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PLoS One. 2019 Mar 14;14(3):e0213175. doi: 10.1371/journal.pone.0213175. eCollection 2019.

Reproducibility of sublingual microcirculation parameters obtained from sidestream darkfield imaging.

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Department of Public Health, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.
Department of Cardiology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.
Department of Clinical Epidemiology and Biostatistics, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.
Department of Vascular Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.



Changes in the microcirculation may be used as a surrogate outcome in studies on cardiovascular disease. We assessed the reliability characteristics of the sublingual microcirculation parameters Vascular Density (VD), Red Blood Cell Filling (RBCF), and Perfused Boundary Region (PBR) as obtained by sidestream darkfield imaging.


For each of the three parameters, the variance components of measurement, the Intraclass Correlation Coefficient (ICC), the Standard Error of Measurement, and the limits of agreement were estimated for the intra-rater setting (N = 50) and the inter-rater setting (N = 48). Subsequently, as a proof of concept, the reliability measures were used for a power analysis to design studies to evaluate the effect of acute stimuli-i.e. having a meal (N = 50) and cigarette smoking (N = 21) on the three parameters.


Reproducibility was poor for all three parameters. The intra-rater ICC for 2 measurements was 0.28 (95% CI: 0.04, 0.53) for the VD, 0.51 (95% CI: 0.27, 0.69) for the RBCF, and 0.33 (95% CI: 0.08-0.56) for the PBR. The standard errors of measurement and the limits of agreement for all three parameters were larger than most statistically significant intra-individual or inter-individual differences reported in previous studies. The proofs of concept showed that sample sizes in excess of 600 subjects are necessary to reach statistical significance for the observed effects of having a meal or smoking on VD and PBR.


The reliability of the three sublingual microcirculation parameters in their current form appears to be low and a large sample size is advisable for their use in conditions similar to those we describe.

Conflict of interest statement

The authors have declared that no competing interests exist.

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