METHOD AND SYSTEM FOR PACKED BED CELL BUOYANCY SEPARATION

Abstract

A method of performing a separation of a sample of a disperse fluid comprises the steps of: i. providing a sample of a disperse fluid comprising particles dispersed in a fluid, wherein the particles comprises at least a first type of particle and at least a second type of particles, wherein the absolute value of the acoustic contrast of the first type of particle, relative to the fluid, is lower than the absolute value of the acoustic contrast of the second type of particle relative to the fluid, and wherein the first and second type of particle either both have a positive acoustic contrast, or alternatively a negative acoustic contrast, relative to the fluid, ii. positioning the sample in a microfluidic cavity, iii. subjecting the sample, in the microfluidic cavity, to an acoustic standing wave configured for causing the first and second type of particle to congregate in at least one first region of the cavity, thereby causing the fluid to occupy at least one second region of the cavity, and thereby defining at least one interface between the first region and the second region, and iv. collecting at least a portion of the first region adjacent and along the at least one interface to obtain the first type of particles. A system is also disclosed.

Claims

1-10. (canceled)

11. A method of performing a separation of a sample of a disperse fluid, comprising the steps of: i. providing a sample of a disperse fluid comprising particles dispersed in a fluid, wherein the particles comprise at least a first type of particle and at least a second type of particle, wherein the first type of particle has a first acoustic contrast relative to the fluid with a first absolute value, and the second type of particle has a second acoustic contrast relative to the fluid with a second absolute value, wherein the first absolute value is lower than the second absolute value, and wherein the first type of particle and the second type of particle either both have a positive acoustic contrast or both have a negative acoustic contrast; ii. positioning the sample in a microfluidic cavity; iii. subjecting the sample in the microfluidic cavity to an acoustic standing wave configured for causing the first type of particle and the second type of particle to congregate in at least one first region of the cavity, thereby causing the fluid to occupy at least one second region of the cavity, and thereby defining an interface between each first region and each second region; and iv. collecting at least a portion of each first region adjacent and along each interface to obtain the first type of particle.

12. The method according to claim 11, wherein the first acoustic contrast and the second acoustic contrast are positive relative to the fluid, and wherein the acoustic standing wave is configured for causing the first type of particles and the second type of particles to congregate in one central first region of the microfluidic cavity.

13. The method according to claim 11, wherein the first acoustic contrast and the second acoustic contrast are negative relative to the fluid, and wherein the acoustic standing wave is configured for causing the first type of particles and the second type of particles to congregate in two peripherally located first regions of the microfluidic cavity.

14. The method according to claim 11, further comprising the step of v. generating a flow of the sample through the microfluidic cavity.

15. The method according to claim 14, wherein the cavity is elongated and fluidly connected at one end to an inlet and at another opposite end to at least one outlet.

16. The method according to claim 11, wherein the cavity is formed in a substrate.

17. The method according to claim 16, wherein ultrasound energy, for causing the acoustic standing wave, is transferred to the substrate from at least one ultrasound transducer connected to the substrate.

18. The method according to claim 11, wherein the sample is a blood sample, whereby the particles comprise cells and the fluid comprises blood plasma.

19. A microfluidic system for performing a separation of a sample of a disperse fluid, the disperse fluid comprising particles dispersed in a fluid, and the particles comprising at least a first type of particle and at least a second type of particle, wherein the first type of particle has a first acoustic contrast relative to the fluid with a first absolute value, and the second type of particle has a second acoustic contrast relative to the fluid with a second absolute value, wherein the first absolute value is lower than the second absolute value, and wherein the first type of particle and the second type of particle either both have a positive acoustic contrast or both have a negative acoustic contrast, wherein the system comprises: a substrate with a microfluidic cavity formed in the substrate, the microfluidic cavity having an inlet configured for allowing the sample into the microfluidic cavity; an ultrasound transducer connected to the substrate and configured for generating an acoustic standing wave in the microfluidic cavity; a drive circuit connected to the ultrasound transducer and configured to drive the ultrasound transducer with a frequency that causes the particles to congregate in at least one first region of the cavity, thereby causing the fluid to occupy at least one second region of the cavity and thereby defining at least one interface between the at least one first region and the at least one second region; and a collecting device arranged and configured to collect at least a portion of each first region adjacent and along the at least one interface to obtain the first type of particle.

20. The system according to claim 19, wherein the collecting device comprises: a branching point arranged and configured to separate different spatial parts of the contents of the cavity into different secondary cavities or channels; and flow regulating means arranged and configured for regulating the flow into each of the secondary cavities or channels.

21. The system according to claim 20, wherein the flow regulating means comprises at least one pump and at least one valve.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND DETAILED DESCRIPTION

[0097] A more complete understanding of the abovementioned and other features and advantages of the technology proposed herein will be apparent from the following detailed description of preferred embodiments in conjunction with the appended drawings, wherein:

[0098] FIG. 1A-B are bright field and corresponding fluorescence microscopy images of a sample of whole blood in stopped flow being subjected to an acoustic standing wave in a cavity. The red blood cells are shown packed into a first region flanked by second regions of blood plasma, and cancer cells of the DU 145 prostate cancer cell lines are revealed adjacent and along the interface of the first and second regions due to their capability of fluorescing,

[0099] FIGS. 1C and D are bright field and corresponding fluorescence microscopy images of a sample of whole blood being subjected to an acoustic standing wave in a cavity under the same conditions as in FIGS. A and B, but with the sample flowing through the cavity,

[0100] FIG. 1E is a flowsheet of the method according to the first aspect of the technology proposed herein,

[0101] FIG. 2 is a schematic image of the forces affecting packed red blood cells and white blood cells in the packed red blood cells,

[0102] FIG. 3A is a schematic view from above of a substrate and microfluidic cavity of a system according to the second aspect of the technology proposed herein, and

[0103] FIG. 3B is a schematic view from above of a substrate and microfluidic cavity of an alternative system according to the second aspect of the technology proposed herein.

[0104] In the figures and the description the same reference numeral is used to refer to the same feature. A ‘ added to a reference numeral indicates that the feature so referenced has a similar function, structure or significance as the feature carrying the reference numeral without the’, however not being identical with this feature.

[0105] FIG. 1A-B are bright field and corresponding fluorescence microscopy images of a sample of whole blood in stopped flow being subjected to an acoustic standing wave in a cavity. The red blood cells are shown packed into a first region flanked by second regions of blood plasma, and cancer cells of the DU 145 prostate cancer cell lines are revealed adjacent and along the interface of the first and second regions due to their capability of fluorescing. The red blood cells are shown packed, i.e. congregated, into a first region flanked by second regions of blood plasma. The whole blood comprises cultured prostate cancer cells DU145 which were fluorescently labeled and spiked in the whole blood from a healthy volunteer. The blood was drawn into a syringe and then infused through a silicon and glass micro channel with a rectangular cross-section (width 375 μm, height 150 μm). After the flow had come to rest, a sound field of a frequency of 2 MHz was activated by an electrical signal submitted to a piezoelectric transducer from a signal generator and amplifier. The final stage of cell congregation was recorded by bright field and fluorescence microscopy after a few seconds of actuation. FIG. 1B, which is an overlay of 10 consecutive fluorescence images, shows that the cancer cells are congregated adjacent and along the interface of the first and second. In other words the DU145 cells have congregated at the interfaces between the packed red blood cells and the plasma. The fluorescing cancer cells have a lower absolute value of the acoustic contrast relative to the blood plasma (in this case a lower density and or higher compressibility) than the red blood cells and are therefore, when present in the almost continuous phase or environment of red blood cells, affected by buoyancy forces causing the cancer cells to rise out of the red blood cells.

[0106] FIGS. 1C and D are bright field and corresponding fluorescence microscopy images of a sample of whole blood being subjected to an acoustic standing wave in a cavity under the same conditions as in FIGS. A and B, but with the sample flowing through the cavity. Here the flow rate was 20 μL/min flow rate. FIG. 1D, similarly to FIG. 1B, shows that the DU145 cancer cells are congregating at the cell plasma interface due to cell-cell interactions.

[0107] FIG. 1E is a flowsheet of the method according to the first aspect of the technology proposed herein.

[0108] FIG. 2 is a schematic image of the forces affecting the packed red blood cells and white blood cells in the packed red blood cells. In the blood the blood cells have a positive acoustic contrast relative to the blood plasma. As shown the acoustic standing wave has a central pressure node. The red blood cells 6, which have a positive acoustic contrast in relation to the blood plasma, are forced towards the center of the cavity, see dotted arrows. This forms an almost continuous phase or packed bed of red blood cells. In this environment the white blood cells 4, which are also forced by the acoustic field towards the center due to their positive acoustic contrast in relation to the plasma, instead, due to having a lower absolute value of acoustic contrast, in relation to the plasma, than the absolute value of acoustic contrast of the red blood cells relative to the plasma, are affected by even larger net forces (dashed arrows) which causes the white blood cells to rise through the red blood cells towards the interface between the packed red blood cells 6 and the surrounding blood plasma 8. The white blood cells are therefore positioned in the portion of the first region that is adjacent and along the interface between the packed red blood cells and the blood plasma. The white blood cells can then be obtained by collecting the portion of the first region. If the objective is to collect as many white blood cells as possible, then the portion of the first region is defined larger, i.e. a larger proportion, indicated with a in the figure, of the width of the first region is collected. This however increases the risk that red blood cells are also collected with the first portion, i.e. this increases the quantity of the separation but reduces the quality.

[0109] If on the other hand the requirement is that as few as possible red blood cells should be collected together with the white blood cells then the portion is made smaller, i.e. a smaller proportion, indicated with b in the figure, of the width of the first region may be collected.

[0110] FIG. 3A is a schematic view from above of a substrate and microfluidic cavity of a system according to the second aspect of the technology proposed herein. FIG. 3 shows a silicon substrate 10 which has formed in it a microfluidic channel 12, defining a cavity, having an inlet 14, a central outlet 16, and two side outlets 18 and 20. An ultrasound transducer 22 is attached to the underside of the substrate 10, and the channel is delimited vertically by a glass sheet 24 bonded to the top of the substrate 10. The ultrasound transducer 22 may also be attached to the side of the substrate 10. The ultrasound transducer 22 is driven by a drive circuit 23. A sample of whole blood 2 is admitted into the channel 12 through inlet 14 and caused to flow towards the outlets 16, 18, and 20. The sample comprises a first type of particle, i.e. white blood cells, one of which is designated the reference numeral 4, which are mixed with the red blood cells generally designated the reference numeral 6. An ultrasound transducer 22 is connected to the substrate 10 and activated with a frequency which provides an acoustic standing wave in the cavity 12. This causes the red blood cells 6 to congregate towards the central pressure node of the acoustic standing wave thus causing the red blood cells 6 to congregate in a first region, corresponding to the center portion 26, of the channel 12. At the same time the white blood cells 4, which are now dispersed in an almost continuous phase of red blood cells 6, start to rise, due to lower density and higher compressibility in relation to the red blood cells, towards the interface of the first region and the second regions 28a and 28b containing the plasma 8. The white blood cells 4 are thus concentrated towards a portion of the first regions lying adjacent and along the interface between the first and second regions 26 and 28a, 28b.

[0111] A collecting device, here implemented as the branching of the cavity into the central and side outlets 16, 18, 20, skims off the portion of the first region 26, for example the outermost 5 to 10% of the first region, and leads this portion into the side outlets 18 and 20. This causes the white blood cells 4, which were driven towards the interface of the first region 26 and the second regions 28a, 28b, to be diverted, together with the majority of the blood plasma 8 in the second regions 28a and 28b, into the side outlets 18 and 20 from which the white blood cells 4 can be obtained.

[0112] If desired further features, such as guide wall 30, may be introduced into the side outlets to reduce the amount of plasma that is collected together with the white blood cells.

[0113] Whereas some red blood cells 6 will be collected together with the white blood cells, the method is capable of collecting all of the white blood cells 4 while collecting less than 10% of the red blood cells 6. Further, the central outlet 16 provides for collecting the red blood cells 6.

[0114] The portion of the first region that is collected is can be set according to the flow and volume of the sample, or alternatively a fixed geometry can be used and the flow of sample adjusted until the total width of the first region gives a suitable portion or “skimming depth”.

[0115] This is inter alia shown in FIG. 3B which is a schematic view from above of a substrate and microfluidic cavity of an alternative system according to the second aspect of the technology proposed herein. Here the collecting device is represented by a branching of the cavity into the central and side outlets 16′, 18′, 20′, which, in contrast to central and side outlets 16, 18 and 20 of FIG. 3A do not physically interfere with the flow of the sample, rather the flow rates of the inlet 14 and in the central and side outlets 16′, 18′ and 20′ are set so as to cause the portion of the first region together with the second regions 28a, 28b containing the plasma to be led out through the side outlets 18′ and 20′ whereas the remainder of the first region 26 is led out through the central outlet 16′. Thus whole blood 2 is led from a container 32 via first pump 34 and tubing 36 to the inlet 14. The remainder of the first region 26 containing the red blood cells 6 is collected from the central outlet 16′ into container 38 using second pump 40 and tubing 42. The portion of the first region containing the white blood cells 4 together with the second regions 28a, 28b containing the plasma 8 are collected through side outlets 18′ and 20′ into container 44 using third pump 46 and tubing 48. The second pump 40 is set to a flow that is approximately the same as the content of blood cells (both red and white) in the whole blood 2. A microscope of camera 50 may be placed to image the branching of the channel 12 into the central and side outlets 16′, 18′ and 20′, and to thereby provide information on the separation for adjusting the second and third pumps 40 and 46. As an alternative the containers 32, 38 and 44 may be pressurized differently to drive the flow and set the flow rates instead of, or in addition to, using the pumps 34, 40 and 46.

Example—Separation of Whole Blood

[0116] Whole blood of 42% HTC was used as sample input in a device similar to that shown in FIG. 3B.

[0117] The flow rates through the outlets 16 (central outlet) and 18, 20 (side outlets) was varied as given in table 1 below.

TABLE-US-00001 TABLE 1 Flow rates Side outlet Sample Central outlet Side outlets flowrate no. [μl/min] [μl/min] split ratio [%] 1 30 20 40 2, 3 35 15 30 4, 5 37 13 26

[0118] The total flow into the cavity through the inlet 14 was 50 μl/min.

[0119] An ultrasound transducer was used to provide an acoustic standing wave with a force maximum (node) at the center of the channel as shown in FIG. 3B. This caused all cells in the whole blood to congregate in a first region (26) around the center axis of the cavity, while the plasma occupied two second regions (28a, 28b) along the respective walls of the cavity. Sample flowing out of the respective outlets (i.e. output fractions) was collected for further study.

[0120] The output fractions were stained for white blood cells (WBC) and platelets. Red blood cells (RBC) were counted without staining, after removal/inverse gating of WBCs and platelets.

[0121] Table 2 below shows the cell concentration in the side outlet fraction compared to the cell concentrations in the whole blood input sample. 67-76% of the mononuclear cells (MNC) are recovered through the side outlet, showing no difference between 30% or 26% flowrate split ratio. However, the amounts of RBCs in the side fraction decreases to 10-13% for the 26% flowrate split ratio (side outlet: 13 μl/min) compared to 22-23% for 30% split ratio (side outlet: 15 μl/min).

TABLE-US-00002 TABLE 2 cell concentrations [%] in side outlet fraction relative input whole blood Sample Granulocytes MNCs Lymphocytes Monocytes Platelets RBCs 1 43 106 107 116 107 41 2 21 74 80 46 69 22 3 20 77 76 81 49 23 4 11 67 71 54 84 10 5 17 76 79 66 83 13

[0122] As shown by table 2, the method according to the first aspect of the technology proposed herein allows to separate out a large proportion of a first type of particle (in this example MNCs) from another type of particle (in this case RBCs) by suitably collecting a portion of the first region along and adjacent the interface between the first region and second region. As further shown, the size of the portion, and thereby the proportion of particles that are separated out and retained, respectively, can be adjusted by adjusting the flow rate through each outlet.

[0123] Feasible Modifications

[0124] The technology proposed herein is not limited only to the embodiments described above and shown in the drawings, which primarily have an illustrative and exemplifying purpose. This patent application is intended to cover all adjustments and variants of the preferred embodiments described herein, thus the present invention is defined by the wording of the appended claims and the equivalents thereof. Thus, the equipment may be modified in all kinds of ways within the scope of the appended claims.

[0125] For instance, it shall be pointed out that structural aspects of embodiments of the method according to the first aspect of the technology proposed herein shall be considered to be applicable to embodiments of the system according to the second aspect of the technology proposed herein, and conversely, methodical aspects of embodiments of the system according to the second aspect of the technology proposed herein shall be considered to be applicable to embodiments of the method according to the first aspect of the technology proposed herein.

[0126] It shall also be pointed out that all information about/concerning terms such as above, under, upper, lower, etc., shall be interpreted/read having the equipment oriented according to the figures, having the drawings oriented such that the references can be properly read. Thus, such terms only indicates mutual relations in the shown embodiments, which relations may be changed if the inventive equipment is provided with another structure/design.

[0127] It shall also be pointed out that even thus it is not explicitly stated that features from a specific embodiment may be combined with features from another embodiment, the combination shall be considered obvious, if the combination is possible.

[0128] Throughout this specification and the claims which follows, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or steps or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.