Microfluidic Red Blood Cell Separation for Rapid Blood Testing - Rune Percy, Alex Smith

Blood Characteristics and Benefits of Microfluidics

Figure 1: Various blood contents and their volume percentages in blood.

Blood is responsible for transporting oxygen and other nutrients to tissues around the body as well as helping regulate the body’s pH and temperature. Whole blood is a nonhomogeneous fluid composed of four main constituents: red blood cells (RBCs), plasma, white blood cells, and platelets [Fig 1][¹]. RBCs, however, make up 99% of the cells that are in whole blood [²]. Since most assays are performed on plasma, fractionation of red blood cells is vital for accurate measurements [¹]. Fractionation, or separation of blood into individual components, is both a challenge and an opportunity for much more comprehensive analyses of blood both in medicine and in the laboratory. [¹].

There are many benefits to biological processing using microfluidics as opposed to at the macro-scale. Low costs, small sample volumes, rapid processing times, and novel separation and detection methods are among the chief benefits of analyzing blood on this scale [³]. Toner et. al. as well as Huo et. al. have published comprehensive reviews of microchip blood processing techniques, as well as associated challenges [¹,⁴]. They found that in order to accurately quantify plasma components, it is vital to remove the high volume of RBCs [¹,⁴]. Traditional lab benchtop centrifuges are still the most common method of plasma separation. However, this process is time-consuming, expensive, and labor intensive [⁴]. Microfluidics can therefore offer a much more attractive solution to current fractionation challenges.

Once blood has been fractionated, there are many different assays that can be performed based on the molecules of interest. Two of the most widely used methods to measure common molecules, such as proteins and electrolytes, are optical and electrochemical detection. Optical methods include flame photometry, UV absorbance, near-infrared spectroscopy, and laser-induced breakdown spectroscopy, among others [⁵]. Using a light source and a detector, optical systems are able to detect changes in the optical response of the system due to the presence of molecules of interest, and calibrate this based on concentration [Fig 2]. This is often done using molecules that fluoresce under controllable conditions.

Figure 2: Example of a simple optical system which could be used with microfluidic technology to measure molecules using a light source and detector.

Electrochemical, or potentiometric methods, use electrodes which produce a detectable electrical signal in the presence of certain molecules; for example, ion-selective electrodes can be used to measure electrolytes [⁵]. The combination of these assays and a microfluidic plasma separation mechanism is an example of a “lab-on-a-chip” (LOC).

Microfluidic LOC systems could dramatically improve care in certain applications by providing more frequent diagnostics at a low cost, and with small sample volumes [⁶]. For example, in hemodialysis treatment, a fluid called dialysate, which diffusively filters blood through a semipermeable membrane to remove toxins and stabilize electrolyte levels, is prescribed based on blood tests of patients [⁵]. Traditional blood analyses are performed at an off-site lab, require large volumes of blood, and are expensive; therefore they are only performed about once per month on average to adjust treatment [⁵]. This is not efficient enough, as the kidneys continuously filter blood using bio-feedback loops in the body, and many patients develop treatment complications. An on-line microfluidic diagnostic technology could potentially monitor molecular concentrations continuously using small blood volumes, which could help personalize treatment and reduce costs [⁵].

Some additional applications include drug and disease screening, or neonatal care [¹,⁶]. In an expert review, Chin et al discuss the LOC field and stress that researchers should focus on point-of-care diagnostic applications to make the most impact. They also discuss how relatively few companies have been able to break out of the lab, despite the growing list of applications for these systems [⁶]. They cite the need for focused research that fits a specific diagnostic application to increase chances of success [⁶].

Microfluidic Methods of Separation

Microfluidics offers a wide range of possible methods to separate plasma from small volumes of whole blood samples. The small length scales of microfluidic systems often yield Reynolds Numbers below 100. This results in highly controllable laminar flow, which allows easy manipulation of the fluid streamlines [⁷]. Plasma separation has been achieved through numerous mechanisms, including acoustophoresis, dielectrophoresis, electrohydrodynamics, and physical hydrodynamic manipulation [¹]. Acoustophoresis involves the superimposition of multiple ultrasonic waves which intersect at nodes to create an acoustic standing wave. A radial acoustic pressure force induces the aggregation of suspended particles in the fluid at the nodes [Fig 3A] [¹]. By designing a microfluidic channel to generate acoustic nodes at its center, RBCs can be aggregated in the center of the channel, leaving clean plasma to be removed from the sides [⁸]. Dielectrophoresis uses a non-uniform electric field to polarize RBCs and move them to common locations using a net electrostatic force [Fig 3B] [¹,⁹]. Electrohydrodynamics works by converting the electrical energy of an electrostatic field to kinetic energy in the form of flow vortices formed by hydrostatic pressure [Fig 3C] [¹]. This principle has been utilized in a microfluidic device to separate plasma from RBCs by generating electrodynamic vortices, or ionic winds [¹⁰].

Figure 3: A) Acoustophoresis induced particle aggregation, B) Dielectrophoresis induced particle aggregation, C) Electrohydrodynamics induced particle aggregation.

Figure 4: Cross-flow design used to separate plasma from whole blood through the use of side channels that restrict the flow of red blood cells.

A number of microfluidic systems sort components of blood by physically manipulating the flow to retain particles of interest. Plasma can be separated from blood by restricting the path of undesired cells (RBCs) in flow, like when using cross-flow designs, or by displacing various components of blood in flow, which can be achieved using deterministic lateral displacement. Cross-flow designs function by flowing plasma through side-channels that restrict the passage of RBCs from the main flow channel. When larger particles like RBCs approach a bifurcation, they are more likely to flow through the channel with the higher flow rate. It has also been shown that RBCs aggregate in the center of channels with pressure-driven blood flow, creating a flow layer on the periphery of a channel that is less concentrated with cells [¹¹,¹²]. Microfluidic devices have been designed to remove plasma from the periphery of flow using channels with a small enough diameter. Yang et. al demonstrated that RBCs could be efficiently separated from whole blood by designing a main channel that had a flow rate that was six times greater than the side channel slower flow rate [Fig. 4][¹³].

Deterministic lateral displacement (DLD) Brownian Ratchet/Deterministic Lateral Displacement - Matthew Tiller is a technique that uses microposts to laterally sort components of fluid based on size. This technique operates much like a coin sorter or the game Plinko from the television show, "The Price is Right." Essentially, depending on the spacing between posts (λ), the gap size (d), and the shift row fraction (ε), a critical diameter (CD) is determined [Fig 5][¹⁴]. A particle whose diameter is greater than this CD will displace laterally, whereas particles smaller than CD will continue in a straighter pattern through the posts, following the streamlines around obstacles[¹⁵-¹⁷]. The displacement angle, Φ, is dependent on ε [¹⁵-¹⁷]. These devices can operate in continuous flow systems with multiple arrays for creating reservoirs of components by diameter [¹⁶]. The success of separation is correlated to flow rate, and to minimize diffusion effects, higher flow velocity is recommended by Huang et. al [¹⁷].

Figure 5: Deterministic Lateral Displacement (DLD) concept. Larger particles are displaced to the right while smaller particles continue flowing straight through the pillars.

Other forms of physical plasma microfluidic separation include lateral flow assays (LFAs) and micro-centrifuge devices. LFAs are simple and robust tools used to separate components of blood based on size using a paper strip. These can be used to perform molecular assays on plasma, which is designed to be absorbed in isolation by a pad [¹⁸]. Micro-centrifuge devices, such as the paperfuge, are able to generate incredible centrifugal forces which can separate RBCs similarly to a traditional centrifuge, except they use capillary tubes and are powered by lateral hand movements [¹⁹]. The benefits of using paper-based microfluidic separation are numerous. These devices are inexpensive, disposable and easy to use, making them very useful for providing healthcare options to developing nations that are resource-deprived [²⁰].

References

[1] Hou, H. W., Bhagat, A. A. S., Lee, W. C., Huang, S., Han, J., & Lim, C. T. Microfluidic Devices for Blood Fractionation. Micromachines, 2011, 2, 4, 319-343. DOI https://doi.org/10.3390/mi2030319

[2] Manzone, T.A., Dam, H.Q., Soltis, D., & Sagar V.V. Blood Volume Analysis: A new Technique and New Clinical Interest Reinvigorate a Classic Study. Journal of Nuclear Medicine Technology, 2007, 35, 2, 55-63. DOI https://doi.org/10.2967/jnmt.106.035972

[3] Whitesides, G.M. The origins and the future of microfluidics. Nature, 2006, 442, 368-373. DOI https://doi.org/10.1038/nature05058

[4] Toner, M.; Irimia, D. Blood-on-a-chip. Annu. Rev. Biomed. Eng. 2005, 7, 77-103. DOI https://doi.org/10.1146/annurev.bioeng.7.011205.135108

[5] Sharma M.K., Wieringa F.P., Frijns A.J.H., Kooman J.P. On-line monitoring of electrolytes in hemodialysis: on the road towards individualizing treatment. Expert Review of Medical Devices. 2016;13(10):933-943. DOI https://doi.org/10.1080/17434440.2016.1230494

[6] Chin, C.D., Linder, V., Samuel K. Sia, S.K.. Commercialization of microfluidic point-of-care diagnostic devices. Lab Chip, 2012, 12, 2118-2134. DOI https://doi.org/10.1039/C2LC21204H

[7] Bhatia S. N. & Ingber D. E. Microfluidic organs-on-chips. Nature biotechnology 32, 760–772. DOI https://doi.org/10.1038/nbt.2989

[8] Lenshof, A., Ahmad-Tajudin, A., Järås, K., Swärd-Nilsson, A. M., Aberg, L., Marko-Varga, G., Malm, J., Laurell, T. Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics. Analytical Chemistry, 2009, 81, 15, 6030-7. DOI https://doi.org/10.1021/ac9013572

[9] Nakashima, Y., Hata, S., & Yasuda, T. 2010. Blood plasma separation and extraction from a minute amount of blood using dielectrophoretic and capillary forces. Sensors & Actuators: B. Chemical, 145, 1, 561-569. DOI https://doi.org/10.1016/j.snb.2009.11.070

[10] Arifin, D.R.; Yeo, L.Y.; Friend, J.R. Microfluidic blood plasma separation via bulk electrohydrodynamic flows. Biomicrofluidics, 2007, 1, 014103. DOI https://doi.org/10.1063/1.2409629

[11] Jäggi, R. D., Sandoz, R., & Effenhauser, C. S. Microfluidic depletion of red blood cells from whole blood in high-aspect-ratio microchannels. Microfluidics and Nanofluidics, 2007, 3, 1, 47-53. DOI https://doi.org/10.1007/s10404-006-0104-9

[12] Bayliss LE (1959) The axial drift of the red cells when blood flows in a narrow tube. J Physiol, 149:593–613. DOI https://doi.org/10.1113/jphysiol.1959.sp006363

[13] Yang, S., Undar, A., & Zahn, J. D. A microfluidic device for continuous, real time blood plasma separation. Lab on a Chip, 2006, 6, 7, 871-80. DOI https://doi.org/10.1039/B516401J

[14] Sturm, J.C., Cox, E.C., Comella, B., Austin, R.H. Ratchets in hydrodynamic flow: more than waterwheels. Interface Focus, 2014, 4, 6. DOI https://doi.org/10.1098/rsfs.2014.0054

[15] McGrath, J., Jimenez, M. & Bridle, H. Deterministic lateral displacement for particle separation: a review. Lab on a Chip, 2014, 14, 4139. DOI https://doi.org/10.1039/C4LC00939H

[16] Inglis D.W., Davis J.A., Austin R.H., Sturm J.C. Critical particle size for fractionation by deterministic lateral displacement. Lab on a Chip, 2006, 6, 5, 655-8. DOI https://doi.org/10.1039/B515371A

[17] Huang, L.R., Cox, E.C., Austin, R.H., Sturm, J.C. Continuous particle separation through deterministic lateral displacement. Science, 2004, 304, 5673, 987-990. DOI 10.1126/science.1094567

[18] Sajid, M., Kawde, A.-N., & Daud, M. Designs, formats and applications of lateral flow assay: A literature review. Journal of Saudi Chemical Society, 2015, 19, 6, 689-705. DOI https://doi.org/10.1016/j.jscs.2014.09.001

[19] Bhamla, M. S., Benson, B., Chai, C., Katsikis, G., Johri, A., & Prakash, Hand-powered ultralow-cost paper centrifuge. Nature Biomedical Engineering, 2017, 1, 1, 9. DOI https://doi.org/10.1038/s41551-016-0009

[20] Yetisen, A. K., Akram, M. S., & Lowe, C. R. Paper-based microfluidic point-of-care diagnostic devices. Lab on a Chip, 13, 2013, 12, 2210-51. DOI https://doi.org/10.1039/C3LC50169H