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Despite the fact that assessment of right ventricular longitudinal strain (RVLS) carries important implications for patient diagnosis, prognosis, and treatment, its implementation in clinical settings has been hampered by the limited reference values and the lack of uniformity in software, method, and definition used for measuring RVLS. Accordingly, this study was designed to establish (1) the reference values for RVLS by 2-dimensional speckle-tracking echocardiography; and (2) their relationship with demographic, hemodynamic, and cardiac factors.In 276 healthy volunteers (55% women; age, 18–76 years), free wall and septum RVLS (6 segments) and free wall RVLS (3 segments) using both 6- and 3-segment regions of interest were obtained. Feasibility of 6-segment RVLS was 92%. Free wall RVLS from 3- versus 6-segment regions of interest had similar values, yet 6-segment region of interest was more feasible (86% versus 73%; P<0.001) and reproducible. Reference values (lower limits of normality) were as follows: 6-segment RVLS, −24.7±2.6% (−20.0%) for men and −26.7±3.1% (−20.3%) for women; 3-segment RVLS, −29.3±3.4% (−22.5%) for men and −31.6±4.0% (−23.3%) for women (P<0.001). Free wall RVLS was 5±2 strain units (%) larger in magnitude than 6-segment RVLS, 10±4% larger than septal RVLS, and 2±4% larger in women than in men (P<0.001). At multivariable analysis, age, sex, pulmonary systolic pressure, right atrial minimal volume, as well as right atrial and left ventricular longitudinal strain resulted as correlates of RVLS values.This is the largest study providing sex- and method-specific reference values for RVLS. Our data may foster the implementation of 2-dimensional speckle-tracking echocardiography–derived RV analysis in clinical practice.