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Membrane-based, sedimentation-assisted plasma separator for point-of-care applications Changchun Liu, Michael Mauk, Robert Gross, Frederic D. Bushman, Paul H. Edelstein, Ronald G. Collman, and Haim H Bau Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402459h • Publication Date (Web): 05 Oct 2013 Downloaded from http://pubs.acs.org on October 10, 2013
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Analytical Chemistry
Membrane-based, sedimentation-assisted plasma separator for point-of-care applications Changchun Liu1*, Michael Mauk1, Robert Gross2,5, Frederic D. Bushman3, Paul H. Edelstein4, Ronald G. Collman3,5, and Haim H. Bau1, 1
Department of Mechanical Engineering and Applied Mechanics,
2
Center for Clinical Epidemiology and Biostatistics,
3
Department of Microbiology,
4
Department of Pathology and Laboratory Medicine, and
5
Department of Medicine
University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
*
Corresponding author
Dr. Changchun Liu Department of Mechanical Engineering and Applied Mechanics University of Pennsylvania 216 Towne Building 220 South 33rd St. Philadelphia, Pennsylvania 19104-6315, USA Phone: (215)898-1380 E-mail:
[email protected] 1
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ABSTRACT Often, high sensitivity, point of care, clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low abundance target molecules.
We
report
on
a
simple
to
use,
low-cost,
pump-free,
membrane-based,
sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a “blood in-plasma out” capability, consistently extracting 275 ±33.5 µL of plasma from 1.8 mL of undiluted whole blood in less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3,500 and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse transcriptase loop mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high efficiency nucleic acid amplification.
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Analytical Chemistry
Introduction Over two-thirds of the estimated 34 million people living with HIV/AIDS worldwide reside in developing countries, and nearly three-fourths of the 2.5 million new HIV infections in 2011 occurred in these countries.1,2 HIV viral load testing plays a critical role in clinical decisions on when and whether to switch to second-line treatment; in optimizing the duration of first-line treatment by detecting occult non-adherence; in diagnosing HIV infection in babies under 18 months of age, born to HIV-infected mothers, in whom the presence of HIV antibodies is not indicative of the disease; and in detecting early newly-infected individuals during the seroconversion window period when antibodies are present at undetectable concentrations.3-7 Although a standard practice in developed countries, HIV viral load determination is not widely used in low and middle income countries because of technical constraints, lack of testing facilities, lack of trained personnel, and cost. There is an urgent need to develop an affordable, simple, easy to use point-of-care (POC) diagnosis technology for HIV viral load testing in resource-constrained settings. 8-11 Usually, plasma separation from raw whole blood is required for HIV viral load testing since the presence of blood cells and components in the sample, such as hemoglobin and lactoferrin, may inhibit DNA polymerase and lead to low amplification efficiency, inaccurate quantification, and even amplification failure.12 Additionally, prevailing HIV viral load standards are based on the number of virus copies in a unit volume of plasma – not whole blood. In clinical laboratories, plasma separation is typically carried out with a bench-top centrifuge. Separation of relatively large volumes of plasma from whole blood remains a challenge in resource-constrained settings due to lack of laboratory infrastructure.13-15 Various microfluidic approaches have been developed to separate plasma from whole blood at the point of care,16-26 including capillary imbibition,16,17 blood cell sedimentation,18,19 cross-flow filtration,20-22
and
on-chip
centrifugation.23,24
VanDelinder
et
al.21
reported
on
a
poly(dimethylsiloxane) (PDMS)-based microfluidic device for separation of plasma from whole human blood by size exclusion in a cross-flow. The device can operate for at least 1 h, extracting ~8% of the blood volume as plasma at an average rate of 0.65 µL/min. Shim et al.16 demonstrated a heterogeneous packed bed filter, where small beads filter the whole blood and larger beads prevent the smaller beads from leaving the separation device. Capillary forces drive the plasma through a 3
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microchannel with a cross-section of 100 µm × 100 µm at a flow rate of 0.19 µL/min. Dimov et al.19 presented a self-powered, integrated microfluidic blood analysis system (SIMBAS) with nearly 100% blood cell filtration efficiency for low blood flow rates (
