Evaluation of the number of laser-Doppler measurements in assessing regional diaphragmatic microcirculation.

As regional diaphragmatic microvascular blood flow varies widely, the aim in this study was to estimate the number of repeated measurements, obtained by Laser-Doppler flowmetry (LDF), required to achieve a standard level of precision. In 40 urethane-anesthetized Sprague-Dawley rats, computer-aided LDF scanning coupled with a microscope generated diaphragmatic blood flow (Qdi) ranging between 94 and 944 mV with the frequency histogram displaying non-Gaussian distributions. A sampling technique was used to assess the number of measuring sites required for valid estimates of the regional diaphragmatic microvascular flow. From a total of 1,000 Qdi values, random samples of sizes between 5 and 100 were repeatedly drawn to estimate the variability of median flow. Our data shows that the 95th percentile decreased gradually, from a +30% error at n = 5 down to +20% at n = 15-20, remained between +20 and +15% up to n = 35, and reached +10% at n = 50. Moreover, by expressing the precision level of measurements as the length of a 95% confidence interval (beta), a linear relationship between beta values obtained either by the sampling method or repeated measures analysis of variance can be shown (r = 0.902, p < 0.001); beta values by either method were within +/-20% error of the mean values at sample sizes above n = 15. It is therefore recommended that for microscope-guided LDF scanning in the assessment of the distribution of diaphragmatic microvascular blood flow, at least 15 repeated measurements should be done to reach an acceptable standard level of precision. However, facing with clinical situations where 'blind' LDF scanning inevitably includes measurements over large vessels, the minimal sample sizes required to represent tissue perfusion demand further exploration.