Apparatus and method for separating plasma from whole blood

Abstract

The invention relates to a microfluidic device having at least one separation chamber with an inlet and an outlet. The inlet opens into the separation chamber at a smaller height than the outlet. In order to separate plasma (21) from whole blood (20), the whole blood (20) is introduced into the separation chamber of the microfluidic device, the blood cells are sedimented from the whole blood (20) and the thus separated plasma (21) is undercoated with a carrier agent (30) having a higher density than the plasma (21).

Description

Technical Field

[0001] This invention relates to a microfluidic device. Furthermore, this invention also relates to a method for separating plasma from whole blood using the microfluidic device. Background Technology

[0002] In many in vitro diagnostic tests, blood is used as the initial sample material. Blood is processed to, for example, measure specific DNA segments or specific proteins. For many diagnostics, analyzing the composition of plasma is of interest.

[0003] For in vitro diagnostics (IVD) where measurements are performed at the patient's site without sending samples to a central laboratory, sample preparation is performed by personnel close to the patient. Because these personnel typically lack extensive knowledge of laboratory procedures, the number of fluidic steps must be reduced to a minimum of simple manual procedures. This can be achieved using microfluidic chip laboratory devices. These can be pneumatically based and can integrate multiple fluid handling and measurement steps. However, in these pneumatically operated microfluidic devices, the centrifugation step, which is routinely used in laboratories to obtain plasma, is often not feasible.

[0004] DE 10 2018 216 308 A1 describes a microfluidic system in which particles can be precipitated from a fluid volume. This can be used to precipitate hemorrhagic cells from serum. The precipitation takes place in a cavity with multiple channels leading into it, and this cavity can be divided into multiple subspaces. Summary of the Invention

[0005] The microfluidic device has at least one separation chamber with an inlet and an outlet. The inlet enters the separation chamber at a lower height than the outlet. In other words, when the device is used as intended, the outlet is arranged higher than the inlet, particularly with respect to gravity, within the chamber. The advantage of this device is that, based on the greater height of the outlet, the first phase of the liquid in the separation chamber is partially discharged via the outlet in a simple manner from the separated phase located below it.

[0006] This device is preferably used to automatically and quantitatively separate whole blood into plasma and cellular components based on sedimentation. The difference in inlet and outlet heights has the advantage that, after blood sedimentation, plasma can be separated from the blood residue and discharged from the separation chamber via the outlet, preferably by means of a carrier introduced through the inlet to displace the plasma. Thus, plasma can be advantageously removed from the separation chamber via the outlet, while the cellular components remain in the separation chamber.

[0007] To monitor this transport process, it is preferable to arrange at least one sensor in the separation chamber. The sensor is particularly preferably an optical sensor, and more particularly preferably a brightness sensor or a camera. With the aid of such a sensor, it is possible to monitor when the phase boundary between the plasma and the remaining whole blood, which is now rich in sedimented cellular components and can also be referred to as residual blood, moves into the sensor's imaging area, thereby ensuring that as much plasma as possible, but no remaining whole blood or cellular components, enters the discharge outlet.

[0008] To ensure that no cellular components enter the outlet, it is also preferable to arrange a filter at and / or in the outlet. Although plasma can pass through such a filter, the cellular components of the blood are retained. Due to the high cellular component content in whole blood, filter-based methods are not suitable for separating large volumes of whole blood because the filter will clog. In other words, the present invention advantageously enables the acquisition of large quantities of plasma that is as free of particles as possible through a combination of sedimentation-based phase separation of plasma and the use of a filter, without causing premature filter clogging. The combination of sedimentation and filtration described herein thus enables the automated separation of larger volumes of plasma, for example, exceeding 50 μL. The filter here is preferably a filter for separating plasma from blood, especially a membrane filter suitable for this purpose, such as Vivid. TM Plasma separation membrane.

[0009] In principle, the inlet and outlet can be arranged such that the inlet is located on the lower side of the separation chamber and the outlet on the upper side. However, the outlet is preferably located in the side wall of the separation chamber. This allows for a sample input port to be located on the upper side of the separation chamber. The sample input port can function as a world-to-chip interface when the microfluidic device is designed as a lab-on-a-chip, allowing whole blood to be directly introduced into the separation chamber. Then, there is no need to first transport the whole blood through the channels of the microfluidic device, so that the whole blood can be immediately introduced into the separation chamber through the inlet.

[0010] Furthermore, it is preferable that the outlet leads to another cavity. This other cavity may, for example, have a volume as large as or nearly as large as the separation cavity. The other cavity also shares a common wall with the separation cavity. The separation cavity and the other cavity can therefore be designed as parts of a higher-level cavity, divided by a common wall. The other cavity facilitates the accumulation of plasma displaced from the separation cavity. By eliminating the need for any tubing between the outlet of the separation cavity and the other cavity, and instead using the outlet of the separation cavity simultaneously as the inlet of the other cavity, a highly space-efficient arrangement of the two cavities is achieved.

[0011] To improve transport efficiency from the separation chamber to the other chamber, it is also preferable that the separation chamber and the other chamber are inclined together toward the other chamber, particularly toward the lower side of the device. This inclination is preferably at least 20°. When the device is used as intended, after the carrier is introduced into the separation chamber, this advantageous inclination facilitates the gravity-driven transport of plasma to the other chamber. According to a preferred design, the bottom of the separation chamber can be designed to be inclined toward the lower side of the device. In other words, the flat bottom surface of the separation chamber is not parallel to the flat lower side of the device, but is arranged at an angle to each other, for example, between 5 and 30 degrees, preferably between 10 and 25 degrees, and most preferably between 15 and 25 degrees.

[0012] The pumping device and valves for the microfluidic system can be implemented, for example, by pneumatically actuated deflection of a polymer membrane within a void in a polymer substrate, where the microfluidic channels and separation chambers are also located. Suitable materials for the separation chambers are polymers such as polycarbonate (PC), polypropylene (PP), polyethylene (PE), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), or thermoplastic elastomers (TPE). Polyurethane (TPU) or styrene block copolymers (TPS) can be used, in particular, as thermoplastic elastomers. These polymers can be processed into separation chambers, particularly by high-throughput methods such as injection molding, thermoforming, stamping, or laser transmission welding.

[0013] The volume of the separation chamber is preferably in the range of 20 μl to 1000 μl, and particularly preferably in the range of 50 μl to 100 μl.

[0014] In a method for separating plasma from whole blood, whole blood is first introduced into the separation chamber of a microfluidic device. If the microfluidic device has a sample inlet, the introduction can be completed through this inlet. Otherwise, whole blood is introduced into the separation chamber through an inlet.

[0015] After whole blood enters the separation chamber, blood cells are separated from the whole blood there. Sedimentation can, in principle, be carried out by gravity, in which the whole blood remains in the separation chamber for a predetermined period of time. However, to accelerate sedimentation, it is also preferable to add magnetic beads to the whole blood and then intensify the sedimentation of blood cells by applying an external magnetic field.

[0016] After the sedimentation of blood cells from whole blood is completed, at least a portion of plasma is discharged through the outlet of the device, wherein the plasma exists as a separated phase above the residual blood due to sedimentation. According to a preferred embodiment of the method, the discharge can be accomplished by forming an underlayer with a carrier. The carrier preferably has a higher density than plasma. Suitable carriers with a higher density than plasma and that are immiscible with such plasma are, in particular, fluorinated hydrocarbons. Particularly suitable carriers are selected from the group consisting of bis(non-fluorobutyl)(trifluoromethyl)amine (FC40), perfluorotripentylamine (FC70), 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecano-2-(trifluoromethyl)hexane (HFE7500) and 1,1,1,2,2,3,4,5,5,5-decafluoro-methoxy-4-(trifluoromethyl)pentane (HFE7300). However, alternative carriers can be other blood or other liquids, which are introduced into the separation chamber through the inlet.

[0017] By forming a lower layer, plasma can be transported towards the outlet through the separation chamber and eventually displaced through the outlet. The remaining whole blood (where sedimented blood cells accumulate) forms a phase separate from the plasma in the separation chamber, which, when the lower layer is formed, can also be transported through the separation chamber by a carrier.

[0018] To prevent residual whole blood from entering the outlet, in one embodiment of the method, it is preferable to continue forming the lower layer until the phase boundary between the plasma and whole blood residue reaches a predetermined height in the separation chamber. This can also be detected, as explained above, particularly by means of sensors in the separation chamber.

[0019] In another embodiment of the method, the lower layer continues to be formed until a predetermined amount of carrier is introduced into the separation chamber. This predetermined amount is selected in particular based on the volume of the separation chamber, the height at which the outlet is positioned, and the volume of whole blood introduced into the separation chamber. For this embodiment of the method, no sensor is required. Furthermore, the volume of plasma transported is known by the predetermined amount of carrier and can be included as a parameter for subsequent analysis.

[0020] The remaining whole blood is then preferably transported from the separation chamber by means of a carrier, so that the separation chamber is ready for reuse. In one embodiment of the method, this can be specified by introducing another carrier into the separation chamber after the plasma continues to be transported at a predetermined location in the microfluidic device, thereby displacing the remaining whole blood through an outlet. The whole blood guided through the outlet can then be transported, for example by means of a valve, to another line for further analysis of cellular components.

[0021] In another embodiment of the method, the carrier is pumped back through the inlet of the separation chamber. The remaining whole blood retained on the carrier then follows the carrier and exits the separation chamber through the inlet as well. Attached Figure Description

[0022] Embodiments of the present invention are shown in the accompanying drawings and are explained in more detail in the following description.

[0023] Figure 1 A schematic cross-sectional view of the separation chamber of a microfluidic device according to an embodiment of the present invention is shown;

[0024] Figures 2a to 2d The steps of a method according to an embodiment of the invention are shown, the steps being performed in accordance with... Figure 1 It operates in the separation chamber;

[0025] Figures 3a to 3c Flowcharts illustrating different embodiments of the method according to the invention are shown;

[0026] Figure 4 A cross-sectional view of the separation chamber is shown in another embodiment of the microfluidic device invented according to the present method;

[0027] Figure 5 A cross-sectional view of the separation chamber is shown in yet another embodiment of the microfluidic device invented according to the present method;

[0028] Figures 6a to 6e The steps of a method according to an embodiment of the invention are shown, the method being carried out in accordance with... Figure 5 It operates in the separation chamber;

[0029] Figure 7 A cross-sectional view of the separation chamber in a microfluidic device according to yet another embodiment of the invention is shown;

[0030] Figure 8 A cross-sectional view of the separation chamber in a microfluidic device according to yet another embodiment of the invention is shown. Detailed Implementation

[0031] The microfluidic device for analyzing blood samples has a separation chamber 10 in a first embodiment of the invention. Figure 1 As shown in the figure. The volume of the separation chamber 10 in this embodiment is 75 μl. The separation chamber has an inlet 11 at its lower side and an outlet 12 at its upper side. The transport of liquid through the inlet 11 and the outlet 12 is accomplished in the microfluidic device by a pneumatically actuated deflection of the polymer membrane into a void in the polymer substrate.

[0032] Figures 2a to 2dThis demonstrates how plasma can be separated from whole blood in the separation chamber 10 of a microfluidic device according to a first embodiment of the method of the invention. (As shown in...) Figure 2a As shown, the separation chamber 10 is initially empty. Whole blood 20 is then introduced into the separation chamber 10 through inlet 11 until the separation chamber is empty as shown in the diagram. Figure 2b As shown, it is completely filled with 20 whole blood. Figure 2c This indicates that after a period of time, blood cells settle out of whole blood 20 driven by gravity g, thus forming a phase composed of plasma 21 above whole blood 20. Figure 2d This demonstrates how the carrier 30, which is FC40 in this embodiment, is finally introduced into the separation chamber 10 through inlet 11 to form a sublayer for whole blood 20 and plasma 21. Here, plasma 21 is gradually extruded from the separation chamber 10 through outlet 12. Sublayer formation ends after a predetermined volume of carrier 30 has been introduced into the separation chamber 10.

[0033] exist Figure 3a The procedure is illustrated below. After whole blood 20 is introduced into separation chamber 10, blood cells are first precipitated from whole blood 20, and then plasma and the remaining whole blood 20 are mixed with carrier 30 to form a lower layer 42. After plasma 21 enters outlet 12 in this manner, plasma is pumped into another part of the microfluidic device in final step 43.

[0034] Figure 3b A second embodiment of the method according to the invention is shown. Figure 3a The method flow has been modified. Sedimentation 41 is divided into two sub-steps 411 and 412. In step 411, magnetic beads are introduced into whole blood 20 through inlet 11, where they bind to blood cells. For this purpose, in the current embodiment, the magnetic beads are specified to have CD45 antibodies, which bind to leukocytes. In the subsequent step 412, an electromagnet disposed below the separation chamber 10 is activated to initiate accelerated sedimentation of blood cells.

[0035] exist Figure 3cA third embodiment of the method according to the invention for separating plasma 21 from whole blood 20 is shown. Following the already described method steps 40 to 43 (in which method step 41 may be replaced by sub-steps 411 and 412 if necessary), the remaining whole blood 20 is removed from the separation chamber 10 44. This can be optionally accomplished either by introducing additional carrier 30 into the separation chamber 10 through inlet 11 to expel the remaining whole blood 20 from the separation chamber 10 through outlet 12, or by pumping the carrier 30 back, thus the carrier leaves the separation chamber 10 through inlet 11 carrying the remaining whole blood 20 with it. The remaining whole blood 20 is then transferred 45 to another part of the microfluidic device by means of a pumping process.

[0036] Figure 4 A second embodiment of the microfluidic device is shown, illustrating the design of the separation chamber 10. Here, the inlet 11 and outlet 12 are not located on the lower and upper sides of the separation chamber 10, respectively. Instead, the inlet 11 extends into the lower end of the sidewall of the separation chamber 10. The outlet 12 terminates above the inlet 11 in the sidewall of the separation chamber 10. A sample inlet 13, in the form of an opening, is provided on the upper side of the separation chamber 10, connecting it to an interface region (not shown), which in this embodiment has a volume of 1 ml. Whole blood 20 can be directly introduced into the separation chamber 10 via the inlet 11 without detour through this interface region.

[0037] Figure 5 A separation chamber 10 is shown in a third embodiment of the microfluidic device. The upper chamber is divided into separation chamber 10 and another chamber 50 by a common wall 51. When the inlet 11 is designed in the same manner as in the second embodiment of the microfluidic device, the outlet 12 is a free-flowing area above the common wall 51 that connects separation chamber 10 to the other chamber 50. The other chamber 50 has another outlet 52 on the lower side of its sidewall. In this embodiment of the microfluidic device, a sample inlet 13 is also provided, allowing direct access to separation chamber 10 via the upper side of the separation chamber.

[0038] Figures 6a to 6e A process according to a fourth embodiment of the method of the invention is shown when using a microfluidic device according to the third embodiment. (See also...) Figure 6a As shown in the diagram, the separation chamber 10 is first filled with whole blood 20 up to the upper edge of the common wall 51. Then proceed with... Figure 6b The sedimentation shown is a phase of plasma 21 deposited above whole blood 20. When now as in Figure 6cAs shown, when the carrier 30 is introduced into the separation chamber 10 through inlet 11, the liquid level in the separation chamber 10 rises until the plasma 21 overflows into the other chamber 50. The introduction of the carrier 30 terminates after one volume has been introduced, in which the upper edge of the whole blood 20 phase is, as intended, approximately below the upper edge of the common wall 51. This achieves the condition that the whole blood 20 is in the separation chamber 10 and the plasma 21 is in the other chamber 50. Figure 6d The separation shown in the figure. Figure 6e This indicates how plasma 21 is eventually pumped out of the other cavity 50 through outlet 12.

[0039] Figure 7 This illustrates how the separation chamber 10 is designed in a fourth embodiment of the microfluidic device. This separation chamber is as similar as possible to the separation chamber 10 according to the third embodiment of the invention. However, a filter is arranged in the outlet 12 to prevent blood cells from entering the other chamber 50 from the separation chamber 10. In this way, a larger volume of carrier 30 can be introduced into the separation chamber 10 without the risk of whole blood 20 being transported into the other chamber 50. While the transport of whole blood 20 is also prevented by the filter 14 after the plasma is removed from the other chamber 50, it is not possible in this embodiment of the microfluidic device to remove the remaining whole blood 20 from the separation chamber 10 by introducing another carrier 30; instead, the remaining whole blood must be aspirated along with the carrier 30 through the inlet 11. The filter here can preferably be a plasma filter 14 for separating plasma from the blood, especially a membrane filter suitable for this purpose, such as Vivid. TM Plasma separation membrane. Filter 14 may have an area, for example, between 25 and 400 square millimeters, such as 100 square millimeters.

[0040] Finally, Figure 8 The design of the separation chamber 10 in a fifth embodiment of the microfluidic device is shown. This is also a slight modification of the separation chamber 10 according to the third embodiment of the microfluidic device. Instead of filter 14, or alternatively, a sensor 15 in the form of a brightness sensor is arranged in the separation chamber 10. This sensor is defined such that it detects the upper edge of the common wall 51. Instead of introducing a predetermined amount of carrier 30 into the separation chamber 10, in this embodiment of the microfluidic device, it is stipulated that the introduction of carrier 30 continues for a prolonged period until sensor 15 detects that the phase boundary between plasma 21 and whole blood 20 has reached the upper edge of the common wall 51.

Claims

1. A method for separating plasma (21) from whole blood (20), comprising the following steps: - Whole blood (20) is introduced (40) into the separation chamber (10) of the microfluidic device, wherein the separation chamber (10) has an inlet (11) and an outlet (12), wherein the inlet (11) enters the separation chamber (10) at a lower height than the outlet (12). - To precipitate blood cells from whole blood (20) (41), and - At least part of the plasma (21) separated by sedimentation (41) is discharged (42) via outlet (12), wherein the plasma (21) is discharged (42) by forming a lower layer with a carrier (30).

2. The method according to claim 1, characterized in that, At least one sensor (15) is arranged in the separation chamber (10).

3. The method according to claim 1 or 2, characterized in that, The filter (14) is arranged at the outlet (12) and / or in the outlet (12).

4. The method according to claim 1 or 2, characterized in that, The outlet (12) is arranged in the side wall of the separation chamber (10) and the sample input (13) is arranged in the upper side of the separation chamber (10).

5. The method according to claim 4, characterized in that, The outlet (12) leads into another cavity (50), which has a common wall (51) with the separation cavity (10).

6. The method according to claim 5, characterized in that, The separation chamber (10) and the other chamber (50) are inclined together toward the other chamber (50).

7. The method according to claim 1, characterized in that, The carrier has a higher density than the plasma (21).

8. The method according to claim 7, characterized in that, The formation of the lower layer continues until the phase boundary between the plasma (21) and the residue of the whole blood (20) reaches a predetermined height in the separation chamber (10).

9. The method according to claim 7 or 8, characterized in that, Continue forming the lower layer until a predetermined amount of carrier (30) is introduced into the separation chamber (10).

10. The method according to claim 1 or 2, characterized in that, The residue of the whole blood (20) is transported out (44) from the separation chamber (10) by means of a carrier (30).

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