SAMPLE

Technical Field

The present disclosure generally relates to a microfluidic device and a method for cell separation of a blood sample. More particularly, the present disclosure describes various embodiments of a microfluidic device for cell separation of the blood sample, as well as a method for cell separation of the blood sample using the microfluidic device.

Background

Analysis of blood samples has been demonstrated for different applications such as study of deformability of red blood cells (RBCs), and separation / isolation of cells from blood, such as platelets, plasma, and leukocytes, as well as rare cells such as circulating tumour cells (CTCs) and fetal cells. Analysis of fetal cells in maternal blood is important for prenatal diagnosis.

Current methods of prenatal diagnosis require fetal cells to be obtained by invasive procedures including chorionic villus sampling (CVS) and amniocentesis / amniotic fluid test (AFT), which carry associated risks for both fetus and mother. The invasive procedures are restricted to patients who have been assessed to be at higher risks of fetal chromosomal or genetic abnormalities / disorders. The invasive procedures are not suitable for routine screening of the general pregnant female population.

Efforts have been spent to develop non-invasive prenatal diagnosis (NIPD) alternatives to these standard invasive methods of prenatal diagnosis. One current alternative is non-invasive prenatal genetic testing (NIPT) which is based on the analysis of cell-free circulating fetal DNA. NIPT is not a diagnostic test but its evaluation results in a screening value which estimates the risks of fetal chromosomal or genetic abnormalities / disorders. Patients who have been evaluated as being at higher risks have to be validated by the standard invasive methods, e.g. CVS or AC, in order to confirm presence of any fetal genetic defects.

Another alternative to the standard invasive methods is to isolate intact fetal cells from maternal blood and to use the isolated fetal cells for direct analysis of fetal chromosomes or DNA with minimal risks to both mother and fetus [Bianchi, D. W., et at., Prenatal Diagnosis 2002, 22 (7), 609-615 ]. However, fetal cells are rare, there being about 1 to 10 fetal erythroblasts out of 5 billion RBCs in 1 ml of maternal blood. There are thus technical difficulties in the isolation of the fetal cells using conventional sample preparation methods [van Wijk, I. J., et al., American Journal of Obstetrics and Gynecology 2001, 184 (5), 991-997; Vona, G., et al., The American Journal of Pathology 2002, 160 (1), 51-58].

Because fetal cells, as well as other cells such as CTCs and stem cells, are extremely rare in blood, a cell enrichment or cell separation process is usually necessary to isolate them prior to analysis. Known sample preparation methods to separate / isolate fetal cells from maternal blood include gradient centrifugation, immunomagnetic spheres, membrane filtration, fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS). Gradient centrifugation (based on density of cells) and membrane filtration (based on size of cells) enable removal of RBCs in the background, but are prone to fetal cell loss. These two methods also enrich white blood cells (WBCs) in the process which complicates downstream isolation and analysis of fetal erythroblasts. FACS and MACS require the use of specific positive antibodies to bind target cells, but a well-known surface antigen exclusively expressed on fetal cells has not yet been identified, leading to poor enrichment yield and purity. Moreover, these conventional methods are laborious, time-consuming, and may not achieve a high enrichment / purification of fetal cells as these methods are highly dependent on the skill and experience of the technician processing the maternal blood.

In recent years, microfluidic has emerged as an enabling technology for cell separation [Bhagat, A. A. S., et al., Med Biol Eng Comput 2010, 48 (10), 999-1014] and point-of-care assays [Yager, P., et al., Nature 2006, 442 (7101), 412-418] due to its inherent advantages including small length scale, reduced sample and reagent volumes, and low device cost.

Several microfluidics technologies have been developed for size-based isolation of fetal cells. For example, a microfluidic strategy was developed to isolate fetal erythroblasts from maternal blood based on the principle of “deterministic lateral displacement” [Huang, R., et al., Prenatal Diagnosis 2008, 28 (10), 892-899 ]. Briefly, a sample of whole blood is injected into an array of micropillars on a microfluidic device / chip to continuously deflect the larger nucleated cells (WBCs and erythroblasts) from the smaller RBCs. The nucleated cells are then eluted off-chip to further isolate the erythroblasts based on paramagnetic properties. The system can process approximately 5 to 20 ml of blood in 2 to 6 hours, and it demonstrated successful isolation of erythroblasts (approximately 37.44 cells per ml) in 58 samples of maternal blood. However, clogging problems in the micropillars array can affect the performance of cell separation.

Another micropillar microfluidic device / chip was developed for physical entrapment of nucleated cells from maternal blood based on cell size and deformability [Mohamed, H., et ai., Journal of Chromatography A 2007, 1162 (2), 187-192 ]. By using pillar gap sizes ranging from 2.5 to 15 pm, goose RBCs were trapped using 2.5 pm gap channels. Goose RBCs are used as a model for fetal cells as they have similar sizes. However, throughput is very low (approximately 350 pi per hour) and not practical for processing clinically-relevant blood sample volumes (approximately 10 to 20 ml in each blood sample). Moreover, it is non-trivial to retrieve cells which are trapped inside the devices for downstream analysis.

Other approaches to cell separation using microfluidics include using a pneumatic- actuated microfluidic device with a gap size of 1 pm [T. Kumo, et al. , In Concentration and extraction chip of fetal nucleated red blood cell (nRBC) by microgap with diaphragm for fetal DNA diagnosis from maternal blood, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Groningen, The Netherlands, Groningen, The Netherlands, 2010; pp 1583-1585 ]; continuous cross-flow filtration [Lee, D., et al., Journal of Chromatography A 2010, 1217 (11), 1862-1866 ], dielectrophoresis [Guolin, X., et al., Journal of Physics: Conference Series 2006, 34 (1), 1106], and biomimetic plasma skimming integrated with immunomagnetic separation [Kersaudy-Kerhoas, M., et al., Archives of Disease in Childhood - Fetal and Neonatal Edition 2014, 99 (Suppi 1), A2-A2 ]. However, experiments using maternal blood samples are lacking, and more validations are required to assess the efficacy of these methods for clinical testing. An affinity-based microfluidic isolation method was reported to purify fetal cells using hydroxyapatite / chitosan nanoparticles and immuno-agent anti-CD147 [He, Z, et al., Journal of Materials Chemistry B 2017, 5 (2), 226-235 ]. The biochip was validated using cord blood and maternal blood samples, including performing a Fluorescence in situ Hybridization (FISH) analysis of isolated nucleated cells for fetal chromosome disorders (Trisomy 13 and 21 ).

United States Patent Publication 2013-0130226 A1 discloses a microfluidic device having spiral channels for cell separation from samples of whole blood based on size of diseased cells and Dean flow dynamics [Di Carlo, D., et al., P Natl Acad Sci USA 2007, 104 (48), 18892-18897 ]. The microfluidic device is used for separation of cells such as CTCs [Hou, H. W., et al., Sci Rep-Uk 2013, 3], leukocytes [Wu, L. D., et al., Anal Chem 2012, 84 (21), 9324-9331 ], and microorganisms [Hou, H. \N., et al., Lab on a chip 2015, 15 (10), 2297-2307] from whole blood for point-of-care assays and testing. However, while the microfluidic device is somewhat effective in isolating and enriching the target cells from blood by depletion of RBCs as a result of the input blood sample flowing through the microfluidic device, there is still a residual amount of RBCs remaining in the output blood sample. Based on some experimental data, the output blood sample may contain about 1 % to 5% of residual RBCs and this RBC contamination may compromise subsequent analysis thereof.

Therefore, in order to address or alleviate at least one of the aforementioned problems and/or disadvantages, there is a need to provide a microfluidic device and a method for cell separation of a blood sample, in which there is at least an improvement and/or advantage over the prior art.

Summary According to a first aspect of the present disclosure, there is a microfluidic device for cell separation of a blood sample. The microfluidic device comprises a set of inlets for receiving the blood sample; a set of outlets fluidically connected to the inlets, comprising a first waste outlet, an output sample outlet, and a second waste outlet; and a set of microfluidic channels fluidically connecting the inlets to the outlets. The set of microfluidic channels comprises a spiral microfluidic channel for receiving the blood sample from the inlets; and an arcuate microfluidic channel fluidically connected to the spiral microfluidic channel. The spiral microfluidic channel bifurcates into the arcuate microfluidic channel and first waste outlet from an inner side and outer side of the spiral microfluidic channel respectively. The arcuate microfluidic channel bifurcates into the output sample outlet and second waste outlet from an inner side and outer side of the arcuate microfluidic channel respectively. The output sample outlet is arranged for collecting an output sample that is substantially depleted of red blood cells relative to the blood sample.

According to a second aspect of the present disclosure, there is a method for cell separation of a blood sample using the microfluidic device according to the first aspect of the present disclosure. The method comprises: introducing the blood sample into the set of inlets; communicating the blood sample from the set of inlets to the spiral microfluidic channel; bifurcating the blood sample from the spiral microfluidic channel into an intermediary sample communicating to the arcuate microfluidic channel and waste communicating to the first waste outlet; bifurcating the intermediary sample from the arcuate microfluidic channel into an output sample communicating to the output sample outlet and waste communicating to the second waste outlet; and collecting the output sample from the output sample outlet, wherein the output sample is substantially depleted of red blood cells relative to the blood sample.

An advantage of the present disclosure is that the blood sample is bifurcated twice to improve effectiveness of depletion of the red blood cells. The output sample is thus substantially depleted of RBCs relative to the blood sample, such as at least 99.5% depletion. The substantial depletion of RBCs in the output sample effectively separates and isolates the nucleated cells from the RBCs, thereby substantially enriching the nucleated cells in the output sample for improved analysis and testing thereof subsequently.

The microfluidic device and method may thus be used for cell separation of a blood sample. Particularly, the microfluidic device and method may be used to separate and isolate nucleated cells from RBCs in the blood sample by communicating the blood sample firstly through the spiral microfluidic channel and secondly through the arcuate microfluidic channel (see, for example, Figure 1 ). The spiral microfluidic channel performs a first bifurcation of the blood sample, and the arcuate microfluidic channel performs a second bifurcation of the blood sample. More specifically, the blood sample is bifurcated into an intermediary sample and waste, and the intermediary sample is later bifurcated into an output sample and waste.

A microfluidic device and a method for cell separation of a blood sample according to the present disclosure are thus disclosed herein. Various features, aspects, and advantages of the present disclosure will become more apparent from the following detailed description of the embodiments of the present disclosure, by way of non- limiting examples only, along with the accompanying drawings.

Brief Description of the Drawings

Figure 1 is an illustration of a microfluidic device for cell separation of a blood sample, in accordance with various embodiments of the present disclosure.

Figure 2A to Figure 2C are illustrations of inlets, a spiral microfluidic channel, and an arcuate microfluidic channel of the microfluidic device, in accordance with various embodiments of the present disclosure.

Figure 3 is a flowchart illustration of a method for cell separation of a blood sample, in accordance with various embodiments of the present disclosure. Figure 4A and Figure 4B are illustrations of cross-section views of the microfluidic channels, showing Dean vortices and cell separation of larger nucleated cells from smaller red blood cells, in accordance with various embodiments of the present disclosure.

Figure 5A and Figure 5B are illustrations in relation to variations in microchannel height of the spiral microfluidic channel, in accordance with various embodiments of the present disclosure.

Figure 6A and Figure 6B are illustrations in relation to variations in blood sample haematocrit in the spiral microfluidic channel, in accordance with various embodiments of the present disclosure.

Figure 7A and Figure 7B are illustrations in relation to variations in blood sample haematocrit in the arcuate microfluidic channel, in accordance with various embodiments of the present disclosure.

Figure 8A and Figure 8B are illustrations in relation to variations in flow rate in the spiral microfluidic channel, in accordance with various embodiments of the present disclosure.

Figure 9A to Figure 9C are illustrations of a microbeads simulation using the microfluidic device, in accordance with various embodiments of the present disclosure.

Figure 10 is an illustration in relation to variations in radius of curvature of the arcuate microfluidic channel, in accordance with various embodiments of the present disclosure.

Figure 11A to Figure 11 D are illustrations of an application to separate white blood cells from whole blood using the microfluidic device, in accordance with various embodiments of the present disclosure. Figure 12 is an illustration of an application to separate fetal nucleated red blood cells from maternal blood using the microfluidic device, in accordance with various embodiments of the present disclosure.

Figure 13 is an illustration of an application to separate putative fetal erythroblasts from maternal blood using the microfluidic device, in accordance with various embodiments of the present disclosure.

Detailed Description

In the present disclosure, depiction of a given element or consideration or use of a particular element number in a particular figure or a reference thereto in corresponding descriptive material can encompass the same, an equivalent, or an analogous element or element number identified in another figure or descriptive material associated therewith. The use of 7” in a figure or associated text is understood to mean“and/or” unless otherwise indicated. As used herein, the term “set” corresponds to or is defined as a non-empty finite organization of elements that mathematically exhibits a cardinality of at least one (e.g. a set as defined herein can correspond to a unit, singlet, or single element set, or a multiple element set), in accordance with known mathematical definitions. The recitation of a particular numerical value or value range herein is understood to include or be a recitation of an approximate numerical value or value range.

For purposes of brevity and clarity, descriptions of embodiments of the present disclosure are directed to a microfluidic device and a method for cell separation of a blood sample, in accordance with the drawings. While aspects of the present disclosure will be described in conjunction with the embodiments provided herein, it will be understood that they are not intended to limit the present disclosure to these embodiments. On the contrary, the present disclosure is intended to cover alternatives, modifications and equivalents to the embodiments described herein, which are included within the scope of the present disclosure as defined by the appended claims. Furthermore, in the following detailed description, specific details are set forth in order to provide a thorough understanding of the present disclosure. Flowever, it will be recognized by an individual having ordinary skill in the art, i.e. a skilled person, that the present disclosure may be practiced without specific details, and/or with multiple details arising from combinations of aspects of particular embodiments. In a number of instances, known systems, methods, procedures, and components have not been described in detail so as to not unnecessarily obscure aspects of the embodiments of the present disclosure.

Microfluidic Device

Representative or exemplary embodiments of the present disclosure describe a microfluidic device 100 for cell separation of a blood sample, with reference to Figure 1. The blood sample contains a collection or mixture of red blood cells (RBCs) and one or more other types of nucleated cells that are targeted for cell separation. Particularly, the microfluidic device 100 is used for separation and isolation of certain nucleated cells from the RBCs in the blood sample. The nucleated cells may be white blood cells (WBCs) to be separated and isolated from whole blood or fraction thereof, or fetal nucleated cells to be separated and isolated from maternal blood or fraction thereof. It will be appreciated that the RBCs as used herein include both mature RBCs without nuclei and immature RBCs (or reticulocytes). The term“whole blood” as used herein refers to human blood from which no constituent, such as RBCs, WBCs, plasma, or platelets, has been removed. There are approximately 5 billion RBCs in 1 ml of whole blood. The term“maternal blood” as used herein refers to whole blood from a pregnant female person. Whilst the device of the invention is ideally used to separate or enrich particular cells from a whole blood sample, it would be understood that the device could be used on partially fractionated blood providing the cells of interest are in the sample loaded on the device.

The microfluidic device 100 comprises a set of inlets for receiving the blood sample and a set of outlets fluidically connected to the set of inlets. In many embodiments as shown in Figure 1 , the set of inlets comprises an inner inlet 102 and an outer inlet 104. One of the inner inlet 102 and outer inlet 104 is arranged for receiving the blood sample, and the other is arranged for receiving a sheath fluid. In some other embodiments, the set of inlets may consist of a sole inlet for receiving the blood sample or a mixture of the blood sample and sheath fluid. In some other embodiments, the set of inlets may comprise three or more inlets for receiving the blood sample and optionally the sheath fluid.

The microfluidic device 100 further comprises a set of outlets fluidically connected to the set of inlets. The set of outlets comprises a first waste outlet 106, an output sample outlet 108, and a second waste outlet 110. In many embodiments as shown in Figure 1 , the first waste outlet 106, output sample outlet 108, and second waste outlet 110 are fluidically connected to the inner inlet 102 and outer inlet 104.

The microfluidic device 100 further comprises a set of microfluidic channels, specifically microfluidic curvilinear channels, fluidically connecting the inlets 102 to the outlets 104. The microfluidic channels may be referred to as microchannels in various instances herein. In many embodiments as shown in Figure 1 , the set of microfluidic channels comprises a spiral microfluidic channel 112 for receiving the blood sample from the inlets and an arcuate fluidic channel 114 fluidically connected to the spiral microfluidic channel 112. Each of the spiral microfluidic channel 112 and arcuate fluidic channel 114 has a cross-section that allows fluid communication therethrough along a curvilinear path or profile. Accordingly, the blood sample is able to communicate or flow from the inlets to the outlets via the spiral microfluidic channel 112 and arcuate fluidic channel 114.

As used herein, two or more microfluidic elements are fluidically connected by forming a fluidic connection therebetween, such that a fluid is able to communicate or flow continuously through the microfluidic elements and across the fluidic connection(s). As used herein, a“microfluidic element” is defined as a component, structure, or part for use in microfluidics.

With reference to Figure 2A, the set of inlets is arranged for receiving or introduction, e.g. by perfusion, of the blood sample. In many embodiments, the set of inlets comprises an inner inlet 102 and an outer inlet 104 arranged adjacent to each other. The inner inlet 102 is positioned towards the inner side or wall of the spiral microfluidic channel 112, such that a fluid is communicable from the inner inlet 102 along the inner side of the spiral microfluidic channel 112. The inner side refers to the radially inward or convex side or wall of the spiral microfluidic channel 112. The outer inlet 104 is positioned towards the outer side or wall of the spiral microfluidic channel 112, such that a fluid is communicable from the outer inlet 104 along the outer side of the spiral microfluidic channel 112. The outer side refers to the radially outward or concave side or wall of the spiral microfluidic channel 112. One of the inner inlet 102 and outer inlet 104 is arranged for receiving or introduction of the blood sample, and the other is arranged for receiving or introduction of the sheath fluid. The sheath fluid may be 1x PBS (phosphate-buffered saline) which may be supplemented with 0.5% BSA (bovine serum albumin). Alternatively, the sheath fluid may be culture media suitable for studies of RBCs, such as Ringer solution RPMI, DMEM, etc.

With reference to Figure 2B, the spiral microfluidic channel 112 is arranged for discharging the blood sample via a first bifurcation junction 116 to the arcuate microfluidic channel 114 and first waste outlet 106. Specifically, the blood sample is discharged from the spiral microfluidic channel 112 by bifurcating from the first bifurcation junction 116 into an intermediary sample communicating / flowing to the arcuate microfluidic channel 114 and waste communicating / flowing to the first waste outlet 106. As shown in Figure 2B, the arcuate microfluidic channel 114 is positioned towards the inner side or wall (radially inward or convex side) of the spiral microfluidic channel 112, and the first waste outlet 106 is positioned towards the outer side or wall (radially outward or concave side) of the spiral microfluidic channel 112. In some embodiments, at the first bifurcation junction 116, the arcuate microfluidic channel 114 and first waste outlet 106 are arranged to have an angular separation ranging from 30 to 150 degrees (or more preferably from 60 to 120 degrees). In one embodiment as shown in Figure 2B, the angular separation is approximately 90 degrees. The spiral microfluidic channel 112 thus bifurcates into the arcuate microfluidic channel 114 and first waste outlet 106 from an inner side and outer side of the spiral microfluidic channel 112 respectively.

With reference to Figure 2C, the arcuate microfluidic channel 114 is arranged for discharging the intermediary sample via a second bifurcation junction 118 to the output sample outlet 108 and second waste outlet 110. Specifically, the intermediary sample is discharged from the arcuate microfluidic channel 114 by bifurcating from the second bifurcation junction 118 into an output sample communicating / flowing to the output sample outlet 108 and waste communicating / flowing to the second waste outlet 110. As shown in Figure 2C, the output sample outlet 108 and second waste outlet 110 are positioned adjacent to each other. Specifically, the output sample outlet 108 is positioned towards the inner side or wall (radially inward or convex side) of the arcuate microfluidic channel 114, and the second waste outlet 110 is positioned towards the outer side or wall (radially outward or concave side) of the arcuate microfluidic channel 114. In some embodiments, at the second bifurcation junction 118, the output sample outlet 108 and second waste outlet 110 are arranged to have an angular separation ranging from 30 to 150 degrees (or more preferably from 60 to 120 degrees). In one embodiment as shown in Figure 2C, the angular separation is approximately 90 degrees. The arcuate microfluidic channel 114 thus bifurcates into the output sample outlet 108 and second waste outlet 110 from an inner side and outer side of the arcuate microfluidic channel 114 respectively.

The first waste outlet 106 comprises a first waste channel 120 and a first waste collector 122 (01 ) leading therefrom for collecting waste bifurcated from the blood sample. The output sample outlet 108 is comprises an output sample channel 124 and an output sample collector 126 (02) leading therefrom for collecting the output sample bifurcated from the intermediary sample. The second waste outlet 110 comprises a second waste channel 128 and a second waste collector 130 (03) leading therefrom for collecting waste bifurcated from the intermediary sample.

In many embodiments, the first waste channel 120 is longer than each of the output sample channel 124 and second waste channel 128. The excess length allows the waste from the first waste outlet 106 to take more time to flow along the first waste channel 120. The additional residence time in the first waste channel 120 allows the waste to slow down so that they may be properly collected at the first waste collector 122 (01 ). In contrast, the flow rates in the output sample channel 124 and second waste channel 128 is lower than in the first waste channel 120. Less residence time is required and as such their lengths are shorter. Moreover, at lower flow rates, the output sample and waste can be properly collected at the output sample collector 126 (02) and second waste collector 130 (03) respectively.

In one embodiment, the first waste channel 120 has a sinusoidal shape or profile, and each of the output sample channel 124 and second waste channel 128 is linear. The sinusoidal first waste channel 120 provides the excess length required to reduce the flow rate. Furthermore, the sinusoidal shape allows the first waste channel 120 to be positioned on a smaller area of the microfluidic device 100, utilizing the limited space of the microfluidic device 100 more effectively. It will be appreciated that the first waste channel 120 may be in other shapes or profiles to provide the excess length while effectively utilizing the limited space of the microfluidic device 100. For example, the first waste channel 120 may have a zig-zag, alternating, meandering, serpentine, or spiral profile.

Method

Various embodiments of the present disclosure describe a method 200 for cell separation of a blood sample using the microfluidic device 100. With reference to Figure 3, the method 200 broadly comprises the following steps:

a. Step 202 of introducing the blood sample into the set of inlets;

b. Step 204 of communicating the blood sample from the set of inlets to the spiral microfluidic channel 112;

c. Step 206 of bifurcating the blood sample from the spiral microfluidic channel 112 into an intermediary sample communicating to the arcuate microfluidic channel 114 and waste communicating to the first waste outlet 106;

d. Step 208 of bifurcating the intermediary sample from the arcuate microfluidic channel 114 into an output sample communicating to the output sample outlet 108 and waste communicating to the second waste outlet 110; and

e. Step 210 of collecting the output sample from the output sample outlet, wherein the output sample is substantially depleted of red blood cells relative to the blood sample. The microfluidic device 100 and method 200 may thus be used for cell separation of a blood sample. Particularly, the microfluidic device 100 and method 200 may be used to separate and isolate nucleated cells from RBCs in the blood sample by communicating the blood sample through the spiral microfluidic channel 112 and arcuate microfluidic channel 114 of the microfluidic device 100. The spiral microfluidic channel 112 performs a first bifurcation of the blood sample at the first bifurcation junction 116, and the arcuate microfluidic channel 114 performs a second bifurcation of the blood sample at the second bifurcation junction 118. More specifically, the spiral microfluidic channel 112 bifurcates the blood sample into an intermediary sample and waste, and the arcuate microfluidic channel 114 bifurcates the intermediary sample into an output sample and waste. The output sample is substantially depleted of RBCs relative to the blood sample. In some experiments, the output sample has at least 99.5% depletion of RBCs relative to the blood sample. The substantial depletion of RBCs in the output sample effectively separates and isolates the nucleated cells, such as WBCs or fetal nucleated cells, from the RBCs, thereby substantially enriching the nucleated cells in the output sample for improved analysis and testing thereof subsequently, such as for downstream genetic analysis / testing.

Dimensional Properties of Microfluidic Device

Each of the spiral microfluidic channel 112 and arcuate microfluidic channel 114 has a length and a cross-section of a cross-section width and cross-section height defining an aspect ratio that enables separation of nucleated cells from the RBCs in the blood sample. As used herein, an aspect ratio is the ratio of the microchannel cross-section height divided by the microchannel cross-section width. The aspect ratio provides the appropriate microchannel cross-section to enable the nucleated cells to flow along one portion of the microchannel cross-section and the remaining RBCs to flow along a distinct portion of the microchannel cross-section. The appropriate aspect ratio causes the nucleated cells to flow along a distinct portion of the microchannel cross-section based on differences in structural characteristics of the nucleated cells and the remaining RBCs in the blood sample. Some examples of such structural characteristics include, but are not limited to, cell size, stiffness, deformability, and adhesiveness (e.g. cytoadhesiveness).

In many embodiments, the spiral microfluidic channel 112 forms a spiral or spiral-like shape / profile from the set of inlets until the first bifurcation junction 116. It will be appreciated that the spiral may be oriented in a clockwise or anti-clockwise manner. The arcuate microfluidic channel 114 has a curvilinear profile extending from the first bifurcation junction 116 to the second bifurcation junction 118. In some embodiments, the curvilinear profile forms an arc which is defined as a part / portion of a curve or a part / portion of the circumference of a circle. In other embodiments as shown in Figure 2C, the curvilinear profile forms an arc portion 114a and a linear portion 114b preceding the arc portion 114a. It will be further appreciated that the orientation of the arc is dependent on the orientation of the spiral, so that the intermediary sample is able to flow from the spiral microfluidic channel 112 to and along the inner side of the arcuate microfluidic channel 114.

In some embodiments as shown in Figure 1 , the spiral microfluidic channel 112 forms a spiral with two complete loops about its centre, and with a radius of curvature of approximately 0.9 cm. The spiral microfluidic channel 112 may have a length of approximately 10 cm. The cross-section of the spiral microfluidic channel 112 may have a width of approximately 500 pm and a height ranging from 85 to 115 pm, resulting in an aspect ratio between 4 and 6. In one embodiment, the cross- section height is approximately 95 pm.

In some embodiments as shown in Figure 2C, the arcuate microfluidic channel 114 has an arc portion 114a and a linear portion 114b. The arc portion 114a is in the form of a half-loop about its centre, with a radius of curvature ranging from 0.1 to 0.9 cm (or more preferably from 0.3 to 0.7 cm). The arc portion 114a subtends a central angle of at least 180 degrees. The linear portion 114b facilitates positioning of the arc portion 114a on the microfluidic device 100 so that there is sufficient space to form the arc portion 114a. The cross-section of the arcuate microfluidic channel 114 may have a width of approximately 150 pm and a height ranging from 85 to 100 pm. In one embodiment, the arcuate microfluidic channel 114 has a radius of curvature of approximately 0.5 cm, central angle of approximately 180 degrees, and a cross- section height of approximately 95 pm. The arc length of the arc portion 114a is thus approximately 1.57 cm. In addition, the linear portion 114b has a length of approximately 0.4 cm, resulting in a total length of approximately 2 cm for the arcuate microfluidic channel 114.

The arcuate microfluidic channel 114 is shorter in length than the spiral microfluidic channel 112 because the flow rate of the blood sample through the arcuate microfluidic channel 114 is lower than the flow rate through the spiral microfluidic channel 112. However, a reduction in flow rate from the spiral microfluidic channel 112 to the arcuate microfluidic channel 114 may result in a reduction in flow velocity. In many embodiments, the arcuate microfluidic channel 114 has a cross-section width (e.g. 150 pm) smaller than a cross-section width (e.g. 500 pm) of the spiral microfluidic channel 112. More specifically, the cross-section widths are calculated to enable substantially constant flow velocities through the spiral microfluidic channel 112 and arcuate microfluidic channel 114. Accordingly, the flow velocities can be maintained at or near a constant value in the spiral microfluidic channel 112 and arcuate microfluidic channel 114, without implementing another device such as a fluid pump to boost the flow velocity in the arcuate microfluidic channel 114.

It will be appreciated that with substantially constant flow velocities, the flow Reynolds number Re along the spiral microfluidic channel 112 and arcuate microfluidic channel 114 is substantially constant. The Reynolds number Re is a dimensionless parameter affecting the separation of cells in the blood sample, as described below.

In one embodiment, the first waste outlet 106 has the same cross-section height of approximately 95 pm and a cross-section width of approximately 350 pm. Furthermore, each of the output sample outlet 108 and second waste outlet 110 has the same cross-section height of approximately 95 pm and a cross-section width of approximately 75 pm. It will be appreciated that various dimensions of the microfluidic device 100 may be modified based on how the microfluidic device 100 will be used for cell separation of a blood sample, such as based on the type of nucleated cells being targeted for separation.

In many embodiments, the microfluidic device 100 is fabricated or manufactured in a polydimethylsiloxane (PDMS) material using patterned SU-8 silicon wafers and standard soft lithography techniques, as will be readily understood by the skilled person. Other materials that may be used to fabricate or manufacture the microfluidic device 100 include, but are not limited to, glass, polycarbonate (PC), polystyrene (PS), poly(methyl methacrylate) (PMMA), and Cyclic Olefin Copolymer (COC).

Fluidic Properties of Microfluidic Device

As used herein, a microchannel is a microfluidic curvilinear channel such as the spiral microfluidic channel 112 arcuate microfluidic channel 114. When a fluid flows through a microchannel, particularly one with a spiral shape or profile such as the spiral microfluidic channel 112, the laminar Poiseuille flow is subjected to centrifugal forces. The centrifugal forces disturb the parabolic fluid velocity profile of the laminar flow and the position of maximum fluid velocity shifts from the cross-section centre of the microchannel towards the outer side of the microchannel, causing a sharp velocity gradient to develop between the maximum fluid velocity position and the outer side. The sharp velocity gradient increases fluid pressure and the localized fluid velocity near the outer side is not sufficient to balance this pressure differential. This imbalance is known as Dean instability and leads to recirculation of fluid in the form of two counter rotating Dean vortices in the upper and lower halves of the microchannel [Dean, W. R., Philos Mag 1928, 5 (30), 673-695 ]. Each Dean vortex loops between the cross-section centre and outer side of the microchannel in order to balance the pressure differential. The Dean vortices or secondary flows are defined by a dimensionless parameter, known as Dean number De, representing the Dean forces due to the secondary flows in the microchannel.

The Dean number De is given by the Dean Number Equation:

where Re is the flow Reynolds number, R is the radius of curvature of the microchannel, and D _h is the microchannel hydraulic diameter. In some studies, D _h is the smallest dimension of the microchannel which is usually the cross-section height of the microchannel.

Fluid particles, e.g. cells in a blood sample, flowing in the microchannel experience Dean drag forces due to the presence of lateral Dean vortices or secondary flows entraining the fluid particles along the flow direction of the Dean vortices. Thus, the fluid particles move repeatedly between the inner and outer sides of the microchannel as the fluid flows along the microchannel. The velocity with which the fluid particles migrate laterally when flowing in the microchannel is dependent on the Dean number De. The lateral distance traversed by a fluid particle can be defined in terms of “Dean cycle”. For example, a fluid particle which is initially positioned near the inner side of the microchannel and migrates to the outer side of the microchannel at a distance downstream is considered to have completed half a Dean cycle. The fluid particle completes one Dean cycle if it returns back to the original position near the microchannel inner side further downstream. Thus, depending on the length of the microchannel, the fluid particles can undergo one or more Dean cycles or a fraction of a Dean cycle.

In addition to the Dean drag forces, fluid particles may also experience inertial lift forces, which may be shear-induced due to the parabolic fluid velocity profile and/or wall-induced due to interactions between the fluid and the sides or walls of the microchannel. The fluid particles flowing in the microchannel is thus subjected to a combination of inertial lift forces and Dean drag forces. Larger fluid particles are more significantly influenced by the inertial lift forces while smaller fluid particles are more significantly influenced by the Dean drag forces. As the ratio of the inertial lift forces and Dean drag forces varies for varying particle sizes, the fluid particles can be equilibrated at distinct positions along the microchannel cross-section based on their sizes. Particularly, under the influence of the Dean drag forces which are dominant over inertial lifting forces for smaller fluid particles, the smaller fluid particles initiate migration along the Dean vortices and equilibrate closer to the outer side of the microchannel. For larger fluid particles, the inertial lift forces experienced by the larger fluid particles are dominant over the Dean drag forces, preventing them from migrating laterally and causing them to equilibrate closer to the inner side of the microchannel. This results in the formation of two distinct streams which are then collected in two separate outlets.

Studies indicate that for fluid particles to focus and equilibrate at distinct positions along the microchannel cross-section, the ratio of the particle size ap and smallest microchannel dimension Dh should be at least 0.07 [Bhagat, A. A. S., et al. , Physics of Fluids 2008, 20, 101702] The larger fluid particles results in a ratio that is more than 0.07 such that the inertial lift forces experienced by the larger fluid particles are significantly large to result in particle equilibration. The ratio may also be referred to as the inertial lift ratio and stated as:

dp

> 0.07

Dh

In many embodiments with reference to Figure 2A, in the method 200, the blood sample and a sheath fluid is perfused into the microfluidic device 100. The blood sample and sheath fluid may be introduced / perfused into the inlets using known device such as syringes and/or fluid pumps. Particularly, the blood sample and sheath fluid may be introduced into the outer inlet 104 and inner inlet 102 respectively, and at an aggregated flow rate of 1000 to 2000 mI/min. In some embodiments, the flow rate of the sheath fluid is ten times the flow rate of the blood sample. For example, the blood sample may be introduced into the outer inlet 104 at a flow rate ranging from 100 to 200 mI/min, and the sheath fluid may be introduced into the inner inlet 102 at a higher flow rate ranging from 900 to 1800 mI/min. In one embodiment, the flow rate of the blood sample is 160 mI/min and the flow rate of the sheath fluid is 1600 mI/min. The sheath fluid is introduced to pinch the blood sample at the outer inlet 104 and to confine the blood sample stream near the outer side of the spiral microfluidic channel 112. The blood sample may be diluted prior to perfusion into the microfluidic device 100. For example, the blood sample may be diluted to contain approximately 10 million RBCs per ml, wherein the RBCs are suspended in 1x PBS and 0.5% BSA. Alternatively, the blood sample may be diluted by mixing together with the sheath fluid during perfusion into the microfluidic device 100.

In another embodiment, the blood sample is introduced into the inner inlet 102 and the sheath fluid is introduced into the outer inlet 104. The sheath fluid thus pinches the blood sample at the inner inlet 102 and confines the blood sample stream near the inner side of the spiral microfluidic channel 112. By confining the blood sample stream near either side of the spiral microfluidic channel 112, cells in the blood sample may migrate laterally from approximately the same location.

With reference to Figure 2A and Figure 4A, as the blood sample flows through the spiral microfluidic channel 112, smaller RBCs experience Dean drag forces F _D due to the Dean vortices and recirculate laterally towards the outer side of the spiral microfluidic channel 112 as the blood sample flows downstream along the spiral microfluidic channel 112. In one embodiment, the blood sample is introduced into the inner inlet 102. Cells in the blood sample recirculate laterally from the inner side towards the outer side, completing an odd number of half Dean cycles. In another embodiment, the blood sample is introduced into the outer inlet 104. Cells in the blood sample recirculate laterally from the outer side towards the inner side and back to the outer side again, completing an integer number of Dean cycles.

The smaller RBCs are communicated along the outer side of the spiral microfluidic channel 112 to the first waste outlet 106 and collected as waste. Larger nucleated cells experience stronger inertial lift forces F _L and equilibrate closer to the inner side of the spiral microfluidic channel 112 and are communicated to the arcuate microfluidic channel 114. Accordingly, the spiral microfluidic channel 112 performs a first bifurcation of the blood sample. Specifically, the blood sample is bifurcated at the first bifurcation junction 116 into an intermediary sample (containing larger nucleated cells) flowing to the arcuate microfluidic channel 114 and waste (containing smaller RBCs) flowing to the first waste outlet 106. With reference to Figure 2B and Figure 4B, as the intermediary sample flows through the arcuate microfluidic channel 114, there may be some residual RBCs in the intermediary sample. The residual RBCs equilibrate further away from the inner side and closer to the outer side due to stronger Dean drag forces F _D and are communicated along the outer side of the arcuate microfluidic channel 114 to the second waste outlet 110 and collected as waste. The larger nucleated cells experience stronger inertial lift forces F _L and equilibrate closer to the inner side of the arcuate microfluidic channel 114 and are communicated to the output sample outlet 108 and collected as an output sample. Accordingly, the arcuate microfluidic channel 114 performs a second bifurcation of the blood sample. Specifically, the intermediary sample is bifurcated at the second bifurcation junction 118 into the output sample (containing larger nucleated cells) flowing to the output sample outlet 108 and waste (containing residual RBCs) flowing to the second waste outlet 110.

As described above, the inertial lift forces F _L and Dean drag forces F _D vary for different particle sizes. Particularly, both forces F _L and F _D scale non-linearly with particle size, and the superposition of F _L and F _D determines the equilibrium position within the microchannel cross-section. Thus, by calibrating the microchannel dimensions and fluid velocity according to the particle size of the targeted nucleated cells, the F _L and F _D experienced by the targeted nucleated cells can be estimated. The targeted nucleated cells can be equilibrated at distinct positions along the microchannel cross-section based on the superposition of F _L and F _D , thereby facilitating separation and isolation of the targeted nucleated cells.

Microchannel Fleight Variation

In many embodiments, the cross-sections of the spiral microfluidic channel 112 and arcuate microfluidic channel 114 have a microchannel height ranging from 85 to 115 pm. In one embodiment, the microchannel height is 95 pm. As stated above, particle equilibration depends on particle size and the shortest microchannel dimension (microchannel height) in the relation according to the inertial lift ratio above. At a microchannel height of 95 pm, the larger nucleated cells, such as leukocytes and erythroblasts which are around 10 to 12 pm, equilibrate near the inner side of the microchannel. Smaller RBCs, which are around 6 to 8 pm in size, equilibrate near the outer side of the microchannel.

Different microchannel heights for the spiral microfluidic channel 112 were experimented on to characterize the depletion of RBCs as a result of the first bifurcation. Particularly, three microchannel heights were tested - approximately 85 to 90 pm, approximately 95 to 100 pm, and approximately 110 to 115 pm. Blood samples were perfused into the microfluidic device 100 at a flow rate of 160 mI/min to characterize the streamflow of the RBCs along the spiral microfluidic channel 112.

High-speed imaging at the first bifurcation junction 116 showed that at a flow rate of 160 mI/min, RBCs tend to equilibrate further away from the inner side of the spiral microfluidic channel 112 with increasing microchannel heights. With reference to the composite high-speed photograph images in Figure 5A, at the microchannel height of 85 to 90 pm, there is a significant amount of RBCs entering the arcuate microfluidic channel 114 which indicates the presence of inertial lifting forces on the RBCs. At greater microchannel heights of 95 to 100 pm and 110 to 115 pm, there are smaller amounts of RBCs entering the arcuate microfluidic channel 114 as the RBCs are subjected to more dominant Dean drag forces in the direction towards the outer side of the spiral microfluidic channel 112. Accordingly, a microchannel height of at least 95 pm is preferred for effective and substantial depletion of RBCs into the first waste outlet 106.

Figure 5B illustrates an intensity linescan chart of the RBCs equilibration positions at the first bifurcation junction 116 and across the cross-section width of the spiral microfluidic channel 112 at a flow rate of 160 mI/min. The origin is defined as the inner side of the spiral microfluidic channel 112 and the shaded region corresponds to the bifurcation into the arcuate microfluidic channel 114. It can be seen that there is substantial depletion of RBCs and the amount of RBCs entering the arcuate microfluidic channel 114 is minimal, thereby substantially enriching the nucleated cells in the blood sample. Haematocrit Variation

As interactions between cells in the blood sample may affect equilibration of the RBCs and influence the effectiveness of RBC depletion, the RBC concentration in the blood sample was varied to determine an optimal sample haematocrit at a flow rate of 160 mI/min for the spiral microfluidic channel 112. Particularly, four sample haematocrits were tested - approximately 5 million, 10 million, 50 million, and 100 million RBCs per ml of the blood sample.

With reference to Figure 6A illustrating the composite high-speed photograph images taken at the first bifurcation junction 116, at a flow rate of 160 mI/min, the focusing band of RBCs tended to broaden across the spiral microfluidic channel 112 with increasing sample haematocrits. This resulted in a significant amount of RBCs entering the arcuate microfluidic channel 114 after the first bifurcation at the first bifurcation junction 116.

Figure 6B illustrates an intensity linescan chart of the RBCs equilibration positions at the first bifurcation junction 116 and across the cross-section width of the spiral microfluidic channel 112 at a flow rate of 160 mI/min. The origin is defined as the inner side of the spiral microfluidic channel 112 and the shaded region corresponds to the bifurcation into the arcuate microfluidic channel 114. Based on the intensity linescan chart, the optimal sample haematocrit is approximately 10 million RBCs per ml of the blood sample. At this RBC concentration, there was minimal RBC contamination entering the arcuate microfluidic channel 114 for the second bifurcation at the second bifurcation junction 118.

The effectiveness of RBCs depletion after each of the first bifurcation and second bifurcation may be similarly assessed based on sample haematocrit variations at the same flow rate of 160 mI/min. Particularly, four sample haematocrits were tested - approximately 5 million, 10 million, 50 million, and 100 million RBCs per ml of the blood sample. Fligh-speed photographic imaging was performed at the second bifurcation junction 118 to generate composite high-speed photograph images as shown in Figure 7A. To quantify the depletion of RBCs, the wastes in the first waste collector 122 (01 ) and second waste collector 130 (03), as well as the output sample in the output sample collector 126 (02), were collected and analyzed. With reference to Figure 7B, at low sample haematocrits of 5 to 10 million RBCs per ml, there was substantial depletion of RBCs after the first bifurcation at the first bifurcation junction 116 with a range of 99.5% to 99.8% depletion as waste into the first waste collector 122 (01 ).

At higher sample haematocrits of 50 to 100 million RBCs per ml, the depletion of RBCs after the first bifurcation decreased to a range of 95.3% to 98.1 % depletion. This may be expected due to broadening of the band of RBCs mentioned above. Residual RBCs, which entered the arcuate microfluidic channel 114 after the first bifurcation, were further depleted by a range of 25.4% to 25.9% as the smaller RBCs migrated towards the outer side of the arcuate microfluidic channel 114. The smaller RBCs are subsequently bifurcated as waste into the second waste collector 130 (03) after the second bifurcation at the second bifurcation junction 118.

Thus, there was an overall improvement in the depletion of RBCs with the inclusion of the second bifurcation junction 118 for second bifurcation of the blood sample, in addition to the first bifurcation at the first bifurcation junction 116. The output sample collected from the output sample collector 126 (02) contained significantly less RBCs relative to the initial blood sample, indicating effective depletion of RBCs with the microfluidic device 100.

Flow Rate Variation

In many embodiments, the blood sample is introduced into the inner inlet 102 or outer inlet 104 at a flow rate ranging from 100 to 180 mI/min. Corresponding flow rate of the sheath fluid ranges from 1000 to 1800 mI/min. In one embodiment, the sample flow rate is 160 mI/min and the sheath fluid flow rate is 1600 mI/min.

Different sample flow rates were tested to characterize the depletion of RBCs after the first bifurcation at the first bifurcation junction 116. Particularly, four sample flow rates were tested - approximately 100 pl/min, approximately 130 pl/min, approximately 140 pl/min, and approximately 160 pl/min. Corresponding sheath fluid flow rates were set accordingly within the range of 1000 to 1600 pl/min.

With reference to the composite high-speed photograph images in Figure 8A, high- speed photographic imaging at the first bifurcation junction 116 showed that at the sample flow rate of 100 pl/min, the RBC band was close to the inner side of the spiral microfluidic channel 112. As the sample flow rate increased, the RBCs migrated laterally and the RBC band broadened towards the outer side of the spiral microfluidic channel 112. This was due to the Dean drag forces F _D becoming more dominant on the smaller RBCs at higher sample flow rates. Notably, the smaller RBCs have a lower inertial lift ratio and negligible inertial lifting forces F _L.

Figure 8B illustrates an intensity linescan chart of the RBCs equilibration positions at the first bifurcation junction 116. The origin is defined as the inner side of the spiral microfluidic channel 112 and the shaded region corresponds to the bifurcation into the arcuate microfluidic channel 114. It can be seen that, as a result of F _D being dominant over F _L especially at higher sample flow rates, there was more substantial depletion of RBCs as waste into the first waste collector 122 (01 ) and the amount of RBCs entering the arcuate microfluidic channel 114 was minimal, thereby substantially enriching the nucleated cells in the blood sample.

Microbeads Simulation

In some embodiments, polystyrene microbeads 132 are mixed into a fluid sample to simulate nucleated cells in whole blood. The microbeads 132 may have a size of approximately 10 pm to represent nucleated cells such as leukocytes and erythroblasts which have sizes approximately from 10 to 12 pm. The microbeads 132 may be fluorescent, such as by fluorescein isothiocyanate (FITC) conjugation, to better illuminate their streamflow through the first and second bifurcations. Figure 9A is a fluorescent photograph image illustrating the streamflow of the microbeads 132 at the first bifurcation junction 116, and Figure 9B is a fluorescent photograph image illustrating the streamflow of the microbeads 132 at the second bifurcation junction 1 18.

A fluid mixture containing the microbeads 132 was perfused into the microfluidic device 100 at a flow rate of 160 mI/min. It was observed that the microbeads 132 equilibrated as a tight band near the inner side of the spiral microfluidic channel 1 12 and bifurcated into the arcuate microfluidic channel 1 14 at the first bifurcation junction 1 16, as shown in Figure 9A. The equilibration was due to the inertial lifting forces F _L being dominant over the Dean drag forces F _D on the microbeads 132. At the second bifurcation junction 1 18 as shown in Figure 9B, it was observed that the microbeads 132 remained equilibrated near the inner side of the arcuate microfluidic channel 1 14 and were discharged through the output sample outlet 108 and collected at the output sample collector 126 (02).

Figure 9C illustrates a fluorescence intensity linescan chart of the microbeads 132 at the start and end of the arcuate microfluidic channel 1 14. The origin is defined as the inner side of the arcuate microfluidic channel 1 14. It can be seen that there was a shift in the distribution of the microbeads 132 towards the inner side of the arcuate microfluidic channel 1 14 as the microbeads 132 flow downstream along the arcuate microfluidic channel 1 14. This indicated there was sufficient inertial lifting forces F _L experienced by the microbeads 132 for them to migrate laterally towards the inner side of the arcuate microfluidic channel 1 14 the second bifurcation at the second bifurcation junction 1 18.

MicroChannel Radius Variation

As described above, the microbeads 132 experience Dean drag forces F _D due to Dean vortices. The Dean vortices are defined by the Dean number De as stated in the Dean Number Equation above. In many embodiments, the Dean number De for the spiral microfluidic channel 1 12 ranges from 5 to 10. In one embodiment, the Dean number De for the spiral microfluidic channel 1 12 is approximately 6.8. In many embodiments, the arcuate microfluidic channel 114 has a radius of curvature ranging from 0.1 to 0.9 cm (or more preferably from 0.3 to 0.7 cm). Different curvature radii for the arcuate microfluidic channel 114 were tested to characterize the equilibrating effects on the microbeads 132 as a result of inertial lifting forces F _L. Particularly, three curvature radii were tested - 0.3 cm, 0.5 cm, and 0.7 cm. The three curvature radii correspond to Dean numbers De of approximately 9.0, 7.0, and 5.9, respectively. The microbeads fluid mixture was perfused into the microfluidic device 100 at different flow rates - 140 pl/min, 150 mI/min, and 160 mI/min. Each curvature radius corresponds to each flow rate, resulting in nine combinations. Figure 10 shows fluorescent photograph images for each combination, each fluorescent photograph image illustrating the streamflow of the microbeads 132 at the second bifurcation junction 118.

Similar to previous observations, for all combinations, the microbeads 132 initially equilibrated as a tight band near the inner side of the spiral microfluidic channel 112 and bifurcated into the arcuate microfluidic channel 114 at the first bifurcation junction 116. Thus, the microbeads 132 enter the arcuate microfluidic channel 114 in closer to the inner side thereof.

At the curvature radius of 0.3 cm, the microbeads 132 experience the most significant F _D due to the highest De, resulting in rapid lateral migration of the microbeads 132 from the inner side towards the outer side of the arcuate microfluidic channel 114. Particularly, at higher flow rates of 150 and 160 mI/min, the microbeads 132 were undesirably bifurcated into the second waste outlet 110 (which normally collects waste) instead of the output sample outlet 108.

At the curvature radius of 0.7 cm, the microbeads 132 experience the least significant F _D due to the lowest De, resulting in slow or negligible lateral migration of the microbeads 132 from the inner side towards the outer side of the arcuate microfluidic channel 114. However, as the length of the arcuate microfluidic channel 114 at curvature radius 0.7 cm is approximately 2.3 times the length at curvature radius 0.3 cm, the microbeads 132 required more time to flow through the arcuate microfluidic channel 114. This additional residence time in the arcuate microfluidic channel 114 allowed the microbeads 132 to migrate laterally towards the outer side, resulting in their eventual undesired bifurcation into the second waste outlet 110. Thus, the curvature radii of 0.3 cm and 0.7 cm are not optimal for cell separation as the microbeads 132 are not successfully separated and isolated from the initial fluid mixture.

At the curvature radius of 0.5 cm, the F _D , De, and length of the arcuate microfluidic channel 114 are intermediate relative to those for the other curvature radii. The microbeads 132 remained equilibrated at the inner side of the arcuate microfluidic channel 114 and subsequently bifurcated into the output sample outlet 108 at the second bifurcation junction 118 as desired, regardless of the flow rate. The curvature radius of 0.5 cm and a flow rate of 160 mI/min were observably optimal conditions for cell separation as the microbeads 132 were successfully separated and isolated from the initial perfused mixture.

Method Application - White Blood Cells

WBCs or leukocytes are distinguished from RBCs as they have nuclei. Nucleated WBCs are around 12 to 15 pm in size while RBCs are smaller at around 6 to 8 pm in size. In some embodiments, the method 200 is used for enriching WBCs isolated from a blood sample comprising whole blood. Specifically, the method 200 is performed using microfluidic devices 100 to separate and isolate WBCs from whole blood.

Samples of diluted whole blood containing approximately 10 million RBCs per ml were perfused into the microfluidic devices 100 at various flow rates (140, 150, and 160 mI/min). With reference to the composite high-speed photograph images in Figure 11 A, high-speed photographic imaging at the first bifurcation junction 116 showed that the majority of the RBCs were bifurcated as waste into the first waste outlet 106, flowing towards the first waste collector 122 (01 ). The larger WBCs equilibrated near the inner side of the spiral microfluidic channel 112 and bifurcated into the arcuate microfluidic channel 114. With reference to the composite high-speed photograph images in Figure 11 B, high- speed photographic imaging at the second bifurcation junction 118 showed that the WBCs remain equilibrated near the inner side of the arcuate microfluidic channel 114, and were bifurcated as an output sample into the output sample outlet 108 and collected at the output sample collector 126 (02). This is similar to the observations in the microbeads simulation. Residual RBCs, which may be present in the streamflow from the first bifurcation junction 116, were bifurcated at the second bifurcation junction 118 as waste into the second waste outlet 110, flowing towards the second waste collector 130 (03).

Figure 11 C illustrates a brightfield intensity linescan chart of the WBCs at the start and end of the arcuate microfluidic channel 114. The origin is defined as the inner side of the arcuate microfluidic channel 114. Similar to the observations in the microbeads simulation, there was a shift in the distribution of the WBCs towards the inner side of the arcuate microfluidic channel 114 as the WBCs flowed downstream along the arcuate microfluidic channel 114.

Figure 11 D illustrates a flow cytometry analysis (e.g. fluorescence-activated cell sorting (FACS)) performed at the various flow rates for various types of WBCs, specifically neutrophils (CD66b+), monocytes (CD14+), and lymphocytes (CD3+CD19+). The flow cytometry analysis indicates that significant amounts of all types of WBCs were recovered in the output samples. Although the flow cytometry analysis also indicates that some WBCs were undesirably discharged into the first waste collector 122 (01 ) and second waste collector 130 (03), the majority of the WBCs were successfully separated and isolated from the initial blood sample.

Method Application - Fetal Nucleated Red Blood Cells

There are several types of fetal nucleated cells circulating in maternal blood, or more specifically maternal peripheral blood circulating within the body. The types of fetal nucleated cells include trophoblasts, erythroblasts, haematopoietic progenitor cells, and lymphocytes. Erythroblasts are nucleated RBCs and are ideal for non-invasive prenatal diagnosis (NIPD) due to their short lifespan in maternal blood, distinct morphology from WBCs, and are detectable in early pregnancy. Erythroblasts are around 10 to 18 pm in size and each contains a nucleus around 4 to 6 pm in size enclosed by a highly deformable membrane. Erythroblasts can be enriched based on CD71 (transferrin receptor), glycophorin A (GPA), and CD36 (thrombospondin) surface markers [Bianchi, D. W., et at., Prenatal Diagnosis 1993, 13 (4), 293-300 ]. However, they are rare as there are only around 1 to 10 erythroblasts per ml of maternal blood [Takabayashi, H., et ai, Prenatal Diagnosis 1995, 15 (1), 74-77]. The enriched population of erythroblasts may contain up to 50% of maternal origin instead of fetal origin [Troeger, C., et ai, Prenatal Diagnosis 1999, 19 (6), 521-526 ].

In some embodiments, the method 200 is used for enriching fetal nucleated cells isolated from a blood sample comprising maternal blood. Specifically, the method 200 is performed using microfluidic devices 100 to separate and isolate fetal nucleated erythroblasts or nucleated RBCs (fnRBCs) from CD45+ depleted whole blood. Enriched fnRBCs from samples of cord blood were pre-labelled with a carboxyfluorescein succinimidyl ester (CFSE) dye and spiked into CD45+ depleted whole blood (diluted to contain approximately 10 million RBCs per ml) to produce blood samples for use in microfluidic devices 100. The blood samples were perfused into the microfluidic devices 100 at various flow rates (140, 150, 160, and 170 pl/min) for microfluidic processing using the method 200. The output samples were then collected from the output sample collectors 126 (02).

A flow cytometry analysis (e.g. FACS) was performed to identify various cell types in the output samples based on surface markers. Specifically, the cell types include fnRBCs identified based on CD71 ⁺ /GPA ⁺ /CD457Hoechst ⁺ , mature RBCs identified based on CD717GPA ⁺ /CD457Hoechst , and immature RBCs (reticulocytes) identified based on CD71 ⁺ /GPA ⁺ /CD457Hoechsf.

Figure 12 illustrates the flow cytometry analysis performed at the various flow rates for various cell types. The flow cytometry analysis indicates the separation and isolation effectiveness of the various cell types at the various flow rates. Based on the flow cytometry analysis, it was observed that, at lower flow rates of 140 and 150 pl/min, 80% to 85% of fnRBCs were recovered in the output samples. However, there were significant contamination amounts (0.5% to 7%) of mature RBCs and reticulocytes, which are undesirable for subsequent analysis. At the flow rate of 160 mI/min, although the contamination amounts were lower (0.1 % to 0.9%), the amount of fnRBCs recovered also decreased to around 60%. At the flow rate of 170 mI/min, the amount of fnRBCs recovered further decreased to around 50% with negligible improvement in the contamination amounts (0.1 % to 0.3%).

The flow cytometry analysis thus show that at the amount of fnRBCs recovered in the output samples and the contamination amounts of mature RBCs and reticulocytes were adequately balanced at the optimal flow rate of 160 mI/min.

Method Application - Putative Fetal Erythroblasts

In some embodiments, the method 200 is performed using microfluidic devices 100 to separate and isolate putative fetal erythroblasts (pfEBs) from maternal blood. Maternal peripheral blood was first obtained and diluted in phosphate-buffered saline (PBS) at a 1 :1 ratio, followed by density gradient centrifugation at 1800 rpm for 15 minutes. The buffy coat at the interface was collected and washed twice in the PBS. Leukocytes were depleted with anti-CD45 magnetic beads. CD45 cells were re- suspended in the PBS (approximately 10 million cells per ml) to produce blood samples for use in microfluidic devices 100. The blood samples were perfused into the microfluidic devices 100 for microfluidic processing using the method 200. The output samples and waste were then collected from the output sample collectors 126 (02) and second waste collectors 130 (03).

The average number of cells collected in the output sample collectors 126 (02) was around 94,000, indicating that the depletion of RBCs was highly effective and substantial - approximately 99.8% of RBCs were depleted. This depletion effectiveness was similar to that in the application to isolate fnRBCs. With reference to Figure 13, a flow cytometry analysis (e.g. FACS) was performed on the output samples (from output sample collectors 126 (02)) and waste (from second waste collectors 130 (03)) to identify various cell types, namely pfEBs, residual WBCs / leukocytes, reticulocytes, and mature RBCs. Based on the flow cytometry analysis as shown in Figure 13 (A and B), it was observed that the amounts of pfEBs and residual WBCs / leukocytes were significantly higher in the output sample collectors 126 (02) than in the second waste collectors 130 (03). In addition, there was considerable removal of contaminant cells (mature RBCs and reticulocytes) as waste into the second waste collectors 130 (03), as shown in Figure 13 (C and D). These results highlight the effectiveness of the microfluidic device 100 to separate, isolate, and enrich rare circulating pfEBs from maternal blood with minimal cell loss and contaminants.

Other Applications

As described in various embodiments above, the microfluidic device 100 uses two microchannels - the spiral microfluidic channel 112 and arcuate microfluidic channel 114 - in a continuous arrangement for performing successive first and bifurcations of samples of whole blood. Through the two bifurcations, there is size-based separation of nucleated cells from the blood samples based on a combination of inertial lifting forces and Dean drag forces. This cell separation, isolation, or sorting technique or Dean Flow Fractionation (DFF) is achieved passively with minimal manual handling of the blood samples. DFF is based on the principles of Dean vortices and fluid particle lateral migration as a result of the combination of inertial lifting forces and Dean drag forces. The lateral Dean drag forces offer superior separation resolution [Kuntaegowdanahalli, S. S., et at., Lab on a chip 2009, 9 (20), 2973-2980; Bhagat, A. A. S., et al., Lab on a chip 2008, 8 (11), 1906-1914] as both inertial lifting forces and Dean drag forces scale non-linearly with particle size, and their superposition determines the equilibrium position within the microchannel cross-section.

The microfluidic device 100 and method 200 are used for cell separation of nucleated cells in a blood sample. For example, WBCs may be separated from whole blood for subsequent immunology studies, or fetal nucleated cells may be separated from maternal blood for subsequent NIPD. Flaving the first bifurcation junction 116 and second bifurcation junction 118 in a continuous arrangement in the microfluidic device 100 advantageously enables substantial removal (e.g. at least 99.5%) of smaller RBCs into waste at high volume throughput or flow rate of 160 mI/min. The substantial depletion of RBCs effectively separates and isolates the nucleated cells from the RBCs, thereby substantially enriching the nucleated cells in the output sample for improved analysis and testing thereof subsequently.

The RBC depletion effectiveness of the microfluidic device 100 was demonstrated by various applications of the method 200 described above. For example, one application achieved successful isolation of rare pfEBs from samples of leukocyte- depleted maternal blood. Thus, the microfluidic device 100 is effective for separation of circulating fetal nucleated cells from maternal RBCs for prenatal diagnosis. Fetal nucleated cells (erythroblasts) are highly enriched with substantial depletion of RBCs (e.g. at least 99.5%) after the first and second bifurcations and at high volume throughput or flow rate of 160 mI/min. The substantial enrichment of fetal nucleated cells obviates the need for RBC lysis, which could otherwise damage and/or alter the morphology of the fetal nucleated cells.

Rapid isolation of rare erythroblasts from maternal blood at high flow rates is important for NIPD. Erythroblasts of fetal origin contain valuable information of the fetal genome that can be used for the diagnosis of fetal aneuploidies and/or monogenic mutations. Furthermore, the possibility to isolate fetal nucleated cells enables the possibility to develop routine clinical prenatal tests that are available to / accessible by the majority, if not all, of the pregnant female population.

In addition, there may be other possible downstream and commercial applications of enriched fetal erythroblasts or fnRBCs. For example, fnRBCs may be enriched for subsequent single-cell isolation using the DEPArray™, a semiconductor-based system for precise isolation of single cells using dielectrophoresis. Briefly, a neutral particle (e.g. cell) can be trapped in stable levitation which allows the system to perform fluorescence-based analysis to discriminate a target cell (fetal cell such as fnRBC) from tens of thousands of contaminating cells (maternal cells such as maternal RBCs). The target cells are then selected and automatically recovered as single cells for downstream genetic analysis, avoiding the possibility of having contaminant maternal cells that can compromise the genetic analysis. Various embodiments of the present disclosure describe a microfluidic device 100 with a spiral microfluidic channel 112 for performing a first bifurcation at a first bifurcation junction 116, as well as an arcuate microfluidic channel 112 for performing a second bifurcation at a second bifurcation junction 118. It will be readily appreciated that there may additional sets of microfluidic channels, e.g. microfluidic curvilinear channels, to perform additional bifurcations at additional bifurcation junctions. In the foregoing detailed description, embodiments of the present disclosure in relation to a microfluidic device and a method for cell separation of a blood sample are described with reference to the provided figures. The description of the various embodiments herein is not intended to call out or be limited only to specific or particular representations of the present disclosure, but merely to illustrate non- limiting examples of the present disclosure. The present disclosure serves to address at least one of the mentioned problems and issues associated with the prior art. Although only some embodiments of the present disclosure are disclosed herein, it will be apparent to a person having ordinary skill in the art in view of this disclosure that a variety of changes and/or modifications can be made to the disclosed embodiments without departing from the scope of the present disclosure. Therefore, the scope of the disclosure as well as the scope of the following claims is not limited to embodiments described herein.