98. Biomechanical phenotype of circulating myeloid cells is altered in ANCA associated vasculitis

Background: Real Time-Deformability Cytometry (RT-DC) is a novel technique able to identify morpho-rheological characteristics of individual cells such as size, deformability, and elasticity. Cells in suspension flow through a microfluidic channel at a set rate while hydrodynamic force is applied, leading to reversible deformation of individual cells from shear stress and pressure gradients. Cell brightness and area can be used to identify cell populations within whole blood. It is possible to characterise a morpho-rheological phenotype of major leucocyte subsets simultaneously using only 50μl of whole blood. Pathological activation of immune cells in vivo can lead to decreased cell deformability, reducing their ability to traffic through microvasculature and potentially increasing the capacity for endothelial damage and vascular inflammation. The morpho-rheological characteristics of immune cells in ANCA associated vasculitis (AAV) are unknown. Methods: Whole blood from healthy controls (HC), patients with active AAV, and patients with AAV in remission was analysed using RT-DC. Anticoagulated blood was diluted 1:20 in CellCarrierB (1xPBS/0.6% methylcellulose). Cell suspensions were drawn into a 1ml sample syringe on a syringe pump (neMESYS, Cetoni) of the AcCellerator (Zellmechanik), an extension for an inverted microscope (Zeiss). Syringes connected to polymer tubing were attached to the sample inlet of a Flic20 PDmicrofluidic chip (Zellmechanik), containing reservoirs connected by a measurement channel with a 20x20 μm2 cross-section. RT-DC measurements were collected at a flow rate of 0.12 μL/s using a high-speed CMOS camera (Mikrotron) to capture images of cells as they reached the end of the microfluidic channel at a frame rate of 2000 frames/second. ShapeOut software was used to calculate cell size, deformation, and elasticity. Results: RT-DC analysis of whole blood showed no difference in neutrophil size between patients with active AAV (n=8), patients with AAV in remission (n=16), and HC (n=10) (Figure 1A). Neutrophils from patients with active AAV were significantly stiffer than patients in remission or HC, displaying decreased deformation (Figure 1B; median 0.078, 0.086, and 0.087 au respectively, p=0.007) and increased elasticity (Figure 1C; median 1.005, 0.968 and 0.968 au respectively, p=0.03). In patients with AAV (active and remission), there was a strong inverse correlation between neutrophil deformation and disease activity as measured by BVAS score (r=-0.77, p<0.0001). There was no correlation of neutrophil physical properties with renal function or neutrophil count. Monocytes demonstrated morpho-rheological properties similar to neutrophils, with no difference in cell size between patients with active AAV (n=6), patients with AAV in remission (n=16), and HC (n=9) (Figure 1D). Monocytes from patients with active AAV had lower deformation (Figure 1E; median deformation 0.066, 0.073, and 0.076 for active AAV, remission AAV, and HC respectively) and higher elasticity than those from patients in remission or HC (Figure 1F; median elasticity 1.072, 0.999 and 0.989 for active AAV, remission AAV, and HC respectively, p=0.04). There was no difference in morpho-rheological characteristics of lymphocytes between the three groups. Conclusion: Neutrophils and monocytes from patients display distinct physical properties in active AAV which correlate with disease activity. This phenotype of increased cell stiffness may lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. These parameters can be rapidly measured using a small volume of whole blood; thus RT-DC may be a useful technique to aid identification of disease activity in AAV and to guide treatment. Disclosures: None Figure 1. Morpho-rheological properties of circulating leucocytes in AAV (A)-(C) Neutrophils, (D)-(F) Monocytes. Data are shown as median with IQR, statistical comparison by Kruskal-Wallis test, with Dunn’s post-test comparison; *p<0.05 **p