Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs

Abstract

Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic “Lab-on-a-Disc” cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are ‘low-pass’, i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both ‘hydrostatically’ and ‘hydrodynamically’ triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, ‘dual siphon’ configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.

Centrifugal microfluidics for sorting immune cells from whole blood

“…While there are numerous sophisticated microfluidic cell sorting and imaging platforms reported in the literature [1, 9, 10, 27–29], however, very few of them can be used directly for whole blood samples due to intricate design, bulky setup, and complicated control and operations. The CMC can process a few microliters of blood, reducing blood consumption to a thousandth of conventional methods, which is especially advantageous for pediatric patients.…”

“…However, existing microfluidic centrifugation systems involving a DGM still fall short of integrating both blood cell sorting and in situ cellular imaging and enumeration [5–8]. Specifically, designs of existing microfluidic centrifugation systems, with complex microfluidic networks to handle blood and the DGM, are suboptimal for integration with optical detection due to large device thickness, bulky setups, intricate units, and complicated operations that prohibit optical accessibility [9, 10]. Thus, for existing microfluidic centrifugation systems using the DGM, cellular analysis is commonly achieved by an external platform to collect blood cells after centrifugation for downstream cellular analysis.…”

Wireless closed-loop control of centrifugo-pneumatic valving towards large-scale microfluidic process integration

“…Initially we extract the blood from a finger prick sample using a protocol described previously. We dilute these samples 1:1 with a dilution buffer using a protocol described previously [5]. To process the blood, we first load 40 μl of density gradient media (DGM) (Histopaque 1077, Sigma-Aldrich) onto the disc and centrifuge at 30 Hz; this volume is choses so the liquid reaches the level of our inlet channel.…”

Siphon-Induced Droplet Break-Off for Enhanced Mixing on a Centrifugal Platform

“…The use of centrifugation for pumping also reduces the cost and complexity of support instrumentation as just low-cost spindle motors are required rather than specialized micropumps. Similarly, inherent centrifugation of samples [21][22][23] applied to sample preparation is a particular strength.The technological precursor of these systems are the centrifugal analyzers which became popular in the 1970s and 1980s [24] for automating a very limited number of simple assay steps, on rather macroscopic sample volumes, and readout on a rotor-based instrument. Currently the main areas of application are immunoassays [25][26][27][28], nucleic acid testing [29][30][31][32][33][34][35][36], and cell sorting/identification [37][38][39][40][41][42][43].…”

“…The use of centrifugation for pumping also reduces the cost and complexity of support instrumentation as just low-cost spindle motors are required rather than specialized micropumps. Similarly, inherent centrifugation of samples [21][22][23] applied to sample preparation is a particular strength.…”