Proceedings of the 2nd IEEE International
Conference on Nano/Micro Engineered and Molecular Systems
January 16 - 19, 2007, Bangkok, Thailand
On-Chip Continuous Blood Cell Subtype Separation by
Deterministic Lateral Displacement
Nan Li', Daniel T. Kamei2, and Chih-Ming Ho'
'Department ofMechanical & Aerospace Engineering, University ofCalifornia, Los Angeles, CA, USA
2
DepartmentofBioengineering, University of California, Los Angeles, CA, USA
Abstract-This paper presents a microfluidic device for
continuous human blood cell subtype separation using the
deterministic lateral displacement principle. Based on their
significant size and shape differences, three major cell types of
human whole blood - platelets, red blood cells and white blood
cells - were demonstrated to be directly separated using a two-
stage separation strategy. Even though all white blood cells are
spherical and have diameters within a narrow range (8 - 20pm),
the initial limitation for using this principle to separate white
blood cell subtypes was conquered by attaching larger
polystyrene microbeads to one of the subtypes to amplify the size
differences. Specifically, continuous separation of human CD4+ T
helper lymphocytes from other white blood cell subtypes was
achieved with high purity and recovery due to the underlying
high affinity and high specificity of the antigen-antibody
interaction used to attach the microbeads to the lymphocytes.
With our novel approach, the pure population of one blood cell
subtype can be effectively isolated by exploring the deterministic
lateral displacement principle, which has the advantages of the
simplicity, high speed and high resolution. Because many cells
express unique surface markers, this method can theoretically be
applied to separate any target cell type from a heterogeneous
mixture for downstream analysis.
Keywords-microfluidics; blood cell separation; deterministic
lateral displacement
I. INTRODUCTION
Isolation of a pure biological cell population from a
heterogeneous mixture of different cell types plays a very
important role in both clinical diagnosis and academic research.
Microfluidic cell separation devices provide more advantages
compared with their conventional counterparts in smaller
sample volume consumption, lower cost and easier for
integration with downstream analytical components [1, 2].
Integrated microchips for separating and isolating cells
utilizing hydrodynamic, dielectrophoretic (DEP) and magnetic
forces have been demonstrated within recent years [3-10].
Relative to these strategies, however, cell separation based
purely on size differences is the simplest approach and
possesses the advantages of low cost and high throughput.
Deterministic lateral displacement is a novel size-based
separation mechanism with high resolution (up to 0.1ILm) [11,
12]. This principle has been explored for separating human red
blood cells (RBCs, 6-7pm) from white blood cells (WBCs, 8-
20jim) based on their distinct size differences [13, 14].
However, most clinical diagnosis and academic research start
with whole blood separation and are focused on specific white
This project was funded by NASA National Space Biomedical Research
Institute (NSBRI). The co-operative agreement number is NCC 9-58-317.
* Contacting Author: Engineering IV 18-111, 420 Westwood Plaza, Los
Angeles, CA 90095, USA (Email: nancyli@ucla.edu)
1-4244-0610-2/07/$20.00 C)2007 IEEE
blood cell subtype, since that would yield information
regarding the human body infectious status and immune
responses. For example, the diagnosis of HIV infection
depends on isolation of human CD4+ lymphocytes from whole
blood [2]. Besides RBCs and WBCs, another major component
of whole blood is the platelets with a diameter between 2pim to
3ptm. The ratio of platelets to RBCs to WBCs in normal human
whole blood is about 50:500:1. The large amount and apparent
small size of platelets makes it possible to directly separate
them from RBCs and WBCs using the deterministic lateral
displacement technique. Furthermore, WBCs include five
subtypes (monocytes, lymphocytes, neutrophils, basophils and
eosinophils) and their diameters are all within a narrow range
between 8pm and 20pm which poses limitations for using the
deterministic lateral displacement technique to separate them
from each other. However, all of these WBC subtypes express
their unique surface antigens, which can be specifically
targeted by antibodies. This property provides the possibility
for attaching microbeads to each WBC subtype through
specific antigen-antibody interaction so that size differences
can be "amplified". Although attaching microbeads to cells has
been previously demonstrated to effectively separate one
specific cell type from others by both electrical and optical
methods [15, 16], the novelty of this approach is combining the
specificity of microbeads attachment with the simplicity of the
deterministic lateral displacement technique. Because of the
high affinity and specificity associated with antigen-antibody
interaction, high recovery ratio and purity may be achieved.
In this paper, we first demonstrate successful separation of
human whole blood cells into three major groups using a two-
stage deterministic lateral separation strategy. By attaching
25pm diameter polystyrene microbeads, CD4+ T helper
lymphocytes are successfully isolated from other WBC
subtypes using deterministic lateral displacement technique.
Disposable microfluidic devices using this method are capable
of achieving continuous WBC subtype separation with high
throughput, recovery and purity.
II. PRINCIPLE
Deterministic lateral displacement separation method
utilizes the asymmetric bifurcation of laminar flow
phenomenon around an array of micro obstacles. These micro
obstacles are shifted a specific distance AX from the previous
row. This shifted distance AX, together with the gap g and the
center-to-center distance between two adjacent obstacles i,
determines the critical separating particle size. When particles
with different sizes flow through this micro obstacles array,
932
they will follow specific pathways "deterministically".
Particles smaller than the critical separating size will change
the path lane periodically and thus follow a "zigzag" mode,
whereas particles larger than the critical separating size will
keep getting "bumped" when they travel to the next row of the
obstacles and thus be displaced to one direction. Assuming the
flow profile between the micro obstacles is parabolic which
holds true at low Reynolds number, the critical separation
diameter D can be numerically determined by [12]:
I
D=g 1+2w+
2w]
where
W=
I
-+ 168 (e 1) ]1 (113)(- 21 i 2
8
-.3)
-8 4
and £ is defined as the row shift fraction which equals AA / A .
c is not linearly proportional to the product of £ and g is
because of the non-uniform flow flux between the micro
obstacles.
III. MATERIALS AND METHODS
Device Fabrication
Microfluidic separation devices are fabricated using soft
lithography with polydimethylsiloxane (PDMS, Sylgard 184,
Dow Coming, MI) polymer from deep reactive ion etched
(DRIE) silicon mold. The effective separation area is 6.8mm
by 1.6mm. At the inlet, samples are hydrodynamically focused
by two streams of sheath buffer (Figure 1(A)). A separation
distance measurement ruler near the outlet is used to quantify
the separation (Figure 1(B)). The important device parameters
for separation, row-to-row shift distance AX, gap g and center-
to-center distance between two adjacent obstacles i, are
illustrated in Figure 1(C).
k mAbs was performed. 10Og FITC conjugated anti-human
Flow
Figure 1. (A) Device inlet. (B) Device outlet. (C) High magnification of the
micro obstacles with critical parameters labeled.
Microbeads and Reagent
Polybead® microbeads used in this study were obtained
from Polysciences Inc. (Warrington, PA). Monoclonal
antibodies (mAbs) (FITC-conjugated anti-human CD4) and red
blood cell lysis buffer were obtained from eBioscience (San
Diego, CA). Human whole blood with heparin as anticoagulant
was obtained from Innovative Research, Inc. (Southfield,
Michigan) and used while fresh. Phosphate buffered saline
(PBS), bovine serum albumin (BSA), heparin sodium and
glycerol were obtained from Sigma (St. Louis, MO).
(1) Antibody Coating on Microbeads
Coating of the microbeads with mAbs was achieved by
pure protein adsorption on the polystyrene surface. 0.5mL of a
25ptm bead suspension at a concentration of 5.68X 106 beads/ml
were mixed with 100pg FITC conjugated anti-human CD4
antibody in lmL PBS and incubated overnight with gentle
vortexing at room temperature. This corresponded to 100pg
mAbs per 2.2 x 10'° Rm2 of bead surface area following the
protocol of Fortin and Hugo [16]. The beads were then washed
twice with 1XPBS buffer. The excess hydrophobic binding
sites on the beads were blocked by incubating with 1XPBS
containing 2% bovine serum albumin (PBS/BSA) for 30min at
room temperature. Finally, these mAbs-coated beads were
washed and suspended in 1 XPBS to a concentration of 1 x 106
beads/mL. The degree of mAbs adsorption was visualized with
a fluorescence microscope.
Attaching mAbs-Coated Microbeads to Cells
Human blood cells were first harvested by centrifuging
lmL of human whole blood at 1,500 x g for 5min and the
upper plasma layer was disposed. Red blood cells were lysed
two times using lysis buffer following manufacture-supplied
protocol. The remaining white blood cells were then suspended
in IXPBS to a final concentration of 1 X 106 cells/mL. 50[tL of
this processed white blood cell suspension was then mixed
with 500[tL of the mAbs-coated 25ptm microbeads solution and
incubated for 60min with gentle vortexing at room temperature.
The samples were then washed twice and resuspended in lmL
PBS.
Cell Staining with Fluorochrome-Conjugated mAbs
To measure the microbeads-cell attachment efficiency and
specificity, a second round cell staining with FITC conjugated
CD4 were added to lmL cell-beads mixture and incubated for
30min at room temperature. Then bead-cell mixture was
washed twice with PBS. Staining effect was visualized with
fluorescence microscope. Quantitation of the cell-beads
binding efficiency and specificity was achieved by counting
how many percent of fluorescently labeled cells were bonded
to microbeads and how many percent of beads attached cells
were fluorescently labeled using a hemocytometer.
Separation Experiments Setup
For the separation experiments, Tygon tubings with inner
diameter of 0.02 inches (Fisher Scientific, Tustin, CA) were
inserted into the PDMS device at the inlet and outlet. The
933
device was placed onto a Leica inverted microscope equipped
with a MotionPro MP1 000 high speed camera (Redlake MASD
LLC, Tucson, AZ) for visualization and image capture. All
videos were captured at a speed of 60 frames/second. Two
syringe pumps (Harvard Apparatus, Holliston, MA) were used
to drive the cell suspension and sheath buffer into the device at
a constant flow rate. For the microbeads separation
experiments, microbeads were first incubated with PBS/BSA
for 30min to prevent non-specific adhesion on device surface.
For cell separation experiments, 1 XPBS containing 1% heparin
sodium (PBS/heparin) was first pumped into the device from
the sample inlet and sheath inlets to drive out the air bubbles.
Then this solution was kept flowing at l1tL/min for 10min to
allow heparin to adsorb on the device surface in order to
prevent cell adhesion. To prevent microbeads and cells from
settling during the separation experiment, 20% glycerol was
added into each sample solution to adjust the solution density.
IV. RESULTS
Separation of Microbeads
Polystyrene microbeads with diameter of 8, 9 and 10~Lm
were separated from each other. The device parameters for
8pm and 9pm beads separation were AX =6Rm, X=80Rm and
g=25jm with a theoretical critical separation particle diameter
of 8.4pm. Experimental results show that the 8pm beads follow
the zigzag mode whereas the 9pm beads follow the
displacement mode ((Figure 2(A)). The device parameters for
9pm and IO0m beads separation were AX =8Rm, X=90%m and
g=25jm with a theoretical critical separation particle diameter
of 9.2pm. Experimental results show that, in this case, the 9pm
beads follow the zigzag mode whereas the IO0m beads follow
the displacement mode. ((Figure 2(B)). The experimental
results are in well agreement with the theoretical predictions.
At the outlet of the device, each group can be effectively
separated by at least a 20Opm distance. Flow velocity was also
investigated and found to have negligible influence on the
separation results (Figure 3). Accordingly, the high velocity is
Figure 2. Separation of polystyrene microbeads. (A) Trace of 8pm and 9pim
diameter beads. (B) Trace of 91im and 1Oim diameter beads. Sample and
sheath flow rates were 0.24L min and 1.8iL min respectively. Picture was
generated by superimposing multiple video frames.
934
200
I +GOullm s
150
I 1.Oins11lW lI
100
- - - ------------------- - - -1 - -t -------------
0
-20 40 100 160 220 280 340 400
separatio n dlista nce at th e ouitlet ({ ni)
Figure 3. Separation profiles of 8ptm and 9ptm polystyrene beads at two
different flow velocities.
preferred because higher throughput can be achieved as long as
the device is still operated at the laminar flow regime.
Separation ofHuman Blood Cells
Fresh human whole blood was directly diluted 50 times
with PBS/heparin and separated immediately to prevent
coagulation. A two-stage separation device was used to
separate whole human blood cells into three major groups:
platelets, RBCs and WBCs. The first stage of the separation
device includes 36 rows of micro obstacles with AX =2Rm
distance shifted for each row. The second stage includes 49
rows of micro obstacles with AX=5Rm distance shifted for each
row. The center-to-center distance i (80pm) and the gap g
(20pm) are the same for both stages. Theoretically, the critical
separation diameters for the first and the second stages are
3.8pm and 6.1 pm respectively. Therefore, separation of
platelets from RBCs and WBCs are expected in the first stage,
whereas further separation of the platelets, RBCs and WBCs
from each other are expected in the second stage.
Figure 4 shows the separation results of human whole
blood cells. In the first stage, platelets follow the zigzag mode
due to their smaller size compared with the critical separation
diameter, whereas both RBCs and WBCs were kept displaced
as they passed each row. At the transition from the first stage to
the second stage, platelets were separated by about 10Oim
distance from RBCs and WBCs. When they entered the second
stage, RBCs changed their pathways from displacement mode
to zigzag mode whereas WBCs still proceeded in displacement
mode.
Statistical analysis of the separation effects is shown in
Figure 5. The effective separation distance between platelets
and RBCs was about 200pm and that between RBCs and
WBCs was about 250pm. However, the distribution profile
associated with the platelets was larger than that associated
with the RBCs and WBCs. This resulted from their higher
diffusion rates throughout the separationg process due to their
smaller sizes (2-3pm). The ratio of separated RBCs to platelets
to WBCs was found about 470:36:1, which is similar to the
ratio of 500:50:1 in normal whole blood. A lower proportion of
platelets were quantified, and this was most probably attributed
to the fact that some of the platelets were out of focus and
therefore were not counted accordingly. Note that the total
separation throughput is about 1,000 cells per second.
__1E.2.2.m.E. * iS.g..1.
Figure 4. Trace of platelets, RBCs, amd WBCs through the two stages of the
whole blood separation device. The sample and sheath flow rates wuie
0.1IL min and 1 iLtmin respectively. Picture was generated by superimposing
multiple video frames. (A) First stage. Separation of platelets (top pathway-
zigzag mode) from RBCs and WBCs (bottom pathway-displacement mode).
20X maginification. (B) Second stage. Separation of platelets (top pathway-
zigzag mode), RBCs (middle pathway-zigzag mode), and WBCs (bottom
pathway-displacement mode) from each other. I OX magnification.
Figure 5. Statistical analysis of the separation of three major human whole
blood cells: platelets, red blood cells (RBCs) and white blood cells (WBCs).
Separation of White Blood Cell Subtypes
To demonstrate the ability to separate WBC subtypes using
the deterministic lateral displacement technique, we attached
25ptm diameter polystyrene beads to the CD4+ T helper
lymphocytes through their unique surface marker. The device
parameters for this separation were designed as AX=9pm,
X=6Qim and g=47Rm so that the theoretical critical separation
935
diameter was about 23pim. Because the 25p.m beads are larger
than almost all of WBCs, both beads and bead-cell complexes
were expected to be isolated from the other WBC subtypes.
After separation, the isolated cells can be detached from the
microbeads by adding a second antibody as a competitor and
pure T helper lymphocytes can be harvested. Meanwhile, the
other WBC subtypes can also be harvested with this specific
lymphocytes depleted.
Since the deterministic lateral displacement principle
allows all of the 25pim beads and bead-cell complexes to be
isolated, the purity and recovery ratio for the target cells are
mainly determined by the beads attachment procedure. By
performing a second round cell staining using FITC conjugated
CD4 mAbs, we quantitatively determined that about 91% of T
helper lymphocytes were attached to the 25ptm beads and thus
could be separated afterwards. Also, about 98% of cells
attached to the microbeads were stained fluorescently, which
indicated that almost all of the cells that attached to the beads
were T helper lymphocytes. The non-specific binding of other
cell types to the beads was therefore negligible. This high
purity is due to the especially high affinity and specificity
associated with the antigen-antibody interaction. Figure 6
shows a CD4+ T lymphocyte attached to a 25ptm bead and its
associated fluorescence.
Figure 6. FITC stained CD4+ T helper lymphocyte attached to a 25pim
polystyrene beads.
Once target cells were attached to microbeads, they were
run through the separation device. Figure 7 is the trace of a
WBC without bead bonded and a T helper lymphocyte bonded
to a 25pim bead. Clearly, since the gap is much larger than the
size of a WBC, the non-bead-bonded WBCs flowed through
the micro obstacles in a straight line with no direction changes
at all, whereas the bead-lymphocyte complex was kept
displaced by the micro obstacles.
Figure 7. Separation of a T helper lymphocyte bonded to a 25pm bead from
other WBC subtypes. Sample and sheath flow rates were 0.2iL min and
1.2iL min respctively. Picture was generated by superimposing multiple
video frames.
Summary
Deterministic lateral displacement technique has been
demonstrated to successfully separate polystyrene microbeads
with different sizes, three major components of human whole
blood, and WBC subtypes with surface marker bound to
microbeads. Figure 8 is the summary of all our experiments.
Particles that followed the displacement mode are plotted as
filled squares, while particles that followed the zigzag mode
are plotted as filled circles. All experiments were performed
under laminar flow conditions so that the flow profile between
the micro obstacles was parabolic. All "zigzaged" particles
were below the critical separation line, while all "displaced"
particles were above the critical separation line. The
experimental results are therefore consistent with the theory.
ci_
.__ __.
..q
El iFn 0
Ilfiffll.
0
4A '01
oilS p'Ar fat
- ofi 1icfCp
urifor fiot rofle
0 6 101i 20O 2 30 35
Rw Shft Fractioni
Figure 8. Separation summary for microbeads and human blood cells using
the deterministic lateral displacement technique. Square: particles follow
displacement mode; Circle: particles follow zigzag mode.
CONCLUSION AND DISCUSSION
V.
A disposable MEMS device for continuous separation of
human whole blood cells (platelets, RBCs and WBCs) and
white blood cell subtypes was demonstrated. The deterministic
lateral displacement principle was explored for successful
separation with high throughput and resolution. For whole
blood cells separation, the ratio of each separated group
collected at the outlet matches very well with the ratio in
normal whole blood. Furthermore, by attaching microbeads to
a specific cell group, this technique can be extended to separate
target cells from other groups with minimal size differences
between them. Due to the high affinity and specificity
associated with the antigen-antibody interaction, high recovery
and purity can be achieved using this method. In principle, this
strategy can be utilized for other cell separation applications
because many cells express unique surface markers.
Coating the device surface with heparin before experiments
was found to significantly decrease the cell adhesion and
clogging problem. However, clogging became severe when the
blood was not fresh, since coagulant factors were released due
to platelets activation. In addition, the flow rate was found to
have minor effects on the separation effects as long as the flow
was in the laminar flow regime. Too high of an operating flow
rate, however, may also pose a problem, since it can cause
some cells to deform to some extent and not follow the
predicted pathway.
In the future, the WBC subtype separating efficiency will
be further characterized using fluorescence activated cell
sorting (FACS). Also, a micro cell Coulter counter, which has
already been demonstrated in our lab [17], will be incorporated
into current device to count the separated cells.
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