AN INTEGRATED MICRODEVICE FOR RAPID NEUTROPHIL PURIFICATION AND PHENOTYPING USING A DROP OF BLOOD

In this work, we introduce a novel microdevice capable of single-step neutrophil sorting from whole blood directly for multiplexed functional phenotyping (chemotaxis and NETosis) (Fig. 1A). Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycaemia, resulting in increased oxidative stress, chronic low-grade inflammation and endothelial dysfunction [1]. Neutrophils are key effector cells of the innate immunity, and are known to play a pivotal role in diabetic pathophysiology [2]. However, study of neutrophil dysfunctions remains technically challenging due to difficulties in isolating “touch-free” neutrophils in their native state using conventional leukocyte isolation methods (density gradient centrifugation and blood lysis). Microfluidics technologies have been recently developed for neutrophil studies, but require additional leukocyte preparation or manual washing steps to efficiently purify neutrophils from whole blood [3, 4].  Herein, we report an integrated microfluidics biochip for single step neutrophil purification and functional phenotyping using small blood volume (fingerprick) for point-of-care diabetes testing.

The microdevice is fabricated using polydimethylsiloxane (PDMS) and consists of a margination channel, 20×20 µm (W×H) with 2 side chambers 400×20 µm (W×H). As whole blood (~10 µL) is pumped through the straight channel, deformable red blood cells (RBCs) migrate laterally towards the axial centre (Fahraeus effect), resulting in margination of other cell types (platelets and leukocytes) into the smaller side channels (Fig. 1B). Neutrophils are selectively captured in the anti-CD66b-functionalized surface and then exposed to a stable diffusion-based gradient of either chemoattractant (fMLP) or calcium ionophore (AZ23187), to study chemotaxis or formation of neutrophil extracellular traps (NETosis) respectively (Fig. 1C). Time-lapse imaging and single cell image analysis are performed to probe chemotaxis (migrating cells and chemotactic velocity) and NETosis (nuclear membrane degradation) phenotypes.

A 10-fold leukocyte enrichment was achieved at the side outlets (Fig. 2A) and leukocyte differential count using flow cytometry analysis also showed ~50% decrease in neutrophil concentration in the eluent collected from anti-CD66b-coated side outlets, indicating efficient on-chip neutrophil capture (Fig. 2B). We next measured the fluorescence intensity along the side chamber using FITC dye preloaded in the reservoir proximal to the connecting side channels, and linescans at various time points showed that the diffusion gradient remained linear and stable for 2 hours (Fig. 3B). For chemotaxis assay we successfully demonstrated migration of captured healthy neutrophils towards the chamber preloaded with fMLP (200 nM) within 2 hours (Fig. 3C). For NETosis assay, glucose-treated neutrophils (30 mM) exhibited an increase in NETosis (P < 0.05) than untreated and mannitol-treated (30 mM) neutrophils (Fig. 4A, B).

The unique strategy of integrating microfluidic neutrophil sorting with cellular functional assays facilitates user operation and the developed diagnostic platform can be further advanced for point-of-care inflammatory profiling and precision medicine approaches. We envision that characterization of neutrophil chemotaxis in diabetes patients can provide direct evidence in microvascular compilations in diabetes and be used as surrogate biomarkers for monitoring endothelial dysfunction, arterial stiffness, peripheral markers of inflammation and oxidative stress in metabolic diseases.