Repeatability, reproducibility and standardisation of a laser doppler imaging technique for the evaluation of normal mouse hindlimb perfusion

Background. Preclinical perfusion studies are useful for the improvement of diagnosis and therapy in dermatologic, cardiovascular and rheumatic human diseases. The Laser Doppler Perfusion Imaging (LDPI) technique has been used to evaluate superficial alterations of the skin microcirculation in surgically induced murine hindlimb ischemia. We assessed the reproducibility and the accuracy of LDPI acquisitions and identified several critical factors that could affect LDPI measurements in mice. Methods. Twenty mice were analysed. Statistical standardisation and a repeatability and reproducibility analysis were performed on mouse perfusion signals with respect to differences in body temperature, the presence or absence of hair, the type of anaesthesia used for LDPI measurements and the position of the mouse body. Results. We found excellent correlations among measurements made by the same operator (i.e., repeatability) under the same experimental conditions and by two different operators (i.e., reproducibility). A Bland-Altman analysis showed the absence of bias in repeatability (p = 0.29) or reproducibility (p = 0.89). The limits of agreement for repeatability were -0.357 and -0.033, and for reproducibility, they were -0.270 and 0.238. Significant differences in perfusion values were observed in different experimental groups. Conclusions. Different experimental conditions must be considered as a starting point for the evaluation of new drugs and strategic therapies.