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

PLoS One. 2016 May 11;11(5):e0155545. doi: 10.1371/journal.pone.0155545. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / instrumentation
  • Biological Assay / methods
  • Centrifugation, Isopycnic / instrumentation*
  • Centrifugation, Isopycnic / methods
  • Equipment Design*
  • Humans
  • Hydrodynamics
  • Leukocytes, Mononuclear / chemistry*
  • Microfluidic Analytical Techniques / instrumentation*
  • Microfluidic Analytical Techniques / methods
  • Pressure

Grants and funding

This work was funded by Science Foundation Ireland (www.sfi.ie) under Grant No 10/CE/B1821 and Enterprise Ireland (www.enterprise-ireland.com) under Grant No CF/2011/1317. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.