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Background Through noninvasive imaging of live, individual immune cells of patients post allogeneic hematopoietic cell transplantation (HCT), we characterized differences in microvasculature among patients with cutaneous acute graft-versus-host disease (aGVHD) compared to post-HCT patients without any organ aGVHD (controls). Traditional histopathology is often non-specific for cutaneous aGVHD. We present parameters characteristic of cell motion within upper dermal capillaries, which could enable noninvasive tracking of disease development. Objective Test the feasibility of eight quantitative parameters extracted from reflectance confocal videos to distinguish between patients with and without cutaneous aGVHD following HCT. Methods We used a clinical reflectance confocal microscope to noninvasively image left volar forearm dermal capillaries of 5 cutaneous aGVHD patients and 5 post-HCT controls prior to topical and systemic therapies. All diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB) based on initiation and response to therapy. Two raters (JRP, IS) independently estimated capillary density and size, and the number and size of paused (temporarily stopping), adherent (stationary for 30+ sec), and rolling leukocytes. The mean value per two raters was calculated for further analysis. Due to the small sample size, descriptive statistics were used. Results Patients in both study groups were similarly distributed in terms of gender (40% female), age (median 63 and 63, respectively), days post-HCT (median 44 and 41), and underlying disease (60% and 40% acute myeloid leukemia; the rest: myelodysplastic syndrome). 4 patients had only skin involvement: grade 1 (N=1 patient), grade 2 (N=3), whereas one patient had grade 2 cutaneous with symptoms of chronic ocular GVHD. One patient required therapy with systemic steroids. We found an increased number of rolling (median: 2 vs 1), adherent (3 vs 1) and paused (2 vs 1) leukocytes in aGVHD patients, compared to post-HCT controls (Figure 1). The average time of a leukocyte being paused was higher in aGVHD patients (6±7 seconds) than post-HCT controls (2±3 seconds). The size of paused leukocytes was greater in aGVHD patients (9±2¼m) compared to controls (6±4¼m) (Figure 2). Capillary size (8±2 vs. 8±2¼m) and density (4±1 vs. 3±2 capillaries) were similar among both groups. Conclusion In similar-sized capillaries, we observed increased leukocyte interactions with the endothelium in aGVHD patients compared to post-HCT controls. Despite similar sized rolling and adherent leukocytes, the size of paused leukocytes was larger in aGVHD patients. Future studies could evaluate the change of these parameters over time.