Noninvasive Microscopic Imaging Reveals Increased Leukocyte Adhesion and Rolling in Skin of Acute Graft-Versus-Host Disease Patients Compared to Post-Transplant Controls

BACKGROUND. The nature and kinetics of leukocyte migration are intimately connected to the pathophysiology of acute graft-versus-host disease (aGVHD). Real-time visualization of leukocyte-endothelial interactions in patients post hematopoietic transplantation (HCT) may augment clinical diagnosis and patient management. In this cross-sectional pilot study, we aimed to test the feasibility of parameters characteristic of leukocyte motion in skin capillaries as potential imaging biomarkers to detect aGVHD.

STUDY POPULATION. We enrolled 16 post-HCT patients: 8 patients with any organ aGVHD and 8 patients with no organ aGVHD on the day of the imaging. Diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB), who was blinded to the results of the confocal imaging. 7 out of 8 aGVHD patients had only skin involvement: stage 1 (N=2 patients), stage 2 (N=2), and stage 3 (N=3) cutaneous aGVHD, whereas one patient had stage 1 gut aGVHD with no skin involvement. 6 out of 8 aGVHD group patients required systemic therapy with steroids.

METHODS. To noninvasively visualize leukocyte motion in the topmost capillaries of post-HCT patients' skin, we used a clinical reflectance confocal microscope (Vivascope 1500, Caliber I.D.). It enables real-time en face view of individual cells at 9 frames per second. We took twenty 30-second videos of different capillaries in the left volar forearm and left upper chest. We counted the number of adherent and rolling leukocytes per 10 minutes of videos per patient.

RESULTS. We found increased leukocyte rolling in the aGVHD group (average of 3 rolling leukocytes per patient), compared to post-HCT controls (average of 1 rolling leukocyte). Highest leukocyte rolling (>8 rolling leukocytes) was associated with increased all-cause mortality, but not predictive of transplant-related mortality (TRM). Similarly, we found increased leukocyte adhesion in the aGVHD group (average of 3 adherent leukocytes per patient), compared to post-HCT controls (average of 1 adherent leukocyte). Interestingly, we observed 7 adherent leukocytes in one patient who had isolated gut aGVHD and no skin involvement.

DISCUSSION. Our preliminary results show altered leukocyte-endothelial interactions in the skin capillaries of aGVHD patients, suggesting that confocal microscopy may be an important tool in augmenting the clinical diagnosis and managing post-HCT patients. Future studies should test whether there is a difference in the pattern of change in parameter values between patients who did and who did not develop aGVHD.

ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service.