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Objective To investigate the effect of patient positioning on the Duplex ultrasound evaluation of great saphenous vein reflux elicited by the pneumatic cuff method. Methods FFifty great saphenous veins (GSV) with relfux (relfux group) and iffteen with no prior history of venous disease (healthy group) were examined by duplex scanning in the supine, 20 degrees reverse-trendelenburg (RT-20), 40 degrees reverse-trendelenburg (RT-40) and standing position. Each GSV was assessed for relfux at three venous sites:two centimeter below the sapheno-femoral junction (SFJ), the greater saphenous vein in the mid thigh (MGV) and the greater saphenous vein in the upper calf (CGV). Pneumatic cuff compression pressure of conifned 100 mmHg (1 mmHg=0.133 kPa) was used onto the calf to elicit relfux. The incidence of positive venous relfux was calculated. The statistical differences of the peak relfux velocity and duration of relfux in four positions were analyzed. Results TThe relfux elicited in the standing position was set as the gold standard. In healthy group, there was no false positive results of relfux in supine, RT-20 and RT-40 positions. In relfux group, false negative results were found at all venous sites when limbs were examined in supine position [false negative rate:59%(19/32), 22%(11/50), 24%(12/50)]. At RT-20 and RT-40 positions, the incidence of venous relfux reached 100% at MGV and CGV, and false negative cases were only detected at SFJ [false negative rate:12%(4/32), 12%(4/32)]. The relfux time in standing, supine, RT-20 and RT-40 positions were (7.75±3.23) s, (5.27±3.66) s, (8.67±3.72) s, (8.55±3.93) s respectively. There were signiifcant differences among different positions in reflux time (F=56.9, P<0.01). In detail, no significant differences were identified between standing position and RT-20 or RT-40 position (q=1.51, 1.33 respectively, both P > 0.05), except for supine position (q=4.11, P<0.01). Peak relfux velocity in standing, supine, RT-20 and RT-40 positions were (55.26±22.24) cm/s, (22.87±12.03) cm/s, (38.46±16.30) cm/s, (45.13±19.21) cm/s respectively. There were also signiifcant differences among different positions in peak relfux velocity (F=13.7, P<0.01). Comparing the supine, RT-20 and RT-40 positions with standing position, differences of the peak relfux velocity between them were all statistically signiifcant (q=12.71, 6.59, 3.98 respectively, all P<0.01). Conclusions When GSV reflux was examined by pneumatic cuff compression, false negative rate was higher in the supine position. RT-20 and RT-40 position were effective to detect GSV relfux, espically for GSV at mid-thigh and upper calf.