Chapter 2 Blood Cell Separation
2.1 Introduction
2.1.3 Microscale cell separation
Recently, there are increasing efforts toward cell separation in microscale. Field- flow fractionation is a group of technologies that requires continuous elution. An electric field is placed perpendicular to the fluid flow, which is laminar due to the microscale geometry. Particles are levitated by the field to different stream laminae, and separated due to different flow velocity in the parabolic laminar flow profile. Particles can be differentiated by the nature of the field applied. For example, investigators from Giddings’ group use sedimentation field-flow fractionation to separate erythrocytes in less than five minutes [15, 16]. In another attempt, Gascoyne’s group uses electrorotation to measure cell membrane capacitance of T- and B- lymphocytes, monocytes, and granulocytes [17]. The difference of cell membrane capacitance among these cell types, according to the single-shell dielectric model, enables separation of T- (or B-) lymphocytes from monocytes, T- (or B-) lymphocytes from granulocytes, and monocytes from granulocytes in a dielectrophoretic/gravitational field-flow fractionation device[18].
The same approach also demonstrates separation of cultured human breast cancer MDA- 435 cells from normal blood cells [19].
Two magnetic approaches have been applied to blood cell separation in the microscale [20]. In one method, the difference of native susceptibility between
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erythrocytes and other blood cells is exploited. The most paramagnetic cell in the body is the deoxygenated erythrocyte. The approximate 109 iron atoms carried by hemoglobin protein in each erythrocyte make the cell paramagnetic, unlike other cells in blood. Even in its oxygenated state, the erythrocytes are less diamagnetic than other blood cells. Both diamagnetic capture mode [21, 22] and paramagnetic capture mode [23] have been demonstrated. The other approach is a more general method using antibody conjugated magnetic beads. CD45-covered micro magnetic beads were used to label leukocytes and separate them from blood [24].
Several biomimetic microdevices have been demonstrated for cell separation or enrichment. A unique device based on the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, was proposed to separate leukocytes directly from whole blood [25]. 34-fold enrichment was achieved.
In another study, a whole blood sample is forced to flow in a lattice of channels designed to mimic the capillary channels hydrodynamically. The leukocytes self-fractionate into the different types due to a combination of stretch-activated adhesion of cells with the walls, stochastic sticking probabilities, and hetero-avoidance between granulocytes and lymphocytes [26]. Chang et al. fabricated an array of square or slender offset pillars coated with E-selectin IgG chimera inside microfluidic channels to mimic physiological process of leukocyte recruitment to blood vessel walls. HL-60 and U-937 cells can be enriched or partially separated by interaction with the antibody-coated wall of the device [27].
Acoustic standing wave technology has been demonstrated for continuous particle separation in both macroscopic and microscopic domains [28]. It has been applied to
continuously separate particles from medium with high efficiency in microchips assembled by anisotropic etched silicon chamber with glass lid [29]. Separation of erythrocytes from lipid microemboli in whole blood has been reported. In this case, the width of the channel is chosen to correspond to half the ultrasonic wavelength, thereby creating a resonator between the side walls of the flow channel in which a standing wave can be formed. Lipid particles are collected in the pressure antinodes by the side walls and erythrocytes in the pressure node [30, 31].
More closed related to this work are examples that separate cells based on size.
As shown in Table 1 and 2, the sizes of blood cells are different (leukocytes are larger than erythrocytes). Not shown in these tables is the fact that although there is a diameter overlap between erythrocytes and leukocytes (especially T lymphocytes), the volume of leukocytes is at least twice of that of a normal erythrocyte due to the biconcave disk shape of the erythrocyte [6]. Therefore, a separation of erythrocytes and leukocytes by size should use the 3D volume information as part of the criteria, instead of being based solely on the largest dimension of the cell. A microstep device fabricated on silicon was shown to separate microbeads of different sizes by physically blocking the larger one with the steps [32]. Recently there is a group of devices under the general name of hydrodynamic separators that separate particles continuously and passively based on the streamline theory inside microfluidic devices. Sturm’s and Austin’s groups designed a device with an asymmetric shifted pillar array and successfully demonstrated separation of microbeads in submicron sizes and DNAs of different lengths [33]. Later the technology was applied to blood cell separation as described later in this chapter [34, 35].
Seki’s group demonstrated several designs of channel based hydrodynamic separators
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[36−39]. In these devices, particles exit into multiple side branch channels determined by their center positions relative to the streamlines going into the side channels. Beads and cells of different sizes were separated inside the devices. Fluidic resistance calculation was used to calculate the fluidic width inside the main channel that goes into side channel, thus the critical particle sizes.
In the following sections of this chapter, two size-based hydrodynamic separators are presented. The pillar-shaped separator can separate particles with high resolution, while the channel-shaped separator reduces chip footprint significantly and has less requirement for flow control.
2.2 Pillar-shaped separator