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Electrophoresis 2008, 29, 1213–1218
Matthew S. Pommer1
Yanting Zhang1
Nawarathna Keerthi1
Dong Chen2
James A. Thomson3
Carl D. Meinhart1
Hyongsok T. Soh1, 4
Research Article
Dielectrophoretic separation
of platelets from diluted whole blood
in microfluidic channels
1
Department of
Mechanical Engineering,
University of California,
Santa Barbara, CA, USA
2
Division of Hematopathology,
Mayo Clinic,
Rochester, MN, USA
3
Department of Anatomy,
University of Wisconsin-Madison
Medical School,
Madison, WI, USA
4
Materials Department,
University of California,
Santa Barbara, CA, USA
The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted
whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest
cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform sizebased fractionation of blood samples and continuously enrich the platelets in a label-free
manner. Cytometry analysis revealed that a single pass through the two-stage DACS device
yields a high purity of platelets (,95%) at a throughput of ,2.26104 cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free
dielectrophoretic separation of delicate cells from complex samples and such a separation
approach may open a path toward continuous screening of blood products by integrated
microfluidic devices.
Received August 13, 2007
Revised September 30, 2007
Accepted October 1, 2007
Keywords:
Bioseparations / Cell sorting / Dielectrophoresis / Microfluidics / Platelets
DOI 10.1002/elps.200700607
1
Introduction
Platelets are the smallest cell type in blood, and they play a
critical role in hemostasis and thrombosis [1]. They are frequently transfused to patients undergoing a wide variety of
medical procedures including general surgery, and solidorgan transplants as well as in treatment of trauma patients
[2]. Typically, platelets circulate the body in their inactivated
form at a nominal concentration of 1.5–4.56105 cells/mL
within blood [3]. However, upon encountering stimuli
including mechanical shear and soluble agonists such as
thrombin, collagen, and von Willebrand factor (VWF) [4],
they become activated and undergo a large irreversible morphological change [5]. Unlike red blood cells which can be
frozen and stored for extended periods of time [6], platelets
are generally stored at 20–247C with gentle agitation [7]. Even
under such controlled conditions, their shelf-life is only 5–
7 days [8]. In practice, the shelf-life is even shorter, due to the
fact that screening for diseases including hepatitis, HIV, and
syphilis must be completed within that time period [9]. Consequently, maintaining a steady supply of platelets from
donors is an important societal need.
Correspondence: Professor Hyongsok T. Soh, Materials Department, University of California, Santa Barbara, CA 93106-5050,
USA
E-mail: [email protected]
Fax: 1805-893-8651
Abbreviations: DACS, DEP-activated cell sorter; DEP, dielectrophoresis; LEC, low electrical conductivity
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Centrifugation is the most widely used method of platelet purification. A number of protocols have been developed
which include the platelet-rich plasma preparation (PRP),
the buffy-coat preparation (BC), and apheresis [7]. Due to the
fact that platelets become activated by mechanical shear
stress, any separation step that requires high-speed centrifugation (i.e., g.4000) [8] results in significant loss [5].
Both PRP and BC methods have been reported to loose up to
50% of the original number of inactivated platelets [7]. Thus,
there remains a need for high-purity, low-stress platelet
separation technologies from whole blood with integrated
detection capability to rapid screen for residual diseases.
Toward this end, we report the use of dielectrophoresis
(DEP) [10] to separate platelets directly from diluted whole
blood in microfluidic channels. By exploiting the fact that platelets are the smallest cells in blood, we utilize the DEP activated cell sorter (DACS) [11, 12] to perform size-based fractionation of blood samples and enrich the platelets in a labelfree manner (Fig. 1). Cytometry analysis revealed that a single
pass through the two-stage device yields a high-purity population of platelets (,95%) with minimal platelet activation, at
a throughput of ,2.26104 cells/second/microchannel.
2
Materials and methods
2.1 Buffers and cell handling protocol
For the platelet activation study, we purchased concentrated
fresh platelets (,109 cells/mL) with anticoagulant EDTA
from a local blood bank (United Blood Services, Santa Barwww.electrophoresis-journal.com
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M. S. Pommer et al.
Electrophoresis 2008, 29, 1213–1218
IL). To avoid inadvertent activation of the platelets, no washing steps were performed prior to the DACS separation. The
sample was not refrigerated at any point during the experiment, and the temperature of all cell suspensions was
maintained between 20 and 247C. The separation and analysis of the sample were completed within 6 h after the blood is
extracted.
2.2 Methods of measuring the platelet activation
levels
The level of platelet activation was measured by monitoring
the expression of CD 62P surface marker [16–18] using flow
cytometry. The anti-CD 62P mAb conjugated with the phycoerythrin was purchased from BD Biosciences (San Jose,
CA). Approximately 20 mL of the undiluted antibody (concentration = 0.125 mg/20 mL) was added to the 100 mL platelet cell suspension (concentration = 106 cells/mL) as suggested by the manufacturer’s protocol. The sample was
incubated for 30 min in the absence of light before cytometric analysis.
2.3 Flow cytometry
Figure 1. Device architecture of the two-stage DACS chip. (a) An
optical micrograph shows the two inlets (sample and buffer) and
two outlets (collection and waste). The integration of two tandem
purification stages increases the purity of the platelet population
at the collection outlet. (b) The diluted whole blood sample
enters the device through the two side streams. The larger (nonplatelet) cells experience a sufficient DEP force from the electrodes, become deflected into the buffer stream, and exit through
the waste outlet. On the other hand, the smaller platelet cells are
not deflected and exit through the collection outlet.
bara, CA). The platelet concentrate is stored at 20–247C, with
gentle horizontal plate agitation. Two types of buffers are
used to compare the platelet activation levels: (i) standard
Tyrode’s buffer (10 mM HEPES, 2 mM MgCl2, 137.5 mM
NaCl, 12 mM KCl, 5 mM glucose, and 0.1% BSA) and (ii)
low-electrical-conductivity (LEC) buffer [13–15] (8.5% w/v
sucrose and 0.3% w/v dextrose). The final concentration of
platelets in both buffers was ,107 cells/mL. As a positive
control, a set of fully activated platelets was prepared by
obtaining a suspension of expired platelets from the blood
bank, aged for one additional week, agitated with high shear
rates using a vortexer at 3200 rpm for 1 h, and performing
five temperature cycles from 4 to 247C over 2 h periods to
induce activation.
Whole blood was extracted from a healthy adult donor
through venipuncture, and put into a vacutainer (Becton
Dickinson, Franklin Lakes, NJ) containing the anticoagulant
citrate dextrose (ACD), which is commonly used for platelet
storage [8]. The sample was then diluted in the LEC buffer
using a volume ratio of 0.7:10 (sample/buffer). The electrical
conductivity of the resulting suspension was ,50 mS/m
measured with ECTestr (Oakton Instruments, Vernon Hills,
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PAS III flow cytometer (Partec, Münster, Germany) was used
to quantify platelet activation levels and the DACS separation
performance. In order to distinguish the platelet population
from the nonplatelet population, two control samples were
prepared. The first sample, containing only nonplatelet cells,
was obtained by the centrifugation of the whole blood for
10 min at 5006g and aspirating the excess to remove platelets. The process was repeated three times. The second sample, containing only the platelets, was obtained by diluting
fresh platelet concentrate to approximately 16106 cells/mL
using both Tyrode’s and LEC buffer. The gates for cytometric
analysis were set according to the two control populations.
2.4 Device fabrication
The process steps used to fabricate the two-stage DACS device [12] are described elsewhere [11]. Briefly, the quadrupole
electrodes were patterned with 20 nm of titanium and
200 nm of gold on top of 4-inch glass wafers (Pyrex 7740
Borosilicate Glass, Corning, Corning, NY) through e-beam
evaporation. Then, photo-sensitive polyimide (HD4010, HD
MicroSystems, Santa Clara, CA) was spun on the bottom
substrate, and patterned using optical lithography. The polyimide layer serves as the spacer between the two glass substrates. After drilling microfluidic vias on the top substrate
by a computer-controlled milling machine (Flashcut CNC,
Menlo Park, CA) and dicing both substrates, the two pieces
were aligned and bonded at 3007C for 2 min using a FlipChip Aligner Bonder (Research Devices, West Piscataway,
NJ). Then, the device was placed inside a wafer bonder (Karl
Suss SB-6, Suss MicroTec, Munich, Germany) with a nitrogen environment, and the polyimide was cured at 3757C for
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Microfluidics and Miniaturization
Electrophoresis 2008, 29, 1213–1218
40 min to complete the bonding process. The resulting
height of the channels is 20 mm. Finally, the microfluidic
inlets and outlets were manually fixed on the drilled vias
using epoxy.
2.5 Experimental setup and testing protocols
Two card-edge connectors were used to attach the electrodes
of the device to a 206voltage amplifier and a waveform generator (TDS3032, Tektronix, Beaverton, OR). The frequency
and amplitude of the applied voltage was monitored using a
digital oscilloscope (54622A, Agilent Technologies, Palo Alto,
CA). The separation process was monitored using an optical
microscope (E600FN, Nikon, Tokyo, Japan). The device was
oriented with the fluidic connections facing away from the
lens to allow optical access.
Prior to each experiment, the DACS device was cleaned
using ethanol and deionized water, and treated with 20%
BSA to precoat the microchannel. Also, before each experiment, new inlet and outlet capillary tubing was attached to
the device to minimize contamination. The cell suspension
and buffer were injected into the device at 150 mL/h using a
syringe pump (PHD 2000, Harvard Apparatus, Holliston,
MA). Two additional syringe pumps, connected to both the
collection and waste outlets were operated at the same flow
rates to ensure equal mass flux at every port. Once the flow
through the device was stabilized and all bubbles were
removed, a sine wave actuation voltage was applied at
100 Vp–p at 1 MHz.
3
Results and discussion
3.1 Separation method and device architecture
Previously, the DEP phenomenon has been utilized for a
wide variety of bioseparations and bioanalytical applications
[19–28]. In this work, we utilize the two stage DACS device
[12], to purify platelets from diluted whole blood in a labelfree manner (Fig. 1b). The physical basis of the size-based
separation arises from the fact that the DEP force (FDEP) on a
particle has a cubic dependence on its radius (,R3) whereas
the hydrodynamic drag force (FHD) under laminar flow conditions has a linear dependence (,R1). More specifically, the
DEP force generated on a spherical particle by the DACS
electrode geometry [23] is
3 2 D*
E 27
R
rms 2 R
FDEP ðtÞ ¼ p2 em Re½KðoÞrE
1þO 2
(1)
32
h
h
where em is the permittivity
of the medium, K(o) is the
rms is the root-mean-squared
Clausius–Mossotti factor, E
value of the applied electric field, R is the effective radius of
the cell, and h is the channel height. The hydrodynamic drag
force (FHD) experienced by a cell can be approximated as a
rigid sphere translating relative to the surrounding fluid with
*
velocity vp, using Stokes’ drag for low Reynolds number flow
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
*
*
FHD ¼ 6pmRvp
1215
(2)
where m is the dynamic viscosity of the fluid. Subsequently,
the ratio of the FDEP/FHD has an ,R2 dependence. These two
forces can be tuned independently: the FDEP with applied
voltage, and the FHD with flow rate, thereby controlling the
FDEP/FHD ratio. In this experiment, the device is tuned to
deflect all larger cells (erythrocytes, leukocytes and all other
nonplatelet cells) into the waste channel. On the other hand,
we ensure that the FDEP/FHD ratio is sufficiently low for small
cells (i.e., platelets), such that they do not undergo significant
deflection so that they are eluted through the collection
channel.
3.2 Effect of LEC buffer on platelet activation
The Tyrode’s buffer is commonly used to suspend platelet
cells during separation from whole blood [29]. It has an electrical conductivity ss , 1.7 S/m, which is similar to that of
the cells, thus making dielectrophoretic separation difficult
(Eq.1). The use of a sugar-based, LEC buffer has been previously reported by a number of groups for DEP [30–33] and
electrorotation [13, 14]. The LEC buffer used in this work is
an isotonic buffer with an electrical conductivity adjusted
here using excess ions from whole blood to 50 mS/m. To
ensure that the buffer conditions do not cause undesired
activation of the platelets, the expression levels of P-selectin
(CD 62P), a commonly used indicator of platelet activation
[34], was measured with flow cytometry by labeling the platelets with anti-CD 62P mAb conjugated with phycoerythrin.
The cells were suspended in their respective media for approximately 2 h, and the fluorescence levels of platelets suspended in LEC buffer were compared with those suspended
in Tyrode’s buffer using flow cytometry (Fig. 2a). Surprisingly, the platelets suspended in the LEC buffer showed
lower levels of CD 62P expression compared to those in Tyrode’s buffer suggesting that the use of LEC buffer does not
have a detrimental effect on the platelet activation. This
finding is consistent with other reports which found that
sugar-based buffers have a minimal effect on the viability of
other mammalian cell types [35–37].
3.3 Effects of electric field on platelet activation
The use of high voltages generates larger FDEPS Eq. (1) that
enable higher throughput. However, the practical limits on
the voltage amplitude are imposed by the onset of electrolysis (i.e., bubble formation) and increased platelet activation [13, 14, 38]. In the DACS device, using the LEC buffer,
we have found that the applied voltage of Vp–p = 100 V at a
frequency of f = 1 MHz does not induce electrolysis. Under
these conditions, the platelets are separated through the
device at a flow rate of 150 mL/h per microchannel which is
equivalent to processing approximately 86107 cells/h in a
microchannel that is 1.75 mm wide. The average velocity of
the cells in the microchannel is n = 6.6 mm/s and thus the
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Electrophoresis 2008, 29, 1213–1218
CD 62P expression (Fig 2b). Although the cells are exposed to
electric field for a longer period of time compared to electroporation, here, the electric field strength is much lower and
may contribute to the resulting high cell viability. Considering the fact that high speed centrifugation can result in the
activation of ,50% of total platelets [7], the DACS separation
method shows encouraging results toward gentle electrokinetic separation.
3.4 Platelet separation performance of DACS
In whole blood, erythrocytes (diameter = 6–8 mm) constitute
the majority (94%) of the cell population, followed by platelets (diameter = 2–5 mm) [39], which constitute about ,5%
[3]. Leukocytes (diameter = 12–15 mm) constitute only about
0.1% [3]. The cytometric analysis of an unprocessed sample
shows that indeed, the population of whole blood is heavily
biased toward larger cells (Fig. 3a). To identify the population
of platelets within the unprocessed sample, a purified platelet population was analyzed. The region of side scatter (SSC)
and forward scatter (FSC) (often called “gates”) that encompasses the pure platelet population was identified (Fig. 3a,
gate A) and isolated from those containing other, larger cell
types (Fig. 3a, gate B). The percentage of particles which lied
within gate A from the diluted whole blood sample was
,18%, which was higher than a typical value reported in literature [3]. We attribute this discrepancy to cell debris and
other smaller contaminants because the diluted blood sample did not undergo any purification steps before DACS
separation. Then, the sample was separated in the DACS
device in a single pass. The eluted fraction from the collection channel shows a cell population which is significantly
depleted of larger cells, and 95% of the cells lie within gate A
(Fig. 3b), which are assumed to be platelets.
4
Figure 2. Effects of buffers and electric fields on platelet activation. The magnitude of platelet activation is monitored with flow
cytometry where the expression level of CD 62P surface marker is
quantitatively measured with an anti-CD 62P antibody conjugated with phycoerythrin. (a) The LEC buffer shows less activation when compared with Tyrode’s buffer. (b) The platelets
processed through the DACS devices operated at a voltage of
100 Vp–p at a frequency of f = 1 MHz, shows a small but measurable increase in the activation level when compared with a platelet suspension put through the device with no electric field.
cells are exposed to the electric fields for approximately 1.8 s.
The expression levels of CD 62P were compared for: (i)
unprocessed cells (negative control), (ii) fully activated cells
(positive control), and (iii) DACS processed cells. Flow cytometric analysis revealed that the AC electric fields used in the
DACS separation have a small, but measurable increase in
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Concluding remarks
In this work, we demonstrate label-free, dielectrophoretic
purification of platelets from diluted whole blood in a continuous flow manner. In a single pass through the two-stage
DACS device, the percentage of platelet population increased
from 18 to 95%. By monitoring the expression level of CD
62P marker using flow cytometry, we found that the use of a
LEC buffer does not have detrimental effects on platelet
activation. In fact, the isotonic, sugar-based buffer used in
this study showed lower levels of CD 62P expression when
compared to the Tyrodes buffer which is commonly used for
platelet separation. The low conductivity sugar-based buffer
permitted the use of a relatively high voltage of 100 Vp–p at a
frequency of f = 1 MHz for DACS separation, without inducing electrolysis. Under these conditions, a small but
finite increase in the CD 62P expression was measured;
however, the fact that high speed centrifugation can result in
activating ,50% of platelets shows encouraging results toward gentle electrokinetic separation. The volumetric
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Electrophoresis 2008, 29, 1213–1218
1217
devices that combine sample preparation with detection
assays for their use at the point-of-care. This work demonstrates effective, label-free, size-based dielectrophoretic
separation of delicate cells, such as the platelets, from complex samples, such as diluted whole blood. This separation
approach may open a path toward continuous screening of
blood products by integrated microfluidic devices.
We thank the financial support from the ARO Institute for
Collaborative Biotechnologies (DAAD1903D004), National
Science Foundation: NIRT (CTS-0404444), Office of Naval Research (447800-23059), and Beckman Foundation (44255057174). We thank Dr. P. R. C. Gascoyne’s group for helpful discussions, and the United Blood Services of Santa Barbara for
providing the platelets for this study. We also thank Student
Health Services at UC Santa Barbara for providing assistance in
obtaining samples for this study. Microfabrication was carried out
in the Nanofabrication Facility at UC Santa Barbara.
The authors have declared no conflict of interest.
5
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