Guideline for in vivo assessment of adherent and rolling leukocytes in human skin microvasculature via reflectance confocal videomicroscopy

Abstract

Objective To develop a guideline that reliably identifies cutaneous adherent and rolling leukocytes from mimicking scenarios via in vivo reflectance confocal videomicroscopy. Methods We used a clinical reflectance confocal microscope, the VivaScope 1500, to acquire 1522 videos of the upper dermal microcirculation from 12 healthy subjects and 60 patients after allogeneic hematopoietic cell transplantation. Blinded to clinical information, two trained raters independently counted the number of adherent and rolling leukocytes in 88 videos. Based on discrepancies in the initial assessments, we developed a guideline to identify both types of leukocyte-endothelial interactions via a modified Delphi method (without anonymity). To test the guideline’s ability to improve the inter-rater reliability, the two raters assessed the remaining 1434 videos by using the guideline. Results We demonstrate a guideline that consists of definitions, a step-by-step flowchart, and corresponding visuals of adherent and rolling leukocytes and mimicking scenarios. The guideline improved the inter-rater reliability of the manual assessment of both interactions. The intraclass correlation coefficient (ICC) of adherent leukocyte counts increased from 0.056 (95% confidence interval: 0-0.236, n=88 videos, N=10 subjects) to 0.791 (0.770-0.809, n=1434, N=67). The ICC of rolling leukocyte counts increased from 0.385 (0.191-0.550, n=88, N=10) to 0.626 (0.593-0.657, n=1434, N=67). Intra-rater ICC post-guideline was 0.953 (0.886-0.981, n=20, N=12) and 0.956 (0.894-0.983, n=20, N=12) for adherent and rolling, respectively. Conclusion The guideline aids in the manual identification of adherent and rolling leukocytes via in vivo reflectance confocal videomicroscopy.