WO2019207724A1 - Blood component separation device, blood component separation method, and blood component analysis method - Google Patents

Abstract

A blood component separation device including a first flow channel (10) provided with a first introduction port (12) and a filter part (11) in which a filter for separating blood cells and blood plasma is installed, and a second flow channel (20) connected to the filter part of the first flow channel.

Description

Blood component separation device, blood component separation method, and blood component analysis method

The present invention relates to a blood component separation device, a blood component separation method, and a blood component analysis method.

When a blood test is performed, the blood cell component and the plasma component are usually separated and analyzed separately. Examples of the method for separating the blood cell component and the plasma component include a centrifugal separation method and a method using a blood cell separation filter.
However, in the centrifugal separation method, the centrifugal operation is complicated. As a blood cell separation filter, for example, Patent Document 1 describes a glass fiber filter for blood filtration and the like. However, the conventional method using a blood cell separation filter cannot sufficiently prevent the mixing of plasma components into the blood cell fraction.

JP 2006-38512 A

One embodiment of the present invention includes a first flow path including a first introduction port into which a solution containing blood cells and plasma is introduced, and a filter unit in which a filter for capturing blood cells is installed, and the first flow A blood component separation device including a second flow path connected to the filter portion of the path. The second flow path may have a second introduction port, and may intersect the first flow path at the filter portion of the first flow path.

One embodiment of the present invention is a method for separating blood components using the blood component separation device, wherein (a) a solution containing blood cells and plasma is introduced from the first inlet into the first. Introducing into the flow path, causing the filter to capture blood cells in the solution, and moving the plasma in the solution in the first flow path; and (b) from the first inlet or the second inlet. Introducing a hemolytic agent, hemolyzing blood cells in the filter, and moving blood cell components released by hemolysis in the second flow path.

One embodiment of the present invention is a method for analyzing blood components using the blood component separation device, wherein (a) a solution containing blood cells and plasma is introduced from the first inlet into the first. A step of introducing into the flow channel and causing the filter to capture blood cells in the solution; and (b) after the step (a), the cleaning liquid is supplied from the first introduction port or the second introduction port. A step of removing the plasma in the filter by introducing into the passage, and (c) after the step (b), introducing a hemolytic agent from the first inlet or the second inlet, and blood cells in the filter And (d) analyzing the blood cells hemolyzed in the step (c).

One embodiment of the present invention is a method for analyzing hemoglobin A1c, wherein (a) a filter for separating blood cells and plasma captures blood cells in a solution containing blood cells and plasma; and (b) ) After the step (a), passing the washing solution through the filter and removing the plasma in the filter; (c) after the step (b), introducing a hemolytic agent into the filter; And hemolyzing A1c in the blood cells hemolyzed in the step (c), and (d) analyzing the hemoglobin A1c in the blood cells hemolyzed in the step (c).

It is a mimetic diagram showing an example of a blood ingredient separation device concerning one embodiment of the present invention. It is a schematic diagram which shows the modification 1 of the blood component separation device which concerns on this invention. It is a schematic diagram which shows the modification 2 of the blood component separation device which concerns on this invention. It is a schematic diagram which shows the modification 3 of the blood component separation device which concerns on this invention. It is a schematic diagram which shows the modification 4 of the blood component separation device which concerns on this invention. It is a schematic diagram which shows the modification 5 of the blood component separation device which concerns on this invention. It is a schematic diagram which shows the modification 6 of the blood component separation device which concerns on this invention. (A) And (b) is sectional drawing which shows an example of the structure of the pump parts P1 and P2 of the blood component separation device 700 of FIG. (A)-(d) is sectional drawing explaining operation | movement of an example of the pump part of FIG. It is sectional drawing which shows an example of the structure of the two-color molding valve | bulb applicable to the pump parts P1 and P2 of the blood component separation device 700 of FIG. It is sectional drawing which shows an example of the structure of the two-color molding valve | bulb applicable to the pump parts P1 and P2 of the blood component separation device 700 of FIG. In Experimental example 2, it is a graph which shows the result of having evaluated the influence of the plasma content which has on the measured value of HbA1c. FIG. 6 is a schematic diagram showing the structure of a blood component separation device used in Experimental Examples 3 to 7. In Experimental example 3, it is a graph which shows the result of having measured the plasma in a blood cell fraction. In Experimental example 4, it is a graph which shows the result of having measured the plasma in a blood cell fraction. In Experiment 5, it is a photograph of each blood component separation device after hemolyzing and collecting blood cells after washing the filter with 20 μL, 25 μL or 30 μL of washing solution. In Experimental Example 6, it is a graph which shows the result of having evaluated the reproducibility of the measured value of HbA1c. In Experimental Example 7, it is a graph which shows the result of having evaluated the reproducibility of the HbA1c measured value.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant description is omitted. Note that the dimensional ratio in each drawing is exaggerated for the sake of explanation, and does not necessarily match the actual dimensional ratio.

[Blood component separation device]
In one embodiment, the present invention is connected to a first flow path that includes a first inlet and a filter section in which a filter that separates blood cells and plasma is installed, and to the filter section of the first flow path. A blood component separation device including a second flow path.

FIG. 1 is a schematic diagram showing an example of a blood component separation device of the present embodiment. The blood component separation device 100 includes a first channel 10, a second channel 20, a third channel 30, a plasma analyzer 50, and a blood cell analyzer 60. The first flow path 10 has a first introduction port 12 and a filter unit 11. The second flow path 20 and the third flow path 30 are connected to the filter part 11 of the first flow path 10 and intersect the first flow path 10 at the filter part 11.

The first flow path 10, the second flow path 20, and the third flow path 30 may be formed of, for example, a tube such as a glass tube or a resin tube, and a groove is formed between two bonded plates. A flow path may be formed by forming.
The width and height of the first channel 10, the second channel 20, and the third channel 30 are not particularly limited, and may be arbitrarily selected according to the type of sample. For example, the width and height of these channels can be set to such a size that the sample can pass through, and examples thereof include 1 μm or more, 10 μm or more, 50 μm or more, 100 μm or more. The widths of the first flow path 10, the second flow path 20, and the third flow path 30 may be the same or different. For example, when a whole blood sample is used, the diameter of the first channel 10 can be 50 μm or more, 80 μm or more, 100 μm or more, and the like. The upper limits of the width and height of these channels are not particularly limited, and may be appropriately selected according to the size of the blood component separation device. Examples of the upper limit values of the width and height of these channels include 100 mm or less, 50 mm or less, 10 mm or less, 5 m or less, 2 mm or less, and the like. An example is a width of about 0.5 to 2.0 mm and a height of about 0.2 mm to 2.0 mm.
The shapes of the first flow path 10, the second flow path 20, and the third flow path 30 are not particularly limited, and the cross section of the flow path may be circular or rectangular.

(First flow path)
The first flow path 10 includes a first inlet 12, a filter unit 11, and a valve V1.

The first inlet 12 introduces a solution containing blood cells and plasma into the first flow path 10. Moreover, it may be used to introduce other samples, reagents, and the like. The first inlet 12 may have a funnel shape so that the sample can be easily introduced.

The filter unit 11 is a part where a filter for capturing blood cells is installed in the flow path. The filter that captures blood cells selectively captures blood cells in the blood and separates them from components other than blood cells in the blood, including plasma. Therefore, it is possible to separate blood cells and plasma in blood. The filter is not particularly limited as long as it has a material and a structure capable of capturing blood cells and allowing plasma to pass through, and a known blood cell separation filter can be used.
Examples of the filter include a porous film and a glass fiber film. Examples of the filter material include polypropylene, polyethylene, polyethylene terephthalate, polybutylene terephthalate, polystyrene, polyvinyl alcohol, urethane, acrylic, rayon, and glass.
Examples of the filter include, for example, a fiber in which the above-described material is integrated in a nonwoven fabric, a molded body such as an open-cell foam in which continuous pores are formed using the above-described material, and a substantially spherical fine particle formed from the above-described material. Are integrated so as to form a close-packed structure, or are integrally molded by sintering the accumulated material, or are formed by forming a through-hole in a film formed from the above material, or on one or both sides of the film. Examples include a laminate of a large number of sheets that are subjected to corrugated discharge or press working.
The average pore diameter of the filter is not limited so long as it captures blood cells and does not inhibit the passage of plasma, and examples thereof include 2 to 10 μm or 3 to 8 μm. The average pore diameter is measured by a bubble point test method (JIS K 3832), an actual measurement method using an enlarged image by an electron microscope, or the like.
The porosity of the filter is not particularly limited, and examples thereof include 20 to 97% and 30 to 95% in order to ensure a practical filtration time and maintain the filter stability.

The valve V1 is for controlling the first flow path 10 in an open state or a closed state. The valve V <b> 1 is disposed on the opposite side of the first introduction port 12 with respect to the filter unit 11 in the first flow path 10. In the 1st flow path 10, the 1st inlet 12, the filter part 11, and the valve | bulb V1 are located in this order. At this time, the first inlet 12 is on the upstream side of the first flow path 10, and the valve portion V1 is on the downstream side. The solution introduced from the first introduction port 12 passes through the filter unit 11 and flows toward the valve unit V1. The structure of the valve V1 is not particularly limited, and any valve used for a fluid device or the like can be used. As an example, the same valve as that constituting the pump parts P1 and P2 in FIG.
For example, the valve V <b> 1 is disposed in the vicinity of the downstream end of the filter unit 11. In order to reduce the dead volume, for example, the valve V1 and the filter unit 11 are preferably in contact with each other. Alternatively, even when the valve V1 and the filter unit 11 are spaced apart from each other, the distance between the end of the valve V1 and the end of the filter unit 11 is at most about several millimeters. Is preferred. The distance between the end of the valve V1 and the end of the filter unit 11 is about 0 to 10 mm, and is about 0 to 5 mm. By disposing the valve V <b> 1 in the vicinity of the filter unit 11, the blood cell fraction hemolyzed by the hemolysis process described later can be efficiently collected via the second flow path 20 or the third flow path 30.
Note that the blood component separation device of the present embodiment may be configured without the valve V1. In addition, when the first introduction port 12 and the filter unit 11 are arranged apart from each other, a valve may be provided between the first introduction port 12 and the filter unit 11.

The opposite side of the first channel 10 from the first inlet 12 with respect to the filter unit 11 is connected to the plasma analysis unit 50. The plasma analysis unit 50 is for analyzing the plasma that has passed through the filter unit 11. The plasma analysis unit 50 can be configured to analyze any plasma component according to the purpose. For example, the plasma analysis unit 50 may be a glucose analysis unit. The plasma analysis unit 50 analyzes total cholesterol, LDL, HDL, TG, UA, blood creatinine, albumin, total protein, ALT, AST, γGTP, BUN, K ion, Na ion, Ca ion, Cl ion, and the like. It may be a thing. The plasma analysis unit 50 may analyze BV, HCV, CRP, cTnT, cTnI, BNP, H-FAB, CK-MB, IL-6, etc., and analyze tumor markers, miRNA, etc. You may do. The plasma analysis unit 50 may analyze a single item, or may analyze a plurality of items. When the blood component separation device includes the plasma analysis unit 50, the plasma component separated by the filter unit 11 can be directly analyzed in the device. Since the plasma component is sent from the filter unit 11 to the plasma analysis unit 50 through the flow path on the blood component separation device, there is no risk of contamination or contamination of the apparatus, and simple plasma. Analysis of components becomes possible. In addition, since the plasma component can be measured directly from the separation of blood cell components, the loss of non-specific measurement objects is eliminated, the accuracy is improved, and the examination time is further shortened.

The blood component separation device of the present embodiment may be provided with a plasma analysis unit 50 and / or a plasma recovery unit. Alternatively, the blood component separation device of the present embodiment may be configured to provide a plasma recovery unit instead of the plasma analysis unit 50 and recover the plasma that has passed through the filter unit 11. The collected plasma may be analyzed manually or using a plasma analyzer or the like, and may be stored for later analysis. Moreover, it is good also as a structure which provides a plasma collection | recovery part with the plasma analysis part 50. FIG. In this case, the plasma that has been analyzed by the plasma analysis unit 50 can be collected by the plasma collection unit.

The first flow path 10 may further include a fluid control unit that controls the flow of fluid in the flow path. When the 1st flow path 10 is provided with a fluid control part, as an example, a fluid control part is arrange | positioned with respect to a filter part on the opposite side to a 1st inlet. In this case, since the filter unit 11 is disposed between the first introduction port 12 and the fluid control unit, the speed at which the sample introduced into the first flow path from the first introduction port 12 passes through the filter unit 11 is increased. It can be controlled by the fluid control unit.
The fluid control unit may employ any configuration used for controlling the flow of fluid in the flow path by a fluid device or the like. Examples of the fluid control unit include an intake port, a pump, and a valve connected to the intake pump.
In the first flow path 10, the fluid moves in the axial direction of the flow path from upstream to downstream. This direction may be referred to as the first direction or the axial direction of the first flow path. The first direction is a direction from the first inlet 12 toward the filter unit 11, and when the valve V1 is provided, the first direction is a direction from the filter unit 11 toward the valve V1.

In addition, the first flow path 10 may further include a waste liquid recovery unit that recovers the cleaning liquid in a cleaning process described later.

(Second flow path)
The second flow path 20 is connected to the filter unit 11 of the first flow path 10 in a direction different from the axial direction of the first flow path 10. The second flow path 20 intersects the first flow path 10 at the filter unit 11. The second flow path 20, the second introduction port 22, and valves V <b> 2 a and V <b> 2 b disposed on both sides sandwiching the connecting portion between the first flow path 10 are provided.

The angle at which the second flow path 20 intersects the first flow path 10 is not particularly limited, and may be an arbitrary angle. As an example, the second flow path 20 is orthogonal to the first flow path 10. As an example, the second flow path 20 and the first flow path 10 are disposed on substantially the same plane. By arranging these flow paths on substantially the same plane, the blood component separation device can be miniaturized.

The second inlet 22 is used to introduce a reagent such as a hemolytic agent into the second flow path 20. The second inlet 22 may have a funnel shape so that a reagent or the like can be easily introduced. Further, the second introduction port 22 may be configured to be connected to a chemical solution port or the like (not shown).

The valves V2a and V2b are for controlling the second flow path 20 to an open state or a closed state. The structure of the valves V2a and V2b is not particularly limited, and any valve used for a fluid device or the like can be used. As an example, a valve similar to the above-described valve V1 may be employed.
The valves V2a and V2b are arranged in the vicinity of the filter unit 11 as an example. In order to reduce the dead volume, for example, the valves V2a and V2b are preferably in contact with the filter unit 11. Alternatively, even when the valves V2a, V2b and the filter unit 11 are spaced apart, the distance between the ends of the valves V2a, V2b and the filter unit 11 is a few millimeters at most. It is preferable that it is a grade. The distance between the ends of the valves V2a and V2b and the end of the filter unit 11 is about 0 to 10 mm, and is about 0 to 5 mm. By disposing the valves V2a and V2b in the vicinity of the filter unit 11, blood components in the filter unit 11 can be efficiently separated.
Note that the blood component separation device of the present embodiment may be configured not to provide the valves V2a and V2b, or may be configured to provide only one of the valves V2a and V2b. By providing the valve V2a, the valve V2a is closed while the blood cell component and the plasma component are separated in the first flow path 10, and the valve V2a is released after the separation reaction between the blood cell component and the plasma component. It becomes possible. This is effective when a reagent is introduced into the second inlet 22 in advance. In addition, by providing the valve V2b, it is possible to retain a reagent or the like in the second flow path 20. This is effective when it takes time to react the components in the filter unit 11 with the reagents.

The side of the second flow path 20 opposite to the second introduction port 22 with respect to the filter unit 11 may be closed or opened. Alternatively, it may be connected to a waste liquid recovery unit or the like.
In the second flow path 20, the fluid moves in a second direction different from the first direction. The second direction is a direction different from the axial direction of the first flow path, and is the axial direction of the second flow path. The second direction is a direction from the second introduction port 22 toward the filter unit 11.

(Third flow path)
The third flow path 30 is connected to the filter unit 11 of the first flow path 10 in a direction different from the axial direction of the first flow path 10. The third flow path 30 intersects the first flow path 10 at the filter unit 11 at a position different from the second flow path 20. In the first flow path 10, the first introduction port 12, the connection portion with the second flow path 20, and the connection portion with the third flow path 30 are arranged in this order. Moreover, in the 1st flow path 10, the connection part with the 1st inlet 12, the 3rd flow path 30, and the 2nd flow path 20 may be arrange | positioned in this order. The third flow path 30 includes valves V3a and V3b that are arranged on both sides of the connection portion with the first flow path 10.

The angle at which the third flow path 30 intersects the first flow path 10 is not particularly limited, and can be any angle. The second channel 20 and the third channel may intersect the first channel 10 at substantially the same angle. As an example, the third flow path 30 is orthogonal to the first flow path 10. Further, as an example, the third flow path 30 is disposed on substantially the same plane as the first flow path 10 and the second flow path 20. By arranging these flow paths on substantially the same plane, the blood component separation device can be miniaturized.

The valves V3a and V3b are for controlling the third flow path 30 to an open state or a closed state. The valves V3a and V3b are disposed on both sides of the connection portion with the first flow path 10. In the third flow path 30, the valve V3a, the connection portion with the first flow path 10, and the valve V3b are arranged in this order. The structure of the valves V3a and V3b is not particularly limited, and any valve used for a fluid device or the like can be used. As an example, valves similar to the above-described valves V1, V2a, and V2b may be employed.
The valves V3a and V3b are arranged in the vicinity of the filter unit 11 as an example. In order to reduce the dead volume, for example, the valves V3a and V3b and the filter unit 11 are preferably in contact with each other. Alternatively, even when the valves V3a, V3b and the filter unit 11 are spaced apart, the distance between the ends of the valves V3a, V3b and the filter unit 11 is a few millimeters at most. It is preferable that it is a grade. The distance between the ends of the valves V3a and V3b and the end of the filter unit 11 is about 0 to 10 mm, and is about 0 to 5 mm. By disposing the valves V3a and V3b in the vicinity of the filter unit 11, blood components in the filter unit 11 can be efficiently separated.
Note that the blood component separation device of the present embodiment may be configured not to provide the valves V3a and V3b, or may be configured to provide only one of the valves V3a and V3b. By providing the valve V3a, the valve V3a is closed while the blood cell component and the plasma component are separated in the first flow path 10, and the valve V3a is released after the separation reaction between the blood cell component and the plasma component. It becomes possible. In addition, by providing the valve V3b, it is possible to retain a reagent or the like in the third flow path 30. This is effective when it takes time to react the components in the filter unit 11 with the reagents.

The third flow path 30 is connected to the blood cell analysis unit 60. The blood cell analyzer 60 is for analyzing the blood cells captured by the filter unit 11. As will be described later, the blood cells captured by the filter unit 11 are hemolyzed by a hemolyzing agent or the like, and are carried to the blood cell analysis unit 60 via the third flow path 30. The blood cell analyzer 60 can be configured to analyze any blood cell component according to the purpose. For example, the blood cell analysis unit 60 may be configured to analyze hemoglobin A1c (HbA1c) (HbA1c measurement unit). As an example, the blood cell analysis unit 60 includes a configuration for measuring HbA1c by a latex agglutination method. The blood cell analyzer 60 may have a configuration for measuring the amount of hemoglobin. In addition, the blood cell analyzer 60 may have a configuration for measuring the activity of erythrocyte enzymes, typified by glucose 6-phosphate dehydrogenase and pyruvate kinase. The blood cell analysis unit 60 may analyze a single item, or may analyze a plurality of items.
Since the blood component separation device includes the blood cell analysis unit 60, the blood cell component separated by the filter unit 11 can be directly analyzed in the device. Since the blood cell component is fed from the filter unit 11 to the blood cell analysis unit 60 via the flow path on the blood component separation device, there is no risk of contamination and the blood cell component can be easily analyzed. .

The blood component separation device of the present embodiment may include a blood cell analysis unit 60 and / or a blood cell collection unit. The blood component separation device of the present embodiment may have a configuration in which a blood cell collection unit is provided instead of the blood cell analysis unit 60 and the blood cell component captured by the filter unit 11 and hemolyzed is collected. The collected blood cell components may be analyzed using a manual operation, a blood cell analyzer (such as a HbA1c measuring device), or the like, or may be stored for later analysis. Moreover, it is good also as a structure which provides a blood cell collection | recovery part with the blood cell analysis part 60. FIG. In this case, the blood cell component that has been analyzed by the blood cell analysis unit 60 can be collected by the blood cell collection unit.

The third flow path 30 may further include a fluid control unit that controls the flow of fluid in the flow path. Since the third flow path 30 includes the fluid control unit, the flow of fluid from the filter unit 11 to the third flow path 30 can be controlled.
The fluid control unit may employ any configuration used for controlling the flow of fluid in the flow path by a fluid device or the like. Examples of the fluid control unit include an intake port, a pump, and a valve connected to the intake pump.

The other end of the third flow path 30 that is not connected to the blood cell analyzer 60 may be closed or open. Alternatively, it may be connected to a waste liquid recovery unit or the like.
In the third flow path 30, the fluid moves in a third direction different from the first direction. The third direction is the axial direction of the third flow path 30. The third direction is a direction from the filter unit 11 toward the blood cell analysis unit 60 or the blood cell collection unit. The second flow path 20 and the third flow path 30 may be substantially parallel. In this case, the second direction and the third direction are substantially the same.

(how to use)
An example of how to use the blood component separation device 100 having the above configuration will be described.

First, the valve V1 is opened, and the valves V2a, V2b, V3a, V3b are closed. In this state, a blood sample is introduced into the first channel 10 from the first inlet 12. The blood sample is not particularly limited as long as it contains a blood cell component and a plasma component, and an example is a whole blood sample. The blood sample may be a blood sample obtained by removing white blood cells and the like.

The blood sample introduced into the first flow path 10 from the first introduction port 12 passes through the filter unit 11. At this time, blood cells are selectively captured by the filter. Most of the plasma passes through the filter. Some plasma may remain on the filter. When the first flow path 10 includes a fluid control unit, the flow of the blood sample passing through the filter unit 11 may be controlled by the fluid control unit. By controlling the flow of the blood sample, the blood sample passing speed in the filter unit 11 can be shortened.

The plasma that has passed through the filter unit 11 is analyzed in the plasma analysis unit 50 connected to the first flow path 10. In the case where the blood cell component separation device includes a plasma recovery unit instead of the plasma analysis unit 50, the plasma may be recovered in the plasma recovery unit.

Next, the cleaning liquid is introduced into the first flow path 10 from the first introduction port 12. The washing solution is not particularly limited as long as it does not destroy blood cells, and a known cell washing solution or the like can be used. Examples of the washing liquid include phosphate buffered saline (PBS), physiological saline, and the like. Thereby, the plasma remaining in the filter unit 11 can be removed. When the blood component separation device 100 includes a fluid control unit, the flow of the cleaning liquid that passes through the filter unit 11 may be controlled by the fluid control unit. By controlling the flow of the cleaning liquid, the cleaning of the filter unit 11 can be shortened.
The washing solution that has washed the filter unit may be collected by the plasma analysis unit 50, and if the first flow path 10 includes a plasma collection unit or a waste solution collection unit, they may be collected by these.

The above-described cleaning process may be performed by opening the valve V2a and introducing the cleaning liquid from the second introduction port 22. The cleaning liquid introduced from the second introduction port 22 is introduced into the filter unit 11 via the second flow path 20. The plasma remaining in the filter unit 11 can be removed by the washing liquid introduced into the filter unit 11 as described above.

Alternatively, when one end of the third flow path 30 having the valve V3a is connected to the waste liquid recovery unit, the valve V1 is closed, the valve V3a is opened, and the cleaning liquid is supplied from the first introduction port 12. It may be introduced. In this case, the plasma remaining in the filter unit 11 can be collected together with the washing solution in the waste liquid collecting unit connected to the third channel 30 via the third channel 30.
Alternatively, the valves V2a and V3a may be opened and the cleaning liquid may be introduced from the second introduction port 22 into the filter unit 11 via the second flow path. Also in this case, the plasma remaining in the filter unit 11 can be collected together with the washing solution in the waste liquid collecting unit connected to the third channel 30 via the third channel 30.

Next, the valves V1, V2b and V3a are closed, and the valves V2a and V3b are opened. In this state, a hemolytic agent is introduced into the second flow path 20 from the second introduction port 22. Any hemolytic agent may be used as long as it can destroy blood cells (for example, erythrocytes), and any known hemolytic agent can be used without particular limitation. Examples of the hemolytic agent include hypotonic solution, water and the like. The hemolytic agent introduced from the second introduction port 22 reaches the filter unit 11 via the second flow path 20, and the filter unit 11 is in the same direction as the direction in which the blood sample has moved through the filter unit 11 (first direction). To move. At this time, blood cells (for example, red blood cells) captured by the filter are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis moves through the filter unit 11 together with the hemolytic agent, and moves to the third channel 30 when it reaches the connection with the third channel 30.

When the second flow path 20 or the third flow path 30 includes a fluid control unit, the fluid control unit causes the hemolytic agent to flow from the second flow path to the filter unit 11 and the third flow from the filter unit 11 to the third flow. The flow of blood cell components to the path 30 may be controlled. By controlling these flows, the time required for the blood cell component to reach the blood cell analyzer 60 can be shortened.

The blood cell component moved from the filter unit 11 to the third flow path 30 is analyzed in the blood cell analysis unit 60 connected to the third flow path 30. When the blood cell component separation device includes a blood cell collection unit instead of the blood cell analysis unit 60, the blood cell component may be collected by the blood cell collection unit.

The hemolysis step may be performed by closing the valves V1, V2a, V2b, and V3a, opening the valve V3b, and introducing the hemolytic agent from the first introduction port 12. The hemolytic agent introduced from the first introduction port 12 hemolyzes the blood cells captured by the filter unit 11, and the released blood cell components are sent to the blood cell analysis unit 60 via the third flow path in the same manner as described above. And move.

As described above, by using the blood component separation device of the present embodiment, the plasma and blood cells can be separated and then each collected or analyzed. In particular, when the blood component separation device includes both the plasma analysis unit 50 and the blood cell analysis unit 60, plasma analysis and blood cell analysis can be performed simultaneously with one device, so that blood analysis can be performed quickly and easily. It can be performed. Since the blood cell separation part (filter part) and the plasma analysis part, and the blood cell separation part (filter part) and the blood cell analysis part are connected by the flow path, there is an advantage that there is no risk of contamination.

In addition, the conventional blood cell separation method cannot prevent the plasma from being mixed into the blood cell fraction, and the analysis result of the blood cell component may become unstable due to the plasma being mixed. For example, as shown in an example described later, when HbA1c is measured with a blood cell sample in which plasma is present, the measurement value becomes unstable, and a reliable measurement value cannot be obtained.
In the blood component separation device according to the present embodiment, after blood cells are captured by the filter, the plasma remaining on the filter can be removed by the washing liquid, so that the amount of plasma mixed into the blood cell fraction can be reduced. As a result, the influence of plasma in blood cell component analysis can be reduced, and stable blood cell component measurement values can be obtained.

The blood component separation device of the present embodiment is not limited to the aspect described above, and each component can be changed as appropriate without departing from the spirit of the present invention. For example, the following modifications 1 to 6 are also within the scope of the blood component separation device of the present embodiment.

<Modification 1>
FIG. 2 is a schematic diagram showing Modification 1 of the blood component separation device of the present embodiment. Blood component separation device 200 has the same configuration as blood component separation device 100 except that it does not have third flow path 30. The side of the second channel 20 opposite to the second inlet 22 with respect to the connection with the first channel is connected to the blood cell analyzer 60.

In the blood component separation device 200, in the hemolysis step of hemolyzing the blood cells captured by the filter unit 11, the valve V1 is closed, the valves V2a and V2b are opened, and the second flow path 20 is opened from the second introduction port 22. Introduce a hemolytic agent. The hemolytic agent introduced into the second flow path 20 reaches the filter unit 11 via the second flow path 20, and a direction (second direction) different from the direction (first direction) in which the blood sample moves through the filter unit 11. The filter unit 11 is moved in the direction). The blood cells captured in the second direction in which the hemolytic agent moves are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis is transported from the filter unit 11 to the blood cell analysis unit 60 via the second flow path 20 and analyzed by the blood cell analysis unit 60.

The second flow path 20 may include a blood cell collection unit instead of the blood cell analysis unit 60 or in addition to the blood cell analysis unit 60. The second flow path 20 may include a fluid control unit that controls the flow of fluid in the second flow path 20. The same applies to Modifications 4 and 5 described later.

<Modification 2>
FIG. 3 is a schematic diagram showing Modification Example 2 of the blood component separation device of the present embodiment. The blood component separation device 300 has the same configuration as the blood component separation device 100 except that the second flow path 20 and the third flow path 30 do not intersect the first flow path 10. In the blood component separation device 300, the second flow path 20 and the third flow path 30 are configured to protrude from the filter unit 11, respectively.
In the blood component separation device 300, in the hemolysis step of hemolyzing the blood cells captured by the filter unit 11, the valve V1 is closed, the valves V2a and V3a are opened, and the second flow path 20 is opened from the second introduction port 22. Introduce a hemolytic agent. The hemolyzed hemolytic agent introduced into the second flow path 20 reaches the filter unit 11 via the second flow path 20 and is in the same direction as the direction in which the blood sample has moved through the filter unit 11 (first direction). The filter unit 11 is moved. At this time, blood cells captured by the filter are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis moves through the filter unit 11 together with the hemolytic agent, and moves to the third channel 30 when it reaches the connection with the third channel 30. Then, it is carried to the blood cell analyzer 60 via the third flow path 30 and analyzed by the blood cell analyzer 60.

<Modification 3>
FIG. 4 is a schematic diagram showing a third modification of the blood component separation device of the present embodiment. The blood component separation device 400 has the same configuration as the blood component separation device 300 except that the second flow path 20 and the third flow path 30 are arranged in the same direction with respect to the first flow path 10. .
The hemolysis process can be performed in the same manner as the blood component separation device 300.

<Modification 4>
FIG. 5 is a schematic diagram showing a fourth modification of the blood component separation device of the present embodiment. The blood component separation device 500 does not have the third flow path 30, and the second flow path 20 does not intersect the first flow path 10. The second flow path 20 is connected to the blood cell analyzer 60.

In the blood component separation device 500, in the hemolysis step of hemolyzing the blood cells captured by the filter unit 11, the valve V1 is closed, the valve V2a is opened, and the first introduction port 12 is connected to the first flow path 10. Introduce hemolytic agent. The hemolytic agent introduced into the first channel 10 reaches the filter unit 11 and moves the filter unit 11 in the same direction as the direction in which the blood sample has moved the filter unit 11 (first direction). At this time, blood cells captured by the filter are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis moves through the filter unit 11 together with the hemolytic agent, and moves to the second channel 20 when it reaches the connection with the second channel 20. Then, it is carried to the blood cell analyzer 60 via the second flow path 20 and analyzed in the blood cell analyzer 60.

The second flow path 20 may include a blood cell collection unit instead of the blood cell analysis unit 60 or in addition to the blood cell analysis unit 60. The second flow path 20 may include a fluid control unit that controls the flow of fluid in the second flow path 20.

<Modification 5>
FIG. 6 is a schematic diagram showing a fifth modification of the blood component separation device of the present embodiment. The blood component separation device 600 does not have the third flow path 30 and the second flow path 20 does not intersect the first flow path 10. The second flow path 20 is connected to the blood cell analyzer 60. Further, the fourth flow path 40 is connected between the first introduction port 12 of the first flow path 10 and the filter unit 11. The fourth flow path 40 includes a fourth introduction port 42 and a valve V4a.

In the blood component separation device 600, in the hemolysis step of hemolyzing the blood cells captured by the filter unit 11, the valve V1 is closed, the valves V2a and V4a are opened, and the fourth flow path 40 is opened from the fourth inlet 42. Introduce a hemolytic agent. The hemolyzed hemolytic agent introduced into the fourth flow path 40 reaches the first flow path 10 through the fourth flow path 40, moves through the first flow path 10, and reaches the filter unit 11. In the filter unit 11, the hemolytic agent moves through the filter unit 11 in the same direction as the direction in which the blood sample moves through the filter unit 11 (first direction). At this time, blood cells captured by the filter are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis moves through the filter unit 11 together with the hemolytic agent, and moves to the second channel 20 when it reaches the connection with the second channel 20. Then, it is carried to the blood cell analyzer 60 via the second flow path 20 and analyzed in the blood cell analyzer 60.

<Modification 6>
FIG. 7 is a schematic diagram showing Modification 6 of the blood component separation device of the present embodiment. The blood component separation device 700 has a first flow path and a second flow path formed in the plate 70. The first flow path is composed of first flow paths 10a and 10b, and the second flow path is composed of second flow paths 20a, 20b and 20c.

The first flow paths 10a and 10b are connected to each other via a valve V1a.
The first flow path 10 a has a first introduction port 12 and a filter unit 11.
The first channel 10b includes a pump unit P1 as a channel controller and a plasma analyzer 50. The first flow path 10b is connected to the inlet I1 through the valve V1f. The inlet I1 is connected to a reagent reservoir (not shown) disposed below the plate 70. Therefore, the reagent can be introduced from the inlet I1 into the first flow path 10b by opening the valve V1f. The first flow path 10b is connected to the waste liquid recovery unit W1 via the valve V1c or V1d. Therefore, the waste liquid can be discharged from the first flow path 10b to the waste liquid recovery part W1 by opening the valve V1c or V1d.
The first flow path 10b forms a circulation flow path (first circulation path) by closing the valves V1a, V1c, V1d, and V1f and opening the valves V1b and V1e.
In addition, the first flow path 10b and the waste liquid collection unit W1 close the valves V1a, V1b, and V1f, and open the valves V1c, V1d, and V1e, thereby allowing fluid to be discharged from the first flow path 10b. A flow path (first discharge path) for discharging to W1 is formed.

The second flow paths 20a and 20b are connected to each other via a valve V2e. The second flow paths 20b and 20c are connected to each other via valves V2k and V2m.
The second flow path 20 a has a second introduction port 22. The second inlet 22 is connected to a hemolytic agent reservoir (not shown) disposed below the plate 70, and the hemolytic agent can be introduced into the second flow path 20 a from the second inlet 22. The second flow path 20a is connected to the filter portion 11 of the first flow path 10a and intersects the first flow path 10a. Valves V2a and V2b are installed on both sides of the connection portion with the first flow path 10a in the second flow path 20a. When the valves V2a and V2b are opened and the hemolytic agent is introduced from the second inlet 22 into the second flow path 20a, the hemolytic agent moves in the axial direction of the second flow path 20a and passes through the filter unit 11. . The second flow path 20a is connected to the inlet I2 via the valve V2d. The inlet I2 is connected to a reagent reservoir (not shown) disposed below the plate 70. Therefore, the reagent can be introduced from the inlet I2 into the second flow path 20a by opening the valve V2d. Furthermore, the reagent can be introduced into the second flow path 20b by opening the valve V2e. Further, the second flow path 20a is connected to the air inlet A via a valve V2c. Therefore, air can be introduced from the air inlet A into the second flow path 20a by opening the valve V2e. Furthermore, air can be introduced into the second flow path 20b by opening the valve V2e.
The second flow path 20b includes a pump unit P2 as a fluid control unit and a blood cell analysis unit 60. The second flow path 20b is connected to the waste liquid recovery unit W2 via the valve V2h or V2i. Therefore, the waste liquid can be discharged from the second flow path 20b to the waste liquid recovery part W2 by opening the valve V2h or V2i.
The second flow path 20c is connected to the inlet I3 through the valve V2n. The inlet I3 is connected to a reagent reservoir (not shown) disposed below the plate 70. Therefore, the reagent can be introduced from the inlet I3 into the second flow path 20c by opening the valve V2n. Furthermore, the reagent can be introduced into the second flow path 20b by opening the valve V2k or the valve V2m. The second flow path 20c is connected to the waste liquid recovery unit W2 via the valve V2j. Therefore, the waste liquid can be discharged from the second flow path 20c to the waste liquid recovery unit W2 by opening the valve V2j.
The second flow path 20b forms a circulation flow path (second circulation path) by closing the valves V2e, V2h, V2i, V2k, V2m and opening the valves V2f, V2g, V2l. In addition, the second flow paths 20b and 20c are configured so that the valves V2e, V2h, V2i, V2j, V2l, and V2n are closed, and the valves V2f, V2g, V2k, and V2m are opened, so that the circulation flow path (third (Circulation path) is formed.
In addition, the second flow path 20b and the waste liquid collection unit W2 close the valves V2e, V2g, V2k, and V2m and open the valves V2f, V2h, V2i, and V2l, thereby allowing fluid to flow into the second flow path 20b. A flow path (second discharge path) for discharging from the waste liquid to the waste liquid recovery unit W2 is formed. In addition, the second flow paths 20b and 20c and the waste liquid recovery unit W2 close the valves V2e, V2g, V2i, V2k, V2l, and V2n and open the valves V2f, V2h, V2j, and V2m, A flow path (third discharge path) for discharging the fluid from the second flow paths 20b and 20c to the waste liquid recovery unit W2 is formed.

The pump parts P1 and P2 are composed of three valves. Examples of the valve include a diaphragm valve including a diaphragm member, and examples of the diaphragm member include an elastomer material.

8A and 8B are cross-sectional views for explaining an example of the structure of the diaphragm valve.
8A shows the opened state of the diaphragm valve 800, and FIG. 8B shows the closed state of the diaphragm valve 800. As shown in FIGS. 8A and 8B, the diaphragm valve 800 includes a first substrate 310, a diaphragm member 330 made of an elastomer material, and a second substrate 320. The second substrate 320 and the diaphragm member 330 are bonded in close contact. The space between the first substrate 310 and the diaphragm member 330 forms a flow path 315 through which a fluid flows. In addition, a through hole 340 is provided in part of the second substrate 320. Further, the diaphragm member 330 is exposed in the through hole 340.

The diaphragm valve 800 is arranged in the flow path 315 (the first flow path 10b or the second flow path 20b in the blood component separation device 700), and adjusts the flow of fluid inside the flow path 315.

In the opened state of the diaphragm valve 800 shown in FIG. 8A, fluid can flow through the flow path 315. On the other hand, as shown in FIG. 8B, when a valve control fluid is supplied from the through hole 340 of the diaphragm valve 800 and the inside of the through hole 340 is pressurized, the diaphragm member 330 is deformed, and the deformed diaphragm member Part of 330 is in close contact with the first substrate 310. This state is a closed state of the diaphragm valve 800. As a result, the flow of fluid inside the flow path 315 is blocked.

Here, as shown in FIGS. 8A and 8B, in order to make the closed state of the diaphragm valve 800 stronger, a convex portion is formed in the region of the first substrate 310 facing the through hole 340. 311 may be formed.

Examples of the valve control fluid include N2 gas, gas such as air, and liquid such as water and oil. The fluid for valve control can be supplied by, for example, a tube connected to the through hole 340.

The elastomer material forming the diaphragm member 330 is not particularly limited as long as it is a material that can be deformed in the axial direction of the through hole 340 in accordance with a change in pressure inside the through hole 340. For example, polydimethylsiloxane (PDMS) ), Silicone elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.

In the closed diaphragm valve 800 shown in FIG. 8B, when the pressure applied to the inside of the through-hole 340 is reduced, the deformed diaphragm member 330 returns to its original shape, and again shown in FIG. ). As a result, the fluid can flow through the flow path 315 again.

A pump can be formed by three or more valves, that is, by arranging three or more valves. FIGS. 9A to 9D are cross-sectional views illustrating the operation of an example of a pump including three diaphragm valves 800a, 800b, and 800c.

In the state shown in FIG. 9A, all of the three valves 800a, 800b, and 800c are in the open state. In this state, since the flow of the fluid inside the flow path 315 is not controlled, the fluid may flow on the right side or the left side as viewed in FIG. It may be stopped.

Subsequently, as shown in FIG. 9B, the valve 800a is controlled to be closed, and the valves 800b and 800c are left open. As a result, the fluid flow inside the flow path 315 is blocked by the valve 800a.

Subsequently, as shown in FIG. 9C, the valves 800a and 800b are controlled to be closed, and the valve 800c is left open. Here, in the process in which the valve 800b is changed from the open state to the closed state, the diaphragm member 330 is deformed, so that the fluid existing around the valve 800b is pushed away. However, since the valve 800a is in the closed state, the displaced fluid moves to the right in the direction indicated by the arrow in FIG. 9C, that is, FIG. 9C. As a result, the fluid flows in the direction indicated by the arrow.

Subsequently, as shown in FIG. 9D, the valves 800b and 800c are controlled to be closed.
Then, in the process in which the valve 800c changes from the open state to the closed state, the diaphragm member 330 is deformed, so that the fluid existing around the valve 800c is pushed away. However, since the valve 800a is in the closed state, the displaced fluid moves to the right side in the direction indicated by the arrow in FIG. 9D, that is, in FIG. 9D. As a result, the flow of the fluid in the direction indicated by the arrow is further accelerated. At this time, the valve 800a may be controlled to an open state as shown in FIG. 9D, or may be maintained in a closed state.

Subsequently, as shown in FIG. 9A again, the valves 800a, 800b and 800c are all controlled to be opened. Here, the fluid inside the flow path 315 may continue to move to the right as viewed in FIG. 9A due to inertia.

Furthermore, the flow of fluid inside the flow path 315 can be controlled by repeating the above steps.

The above process is an example of a method for controlling a pump including three valves, and the method for controlling a pump including three valves is not limited thereto. For example, in the valve control described above, the flow of the fluid inside the flow path 315 can be controlled in the opposite direction to that described above by reversing the timing of opening and closing the valves 800a and 800c. It is also possible to control the flow rate by adjusting the cycle in which the operations in FIGS. 9A to 9D are repeated to form a pulsed minute solution flow. The flow velocity can also be controlled by adjusting the pressure of the gas driving the valves 800a to 800c and the diameter of the valve.

It is also possible to control the flow of fluid inside the flow path 315 with a pump having four or more valves.

Also, the structure of the diaphragm valve is not limited to that described above. For example, as a diaphragm valve, a cylindrical structure having an outer cylinder part and an inner cylinder part described in International Publication No. 2016/006615, and a thin film part arranged to cover one end of the inner cylinder part, A valve provided with a diaphragm member having a circumference of the thin film portion and an anchor portion closely attached along the inner wall of the outer cylinder portion and the outer wall of the inner cylinder portion may be used.

In the blood component separation device of the present embodiment, other usable valves include, for example, a two-color molded valve obtained by continuously molding a resin portion and an elastomer portion using two molds. Illustrated. The two-color molded valve has advantages that the time required for production is short and the adhesiveness between the resin and the elastomer is high.

Specific examples of the two-color molded valve are illustrated in FIGS. In the valve 900 illustrated in FIG. 10, the substrate 909 includes a recess (eg, a flow path) 940A formed on a lower surface (one surface) 909a, a bottom of the recess 940A (eg, an upper side of the recess 90A in FIG. 10), and the substrate 909. And an opening 952 opening (penetrating) in the upper surface (other surface) 909b. A valve driven unit 970 is provided in the opening 952. The valve driven part 970 is made of the same soft material as the valve part 950 described above, and includes a valve part (deformation part) 971 and a cylinder part (connection part) 973. The valve portion 971 closes the lower surface 909 a side of the substrate 909 in the opening 952. For example, the tubular portion 973 is formed of a single member with the valve portion 971, is provided along the inner peripheral surface of the opening portion 952, and is integrally connected to the valve portion 971 at the lower end. The inner space of the cylinder portion 973 is closed at the lower end by the valve portion 971 and forms an opening 970a having an upper end opened. For example, when the depression 90A in FIG. 10 is a flow path, a fluid (eg, a solution containing a sample substance, a cleaning liquid, etc.) flows from the left side to the right side of the drawing.

The lower surface 909a of the substrate 909 is joined to the upper surface 908b of the lower plate 908. A curved surface (eg, hemispherical) concave surface 980 is formed on the upper surface 908b at a position facing the depression 940A.

The valve driven unit 970 having the above-described configuration is configured such that, for example, when a downward force (eg, air pressure, hydraulic pressure, mechanical force, etc.) is applied to the valve unit 971 through the opening 970a, The open / close state of the flow path (eg, the depression 940A) is controlled by the deformation of 971. As an example, as shown in FIG. 11, the valve portion 971 is deformed and bent toward the recess 940 </ b> A and contacts the concave surface 980 to close the flow path (e.g., the recess 940 </ b> A) (the valve is closed). Further, the valve driven portion 970 is released from the downward force applied to the valve portion 971, so that the deformation (eg, bending) of the valve portion 971 is eliminated and the flow path ( For example, the recess 940A) is opened (the valve is open).

The above-described two-color molded valve can be manufactured, for example, by the method described in International Publication No. 2018/012429. In addition, as the two-color molded valve, known ones such as those described in International Publication No. 2018/012429 can be used without particular limitation.

An example of how to use the blood component separation device 700 having the above-described configuration will be described.

First, the valves V1a, V1b, and V1e are opened, and the other valves are closed, and a blood sample (eg, a whole blood sample or the like) is introduced from the first inlet 12 into the first flow path 10a. The blood sample introduced into the first flow path 10a reaches the filter unit 11, and moves the filter unit 11 in the direction of the valve V1a (first direction). At this time, blood cells in the blood sample are captured by the filter. On the other hand, most of the plasma passes through the filter unit 11 and reaches the first flow path 10b. Here, when the valve V1a is closed and the pump unit P1 is operated as appropriate, the plasma that has reached the first flow path 10b can be circulated through the first circulation path including the first flow path 10b. If necessary, a reagent for plasma analysis is introduced from the inlet I1, and circulated through the first circulation path to be mixed with plasma. The plasma analysis unit 50 can perform plasma analysis while circulating the plasma in the first circulation path. The fluid circulating in the first circulation path can be discharged to the waste liquid recovery unit W1 through the first discharge path with the valves V1b and V1f being closed and the valves V1c, V1d and V1e being opened as necessary. .

Next, the valves V1a, V1c, V1d, and V1e are opened, the other valves are closed, and the cleaning liquid is introduced from the first introduction port 12 into the first flow path 10a. The cleaning liquid introduced into the first flow path 10a reaches the filter unit 11, and moves the filter unit 11 in the valve V1a direction (first direction). At this time, the plasma remaining on the filter moves in the first direction together with the washing liquid, and is introduced into the first flow path 10b through the valve V1a. The washing liquid containing plasma introduced into the first flow path 10b can be discharged to the waste liquid collecting part W1 through the first discharge path with the valves 1b and V1f closed and the valves V1c, V1d and V1e opened. .

Next, the valves V2a, V2b, V2e, V2f, V2g, V2l are opened, the other valves are closed, and the hemolytic agent is introduced from the second inlet 22 into the second flow path 20a. The hemolytic agent introduced into the second flow path 20a reaches the filter unit 11 and moves the filter unit 11 in the valve V2b direction (second direction). At this time, the blood cells captured by the filter are hemolyzed by the hemolytic agent. The blood cell component released by hemolysis moves in the second flow path 20a along with the hemolytic agent in the axial direction, and is introduced from the valve V2b to the second flow path 20b via the valve V2e. Here, the valve V2b is closed, the valve V2c is opened, and air is blown into the second flow path 20a from the air inlet A. It can guide to the flow path 20b. The blood cell component introduced into the second flow path 20b can be circulated through the second circulation path including the second flow path 20b by closing the valve V2e and appropriately operating the pump part P2. If necessary, a reagent for blood cell analysis is introduced from inlet I2, and circulated through the second circulation path to be mixed with blood cell components. Alternatively, if necessary, a reagent for blood cell analysis is introduced from the inlet I3 and circulated through the third circulation path including the second flow paths 20b and 20c to be mixed with the blood cell component. The blood cell analysis unit 60 can perform blood cell analysis while circulating the blood cell component in the second circulation path or the third circulation path. The fluid circulating through the second circulation path or the third circulation path can be discharged to the waste liquid recovery unit W2 through the second discharge path or the third discharge path as necessary.

[Blood component separation method]
In one embodiment, the present invention provides a method for separating blood components using the blood component separation device of the above embodiment. The method of this embodiment includes (a) introducing a blood sample from the first introduction port into the first flow path, and causing the filter to capture blood cells in the blood sample; and (b) the first A hemolysis step of introducing a hemolytic agent from the introduction port or the second introduction port and hemolyzing the blood cells in the filter.

The blood component separation method of the present embodiment can be performed in the same manner as the method exemplified in “(Usage method)” described in the above “[Blood component separation device]”.
As an example, in the method of the present embodiment, after the step (a) and before the step (b), (c) the cleaning liquid is supplied from the first introduction port or the second introduction port. A plasma removal step of introducing into the channel and removing the plasma in the filter.

[Blood component analysis method]
In one embodiment, the present invention provides a method for analyzing blood components using the blood component separation device of the above embodiment. The method of this embodiment includes (a) introducing a blood sample from the first introduction port into the first flow path, and causing the filter to capture blood cells in the blood sample; and (b) the step ( After a), a step of introducing a washing liquid into the first flow path from the first introduction port or the second introduction port to remove plasma in the filter, and (c) after the step (b) Introducing a hemolytic agent from the first inlet or the second inlet and hemolyzing the blood cells in the filter; (d) analyzing the blood cells hemolyzed in the step (c); including.

The blood component analysis method of the present embodiment can be performed in the same manner as the method exemplified in “(Usage method)” described in “[Blood component separation device]” above. When the blood component separation device including the blood cell analysis unit 60 is used in the method of the present embodiment, the blood cell analysis unit 60 can analyze the blood cell component in the step (d). In the case of using a blood component separation device that does not include the blood cell analysis unit 60, it is only necessary to collect the hemolyzed blood cell component from the blood component separation device and analyze the collected blood cell component.

In addition to the steps (a) to (d), the method of the present embodiment further includes (d) a step of analyzing the plasma that has passed through the filter after the step (a) or the step (b). May be included. When using a blood component separation device including the plasma analysis unit 50, the plasma analysis can be performed by the plasma analysis unit 50. In the case of using a blood component separation device that does not include the plasma analysis unit 50, it is only necessary to collect the hemolyzed plasma from the blood component separation device and analyze the collected plasma.

[Method for analyzing hemoglobin A1c]
In one embodiment, the present invention provides a method for analyzing hemoglobin A1c. The method of this embodiment includes (a) a step of capturing blood cells in a blood sample with a filter that separates blood cells and plasma, and (b) a washing liquid is passed through the filter after the step (a), Removing the plasma in the filter; (c) after the step (b), introducing a hemolytic agent into the filter and hemolyzing the blood cells in the filter; and (d) in the step (c) Analyzing hemoglobin A1c in the hemolyzed blood cells.

The method of the present embodiment can be performed in the same manner as the method exemplified in “(Usage method)” described in “[Blood component separation device]” above. Further, the method of this embodiment may be performed using a column or the like equipped with a blood cell separation filter without using the blood component separation device of the above embodiment.

Analysis of HbA1c can be performed using a known HbA1c measurement method. Examples of a method for measuring hemoglobin A1c include latex agglutination.
In the latex agglutination method, latex particles are adsorbed with HbA1c (adsorption reaction), reacted with an anti-HbA1c antibody (mouse IgG or the like), and further reacted with a secondary antibody (anti-mouse IgG antibody or the like). Thereby, HbA1c is cross-linked by the anti-HbA1c antibody and the secondary antibody, and latex particles aggregate (aggregation reaction). The higher the HbA1c concentration, the greater the amount of latex particles aggregated. Non-aggregated latex particles do not reflect light, but when aggregated, light is reflected, so the amount of transmitted light varies depending on the amount of latex particles aggregated. Therefore, the HbA1c concentration can be measured by measuring the absorbance after the aggregation reaction. As an example, the HbA1c concentration can be calculated from a value obtained by subtracting the absorbance at 800 nm from the absorbance at 660 nm.

As shown in the examples described later, the measured value of HbA1c becomes unstable when plasma is present, and a highly reliable measured value cannot be obtained. In the method of this embodiment, blood cells are captured by the filter, and then the filter is washed with a washing solution to remove plasma in the filter. Thereby, the amount of plasma mixed in the blood cell component hemolyzed by the hemolytic agent can be greatly reduced. Therefore, a highly reliable HbA1c measurement value can be obtained.

As described above, the embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes design and the like within a scope not departing from the gist of the present invention.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1] Effect of plasma content on HbA1c measurement value 0 to HbA1c standard test reagent (HbA1c concentration 6.1%, HbA1c concentration 10%; hemoglobin A1c cut-off test certified reference material, laboratory medicine reference material mechanism) Human plasma was added so as to be 60% (v / v). The HbA1c concentration of the HbA1c standard test reagent to which human plasma was added was measured by a latex agglutination method using Lapidia (registered trademark) Auto HbA1c-L (Fujirebio). About each sample, the light absorbency of 660 nm and the light absorbency of 800 nm were measured, and the value of the light absorbency of 660 nm-light absorbency of 800 nm was calculated | required.
The results are shown in Table 1 and FIG.

When the plasma content was 15% (v / v) or less, the coefficient of variation (CV) of the standard test reagent with a HbA1c concentration of 10% was 2.0% or less, and the latex agglutination reaction was stable. Therefore, in order to stably measure HbA1c, it was confirmed that the plasma content in the sample is desirably 15% (v / v) or less.

[Experimental Example 2] Measurement of plasma components in blood cell fraction Cyra-added rabbit whole blood was prepared by adding 1 µL of Cy5 and mixing it with 1000 µL of rabbit whole blood to trace plasma components.
The Cy5-added rabbit whole blood sample was centrifuged (centrifugal force (1000 × g), 5 minutes) to separate a blood cell fraction and a plasma fraction. A dilution series of the blood cell fraction and the plasma fraction was prepared, and a calibration curve for the amount of hemoglobin and the amount of Cy5 was prepared using each dilution series.
Next, the blood cell separation of the Cy5-added rabbit whole blood sample was performed using a blood component separation device having the structure shown in FIG. 15 μL of Cy5-added rabbit whole blood sample is introduced into the first channel from the inlet (atmospheric pressure: 1 minute), and negative pressure is applied for 2 minutes at 5 kPa from the opposite side of the inlet to filter the Cy5-containing rabbit whole blood sample. Passed through. Thereafter, the ports b and d of the third flow path are closed, 100 μL of hypotonic solution (ultra pure water) is introduced from the port c of the second flow path, and the blood cell fraction captured by the filter is dissolved, The hemolyzed blood cell fraction was collected from port a. Similarly, the ports a and c of the second flow path are closed, 100 μL of hypotonic solution is introduced from the port d of the third flow path, the blood cell fraction captured by the filter is dissolved, and hemolysis is performed from the port b. The collected blood cell fraction was collected. The collected blood cell fraction was measured for hemoglobin absorbance and Cy5 fluorescence, and the plasma content in the blood cell fraction was calculated from the calibration curve prepared above.

The results are shown in FIG. When the hypotonic solution was introduced into the second channel and the blood cell fraction was collected, the plasma fraction content was 26% (v / v). On the other hand, when the hypotonic solution was introduced into the third channel and the blood cell fraction was collected, the plasma fraction content was 42% (v / v).

[Experimental Example 3] Measurement of plasma components in blood cell fraction (effect of washing)
Similar to Experimental Example 1, a Cy5-added rabbit whole blood sample was prepared, and the Cy5-added rabbit whole blood sample was separated using a blood component separation device having the structure shown in FIG. (Atmospheric pressure: 1 minute, 5 kPa negative pressure: 2 minutes). Next, 15 μL of PBS was introduced from the inlet into the first flow path (5 kPa negative pressure: 2 minutes), and the filter was washed. Thereafter, the ports b and d of the third flow path are closed, 100 μL of hypotonic solution is introduced from the port c of the second flow path, the blood cell fraction captured by the filter is dissolved, and hemolysis is performed from the port a. The collected blood cell fraction was collected. In the same manner as in Experimental Example 1, the plasma content in the collected blood cell fraction was calculated.

The results are shown in FIG. In FIG. 153, “no washing” is the case where PBS washing was not performed, and is the same as the case where the blood cell fraction was collected in the second flow path of Experimental Example 1. “With washing” was when PBS washing was performed, and the plasma component content was greatly reduced as compared with “without washing”.

[Experimental Example 4] Evaluation of the amount of filter washing liquid Cy5-added rabbit whole blood sample was prepared by adding 1 μL of Cy5 and mixing it with 9 μL of rabbit whole blood in order to trace plasma.
Next, the blood cell separation of the Cy5-added rabbit whole blood sample was performed using a blood component separation device having the structure shown in FIG. 10 μL of Cy5-added rabbit whole blood sample was introduced into the first channel from the inlet, and negative pressure was applied for 2 minutes at 10 kPa from the opposite side of the inlet, and the Cy5-containing rabbit whole blood sample was passed through the filter. Next, 20 μL, 25 μL, or 30 μL of PBS was introduced into the first channel from the inlet (10 kPa negative pressure: 2 minutes), and the filter was washed. Thereafter, the port a of the second channel and the port d of the third channel are closed, 50 μL of hypotonic solution is introduced from the port c of the second channel, and the blood cell fraction captured by the filter is dissolved. The hemolyzed blood cell fraction was collected from port b of the third flow path. When most of the blood fraction was pushed out, the introduction of hypotonic solution was stopped.

FIG. 16 is a photograph of each blood component separation device after the blood cell fraction was collected after washing the filter with each PBS amount. Comparing the color of the liquid immediately after passing through the filter (dotted oval), it was qualitatively shown that the blue component of Cy5 becomes lighter as the amount of cleaning liquid increases. When the blue portion of Cy5 was plasma, it was considered that most of the plasma in the filter was removed with a washing liquid volume of 25 μL.

[Experimental Example 5] Reproducibility evaluation of measured values of HbA1c (Example 1)
Using a blood component separation device having the structure shown in FIG. 11, blood cell separation of human refrigerated blood (normal human whole blood / type O, Kojin Bio) was performed. From the inlet, 10 μL of human chilled blood was introduced into the first flow path, negative pressure was applied at 10 kPa for 2 minutes from the opposite side of the inlet, and the human chilled blood was passed through the filter. Next, PBS 25 μL, 25 μL, or 30 μL was introduced into the first channel from the inlet (10 kPa negative pressure: 2 minutes), and the filter was washed. Thereafter, the port a of the second flow path and the port d of the third flow path are closed, 100 μL of hypotonic solution is introduced from the port c of the second flow path, and the blood cell fraction captured by the filter is dissolved. The hemolyzed blood cell fraction was collected from port b of the third flow path. When about 15 μL of the hemolyzed blood was extruded, the recovery of the hemolyzed was finished.
A latex agglutination method using Lapidia (registered trademark) Auto HbA1c-L was performed using 5 μL of the hemolyzed blood collected as described above, and absorbance at 660 nm and 800 nm was measured. From the measured absorbance, the HbAc1 concentration and the coefficient of variation (CV) were calculated.

(Comparative Example 1)
In a Cobas (registered trademark) c101 (Roche) cartridge, a latex agglutination inhibition method using 2.0 μL of human chilled blood was performed, and the HbAc1 concentration and coefficient of variation (CV) were calculated.

(Comparative Example 2)
In an Affinion (registered trademark) HbAic (Alere) cartridge, hemolysis of 1.5 μL of human chilled blood was performed. Thereafter, color development caused by HbA1c was measured by a boronic acid affinity method. From the measured values of color development, the HbAc1 concentration and the coefficient of variation (CV) were calculated.

(result)
The results of Example 1 and Comparative Examples 1 and 2 are shown in FIG. In Example 1, as compared with Comparative Examples 1 and 2, it was confirmed that the coefficient of variation (CV) was small and measurement with high reproducibility was possible.

[Experimental Example 6] Evaluation of reproducibility of measured values of HbA1c Blood cells of human whole blood (normal human whole blood / type O, Kojin Bio) were separated using a blood component separation device having the structure shown in FIG. From the inlet, 15 μL of human whole blood was introduced into the first channel, and negative pressure was applied for 2 minutes at 10 kPa from the opposite side of the inlet, and the standard test reagent was passed through the filter. Next, 25 μL of PBS was introduced from the introduction port into the first flow path (10 kPa negative pressure: 2 minutes), and the filter was washed. Thereafter, the port a of the second channel and the port d of the third channel are closed, 50 μL of hypotonic solution is introduced from the port c of the second channel, and the blood cell fraction captured by the filter is dissolved. The hemolyzed blood cell fraction was collected from port b of the third flow path. When about 50 μL of the hemolyzed blood was extruded, the recovery of the hemolyzed blood was finished.
Using the hemolyzed blood collected as described above, a latex agglutination method using Lapidia (registered trademark) Auto HbA1c-L was performed, and absorbance at 660 nm and 800 nm was measured. The coefficient of variation (CV) was calculated from the measured absorbance.
Further, the blood cell fraction of human whole blood was separated by centrifugal separation (centrifugal force (1000 × g), 5 minutes), and the absorbance at 660 nm and 800 nm was measured in the same manner.
In addition, when HbAc1 concentration of human whole blood was measured using Affinion (registered trademark) HbAic (Alere), the HbAc1 concentration was 5.06%.

Results are shown in FIG. In FIG. 18, a standard specimen (HbA1c concentration 6.1%, HbA1c concentration 10%; hemoglobin A1c cut-off test certified reference material, laboratory medicine reference material mechanism) was measured using Rapidia (registered trademark) Auto HbA1c-L. The results are also shown. In the blood cell fraction separated from the whole blood using the blood component separation device, the measured HbA1c value equivalent to the blood cell fraction obtained by the centrifugation method was obtained. Moreover, the coefficient of variation (CV) was small compared with the centrifugation method. Furthermore, the blood cell fraction separated using the blood component separation device had a lower measured value by the latex agglutination method than the standard specimen. Generally, the HbA1c concentration of normal human whole blood is 6% or less. Therefore, the reliability of the measured value by this method was shown by comparison with the measured value of the standard specimen (HbA1c concentration 6.1%, HbA1c concentration 10%).

10, 10a, 10b first flow path,
DESCRIPTION OF SYMBOLS 11 Filter part 12 1st inlet 20, 20, 20a, 20b, 20c 2nd flow path 22 2nd inlet 30 3rd flow path 40 4th flow path 42 4th inlet 50 Plasma analysis part 60 Blood cell analysis part 70 Plate 100 , 200, 300, 400, 500, 600, 700 Blood component separation device 310 First substrate 311, 311 a, 311 b, 311 c Convex portion 315 Channel 320 Second substrate 330 Diaphragm member 331 Anchor portion 340, 340 a, 340 b, 340c Through- hole 800, 800a, 800b, 800c Diaphragm valve V1, V1a to V1f, V2a to V2n Valve P1, P2 Pump part I1 to I3 Inlet W1, W2 Waste liquid recovery part A Air inlet

Claims (22)

1. A first flow path comprising: a first introduction port into which a solution containing blood cells and plasma is introduced; and a filter unit provided with a filter for capturing blood cells;
   A second flow path connected to the filter portion of the first flow path;
   A blood component separation device.
2. The blood component separation device according to claim 1, wherein the second flow path has a second introduction port, and intersects the first flow path at the filter portion of the first flow path.
3. The solution introduced from the first introduction port flows inside the filter portion in the axial direction of the first flow path,
   The blood component separation device according to claim 2, wherein the solution introduced from the second introduction port flows in the filter unit in a direction different from an axial direction of the first flow path.
4. A plasma analyzer connected to the first flow path;
   The blood component separation device according to any one of claims 1 to 3.
5. Further comprising a plasma collection unit,
   The blood component separation device according to any one of claims 1 to 4.
6. The first flow path is
   A fluid control unit for controlling the flow of fluid in the first flow path;
   The filter unit is disposed between the first introduction port and the fluid control unit,
   The blood component separation device according to any one of claims 1 to 5.
7. The blood component separation device according to claim 6, wherein the fluid control unit has an intake port, a pump, or a valve connected to an intake pump.
8. The first flow path includes at least one valve disposed on the side opposite to the first introduction port with the filter portion interposed therebetween.
   The blood component separation device according to any one of claims 1 to 7.
9. The second flow path includes at least one valve disposed in one or both flow paths sandwiching a connection portion with the first flow path.
   The blood component separation device according to any one of claims 2 to 8.
10. A third flow path connected to the filter portion of the first flow path;
    In the first flow path, the first introduction port, the connection portion with the second flow path, and the connection portion with the third flow path are arranged in this order.
    The blood component separation device according to any one of claims 1 to 9.
11. In the filter part,
    The connecting portion with the second flow path is disposed at one end of the filter portion,
    The blood component separation device according to claim 10, wherein a connection portion with the third flow path is disposed at the other end of the filter portion.
12. The blood component separation device according to claim 11, wherein the third flow path intersects the first flow path at the filter portion of the first flow path.
13. The blood component according to any one of claims 10 to 12, wherein the third flow path includes at least one valve disposed in one or both of the flow paths sandwiching a connection portion with the first flow path. Separation device.
14. The blood component separation device according to any one of claims 1 to 13, further comprising a blood cell analysis unit for analyzing hemolyzed blood cells.
15. The blood component separation device according to claim 14, wherein the blood cell analyzer is connected to the second flow path or the third flow path.
16. The blood component separation device according to claim 14 or 15, wherein the blood cell analysis unit is a hemoglobin A1c measurement unit that measures hemoglobin A1c.
17. Further comprising a blood cell collection unit,
    The blood component separation device according to any one of claims 1 to 16.
18. A method for separating blood components using the blood component separation device according to any one of claims 1 to 17, comprising:
    (A) A solution containing blood cells and plasma is introduced from the first introduction port into the first flow path, the blood cells in the solution are captured by the filter, and the plasma in the solution is transferred to the first flow path. Moving in
    (B) introducing a hemolytic agent from the first introduction port or the second introduction port, hemolyzing blood cells in the filter, and moving blood cell components released by hemolysis in the second flow path;
    Including a method.
19. (C) After the step (a) and before the step (b), a cleaning solution is introduced into the first flow path from the first introduction port or the second introduction port, and the plasma in the filter The method of claim 18, further comprising a plasma removal step of removing.
20. A method for analyzing a blood component using the blood component separation device according to any one of claims 1 to 16, comprising:
    (A) introducing a solution containing blood cells and plasma into the first channel from the first introduction port, and causing the filter to capture blood cells in the solution;
    (B) after the step (a), introducing a washing liquid into the first flow path from the first introduction port or the second introduction port, and removing plasma in the filter;
    (C) after the step (b), introducing a hemolytic agent from the first inlet or the second inlet, and hemolyzing the blood cells in the filter;
    (D) analyzing the blood cells hemolyzed in the step (c);
    Including a method.
21. (D) after the step (a) or the step (b), further comprising the step of analyzing the plasma that has passed through the filter,
    21. A method for analyzing a blood component according to claim 20.
22. A method for analyzing hemoglobin A1c, comprising:
    (A) capturing a blood cell in a solution containing the blood cell and plasma with a filter that separates the blood cell and plasma;
    (B) after the step (a), passing a washing solution through the filter to remove plasma in the filter;
    (C) after the step (b), introducing a hemolytic agent into the filter and hemolyzing the blood cells in the filter;
    (D) analyzing hemoglobin A1c in the blood cells hemolyzed in the step (c);
    Including a method.

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