- Top of page
- MATERIALSAND METHODS
- DISCUSSIONAND PERSPECTIVES
- LITERATURE CITED
In this article, we demonstrate the potential of a microfluidic chip for the differentiation of immunologically stained blood cells. To this end, white blood cells stained with antibodies typically applied for the determination of the immune status were measured in the micro-device. Relative concentrations of lymphocytes and subpopulations of lymphocytes are compared to those obtained with a conventional flow cytometer. The stability of the hydrodynamic focusing and the optical setup was determined by measuring the variation of the signal pulse height of fluorescence calibration beads, being about 2% for the micro-device. This value and the overall performance of the micro-device are similar to conventional flow cytometers. It follows from our results that such microfluidic structures are well suited as modules in a compact, portable read-out instrument. The production process of the microflow cytometers, which we exploited for immunological cell differentiation, is compatible with mass production technology like injection molding and, hence, low cost disposable chips could be available in the future. © 2011 International Society for Advancement of Cytometry
Microflow cytometry is an emerging field because of its potential to provide low cost, disposable chips for complex cellular-based analyses. Such technology could provide the basis for simple and robust analytical systems being of particular relevance for point-of-care in-vitro diagnostics with applications in intensive care and emergency medicine. The main challenge here, is to design micro-devices featuring functionality which is at least similar, if not superior to their large frame conventional equivalents.
Research in the field of microflow cytometry concentrates on four main areas (1–5): focusing of particles in microfluidic channels, miniaturized fluid handling (6–9), integration of optical elements into the chip (10–14), and applications development, e.g., for biomedical purposes. When analyzing micro particles, focusing of the sample stream is usually applied to restrict their trajectories inside the flow channel (1, 3, 5, 6, 8–11, 15–26). This positioning serves to increase signal stability when measuring particle properties and to avoid surface fouling in microfluidic channels. The latter is of particular importance in hematology since the samples typically contain high concentrations of blood cells. For this purpose, the micro-device studied here uses hydrodynamic focusing as described in the next section.
Differentiation of blood cells is considered as one of the most promising applications of microflow cytometers (9, 20, 27–29). The prospects of high-throughput analysis and separation of blood cells using micro-devices were reviewed by Toner and Irimia (6). In the present article, we explore the potential of a microfluidic chip for differentiation of immunologically stained leukocytes. Our study is based on comparison of the results obtained for the same sample material with the micro-device and a routine instrument. In order to facilitate such a comparison, we implemented a fluid handling similar to commercial flow cytometers and used corresponding detection electronics for the micro-device.
The aim of this article is to demonstrate the equivalence of microflow cytometry and conventional flow cytometry for immunologically, fluorescence-based leukocyte differentiation. Although the signal-to-noise ratio in the microfluidic device is somewhat smaller, we will demonstrate that it is just as well applicable for the reliable identification of cells and the determination of relative blood cell concentrations as conventional flow cytometers. In addition, the performance of the microfluidic device was quantitatively characterized by measuring the dependence of the coefficient of variation (CV) on the relative sample flow rate and as a function of fluorescence intensity. We demonstrate a CV of pulse height distribution of less than 2% for the micro-device, being in the range of conventional flow cytometer. These results open up a broader perspective for future developments of complex microflow systems which integrate, besides the presented analysis chip, sample preparation, and cell sorting modules.
- Top of page
- MATERIALSAND METHODS
- DISCUSSIONAND PERSPECTIVES
- LITERATURE CITED
Most modern instruments can achieve CVs between 1% and 2% in fluorescence measurements of beads (38), although commercial flow cytometers are typically specified with a CV between 2% and 5%. Apart from DNA content measurements for cell cycle analysis to identify different tumor cell lines (39) such a pulse height resolution is sufficient due to significantly larger biologically based variations for light scattering experiments and immunofluorescence detection. This targeted range is achieved exploiting the cascaded hydrodynamic focusing built in the micro-device introduced here.
Several methods have been used to achieve particle focusing in micro-chips. Most commercially available optical flow cytometers use hydrodynamic focusing. The notation regarding the dimension of hydrodynamic focusing is not consistent in literature (18, 19). Most simple arrangements for hydrodynamic focusing in micro-chips enclose the sample only in one lateral dimension and we call this one-dimensional focusing here (3, 8, 9, 11, 20–22). This geometry is frequently used when lithography is applied in chip production. However, such systems will be prone to fouling in the microfluidic channels and it has been argued that this geometry will produce data with relatively large variance (1, 11, 23). Two-dimensional focusing requires to ensheath the sample stream in both lateral dimensions. Previous designs of two-dimensional hydrodynamic focusing used up to six inlets to ensheath the sample flow (1, 18, 24, 25). Such a system was demonstrated by Simonnet and Groisman where the relative sheath flow values of the six inlets were used to fully control the position and size of the sample stream (18). This device achieved a CV of 2.7% in fluorescence measurements at particle count rates up to 1 kHz for the brightest particles tested, which is marginally worse than the CV achieved with the micro-device presented here (Fig. 4c). The possibility to position the sample flow inside the flow channel is an interesting option. However, the sensitive adjustment of all sheath flows can cause some inconvenience in daily use.
A unique feature of the design used in our work is that only a single inlet for the sheath flow is used to achieve two-dimensional (2D) hydrodynamic focusing (10). The micro-device we employed has a robust and reliable hydrodynamic focusing stage for 2D positioning of particles in both directions perpendicular to the direction of the flow. This attribute could turn out to be indispensable for practical flow cytometric analysis to obtain a pulse height resolution significantly lower than the biological variation, for example, in cell size or antibody expression.
The design of hydrodynamic focusing used depends on the technique applied for chip production. Simple one-step lithography is typically used to create 2[1/2]D fluidic structures (i.e. 2D layout and [1/2]D for the channel height). However, such microchips are limited to 1D hydrodynamic focusing only. A distinct advantage of lithography is that it offers smooth surface walls which can be used to create low loss optical waveguides. Such waveguides are sometimes applied to collect optical signals (8, 12). Lithography can also be used to create V-shaped grooves (3). Altendorf et al. used a V-shaped groove to align blood cells by cell adhesion (40). This allowed them to differentiate platelets, lymphocytes, monocytes, and neutrophils in blood samples (depleted of erythrocytes) by small angle and large angle light scattering. More elaborate structures can be built by stacking 2[1/2]D fluidic structures, which permit implementation of 2D hydrodynamic focusing. An example is the chip used by Kim et al., who used sequential lithographic steps to create a master on a silicon wafer featuring flow channels and chevron-shaped grooves (23). This device used two inlets for sheath flow and was applied for detecting bacteria with fluorescent coded microspheres. In contrast to sequential lithography, we exploited ultra-precision milling technique to manufacture the master. This approach, as reported in details in Ref.10, offers higher flexibility in design of fluidics and integrated optical elements.
In hydrodynamic focusing, the sample stream is confined by a sheath stream and focusing is achieved by squeezing the combined stream through a narrowing channel (1, 5, 17). Alternatively, acoustic focusing (15) can be used to analyze particles and cells (16). Dron et al. demonstrated acoustic focusing of particles in a planar microchannel using a standing acoustic wave (41). The aim was to improve imaging of particles for particle image velocimetry (PIV). Shi et al. used a combination of sheath flow and acoustic focusing for particle separation (42). Smaller particles are focused substantially slower by the acoustic field (15). This effect was used by Shi et al. to demonstrate the continuous separation of particles with different sizes. Focusing can also be achieved with dielectrophoretic forces (DEP) and is widely used in microfluidics (1, 3, 6, 19, 26). The advantage of using DEP or acoustic focusing is that the forces exerted can also be used for other purposes in a coherent approach, in particular for particle sorting (7, 19, 42).
Notably a high signal stability can potentially be achieved without focusing the sample stream at all, if the complete fluid channel is measured with sufficiently high spatial resolution, e.g., using a camera or laser scanning (21, 43–45). Bang et al. achieved a CV as low as 1.4% using one-dimensional hydrodynamic focusing applying camera detection. Alternatively, the excitation light for fluorescence detection can be adjusted to be homogenous across the whole microfluidic channel. Recently, Joo et al. used excitation by light emitting diodes to detect fluorescence from beads and cells (46). They demonstrated high signal stability without using particle focusing in their portable flow cytometer. However, the count rate for fluorescent particles demonstrated was limited to about 2 Hz. Such comparatively low count rates are expected for approaches using extended detection regions and may limit their usefulness for application in hematology and immunology.
In hematology, cell concentrations in the original samples are typically high so that the maximum useful particle count rate is usually not limited by sample throughput but by coincidence loss (47, 48). The number of particles counted Nc is approximately given by
where N is the number of particles passing the detection region and τ is the mean dead time of the detection system (Tc: counting time). Thus, the useful count rate is approximately inversely proportional to the mean dead time at a given level of coincidence loss. The mean dead time is ultimately limited by the time needed for the particle to cross the detection region although there might be an additional contribution from electronic signal processing. The basic consequence is that high count rates require high linear particle velocity in the detection region. In this context, a potential drawback of using dielectric focusing (DEP) is that the maximum flow speed may be limited to about 1 cm s−1 (8), whereas with microfluidic hydrodynamic focusing particle velocities above 1 m s−1 have been demonstrated (7, 10, 18).
The required particle count rates depend on the application. For instance, a critical threshold in HIV disease progression is a concentration of CD4 positive T lymphocytes of 200 cells per μL (49). About 10,000 leukocytes should be counted to adequately reduce the contribution of the statistical uncertainty to the overall accuracy when determining this concentration. Conventional optical flow cytometers are specified for maximum count rates ranging from 500 Hz to 100 kHz, which would allow to carry out such measurements in less than a minute.
Recently, blood cell counting using microflow cytometers started to appear in commercial systems. Cheung et al. used a combination of DC and AC impedance counting to measure the concentration of erythrocytes (20). This combination allows to measure cellular properties in addition to size (50, 51). A readout system using this chip is now commercially available from Axetris (Switzerland). However, most applications of microflow cytometers for blood cell counting have not reached that level yet. Holmes et al. demonstrated 3-part differential count in a microfluidic device detecting lymphocytes, monocytes, and neutrophils using AC impedance counting in combination with fluorescence detection (27). Four-part differential blood count was demonstrated by Shi et al. exploiting fluorescence staining with FITC and PI (28). They succeeded in differentiating lymphocytes, monocytes, neutrophils, and eosinophils with particle count rates around 10 Hz. Wang et al. applied a microfluidic system for staining and counting CD4 positive and CD8 positive T lymphocytes (9). They combined a vortex-type micromixer for antibody staining of T lymphocytes with a microfluidic flow cytometer with 1D hydrodynamic focusing and bivariate fluorescence detection demonstrating count rates of a few hertz. On chip staining of T lymphocytes has been reported before by Chan et al. (29), however, without using an integrated micromixer. Chan et al. used chips from Agilent Technologies which were vortexed to achieve mixing of the cell suspension and antibody reagent in the sample wells of the chip.
The micro-device presented in this article was used to determine relative concentrations of fluorescently labeled subpopulations of lymphocytes and other leukocytes at a high count rate. Although the signal-to-noise ratio achieved was smaller compared to MoFlo cell sorter, the performance of the micro-device was similar to the large frame flow cytometer when detecting cells with a high level of fluorescence. Furthermore, the sensitivity was sufficient to identify cells exhibiting moderate fluorescence intensity, e.g., anti-CD4-PE labeling of monocytes. The comparison between conventional instruments and microfabricated devices presented here proves the applicability of adequately designed microflow cytometers for cell-based assays for biomedical research, and also for routine applications. The development of a system composed of a compact read-out instrument to accommodate disposable cartridges with integrated microfluidic chips is underway, although such a combination of optimized microfabricated subunits has not been demonstrated yet.
The results listed in Table 1 are to our knowledge the first comparison of measurements of relative blood cell concentrations using microfluidic and conventional flow cytometry. It indicates the capability of microfluidic devices for immunological cell differentiation and the determination of cell concentrations. However, improved accuracy for measuring cell concentrations is highly desirable. At present, both set-ups used were not optimized to allow precise determination of (relative) cell concentrations. The performance of both instruments could be improved by modifying the sample transport to the flow cell, in particular by reducing tubing length and selecting a material to prevent cell specific adhesion. It should be noted that the occurrence of large inter-platform differences (up to 25%) is sometimes also observed in interlaboratory comparisons for the measurands of the immune status, regularly organized in Germany by the Reference Institute for Bioanalytics (RfB) and the Society for Promotion of Quality Assurance in Medical Laboratories (INSTAND).
Within this limit there is an overall good agreement between the measurements compared here. A deviation below 20% was observed for the relative concentrations of lymphocytes, monocytes, and TS lymphocytes. The values for the relative concentrations of B lymphocytes and NK cells also agree within 25%, but in both cases the uncertainties are large due to the low number of repeat measurements and poor counting statistics. On the other hand, larger deviations are found for the relative concentrations of T lymphocytes as well as for the T-helper/inducer cells. In this context, it is noticeable that the relative concentrations of lymphocytes and lymphocyte subsets determined with the MoFlo cell sorter systematically deviate to smaller values.
At present, conventional flow cytometers using quartz flow cells still offer superior signal-to-noise ratios in fluorescence detection. Although this does not limit the application of micro-devices for counting fluorescently labeled cells as considered in this article, the fluorescence background in micro-device could be reduced using other thermoplastics; e.g., cyclic olefin copolymers are known to allow the production of high quality optical components. Adding optical components for laser beam shaping or more efficient integrated light collection might also help to resolve this issue and make disposable plastic flow cells the better choice even for general purpose flow cytometers. Further developments would aim at the integration of different subassemblies into a compact disposable cartridge mounted in fully automated sample-handling and readout device.