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Multiplexed label-free fractionation of peripheral blood mononuclear cells for rapid identification of monocyte-platelet aggregates Hui Min Tay, Wei-Hseun Yeap, Rinkoo Dalan, Siew-Cheng Wong, and Hanwei Hou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04415 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018
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Analytical Chemistry
Multiplexed label-free fractionation of peripheral blood mononuclear cells for rapid identification of monocyte-platelet aggregates Hui Min Tay,†,# Wei Hseun Yeap,‡,# Rinkoo Dalan,§ Siew Cheng Wong,*,‡,∥ and Han Wei Hou*,†,⊥ School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore ‡ Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 8a Biomedical Grove, 138648, Singapore § Endocrine and Diabetes, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, 308433, Singapore ∥ School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore ⊥ Lee Kong Chian School of Medicine, Nanyang Technological University, Clinical Sciences Building, 11 Mandalay Road, 308232, Singapore †
* Email:
[email protected] (S.C.W.);
[email protected] (H.W.H)
ABSTRACT: Monocytes and platelets play key roles in atherosclerosis and thrombosis, and circulating monocyte-platelet aggregates (MPA) in blood have been widely proposed as surrogate biomarkers for cardiovascular risk stratification and monitoring anti-platelet therapies. However, conventional MPA characterization is based on whole blood fixation and flow cytometry analysis which adversely affect cell viability and detection accuracy due to significant leukocyte and platelet contaminations. Herein, we introduce a rapid and label-free microfluidic approach for complete size-based fractionation of peripheral blood mononuclear cells (PBMCs) into monocytes, lymphocytes and platelets. The developed biochip enables gentle sorting of intact MPA in the enriched monocytes with efficient depletion of lymphocytes and platelets for accurate MPA quantification. We first compared the developed microfluidic technology (Dean Flow Fractionation, DFF) with standard magnetic negative isolation (MACS), and observed that DFF-sorted monocytes had similar viability, purity and key immune functions (phagocytosis, macrophage differentiation) as MACS-sorted monocytes. As proof-of-concept for diabetes testing, we isolated and characterized monocytes/MPA from a cohort of healthy (n=5) and type 2 diabetes mellitus (T2DM) subjects (n=8) in PBMCs and DFF-sorted monocytes. High speed imaging, immunofluorescence and flow cytometry analysis clearly indicated higher levels of MPA in T2DM patients (P
