JPWO2019049204A1 - Fluid devices and their use - Google Patents

Abstract

A flow path provided with a circulation flow path for flowing a fluid containing cells, a pump unit provided in the circulation flow path and controlling the flow of the fluid or the pressure of the fluid, and a cell provided in the flow path. A fluid device comprising an analysis unit, wherein the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of a flow path other than the cell analysis unit.

Description

The present invention relates to fluid devices and their use. More specifically, it relates to a fluid device, a method of disrupting cells, a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample.

In pathological examinations and biological studies, cells may be disrupted. As a method for disrupting cells, there are a physical method, a chemical method and the like. When crushing cells by a physical method, for example, instruments and devices such as microbeads, shakers, centrifuges, and ultrasonic crushers are used. However, in the process of crushing the cells, the worker may come into contact with the sample and be infected with a pathogen or a virus, or the surroundings may be contaminated by the aerosol generated from the sample.

Also, when cells are disrupted by chemical methods, it is usually necessary to control the temperature and dilution of the sample occurs. In addition, since post-treatment is required to stop the chemical treatment, it takes time and the target product may be denatured.

Therefore, an improved cell disruption technique is required.

Japanese Patent No. 3571950

The fluid device according to the embodiment includes a flow path for flowing a fluid containing cells, which is provided with a circulation flow path, and a pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid. , It is provided with an aperture-shaped cell analysis unit provided in the flow path.

A method of disrupting cells according to an embodiment is a fluid device comprising a flow path, a pump section provided in the flow path, and a partition valve for defining a region of the flow path that includes the pump section. A step of flowing a fluid containing the cells through the flow path, a step of closing the partition valve to define a region of the flow path including the pump portion, and a step of operating the pump portion to change the pressure of the fluid. It is provided with a step of crushing the cells by causing the cells to crush.

The method for analyzing the number of erythrocytes and white blood cells in a blood-derived sample according to one embodiment includes a flow path, a pump unit provided in the flow path, and an aperture-shaped cell analysis unit provided in the flow path. A step of flowing the blood-derived sample through the flow path of the fluid device including the above, a step of measuring the number of red blood cells and white blood cells by the cell analysis unit, and a step of operating the pump unit to control the pressure of the blood-derived sample. A step of selectively crushing the erythrocytes by changing the erythrocytes and a step of measuring the number of white blood cells by the cell analysis unit after crushing the erythrocytes are provided.

It is a schematic diagram explaining the structure of the fluid device which concerns on one Embodiment. (A) and (b) are sectional views explaining the structure of a diaphragm valve. (A) and (b) are sectional views explaining the structure of a diaphragm valve. (A) to (d) are cross-sectional views illustrating the operation of a pump including three diaphragm valves. It is a schematic diagram explaining the structure of the fluid device which concerns on one Embodiment. It is a schematic diagram explaining the structure of the fluid device which concerns on one Embodiment. (A) to (c) are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using a fluid device. (A) and (b) are schematic views explaining the structure of the fluid device manufactured in Experimental Example 1. It is a graph which shows the result of Experimental Example 2. It is a graph which shows the result of Experimental Example 3.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. In the drawings, the same or corresponding parts are designated by the same or corresponding reference numerals, and duplicate description will be omitted. The dimensional ratio in each figure is exaggerated for explanation and does not necessarily match the actual dimensional ratio.

[Fluid device]
(First Embodiment)
In one embodiment, the present invention comprises a flow path for flowing a fluid containing cells, and a pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid. Provided is a fluid device including an aperture-shaped cell analysis unit provided in the flow path.

FIG. 1 is a schematic diagram illustrating the structure of the fluid device 100 according to the embodiment. The fluid device 100 includes a flow path 110 having a circulation flow path, a pump unit 130 provided in the circulation flow path, and an aperture-shaped cell analysis unit 120 provided in the flow path 110 having a circulation flow path. As will be described later, the cell analysis unit 120 may be present inside the circulation flow path, or may be present in a region other than the circulation flow path in the flow path 110.

In the fluid device of the present embodiment, the circulation flow path means a flow path through which the internal fluid can circulate. The circulation flow path may form a closed circuit, or may be configured so that a closed circuit can be formed by opening and closing the partition valve 140 described later.

The flow path 110 is for flowing the fluid L containing cells. The fluid L is not particularly limited, but may be, for example, a blood-derived sample. In this case, examples of the cells contained in the fluid L include erythrocytes and leukocytes. The flow of the fluid L can be controlled by operating the pump unit 130. Further, the pressure of the fluid L can be adjusted by operating the pump unit 130. For example, by operating the pump unit 130, the fluid L in the stationary state can be flowed, or the flow velocity of the fluid L can be increased.

It can also be said that the pressure of the fluid L is the pressure received by the cells contained in the fluid L. For example, the pressure of the fluid L can be increased by increasing the flow velocity of the fluid L. Alternatively, the pressure of the fluid L can be reduced by reducing the flow velocity of the fluid L.

Cells can be disrupted using the fluid device of this embodiment. Specifically, the pump unit 130 can crush the cells by changing the pressure of the fluid L flowing through the flow path 110. For example, the cell membrane of a cell can be ruptured by generating a pressure change such as releasing the pressurization after applying a predetermined pressurization to the fluid L. In addition, by controlling the conditions of pressure change, only specific cells in the cell mixture can be disrupted. It is considered that this is because the structure of the cell membrane differs depending on the type of cell, and the resistance to pressure differs. As will be described later in the examples, for example, when the fluid L contains erythrocytes and leukocytes, only erythrocytes can be selectively crushed.

With the fluid device of the present embodiment, cells can be disrupted in an extremely short time without using a conventional cell disruptor or reagent. In addition, since the sample is not diluted and the cell disruption conditions are mild, there is little risk of denaturation of the target product, and there is no need to adjust the temperature for cell disruption. Details of cell disruption will be described later.

Further, according to the fluid device of the present embodiment, since the cells are crushed in the fluid device, it is possible to control the sample from scattering to the outside of the fluid device. Therefore, it is possible to reduce the risk that the worker is infected with a pathogen or a virus or contaminates the surroundings.

The fluid device of this embodiment may be a cartridge. That is, the fluid device of the present embodiment is a component that can be freely attached and detached, and may be attached to and detached from the analyzer. Since the fluid device of the present embodiment is a cartridge, cells can be crushed and analyzed in a closed space inside the cartridge, so that the risk of infection / contamination can be further reduced.

In the fluid device 100, the fluid L inside the circulation flow path included in the flow path 110 is circulated by operating the pump unit 130 in a state where the partition valves 140a and 140b are closed and the flow path 110 forms a circuit. Can be done. This makes it possible to disrupt cells more efficiently.

As will be described later in the embodiment, by operating the pump unit 130 in a state where the circulation flow path is closed, the fluid L flows inside the circulation flow path having a defined volume. Here, "definition" means distinguishing from other regions of the flow path. Then, when the operating conditions of the pump unit 130 are adjusted to change the pressure of the fluid L, the pressure received by the cells contained in the fluid L changes. More specifically, the cells are pressurized or depressurized. It is considered that such a pressure change causes the cell membrane of the cell to rupture inside the defined region 115 in the flow path 110, and the cell is crushed.

As shown in FIG. 1, a part of the flow path 110 may form an introduction flow path 110a for introducing the fluid L into the region 115 including the pump portion 130, or for discharging the fluid from the region 115. The flow path 110b may be formed. As a result, it is possible to easily perform operations such as introducing the fluid L into the region 115 and discharging the fluid L from the region 115.

Further, the introduction flow path 110a preferably includes a partition valve 140a that controls the flow of the fluid L, and the discharge flow path 110b preferably includes a partition valve 140b that controls the flow of the fluid L. preferable. At this time, the region 115 including the pump portion 130 is defined from the introduction flow path 110a by closing the partition valve 140a, and is defined from the discharge flow path 110b by closing the partition valve 140b.

Further, the fluid device 100 may include a waste liquid tank 150. By storing the waste liquid in the waste liquid tank 150, the risk of infection or contamination by pathogens or viruses can be further reduced.

《Pump section》
The pump unit 130 may be composed of, for example, a valve. Further, the above valve is a diaphragm valve including a diaphragm member, and the diaphragm member may be formed of an elastomer material. Then, the diaphragm member may be deformed to control the flow of the fluid L in the flow path 110, and the volume of the region 115 including the pump portion 130 may be changed. Here, the deformation direction of the diaphragm member is not particularly limited as long as the flow of the fluid L can be controlled, and may be, for example, a direction perpendicular to the axis of the flow path 110.

2A and 2B are cross-sectional views illustrating an example of the structure of the diaphragm valve. FIG. 2A shows an open state of the diaphragm valve 200, and FIG. 2B shows a closed state of the diaphragm valve 200. As shown in FIGS. 2A and 2B, the diaphragm valve 200 includes a first substrate 210, a diaphragm member 230 made of an elastomer material, and a second substrate 220. The second substrate 220 and the diaphragm member 230 are adhered to each other in close contact with each other. Further, the space between the first substrate 210 and the diaphragm member 230 forms a flow path 110 through which a fluid flows. Further, a through hole 240 is provided in a part of the second substrate 220. Further, in the through hole 240, the diaphragm member 230 is exposed.

The diaphragm valve 200 is arranged in the flow path 110 and controls the flow of the fluid L or the pressure of the fluid L inside the flow path 110.

In the open state of the diaphragm valve 200 shown in FIG. 2A, the fluid L can flow inside the flow path 110. On the other hand, as shown in FIG. 2B, when a fluid for valve control is supplied from the through hole 240 of the diaphragm valve 200 and the inside of the through hole 240 is pressurized, the diaphragm member 230 is deformed and the deformed diaphragm member is deformed. A part of 230 is in close contact with the first substrate 210. This state is the closed state of the diaphragm valve 200. As a result, the flow of the fluid L inside the flow path 110 is blocked.

Here, as shown in FIGS. 2A and 2B, in order to make the closed state of the diaphragm valve 200 stronger, a convex portion is formed in the region of the first substrate 210 facing the through hole 240. 211 may be formed.

The fluid valve control, N ₂ gas, a gas such as air, water, liquid such as oil and the like. The fluid for valve control can be supplied, for example, by a tube connected to the through hole 240 or the like. Alternatively, the opening and closing of the valve may be controlled by a mechanical force or an electromagnetic force.

The elastomer material forming the diaphragm member 230 is not particularly limited as long as it is a material that can be deformed in the axial direction of the through hole 240 in response to a change in pressure inside the through hole 240. For example, polydimethylsiloxane (PDMS). ), Silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.

In the closed diaphragm valve 200 shown in FIG. 2B, when the pressure applied to the inside of the through hole 240 is reduced, the deformed diaphragm member 230 returns to its original shape, and FIG. 2A again. ) Will be in the open state. As a result, the fluid L can flow inside the flow path 110 again.

The structure of the diaphragm valve is not limited to that described above. For example, the diaphragm valve 300 shown in FIGS. 3A and 3B can also be used. FIG. 3A shows the open state of the valve 300, and FIG. 3B shows the closed state of the valve 300.

As shown in FIGS. 3A and 3B, in the valve 300, the diaphragm member 230 is not arranged on the entire surface of the second substrate 220 as compared with the valve 200 described above, and only around the valve 300. The main difference is that they are locally located.

Since the diaphragm member 230 of the valve 300 is provided with the anchor portion 231 so that the diaphragm member 230 is prevented from being damaged and peeled off from the second substrate 220 even when the diaphragm member 230 is deformed by pressure. Has been done.

<< Pump section including 3 or more valves >>
Like the fluid device 100, the pump unit 130 may be composed of three or more valves. By using three or more valves, that is, by arranging three or more valves, the flow of the fluid L by the pump unit 130 can be controlled more efficiently.

The operation of the triple valve will be described with reference to FIGS. 4A to 4D. 4 (a) to 4 (d) are cross-sectional views illustrating the operation of an example of a pump including three diaphragm valves 200a, 200b and 200c.

In the state shown in FIG. 4A, all three valves 200a, 200b and 200c are in the open state. In this state, since the flow of the fluid L inside the flow path 110 is not controlled, the fluid L may flow to the right side or the left side toward FIG. 4A, or the fluid may flow to the left side. The flow may be stopped (stationary).

Subsequently, as shown in FIG. 4B, the valve 200a is controlled to be in the closed state, and the valves 200b and 200c are left in the open state. As a result, the flow of the fluid L inside the flow path 110 is blocked by the valve 200a.

Subsequently, as shown in FIG. 4C, the valve 200a and the valve 200b are controlled to be in the closed state, and the valve 200c is left in the open state. Here, in the process of changing the valve 200b from the open state to the closed state, the diaphragm member 230 is deformed, so that the fluid L existing around the valve 200b is pushed away. However, since the valve 200a is in the closed state, the displaced fluid moves in the direction indicated by the arrow in FIG. 4C, that is, to the right toward FIG. 4C. As a result, a flow in the direction indicated by the arrow is generated in the fluid L. Further, the fluid L is pressurized.

Subsequently, as shown in FIG. 4D, the valves 200b and 200c are controlled to be in the closed state. Then, in the process of changing the valve 200c from the open state to the closed state, the diaphragm member 230 is deformed, and the fluid L existing around the valve 200c is pushed away. However, since the valve 200a is in the closed state, the displaced fluid L moves in the direction indicated by the arrow in FIG. 4D, that is, to the right toward FIG. 4D. As a result, the flow of the fluid L in the direction indicated by the arrow is further accelerated. Further, the fluid L is further pressurized. At this time, the valve 200a may be controlled to be in the open state as shown in FIG. 4D, or may be maintained in the closed state.

Subsequently, as shown in FIG. 4A again, the valves 200a, 200b and 200c are all controlled to be in the open state. As a result, the pressurization of the fluid L is released. Further, in this state, the fluid L inside the flow path 110 may continue to move to the right side toward FIG. 4A due to inertia.

Further, by repeating the above steps, the flow of the fluid L or the pressure of the fluid L inside the flow path 110 can be controlled.

The above steps are an example of a control method for a pump having three valves, and the control method for a pump having three valves is not limited to this. For example, in the valve control described above, the flow of the fluid L inside the flow path 110 can be controlled in the direction opposite to that described above by reversing the opening and closing timings of the valve 200a and the valve 200c. Further, the flow of the fluid L or the fluid L can also be adjusted by adjusting the cycle of repeating the operations of FIGS. 4 (a) to 4 (d), the pressure of the fluid for controlling the valve for driving the valves 200a to 200c, the diameter of the valve, and the like. Pressure can be controlled.

It is also possible to control the flow of the fluid L inside the flow path 110 by a pump provided with four or more valves.

《Partition valve》
The fluid device 100 may further include a partition valve 140. By closing the partition valves 140 (140a, 140b) to define the region 115 including the pump portion 130 of the flow path 110 and then operating the pump portion 130, the pressure of the fluid L can be controlled more efficiently.

In the fluid device 100, by closing the partition valve 140, the region 115 including the pump portion 130 becomes a defined region. That is, in the fluid device 100, by closing the partition valves 140 (140a, 140b), the region 115 including the pump portion 130 of the flow path 110 becomes a circulation flow path (loop flow path) and becomes independent from other parts. it can.

As a result, the flow velocity of the fluid temporarily slows down or stops in the flow path 110. After that, when the pump unit 130 is operated, the flow velocity increases in the region 115, and the pressure applied to the fluid L increases.

Further, for example, when the pump portion 130 is a diaphragm valve, closing the diaphragm valve deforms the diaphragm member toward the flow path 110 side, and the volume in the region 115 becomes smaller. As a result, the pressure of the fluid L in the region 115 increases. On the other hand, by opening the diaphragm valve, the diaphragm member is deformed in a direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. In this way, the pressure of the fluid L can be adjusted by varying the volume in the region 115.

The partition valve 140 may be a diaphragm valve including a diaphragm member, or may be the same as the valve constituting the pump portion described above.

The partition valve 140 may be a normally closed valve or a normally open valve. A normally closed valve is a valve that is in a closed state in a steady state and is opened by operating the valve. Further, the normally open valve is a valve that is in an open state in a steady state and is closed by operating the valve.

When the partition valve 140 is a normally closed valve, the partition valve 140 is in the closed state in the steady state, and operating the valve releases the region 115 defined by the partition valve 140.

Further, when the partition valve 140 is a normally open valve, the partition valve 140 is in the open state in the steady state and is closed by operating the valve, and a part region 115 of the flow path 110 is the partition valve 140. It becomes the area defined by.

Further, the fluid device 100 may include one partition valve 140 or two or more partition valves 140. When there are two or more partition valves 140, the region 115 including the pump portion can be defined by closing each partition valve 140, and can be temporarily made into an independent space.

《Cell analysis department》
The cell analysis unit 120 is an area for analyzing cells flowing in the flow path 110. Here, cell analysis means analyzing a predetermined parameter of a fluid containing cells. The parameters are not particularly limited, and examples thereof include the number of cells in a fluid containing cells, the size of cells, and the like. The cell analysis unit has, for example, an aperture shape. The aperture shape is a shape in which the cross-sectional area of the flow path cross section orthogonal to the fluid traveling direction of the cell analysis unit 120 is smaller than the cross-sectional area of the flow path cross section orthogonal to the fluid traveling direction of the flow path 110. For example, the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of the flow paths 110 before and after the cell analysis unit 120. For example, when the flow path is annular, the diameter of the cell analysis unit 120 is smaller than the diameter of other regions of the flow path 110. That is, the cross section of the flow path of the cell analysis unit, which has an aperture shape, may have a size suitable for analysis of the analysis target, or may have a size through which the cells to be analyzed pass one by one. As a result, at least in the cell analysis unit 120, cells can be passed one by one. As a result, cells can be detected for each cell, which facilitates cell analysis. The analysis of cells may be performed optically or electrically, for example. Cell analysis items can be set as appropriate. For example, the number of cells may be measured, or the size of cells may be measured.

The cross-sectional shape of the flow path and the aperture shape is not limited to a rectangle, and may be, for example, a circular shape or an arbitrary polygonal shape. For example, the cross section of the flow path 110 may be a rectangle having a width of 1.5 mm and a height of 0.3 mm. Further, the cross section of the cell analysis unit having an aperture shape may be a rectangle having a width of 30 μm and a height of 30 μm. When the cell analysis unit having a flow path and an aperture shape has such a shape and size, for example, leukocytes can pass through alone.

<< Position of cell analysis department >>
In the fluid device 100, the cell analysis unit 120 exists inside the circulation flow path included in the flow path 110. That is, the cell analysis unit 120 exists in the circulation channel. Since the cell analysis unit 120 is present inside the circulation channel, the state of cells inside the circulation channel can be analyzed in real time while the pump unit 130 is operated to crush the cells. As a result, for example, the pump unit 130 can be stopped when the cell crushing rate reaches an arbitrary value, or the cell crushing operation can be continued until the cells are completely crushed.

(Second Embodiment)
FIG. 5 is a schematic view illustrating the structure of the fluid device 500 according to the second embodiment. The fluid device 500 of the second embodiment has a second aspect in that the cell analysis unit 120 exists outside the circuit when the partition valves 140a and 140b are closed to form a circuit in which the circulation flow path included in the flow path 110 is closed. It is mainly different from the fluid device 100 according to one embodiment. That is, in the fluid device 500, the cell analysis unit 120 exists in a flow path other than the circulation flow path.

In the fluid device 500 of the second embodiment, since the cell analysis unit 120 exists outside the circuit, the flow of the fluid L inside the flow path 110 is not affected by the cell analysis unit 120. Therefore, the degree of freedom in controlling the flow of the fluid L by operating the pump unit 130 is higher than that of the fluid device 100 according to the first embodiment.

In the fluid device 500 of the second embodiment, for example, the cell analysis unit 120 analyzes the cells flowing through the flow path 110 before starting the cell crushing or after completing the cell crushing.

In the fluid device 500, the cell analysis unit 120 exists in the discharge flow path 110b. In this case, the cells can be analyzed by flowing the fluid L in the order of the introduction flow path 110a, the circulation flow path, and the discharge flow path 110b in one way. However, the position of the cell analysis unit 120 is not limited to this, and the cell analysis unit 120 may exist in the introduction flow path 110a. In this case as well, the cells can be analyzed by sending the fluid L in the order of the introduction flow path 110a and the circulation flow path, and then flowing the fluid L back into the introduction flow path 110a in the opposite direction.

(Modification example)
FIG. 6 is a schematic diagram illustrating a modified example of the fluid device. The fluid device 600 is mainly different from the fluid device 100 according to the first embodiment in that the circulation flow path included in the flow path 110 forms two or more closed circuits by controlling the opening and closing of the partition valve 140. .. In the fluid device 600, by controlling the opening and closing of the valve 140, the flow of the fluid L inside the flow path 110 can be controlled and cells can be analyzed.

In the fluid device 600, when the partition valves 140a1, 140a2, 140b1, 140b2 are closed and the flow path 110 forms a circuit, the cell analysis unit 120 exists inside the circuit. However, the position of the cell analysis unit 120 is not limited to this, and may be configured to exist outside the circuit.

As shown in FIG. 6, the fluid device 600 includes a first circulation flow path and a second circulation flow path, and a circuit in which the first circulation flow path and the second circulation flow path are closed by closing the partition valve 140. May be configured to form.

Further, as shown in FIG. 6, the first circulation flow path and the second circulation flow path in the fluid device 600 share at least a part of the flow paths, and the shared flow path is provided with a pump unit to provide the first circulation. The cell analysis unit 120 may be provided in the flow path or the second circulation flow path.

Further, as shown in FIG. 6, in the fluid device 600, the first circulation flow path and the second circulation flow path are not shared in the vicinity of both ends of the flow path shared by the first circulation flow path and the second circulation flow path. Each of the flow paths may have a valve.

[How to crush cells]
In one embodiment, the present invention is a method of disrupting cells, a partition valve for defining a flow path, a pump portion provided in the flow path, and a region including the pump portion of the flow path. A step of flowing a fluid containing the cells into the flow path of the fluid device including the above, a step of closing the partition valve to define a region including the pump part of the flow path, and operating the pump part. Provided is a method comprising a step of crushing the cells by changing the pressure of the fluid.

Here, as an example, a method of disrupting cells using the fluid device 100 shown in FIG. 1 will be described. FIG. 1 is a schematic diagram illustrating the structure of the fluid device 100 according to the embodiment. The fluid device 100 includes a flow path 110 having a circulation flow path, a pump unit 130 provided in the circulation flow path, and an aperture-shaped cell analysis unit 120 provided in the flow path 110 including the circulation flow path. There is.

Here, the pump unit 130 may include three or more valves. As described above, the flow of the fluid L can be controlled more efficiently by the pump unit 130 by using three or more valves, that is, by arranging three or more valves.

In the fluid device 100, the partition valves 140a and 140b are closed to form a circuit in which the region 115 of the flow path 110 is closed.

First, the partition valves 140a and 140b of the fluid device 100 are opened, and the partition valves 140c and 140d are closed. In this state, the fluid L containing cells is introduced from the introduction flow path 110a. The fluid L containing cells may be, for example, a blood-derived sample containing red blood cells and white blood cells. As a result, the inside of the flow path 110 is filled with the fluid L containing cells. The surplus fluid L passes through the discharge flow path 110b and is housed in the waste liquid tank 150. Here, the flow of the fluid L containing the cells may be generated by operating the pump unit 130. In this case, the operating condition of the pump unit 130 may be a condition that does not crush the cells.

Subsequently, after the inside of the flow path 110 is filled with the fluid L containing cells, the partition valves 140a and 140b are closed and 140c is opened. As a result, the region 115 of the flow path 110 forms a closed circuit.

Subsequently, the pump unit 130 is operated under the condition that the pressure of the fluid L containing the cells is changed. Here, conditions for changing the pressure of the fluid L include the driving time, driving pressure, frequency, and the like of the pump. As a result, the flow velocity increases in the region 115, and the pressure applied to the fluid L increases. Further, when the pump portion 130 is a diaphragm valve, closing the diaphragm valve deforms the diaphragm member toward the flow path 110 side, and the volume in the region 115 becomes smaller. As a result, the pressure of the fluid L in the region 115 increases. On the other hand, by opening the diaphragm valve, the diaphragm member is deformed in a direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. Such pressure changes disrupt the cells.

Here, by adjusting the conditions for changing the pressure, for example, only erythrocytes in a blood-derived sample containing erythrocytes and leukocytes can be selectively crushed. Further, by operating the pump unit 130 in a state where the region 115 of the flow path 110 is closed to form a circuit, the efficiency of crushing cells can be increased.

[Method of measuring the number of red blood cells and white blood cells in a blood-derived sample]
In one embodiment, the present invention is a method of measuring the number of red blood cells and white blood cells in a blood-derived sample, which comprises a flow path, a pump portion provided in the flow path, and an aperture provided in the flow path. The step of flowing the blood-derived sample through the flow path of the fluid device including the cell analysis section of the shape, the step of measuring the number of red blood cells and white blood cells in the cell analysis section, and the operation of the pump section are described. Provided is a method including a step of selectively crushing the erythrocytes by changing the pressure of a blood-derived sample, and a step of measuring the number of white blood cells by the cell analysis unit after crushing the erythrocytes. It can be said that the method of this embodiment is a method of performing blood cell calculation (blood calculation).

Here, as an example, a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 500 shown in FIG. 5 will be described.

First, the partition valves 140a and 140b of the fluid device 500 are opened, and the partition valves 140c and 140d are closed. In this state, the blood-derived sample is introduced from the introduction flow path 110a. The blood-derived sample may be introduced by pressurization or by operating the pump unit 130. In this case, the operating conditions of the pump unit 130 are preferably conditions that do not disrupt the cells.

As a result, the inside of the flow path 110 is filled with the blood-derived sample. The surplus blood-derived sample passes through the cell analysis unit 120 and the discharge flow path 110b and is stored in the waste liquid tank 150. Here, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. At this time, in the blood-derived sample passing through the cell analysis unit 120, red blood cells and leukocytes are not crushed. Therefore, the number of cells measured at this stage is the total number of red blood cells and white blood cells.

Subsequently, after the inside of the flow path 110 is filled with the blood-derived sample, the partition valves 140a and 140b are closed and the 140c is opened. As a result, the region 115 of the flow path 110 forms a closed circuit.

Subsequently, the pump unit 130 is operated under the condition of selectively crushing the red blood cells in the blood-derived sample, and the pressure of the blood-derived sample is changed to selectively crush the red blood cells.

Subsequently, the partition valve 140b is opened to feed the blood-derived sample. Then, the blood-derived sample inside the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110b and is housed in the waste liquid tank 150.

At this time, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. Here, in the blood-derived sample passing through the cell analysis unit 120, only red blood cells are selectively crushed, and leukocytes are not crushed. Therefore, the number of cells measured at this stage is the number of white blood cells.

By the above steps, the number of white blood cells in the blood-derived sample can be measured. In addition, the number of red blood cells can be obtained more accurately by subtracting the number of only white blood cells measured later from the total number of red blood cells and white blood cells measured first. However, since the number of white blood cells is usually very small with respect to the number of red blood cells, the total number of red blood cells and white blood cells measured first may be used as the number of red blood cells.

The condition for selectively crushing red blood cells in the blood-derived sample may be, for example, a condition in which the driving pressure of the pump portion having the diaphragm member is more than 0 kPa and 110 kPa, for example, 50 to 110 kPa, for example, 70 to 110 kPa. Further, the driving frequency of the pump unit having the diaphragm member may be a condition of more than 0 Hz and 3.3 Hz, for example, 0.5 to 3.3 Hz, for example, 2 to 3.3 Hz. The operating time of the pump unit may be, for example, 1 to 30 minutes, 5 to 20 minutes, or about 10 minutes.

Further, the diameter of the diaphragm member may be, for example, 1 to 3 mm or 2 to 3 mm. Further, the thickness of the diaphragm member may be, for example, 10 to 2000 μm, or may be, for example, 100 to 1000 μm. The thinner the diaphragm member, the lower the driving pressure and the required displacement. Further, the material of the diaphragm member may be a silicone-based elastomer such as polydimethylsiloxane (PDMS), polymethylphenylsiloxane, or polydiphenylsiloxane.

Under the above conditions, red blood cells can be selectively disrupted. The leukocyte crush rate under the above conditions is 2.5% or less.

Subsequently, as another example, a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600 shown in FIG. 6 will be described. 7 (a) to 7 (c) are schematic views illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600.

First, as shown in FIG. 7A, the partition valves 140a2, 140b1, 140c1, 140c2, 140c5 of the fluid device 600 are opened, and the partition valves 140a1, 140b2, 140c3, 140c4 are closed. Subsequently, the blood-derived sample is introduced from the introduction flow path 110a1. The blood-derived sample may be introduced by pressurization or by operating the pump unit 130. In this case, the operating conditions of the pump unit 130 are preferably conditions that do not disrupt the cells.

As a result, as shown by the arrow in FIG. 7A, the blood-derived sample flows inside the flow path 110, and the inside of the flow path 110 is filled with the blood-derived sample. The surplus blood-derived sample passes through the discharge flow path 110b1 and is stored in the waste liquid tank 150. Here, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. At this time, in the blood-derived sample passing through the cell analysis unit 120, red blood cells and leukocytes are not crushed. Therefore, the number of cells measured at this stage is the total number of red blood cells and white blood cells.

Subsequently, after the inside of the flow path 110 is filled with the blood-derived sample, as shown in FIG. 7B, the partition valves 140a1, 140b1, 140c3, 140c5 are closed, and 140a2, 140b2, 140c1, 140c2, 140c4 are closed. Open. As a result, the region 115 of the flow path 110 forms a closed circuit.

Subsequently, the pump unit 130 is operated under the condition of selectively crushing the red blood cells in the blood-derived sample, and the pressure of the blood-derived sample is changed to selectively crush the red blood cells. During this time, as shown by the arrow in FIG. 7B, the blood-derived sample circulates inside the closed circuit formed by the flow path 110.

Further, as shown by the arrow in FIG. 7B, by introducing the drainage fluid from the introduction flow path 110a2, the blood-derived material existing inside the region including the cell analysis unit 120 of the flow path 110 The sample can be discharged. Examples of the discharge fluid include N ₂ gas, a gas such as air, a buffer, a liquid such as oil, and the like. The blood-derived sample passes through the discharge flow path 110b2 and is stored in the waste liquid tank 150.

Subsequently, as shown in FIG. 7C, the partition valves 140a2, 140b1, 140c2, 140c5 are closed, and 140a1, 140b2, 140c1, 140c3, 140c4 are opened.

Subsequently, in this state, the discharge fluid is introduced from the introduction flow path 110a1. The discharge fluid may be introduced by pressurization or by operating the pump unit 130. In this case, the operating conditions of the pump unit 130 are preferably conditions that do not disrupt the cells.

Then, as shown by the arrow in FIG. 7C, the blood-derived sample inside the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110b2 and is housed in the waste liquid tank 150. At this time, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. Here, in the blood-derived sample passing through the cell analysis unit 120, only red blood cells are selectively crushed, and leukocytes are not crushed. Therefore, the number of cells measured at this stage is the number of white blood cells. By the above steps, the number of white blood cells in the blood-derived sample can be measured.

Next, the present embodiment will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Experimental Example 1]
(Manufacturing of fluid devices)

Further, the device shown in FIG. 8B (hereinafter referred to as “device B”) was the same as the device A except that the diameter of each valve was 2 mm. The devices A and B were manufactured for the purpose of evaluating whether or not the cells could be crushed by the operation of the pump unit, and the cell analysis unit did not have them.

[Experimental Example 2]
(Examination of crushing red blood cells)
Cell disruption was examined using devices A and B produced in Experimental Example 1. Rabbit blood was used as a sample.

First, a sample was introduced into each fluid device. The amount of sample introduced was 62.5 μL for each device. Subsequently, the valve 140 of each device was opened and closed to form a closed circuit of the flow path 110. As a result, the sample was sealed in the circuit of each device.

Subsequently, the pump unit 130 was operated to crush the cells. The operating conditions of the pump unit 130 were set to the respective conditions shown in Table 1. The operating time of the pump unit 130 was set to 10 minutes under all conditions.

Subsequently, the valve 140 of each device was opened and closed, and a sample was collected from each device. Subsequently, the absorbance of each collected sample at 576 nm was measured using a spectrophotometer (UV-1800 / Shimadzu Corporation). The absorbance of the physiological saline solution was measured as a negative control. In addition, as a positive control, water for injection (Otsuka Pharmaceutical Co., Ltd.) was added instead of physiological saline, and the absorbance of the completely hemolyzed sample was measured. The hemolysis rate of each sample was calculated using the following formula (1).
Hemolysis rate (%) = (absorbance of sample-absorbance of negative control) / (absorbance of positive control-absorbance of negative control) x 100 ... (1)

FIG. 9 is a graph showing the measurement result of the hemolysis rate. As a result, it was found that the proportion of crushed red blood cells tended to increase as the bulb diameter, driving pressure, and frequency were increased.

[Experimental Example 3]
(Examination of the effect of cell disruption on leukocytes)
In the same manner as in Experimental Example 2, red blood cells in the blood of rabbits were crushed and the effect on white blood cells was examined.

First, a sample was introduced into device A. Rabbit blood was used as a sample. The amount of sample introduced was 62.5 μL. Subsequently, the valve 140 of the device A was opened and closed to form a closed circuit of the flow path 110. As a result, the sample was sealed in the circuit of device A.

Subsequently, the pump unit 130 was operated to crush the cells. The operating conditions of the pump unit 130 were set to the respective conditions shown in Table 2. The operating time of the pump unit 130 was set to 10 minutes under all conditions.

Subsequently, the valve 140 of the device A was opened and closed, and the sample was collected. Subsequently, the hemolysis rate of each collected sample was measured using a spectrophotometer (UV-1800 / Shimadzu Corporation) in the same manner as in Experimental Example 2. In addition, after removing the cells in the sample by passing through a filter with a pore size of 1.2 μm, the amount of DNA in each sample was measured using real-time quantitative PCR (Light Cycler 480 System II / Roche Diagnostics). Was measured. When white blood cells are destroyed, the amount of DNA in the sample increases. The relative value of the amount of DNA in the recovered sample was calculated when the total amount of DNA contained in the original sample was 100.

FIG. 10 is a graph showing the measurement results of the hemolysis rate and the amount of DNA. As a result, it was clarified that even if the red blood cells were destroyed by the device A, the white blood cells were hardly destroyed.

100, 500, 600 ... Fluid device, 110 ... Channel, 110a, 110a1, 110a2 ... Introduction flow path, 110b, 110b1, 110b2 ... Discharge flow path, 115 ... Region, 120 ... Cell analysis unit, 130, P ... Pump section, 140, 140a, 140a1, 140a2, 140b, 140b1, 140b2, 140c, 140c1, 140c2, 140c3, 140c4, 140c5, 140d ... Partition valve, 150 ... Waste liquid tank, 200, 200a, 200b, 200c, 300 ... Diaphragm Valve (valve), 210 ... 1st substrate, 211, 211a, 211b, 211c ... Convex portion, 220 ... 2nd substrate, 230 ... Diaphragm member, 231 ... Anchor portion, 240 ... Through hole, L ... Fluid.

Claims (19)

A flow path for flowing a fluid containing cells, which has a circulation flow path,
A pump unit provided in the circulation flow path and controlling the flow of the fluid or the pressure of the fluid, and
The cell analysis unit provided in the flow path and
With
A fluid device in which the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of a flow path other than the cell analysis unit. The fluid device according to claim 1, further comprising a partition valve for defining a region including the pump portion. The fluid device according to claim 2, wherein the partition valve is provided in a flow path other than the circulation flow path, and by closing the partition valve, a circuit in which the circulation flow path is closed is formed. The fluid device according to any one of claims 1 to 3, wherein the cell analysis unit is present in the circulation channel. The fluid device according to any one of claims 1 to 3, wherein the cell analysis unit exists in a flow path other than the circulation flow path. The fluid device according to any one of claims 1 to 5, wherein the pump unit includes three or more valves. The pump portion has a diaphragm member and has a diaphragm member.
The fluid device according to any one of claims 2 to 6, wherein the diaphragm member is deformed to control the flow of fluid in the flow path and change the volume of the region including the pump portion. The fluid device according to any one of claims 1 to 7, wherein the fluid is a blood-derived sample and the cells contain red blood cells and white blood cells. The circulation flow path includes a first circulation flow path and a second circulation flow path.
The fluid device according to any one of claims 3 to 8, wherein the first circulation flow path and the second circulation flow path share at least a part of the flow path. The pump unit is provided in the shared flow path.
The fluid device according to claim 9, wherein the cell analysis unit is provided in the first circulation flow path or the second circulation flow path. A claim that each of the first circulation flow path and the flow path not shared by the second circulation flow path has valves in the vicinity of both ends of the flow path shared by the first circulation flow path and the second circulation flow path. Item 9. The fluid device according to item 9. The fluid device according to any one of claims 1 to 11 is provided.
The pump unit crushes the flow of the fluid or the pressure of the fluid to the extent that (i) the sample can be circulated without crushing the red blood cells and the white blood cells in the sample, and (ii) only the red blood cells in the sample are crushed and the white blood cells. Is a fluid device for blood calculation that is configured to be controllable to the extent that the sample can be circulated without crushing. A method of crushing cells
A fluid containing the cells is placed in the flow path of a fluid device including a flow path, a pump portion provided in the flow path, and a partition valve for defining a region including the pump section of the flow path. The flow process and
A step of closing the partition valve to define a region of the flow path including the pump portion,
A step of crushing the cells by operating the pump unit to change the pressure of the fluid, and
How to prepare. The fluid device further comprises an aperture-shaped cell analyzer provided in the flow path.
The method according to claim 13, further comprising a step of analyzing cells in the fluid in the cell analysis unit. 13. The method of claim 13 or 14, wherein the compartment valve is closed to form a circuit in which the flow path is closed. The method according to any one of claims 13 to 15, wherein the pump unit includes three or more valves. The method according to any one of claims 13 to 16, wherein the fluid contains a plurality of types of cells, and in the step of crushing the cells, the pump unit is operated under the condition of crushing at least one type of cells. The method according to any one of claims 13 to 17, wherein the fluid is a blood-derived sample and the cells contain red blood cells and white blood cells. A method for measuring the number of red blood cells and white blood cells in a blood-derived sample.
A step of flowing the blood-derived sample through the flow path of a fluid device including a flow path, a pump unit provided in the flow path, and an aperture-shaped cell analysis unit provided in the flow path.
The step of measuring the number of red blood cells and white blood cells in the cell analysis unit, and
A step of selectively crushing the red blood cells by operating the pump unit to change the pressure of the blood-derived sample, and
After crushing the red blood cells, the step of measuring the number of white blood cells in the cell analysis unit and
How to prepare.

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