

The flow-mediated dilation (FMD) test is considered by many to be a functional bioassay of NO bioavailability and endothelial function. Typically, bioassays are measured in duplicate or triplicate which allows for removal of outlying values and subsequent data averaging; however, only a single FMD measurement has traditionally been performed. Although there have been multiple studies evaluating the reproducibility of FMD, concern regarding the reliability of this ultrasonic assessment still exists reflected by its absence in the clinical environment. PURPOSE: This study sought to evaluate the impact of applying the typical assay approach of triplicate measurements to reduce variance in FMD, with the ultimate goal of improving both statistical power in research and clinical relevance. METHODS: Eight, young, apparently healthy, college aged males (ages 23±1 years; BMI 23±1 kg/ht2) participated in this investigation. On three separate days, FMD was assessed following 5 minutes of forearm ischemia and the single outlier was removed (triplicate approach). Reproducibility of FMD was assessed by coefficient of variation (CV %) and intra-class correlation coefficient (ICC). RESULTS: The initial FMD performed on all subjects (single measurement) revealed an FMD of 5.4 ± 0.6, which was not statistically different (p=.532) from the averaged FMD data utilizing the triplicate approach (6.4 ± 1.3). The mean ± SEM CV % for the single FMD assessment was 34.9 ± 8.6% and the intra-class correlation coefficient (ICC) was 0.63; p=0.07. With the triplicate approach the FMD CV % was greatly reduced to 6.8 ± 4.3 while the ICC improved significantly to 0.975; p<0.001. CONCLUSION: Considering the continued skepticism regarding the robust nature of FMD testing, the results of the present study advocates a triplicate approach when assessing FMD which significantly improves reliability, but does not alter the group mean response. With the previously documented efficacy of 30 minutes between FMD measurements without a carry over effect, and the possibility that the time between measurements can be further reduced, the triplicate approach for FMD can improve the statistical power and may even be suitable for clinical use. Supported by TRDRP 15RT-0100 and Parker B Francis Fellowship Program.