Variability of microcirculatory measurements in healthy volunteers

We included healthy non-smoking volunteers aged 18-40. Exclusion criteria were diabetes mellitus type 1 or 2, nicotine consumption and chronic diseases for which chronic medications are used. In addition, volunteers were excluded in case of mouth bleeding, mouth sores and mouth infections as these reduce the quality of the measurement. We recorded the age, sex, height and weight of the volunteers.

Measurements

Measurements were performed using an SDF camera (CapiScope HVCS, KK Technology, Honiton, UK) equipped with GlycoCheck software (Microvascular Health Solutions Inc., Salt Lake City, UT, USA). Two investigators (SEM and BB), both experienced in performing sublingual measurements with this tool, performed three consecutive measurements for each subject. The order of measurement of the two researchers was randomly determined. Measurements were taken with subjects in the supine position, with the researcher standing behind the headboard. The volunteers were asked to swallow saliva before measuring, after which the camera was manually placed and held still in the sublingual region. To limit the movement of the camera, the researchers could put their wrist on the lower jaw of the volunteers. Care was taken to limit pressure artifacts by ensuring that erythrocytes could be seen moving through blood vessels during measurements. Images were recorded only in the absence of air bubbles, excessive amounts of saliva, excessive vessel loops, and excessive amounts of large venules. All measurements in an individual volunteer were performed within 30 minutes.

GlycoCheck Parameters

The GlycoCheck software records 1 s movies composed of 23 images. The recording is launched automatically when the software deems the images of sufficient quality, which means that the intensity and the focus are sufficient for the calculations and that the camera is sufficiently still. Vessels are automatically detected and measurement points are set at 10 µm intervals. GlycoCheck limits its calculations to vessels between 5 and 25 µm wide. A measurement is finished when 3000 measurement points have been acquired.

Perfused border region

The inner layer of the glycocalyx is penetrable by red blood cells and therefore also called the perfused boundary region (PBR)ten. An intact glycocalyx is thicker and less penetrable for red blood cells, leading to a thinner PBR, compared to a damaged glycocalyx. The PBR is therefore an inverse measure of the thickness of the glycocalyx. GlycoCheck software calculates the PBR from the intensity profile at each measurement pointten. A more gradual increase in the intensity profile means a thicker PBR – indicating a thinner glycocalyx.

Red blood cell fill percentage

Percent red blood cell filling, a measure of microvascular perfusion, is defined as the median percentage of time that red blood cells are present at each measurement pointten.

Density of microvascular vessels

GlycoCheck calculates the density of microvascular vessels from the number of measurement points, as each measurement point represents 10 µm of microvessel lengthten. The cumulative length of the microvessels in µm was therefore equal to the number of measurement points multiplied by 10. Density of the microvascular vessels in µm/mm2 was calculated by dividing the cumulative length of the microvessels by the total area recorded in mm2.

Statistics

Data are presented as median [25–75th percentile] or as mean ± standard deviation (SD) as appropriate. Normality was assessed using the Kolmogorov-Smirnov test and Shapiro Wilk’s tests for normality. Data analysis was performed with SPSS (version 27; IBM, Armonk, NY, USA). Values ​​of p

Intra-observer agreement for single measurements was assessed with intraclass correlation coefficients (ICC) using a two-way random model with absolute agreement (ICC(2,1) type according to the Shrout and Fleiss convention14) and reported as ICC (95% confidence interval; CI) for each of the two observers. For the mean of three measurements, ICCs were assessed using a two-way random model with absolute agreement for the mean of multiple measurements (type ICC(2, k); where k = 3). ICCs were found to be poor ( 0.75) according to the guidelines written by Cicchetti15.

Sample size calculation was performed such that an ICC of 0.6 or more (indicating good reproducibility) is detected with a 95% confidence interval width of at most 0 ,3. Based on the method described by Lew et al., we calculated a sample size of 45 volunteers16.

The average values ​​of the 3 measurements taken by each observer were used for the analysis of the interobserver agreement. Interobserver agreement was assessed using Bland-Altman plots and with the ICCs of a two-way random model with absolute agreement for single measurements (ICC type (2,1)). The paired t test was used to assess whether there was a systematic difference between the two observers.

Ethics

The protocol has been reviewed and approved by our Institutional Review Board (METC: #2017-0122). The volunteers were informed about the study in words and in writing. Written consent was obtained from all participants. The study was carried out in accordance with the Declaration of Helsinki.

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