Indirect measurement of the vascular endothelial glycocalyx layer thickness in human submucosal capillaries with a plug-in for ImageJ

Background: The thickness of vascular endothelial glycocalyx layer can be measured indirectly during a spontaneous leukocyte passage from oral submucosal capillaries in humans. The subsequent differences in red blood cell (RBC) column widths, before a spontaneous white blood cell passage (pre-WBC) and after a spontaneous WBC passage (post-WBC) can be used in off-line analysis to measure glycocalyx thickness: [pre-WBC width-post-WBC width]/2. We created and validated a semi-automatic plug-in for ImageJ to measure the endothelial glycocalyx layer thickness.

Methods: Video clips presenting human sublingual microvasculature were created with a side-stream dark field imaging device. Spontaneous leukocyte passages in capillaries were analyzed from video clips with ImageJ. The capillary glycocalyx layer thickness was measured by the indirect approach with two manual and two semi-automatic methods.

Results: There were no statistically significant differences between glycocalyx layer thicknesses measured with different methods, even though small inter-method differences in RBC column thicknesses could be detected. Inter-rater differences were systematically smaller with both semi-automatic methods. Intra-rater coefficient of variation [CV] (95% CI) was largest when measurements were made completely manually [9.2% (8.4-10.0)], but improved significantly with automatic image enhancement prior to manual measurement [7.2% (6.4-8.0)]. CV could be improved further when using semi-automatic analysis with an in-frame median filter radius of 1 pixel [5.8% (5.0-6.6)], or a median filter radius of 2 pixels [4.3% (3.5-5.1)].

Conclusions: Semi-automatic analysis of glycocalyx decreased the intra-rater CV and the inter-rater differences compared to the manual method. On average, each of the four methods yielded equal results for the glycocalyx thickness. Being the only feasible bed side method in most clinical scenarios, indirect measurement of glycocalyx thickness with orthogonal polarization spectral imaging or side-stream dark field imaging device and our plug-in can advance the study of glycocalyx layer pathology in man.