JP6939988B2 - Blood component separation device, blood component separation method, and blood component analysis method - Google Patents

Description

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, it is usually separated into a blood cell component and a plasma component, and each is analyzed. As a method for separating the blood cell component and the plasma component, there are a centrifugal separation method, a method using a blood cell separation filter, and the like.
However, in the centrifugation method, the operation of centrifugation is complicated. Further, as the 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 has not been able to sufficiently prevent the contamination of plasma components into the blood cell fraction.

Japanese Unexamined Patent Publication No. 2006-38512

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, a filter portion provided with a filter for capturing blood cells, and the first flow. A blood component separation device, comprising a second flow path that connects to the filter portion of the pathway. 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.

Further, one embodiment of the present invention is a method of separating blood components using the blood component separation device, wherein (a) a solution containing blood cells and plasma is prepared from the first introduction port. A step of introducing blood cells into the flow path, causing the filter to capture blood cells in the solution, and moving plasma in the solution in the first flow path, and (b) from the first introduction port or the second introduction port. The method includes a step of introducing a hemolytic agent, hemolyzing the blood cells in the filter, and moving the blood cell component released by the hemolysis in the second flow path.

Further, one embodiment of the present invention is a method of analyzing a blood component using the blood component separation device, wherein (a) a solution containing blood cells and plasma is prepared from the first introduction port. After the step of introducing the blood cells into the flow path and causing the filter to capture blood cells in the solution, and (b) the step (a), the cleaning liquid is flowed from the first introduction port or the second introduction port. After the step of introducing into the tract and removing the plasma in the filter, and (c) the step (b), a hemolytic agent is introduced from the first introduction port or the second introduction port, and blood cells in the filter are introduced. The method comprises a step of hemolyzing the blood cells and (d) a step of analyzing the blood cells hemolyzed in the step (c).

Further, one embodiment of the present invention is a method for analyzing hemoglobin A1c, which comprises (a) a step of causing a filter for separating blood cells and plasma to capture blood cells in a solution containing blood cells and plasma, and (b). ) After the step (a), a washing solution is passed through the filter to remove plasma in the filter, and (c) after the step (b), a hemolytic agent is introduced into the filter, and the filter is used. It is a method including a step of hemolyzing the blood cells in the blood cells and (d) a step of analyzing hemoglobin A1c in the blood cells hemolyzed in the step (c).

It is a schematic diagram which shows an example of the blood component separation device which concerns on one Embodiment of this 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) are cross-sectional views showing an example of the structure of the pump portions P1 and P2 of the blood component separation device 700 of FIG. 7. (A) to (d) are cross-sectional views illustrating the operation of an example of the pump unit of FIG. It is sectional drawing which shows an example of the structure of the two-color molded valve applicable to the pump part 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 molded valve applicable to the pump part P1 and P2 of the blood component separation device 700 of FIG. It is a graph which shows the result of having evaluated the influence of the plasma content on the HbA1c measured value in Experimental Example 1. It is a schematic diagram showing a structure of a blood component separation device used in Experimental Example 2-6. It is a graph which shows the result of having measured the plasma in a blood cell fraction in Experimental Example 2. It is a graph which shows the result of having measured the plasma in a blood cell fraction in Experimental Example 3. In Experimental Example 4 , it is a photograph of each blood component separation device after washing the filter with a washing solution of 20 μL, 25 μL or 30 μL, and then hemolyzing and collecting blood cells. It is a graph which shows the result of having evaluated the reproducibility of the HbA1c measurement value in Experimental Example 5. It is a graph which shows the result of having evaluated the reproducibility of the HbA1c measurement value in Experimental Example 6.

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.

[Blood component separation device]
In one embodiment, the present invention connects to a first flow path including a first introduction port and a filter unit provided with a filter for separating blood cells and plasma, and the filter unit of the first flow path. Provided is a blood component separation device including a second flow path.

FIG. 1 is a schematic view showing an example of the blood component separation device of the present embodiment. The blood component separation device 100 includes a first flow path 10, a second flow path 20, a third flow path 30, a plasma analysis unit 50, and a blood cell analysis unit 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 portion 11 of the first flow path 10, and intersect with the first flow path 10 at the filter section 11.

The first flow path 10, the second flow path 20, and the third flow path 30 may be formed of, for example, a glass tube, a resin tube, or the like, and a groove is formed between the two bonded plates. The flow path may be formed by forming.
The width and height of the first flow path 10, the second flow path 20, and the third flow path 30 are not particularly limited and may be arbitrarily selected according to the type of sample. For example, the width and height of these flow paths can be set to a size that allows the sample to pass through, and examples thereof include 1 μm or more, 10 μm or more, 50 μm or more, 100 μm or more, and the like. 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 flow path 10 may be 50 μm or more, 80 μm or more, 100 μm or more, or the like. The upper limit of the width and height of these flow paths is 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 flow paths include 100 mm or less, 50 mm or less, 10 mm or less, 5 m or less, 2 mm or less, and the like. As an example, a width of about 0.5 to 2.0 mm and a height of about 0.2 mm to 2.0 mm can be mentioned.
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 flow path cross section may be circular or rectangular.

(1st flow path)
The first flow path 10 includes a first introduction port 12, a filter unit 11, and a valve V1.

The first introduction port 12 introduces a solution containing blood cells and plasma into the first flow path 10. It may also be used to introduce other samples, reagents, etc. The first introduction port 12 may have a funnel shape so that a sample can be easily introduced.

The filter unit 11 is a portion where a filter for capturing blood cells is installed in the flow path. A filter that captures blood cells selectively captures blood cells in the blood and separates them from non-blood cell components in the blood, including plasma. Therefore, it becomes possible to separate blood cells and plasma in blood. The filter is not particularly limited as long as it has a material and structure that captures blood cells and allows plasma to pass through, and a known blood cell separation filter can be used.
Examples of the filter include a porous membrane and a glass fiber membrane. Examples of the filter material include polypropylene, polyethylene, polyethylene terephthalate, polybutylene terephthalate, polystyrene, polyvinyl alcohol, urethane, acrylic, rayon, glass and the like.
Examples of the filter include a non-woven fabric in which fibers obtained by molding the above material are integrated, a molded body such as an open cell foam in which continuous pores are formed by using the material, and substantially spherical fine particles obtained by molding the material. Are integrated so as to form a finely packed structure, or are integrally molded by sintering the integrated material, through holes are formed in a film formed from the above material, or on one or both sides of the film. Examples thereof include a stack of a large number of sheets that have been textured by corona discharge or press processing.
The average pore size of the filter may be such that it captures blood cells and does not obstruct the passage of plasma, and examples thereof include 2 to 10 μm and 3 to 8 μm. The average pore size is measured by a bubble point test method (JIS K 3832) or an actual measurement method using an enlarged image with an electron microscope.
The porosity of the filter is not particularly limited, and examples thereof include 20 to 97% and 30 to 95% in order to secure a practical filtration time and maintain the stability of the filter.

The valve V1 is for controlling the first flow path 10 to be in an open state or a closed state. The valve V1 is arranged in the first flow path 10 on the side opposite to the first introduction port 12 with respect to the filter portion 11. In the first flow path 10, the first introduction port 12, the filter unit 11, and the valve V1 are arranged in this order. At this time, the first introduction port 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 valves as those constituting the pump units P1 and P2 in FIG. 7, which will be described later, can be used.
As an example, the valve V1 is arranged near the downstream end of the filter unit 11. In order to reduce the dead volume, for example, it is preferable that the valve V1 and the filter portion 11 are in contact with each other. Alternatively, even when the valve V1 and the filter portion 11 are arranged apart from each other, the distance between the end portion of the valve V1 and the end portion of the filter portion 11 is at most several millimeters. Is preferable. The distance between the end of the valve V1 and the end of the filter portion 11 is about 0 to 10 mm, and is about 0 to 5 mm. By arranging the valve V1 in the vicinity of the filter unit 11, the blood cell fraction hemolyzed by the hemolysis step described later can be efficiently collected via the second flow path 20 or the third flow path 30.
The blood component separation device of the present embodiment may be configured not to be provided with the valve V1. Further, when the first introduction port 12 and the filter portion 11 are arranged apart from each other, a valve may be provided between the first introduction port 12 and the filter portion 11.

The side of the first flow path 10 with respect to the filter unit 11 opposite to the first introduction port 12 is connected to the plasma analysis unit 50. The plasma analysis unit 50 is for analyzing plasma that has passed through the filter unit 11. The plasma analysis unit 50 can be configured to be able to analyze any plasma component according to the purpose. For example, the plasma analysis unit 50 may be a glucose analysis unit. 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. Further, the plasma analysis unit 50 may analyze BV, HCV, CRP, cTnT, cTnI, BNP, H-FAB, CK-MB, IL-6 and the like, and analyze tumor markers, miRNA and the like. It may be something to do. The plasma analysis unit 50 may analyze a single item or a plurality of items. Since the blood component separation device includes the plasma analysis unit 50, the plasma components separated by the filter unit 11 can be analyzed as they are in the device. Since plasma components are sent from the filter unit 11 to the plasma analysis unit 50 via the flow path on the blood component separation device, there is no risk of contamination or contamination of the device, and simple plasma is used. The components can be analyzed. In addition, since the plasma component can be measured directly from the separation of the blood cell component, the loss of the non-specific measurement object is eliminated, the accuracy is improved, and the examination time is shortened.

The blood component separation device of the present embodiment may be provided with a plasma analysis unit 50 and / or a plasma collection unit. Alternatively, the blood component separation device of the present embodiment may be configured to provide a plasma collection unit instead of the plasma analysis unit 50 and collect plasma that has passed through the filter unit 11. The collected plasma may be analyzed manually or by using a plasma analyzer or the like, or may be stored for later analysis or the like. Further, a plasma collection unit may be provided together with the plasma analysis unit 50. 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 first flow path 10 includes a fluid control unit, as an example, the fluid control unit is arranged on the side opposite to the first introduction port with respect to the filter unit. In this case, since the filter unit 11 is arranged 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. , Can be controlled by the fluid control unit.
The fluid control unit can adopt any configuration used for controlling the flow of fluid in the flow path in a fluid device or the like. Examples of the fluid control unit include an intake port, a pump, a valve, and the like 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 a first direction or an axial direction of the first flow path. The first direction is a direction from the first introduction port 12 toward the filter unit 11, and when the valve V1 is provided, the direction is from the filter unit 11 toward the valve V1.

Further, the first flow path 10 may further include a waste liquid recovery unit or the like for recovering the cleaning liquid in the cleaning step described later.

(Second flow path)
The second flow path 20 is connected to the filter portion 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 portion 11. It includes valves V2a and V2b arranged on both sides of the connection portion between the second flow path 20, the second introduction port 22, and the first flow path 10.

The angle at which the second flow path 20 intersects with the first flow path 10 is not particularly limited and may be any angle. As an example, the second flow path 20 is orthogonal to the first flow path 10. Further, as an example, the second flow path 20 and the first flow path 10 are arranged 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 introduction port 22 is used to introduce a reagent such as a hemolytic agent into the second flow path 20. The second introduction port 22 may have a funnel shape so that reagents and 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 be in 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 valve V1 described above may be adopted.
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, it is preferable that the valves V2a and V2b are in contact with the filter portion 11. Alternatively, even when the valves V2a and V2b and the filter portion 11 are arranged apart from each other, the distance between the end portions of the valves V2a and V2b and the end portion of the filter portion 11 is at most several millimeters. It is preferably about. The distance between the end portions of the valves V2a and V2b and the end portion of the filter portion 11 is about 0 to 10 mm, and is about 0 to 5 mm. By arranging the valves V2a and V2b in the vicinity of the filter unit 11, blood components can be efficiently separated in the filter unit 11.
The blood component separation device of the present embodiment may be configured not to be provided with valves V2a and V2b, or may be configured to be provided with only one of valves V2a and V2b. By providing the valve V2a, the valve V2a can be closed while the blood cell component and the plasma component are separated in the first flow path 10, and the valve V2a can be released after the separation reaction between the blood cell component and the plasma component. It will be possible. This is effective when the reagent is introduced into the second introduction port 22 in advance. Further, by providing the valve V2b, it becomes possible to retain the reagent and the like in the second flow path 20. This is effective when it takes time for the reaction between the component in the filter unit 11 and the reagent.

The side of the second flow path 20 opposite to the second introduction port 22 with respect to the filter portion 11 may be closed or open. 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 channel)
The third flow path 30 is connected to the filter portion 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 portion 11 at a position different from that of 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. Further, in the first flow path 10, the first introduction port 12, the connection portion with the third flow path 30, and the connection portion with the second flow path 20 may be arranged in this order. The third flow path 30 includes valves V3a and V3b arranged on both sides of the connection portion with the first flow path 10.

The angle at which the third flow path 30 intersects with the first flow path 10 is not particularly limited and may be any angle. The second flow path 20 and the third flow path may intersect the first flow path 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 arranged 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 be in an open state or a closed state. The valves V3a and V3b are arranged 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, the same valves as the above-mentioned valves V1, V2a, V2b may be adopted.
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, it is preferable that the valves V3a and V3b are in contact with the filter portion 11. Alternatively, even when the valves V3a and V3b and the filter portion 11 are arranged apart from each other, the distance between the end portions of the valves V3a and V3b and the end portion of the filter portion 11 is at most several millimeters. It is preferably about. The distance between the end portions of the valves V3a and V3b and the end portion of the filter portion 11 is about 0 to 10 mm, and is about 0 to 5 mm. By arranging the valves V3a and V3b in the vicinity of the filter unit 11, blood components can be efficiently separated in the filter unit 11.
The blood component separation device of the present embodiment may be configured not to be provided with valves V3a and V3b, or may be configured to be provided with only one of valves V3a and V3b. By providing the valve V3a, the valve V3a can be closed while the blood cell component and the plasma component are separated in the first flow path 10, and the valve V3a can be released after the separation reaction between the blood cell component and the plasma component. It will be possible. Further, by providing the valve V3b, it becomes possible to retain the reagent or the like in the third flow path 30. This is effective when it takes time for the reaction between the component in the filter unit 11 and the reagent.

The third flow path 30 is connected to the blood cell analysis unit 60. The blood cell analysis unit 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 hemolytic agent or the like and transported to the blood cell analysis unit 60 via the third flow path 30. The blood cell analysis unit 60 can be configured to be able to analyze any blood cell component according to the purpose. For example, the blood cell analysis unit 60 may have a configuration capable of analyzing 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 analysis unit 60 may have a configuration for measuring the amount of hemoglobin. Further, the blood cell analysis unit 60 may have a configuration for measuring the activity of an erythrocyte enzyme represented by glucose 6-phosphate dehydrogenase, pyrupine kinase and the like. The blood cell analysis unit 60 may analyze a single item or a plurality of items.
Since the blood component separation device includes the blood cell analysis unit 60, the blood cell components separated by the filter unit 11 can be analyzed as they are in the device. Since the blood cell component is sent 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 like, and simple analysis of the blood cell component becomes possible. ..

The blood component separation device of the present embodiment may be provided with a blood cell analysis unit 60 and / or a blood cell collection unit. The blood component separation device of the present embodiment may be configured to provide a blood cell collection unit instead of the blood cell analysis unit 60 and collect the blood cell component captured and hemolyzed by the filter unit 11. The collected blood cell components may be analyzed manually or by using a blood cell growth analyzer (HbA1c measuring device or the like), or may be stored for later analysis or the like. Further, a blood cell collection unit may be provided together with the blood cell analysis unit 60. 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. When the third flow path 30 includes a 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 can adopt any configuration used for controlling the flow of fluid in the flow path in a fluid device or the like. Examples of the fluid control unit include an intake port, a pump, a valve, and the like connected to the intake pump.

The other end of the third flow path 30 that is not connected to the blood cell analysis unit 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, and in this case, the second direction and the third direction are substantially the same.

(how to use)
A method of using the blood component separation device 100 having the above configuration will be described with reference to an example.

First, the valve V1 is opened, and the valves V2a, V2b, V3a, and V3b are closed. In this state, the blood sample is introduced into the first flow path 10 from the first introduction port 12. The blood sample is not particularly limited as long as it contains a blood cell component and a plasma component, and an example thereof is a whole blood sample. Further, the blood sample may be a whole blood sample from which leukocytes and the like have been removed.

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 in the filter. When the first flow path 10 has a fluid control unit, the fluid control unit may control the flow of the blood sample passing through the filter unit 11. By controlling the flow of the blood sample, the passing speed of the blood sample in the filter unit 11 can be shortened.

The plasma that has passed through the filter unit 11 is analyzed by the plasma analysis unit 50 connected to the first flow path 10. When the blood cell component separation device includes a plasma recovery unit instead of the plasma analysis unit 50, the plasma may be collected by the plasma collection 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 cleaning solution include Phosphate buffered saline (PBS), physiological saline and the like. As a result, the plasma remaining in the filter unit 11 can be removed. When the blood component separation device 100 has a fluid control unit, the fluid control unit may control the flow of the washing liquid passing through the filter unit 11. By controlling the flow of the cleaning liquid, the cleaning of the filter unit 11 can be shortened.
The cleaning liquid obtained by washing 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 liquid collection unit, the cleaning liquid may be collected by these.

The above cleaning step 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 in the same manner 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 discharged 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 liquid in the waste liquid collection unit connected to the third flow path 30 via the third flow path 30.
Further, the valves V2a and V3a may be opened, and the cleaning liquid may be introduced into the filter unit 11 from the second introduction port 22 via the second flow path. Also in this case, the plasma remaining in the filter unit 11 can be collected together with the washing liquid in the waste liquid collection unit connected to the third flow path 30 via the third flow path 30.

Next, the valves V1, V2b and V3a are closed and the valves V2a and V3b are opened. In this state, the hemolytic agent is introduced into the second flow path 20 from the second introduction port 22. The hemolytic agent may be any one capable of destroying blood cells (for example, red blood cells), and can be used without particular limitation with known hemolytic agents. 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 blood sample moves in the filter unit 11 (first direction). To move. At this time, the 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 in the filter section 11 together with the hemolyzing agent, and when it reaches the connection portion with the third flow path 30, it moves to the third flow path 30.

When the second flow path 20 or the third flow path 30 has a fluid control unit, the flow of the hemolytic agent from the second flow path to the filter unit 11 and the flow from the filter unit 11 to the third flow by the fluid control unit. The flow of blood cell components to the pathway 30 may be controlled. By controlling these flows, it is possible to shorten the time for the blood cell component to reach the blood cell analysis unit 60.

The blood cell component that has moved from the filter unit 11 to the third flow path 30 is analyzed by 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 above hemolysis step may be performed by closing the valves V1, V2a, V2b, and V3a, opening the valve V3b, and introducing the hemolyzing agent from the first introduction port 12. The hemolytic agent introduced from the first introduction port 12 hemolyzes the blood cells captured in the filter unit 11, and the released blood cell component is 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, plasma and blood cells can be separated and then 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 unit (filter unit) and the plasma analysis unit, and the blood cell separation unit (filter unit) and the blood cell analysis unit are connected by a flow path, there is an advantage that there is no risk of contamination.

Further, the conventional blood cell separation method cannot prevent the contamination of plasma into the blood cell fraction, and the contamination of the plasma may cause the analysis result of the blood cell component to become unstable. For example, as shown in Examples described later, when HbA1c is measured in a blood cell sample in which plasma is present, the measured value becomes unstable and a reliable measured value cannot be obtained.
In the blood component separation device of the present embodiment, after the blood cells are captured by the filter, the plasma remaining in the filter can be removed by the washing solution, so that the amount of plasma mixed in the blood cell fraction can be reduced. This makes it possible to reduce the influence of plasma in the blood cell component analysis and obtain a stable blood cell component measurement value.

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

<Modification example 1>
FIG. 2 is a schematic view showing a modification 1 of the blood component separation device of the present embodiment. The blood component separation device 200 has the same configuration as the blood component separation device 100 except that it does not have the third flow path 30. The side of the second flow path 20 opposite to the second introduction port 22 with respect to the connection part with the first flow path is connected to the blood cell analysis unit 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 in the closed state, the valves V2a and V2b are in the open state, and the second flow path 20 is transmitted 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 the direction (second direction) different from the direction in which the blood sample moves in the filter unit 11 (first direction). The filter unit 11 is moved in the direction). Blood cells trapped in the second direction in which the hemolyzing agent moves are hemolyzed by the hemolyzing agent. The blood cell component released by hemolysis is carried from the filter unit 11 to the blood cell analysis unit 60 via the second flow path 20, and is analyzed by the blood cell analysis unit 60.

The second flow path 20 may include a blood cell collection unit in place of the blood cell analysis unit 60 or in addition to the blood cell analysis unit 60. Further, 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 the modified examples 4 and 5 described later.

<Modification 2>
FIG. 3 is a schematic view showing a modification 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 with 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 in the closed state, the valves V2a and V3a are in the open state, and the second flow path 20 is transmitted 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 blood sample moves in the filter unit 11 (first direction). The filter unit 11 is moved. 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 filter section 11 together with the hemolyzing agent, and when it reaches the connection portion with the third flow path 30, it moves to the third flow path 30. Then, it is carried to the blood cell analysis unit 60 via the third flow path 30 and analyzed by the blood cell analysis unit 60.

<Modification example 3>
FIG. 4 is a schematic view showing a modification 3 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 step can be performed in the same manner as in the blood component separation device 300.

<Modification example 4>
FIG. 5 is a schematic view showing a modification 4 of the blood component separation device of the present embodiment. The blood component separation device 500 does not have a third flow path 30, and the second flow path 20 does not intersect with the first flow path 10. The second flow path 20 is connected to the blood cell analysis unit 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 and the valve V2a is opened, from the first introduction port 12 to the first flow path 10. Introduce a hemolytic agent. The hemolytic agent introduced into the first flow path 10 reaches the filter unit 11 and moves the filter unit 11 in the same direction as the blood sample has moved the filter unit 11 (first 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 filter section 11 together with the hemolyzing agent, and when it reaches the connection portion with the second flow path 20, it moves to the second flow path 20. Then, it is carried 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 in place of the blood cell analysis unit 60 or in addition to the blood cell analysis unit 60. Further, 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 view showing a modification 5 of the blood component separation device of the present embodiment. The blood component separation device 600 does not have a third flow path 30, and the second flow path 20 does not intersect with the first flow path 10. The second flow path 20 is connected to the blood cell analysis unit 60. Further, a 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 and the valves V2a and V4a are opened, and the fourth inlet 42 to the fourth flow path 40. Introduce a hemolytic agent. The hemolyzed hemolyzing agent introduced into the fourth flow path 40 reaches the first flow path 10 via 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 the filter unit 11 in the same direction as the blood sample moves in the filter unit 11 (first 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 filter section 11 together with the hemolyzing agent, and when it reaches the connection portion with the second flow path 20, it moves to the second flow path 20. Then, it is carried to the blood cell analysis unit 60 via the second flow path 20 and analyzed by the blood cell analysis unit 60.

<Modification 6>
FIG. 7 is a schematic view showing a modification 6 of the blood component separation device of the present embodiment. In the blood component separation device 700, a first flow path and a second flow path are formed in the plate 70. The first flow path is composed of the first flow paths 10a and 10b, and the second flow path is composed of the 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 10a has a first introduction port 12 and a filter portion 11.
The first flow path 10b includes a pump unit P1 as a flow path control unit and a plasma analysis unit 50. The first flow path 10b is connected to the inlet I1 via a valve V1f. The inlet I1 is connected to a reagent reservoir (not shown) located below the plate 70. Therefore, by opening the valve V1f, the reagent can be introduced from the inlet I1 into the first flow path 10b. The first flow path 10b is connected to the waste liquid recovery unit W1 via a valve V1c or V1d. Therefore, by opening the valve V1c or V1d, the waste liquid can be discharged from the first flow path 10b to the waste liquid recovery unit W1.
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.
Further, the first flow path 10b and the waste liquid recovery unit W1 collect the fluid from the first flow path 10b by closing the valves V1a, V1b and V1f and opening the valves V1c, V1d and V1e. 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 20a has a second introduction port 22. The second introduction port 22 is connected to a hemolytic agent storage portion (not shown) arranged below the plate 70, and the hemolytic agent can be introduced into the second flow path 20a from the second introduction port 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 of the second flow path 20a with the first flow path 10a. When the valves V2a and V2b are opened and the hemolytic agent is introduced into the second flow path 20a from the second introduction port 22, 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) located below the plate 70. Therefore, by opening the valve V2d, the reagent can be introduced into the second flow path 20a from the inlet I2. Further, by opening the valve V2e, the reagent can be introduced into the second flow path 20b. Further, the second flow path 20a is connected to the air introduction port A via the valve V2c. Therefore, by opening the valve V2e, air can be introduced into the second flow path 20a from the air introduction port A. Further, by opening the valve V2e, air can be introduced into the second flow path 20b.
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 a valve V2h or V2i. Therefore, by opening the valve V2h or V2i, the waste liquid can be discharged from the second flow path 20b to the waste liquid recovery unit W2.
The second flow path 20c is connected to the inlet I3 via the valve V2n. The inlet I3 is connected to a reagent reservoir (not shown) located below the plate 70. Therefore, by opening the valve V2n, the reagent can be introduced from the inlet I3 into the second flow path 20c. Further, by opening the valve V2k or the valve V2m, the reagent can be introduced into the second flow path 20b. Further, the second flow path 20c is connected to the waste liquid recovery unit W2 via the valve V2j. Therefore, by opening the valve V2j, the waste liquid can be discharged from the second flow path 20c to the waste liquid recovery unit W2.
The second flow path 20b forms a circulation flow path (second circulation path) by closing the valves V2e, V2h, V2i, V2k, and V2m and opening the valves V2f, V2g, and V2l. Further, in the second flow paths 20b and 20c, the valves V2e, V2h, V2i, V2j, V2l and V2n are closed and the valves V2f, V2g, V2k and V2m are opened to open the circulation flow path (third). Circulation pathway) is formed.
Further, the second flow path 20b and the waste liquid recovery unit W2 close the valves V2e, V2g, V2k, and V2m, and open the valves V2f, V2h, V2i, and V2l to bring the fluid into the second flow path 20b. A flow path (second discharge path) for discharging from the waste liquid recovery unit W2 is formed. Further, the second flow paths 20b and 20c and the waste liquid collecting 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 units 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.

8 (a) and 8 (b) are cross-sectional views illustrating an example of the structure of the diaphragm valve.
FIG. 8A shows the open 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 adhered to each other in close contact with each other. Further, the space between the first substrate 310 and the diaphragm member 330 forms a flow path 315 through which a fluid flows. Further, a through hole 340 is provided in a part of the second substrate 320. Further, in the through hole 340, the diaphragm member 330 is exposed.

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 regulates the flow of the fluid inside the flow path 315.

In the open state of the diaphragm valve 800 shown in FIG. 8A, a fluid can flow inside the flow path 315. On the other hand, as shown in FIG. 8B, when a fluid for valve control 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 is deformed. A part of 330 is in close contact with the first substrate 310. This state is the 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 fluid for valve control include N2 gas, gas such as air, liquid such as water and oil, and the like. The fluid for valve control can be supplied, for example, by a tube connected to the through hole 340 or the like.

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 response to a change in pressure inside the through hole 340. For example, polydimethylsiloxane (PDMS). ), Silicone-based 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 FIG. 8A again. ) Will be in the open state. As a result, the fluid can flow inside the flow path 315 again.

A pump can be formed by three or more valves, that is, by arranging three or more valves side by side. 9 (a) to 9 (d) 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 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 to the right side or the left side toward FIG. 9A, and the fluid flow may flow. It may be stopped.

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

Subsequently, as shown in FIG. 9C, the valve 800a and the valve 800b are controlled to be in the closed state, and the valve 800c is left in the open state. Here, in the process of changing the valve 800b 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 in the direction indicated by the arrow in FIG. 9 (c), that is, to the right toward FIG. 9 (c). As a result, the fluid has a flow in the direction indicated by the arrow.

Subsequently, as shown in FIG. 9D, the valves 800b and 800c are controlled to be in the closed state.
Then, in the process of changing the valve 800c from the open state to the closed state, the diaphragm member 330 is deformed, and the fluid existing around the valve 800c is pushed away. However, since the valve 800a is in the closed state, the displaced fluid moves in the direction indicated by the arrow in FIG. 9D, that is, to the right toward 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 be in the open state as shown in FIG. 9D, or may be maintained in the closed state.

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

Further, by repeating the above steps, the flow of the fluid inside the flow path 315 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, by reversing the opening and closing timings of the valve 800a and the valve 800c, the flow of the fluid inside the flow path 315 can be controlled in the direction opposite to that described above. Further, it is also possible to adjust the cycle of repeating the operations of FIGS. 9 (a) to 9 (d) to form a pulsed micro solution flow and control the flow velocity. 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 by a pump provided with four or more valves.

Further, the structure of the diaphragm valve is not limited to that described above. For example, as a diaphragm valve, a tubular structure having an outer cylinder portion and an inner cylinder portion described in International Publication No. 2016/006615, and a thin film portion arranged so as to cover one end of the inner cylinder portion. A valve having a diaphragm member having an anchor portion that goes around the peripheral edge of the thin film portion and is in close contact with the inner wall of the outer cylinder portion and the outer wall of the inner cylinder portion can also be used.

In the blood component separation device of the present embodiment, as another valve that can be used, for example, a two-color molded valve obtained by continuously molding a resin portion and an elastomer portion using two molds is used. Illustrated. The two-color molded valve has the advantages that the time required for manufacturing is short and the adhesion between the resin and the elastomer is high.

Specific examples of the two-color molded valve are illustrated in FIGS. 10 and 11. In the valve 900 illustrated in FIG. 10, the substrate 909 has a recess (eg, flow path) 940A formed on the lower surface (one surface) 909a, a bottom portion of the recess 940A (eg, the upper side of the recess 90A in FIG. 10), and the substrate 909. It has an opening 952 that opens (penetrates) on the upper surface (other surface) 909b of the above. The opening 952 is provided with a valved drive unit 970. The valved drive unit 970 is made of the same soft material as the valve portion 950 described above, and includes a valve portion (deformed portion) 971 and a cylinder portion (connecting portion) 973. The valve portion 971 closes the lower surface 909a side of the substrate 909 in the opening 952. For example, the tubular portion 973 is composed of a valve portion 971 and a single member, is provided along the inner peripheral surface of the opening 952, and is integrally connected to the valve portion 971 at the lower end. The inner space of the tubular portion 973 is closed at the lower end by the valve portion 971 to form an opening 970a at which the upper end opens. For example, when the recess 90A in FIG. 10 is a flow path, the fluid (eg, a solution containing a sample substance, a cleaning solution, etc.) is configured to flow 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 recess 940A.

The valved drive unit 970 having the above configuration is, for example, a valve unit when a downward force (eg, pneumatic pressure, hydraulic pressure, mechanical force, etc.) is applied to the valve unit 971 through the opening 970a. The open / closed state of the flow path (eg, recess 940A) is controlled by the deformation of 971. As an example, as shown in FIG. 11, the valve portion 971 is deformed toward the recess 940A and bends to come into contact with the concave surface 980 to block the flow path (eg, the recess 940A) (the valve is closed). Further, in the valved drive unit 970, the deformation (eg, bending) of the valve portion 971 is eliminated by releasing the downward force applied to the valve portion 971, and the flow path (eg, bending) is separated from the concave surface 980. For example, the depression 940A) is opened (the valve is open).

The two-color molded valve described above 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.

A method of using the blood component separation device 700 having the above configuration will be described with reference to an example.

First, the valves V1a, V1b, and V1e are opened, and the other valves are closed, and a blood sample (eg, whole blood sample, etc.) is introduced from the first introduction port 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 valve V1a direction (first direction). At this time, the blood cells in the blood sample are captured by the filter. On the other hand, most of the plasma passes through the filter section 11 and reaches the first flow path 10b. Here, when the valve V1a is closed and the pump unit P1 is appropriately operated, the plasma that has reached the first flow path 10b can be circulated in the first circulation path including the first flow path 10b. If necessary, reagents for plasma analysis are introduced from inlet I1 and circulated through the first circulation pathway to mix with plasma. Plasma analysis can be performed by the plasma analysis unit 50 while circulating plasma in the first circulation pathway. The fluid circulating in the first circulation path can be discharged to the waste liquid recovery unit W1 by the first discharge path with the valves V1b and V1f closed and the valves V1c, V1d and V1e open as needed. ..

Next, with the valves V1a, V1c, V1d, and V1e in the open state and the other valves in the closed state, 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 in the filter moves in the first direction together with the washing liquid and is introduced into the first flow path 10b via the valve V1a. The cleaning liquid containing plasma introduced into the first flow path 10b can be discharged to the waste liquid collection unit W1 by the first discharge path with the valves 1b and V1f in the closed state and the valves V1c, V1d and V1e in the open state. ..

Next, with the valves V2a, V2b, V2e, V2f, V2g, and V2l in the open state and the other valves in the closed state, the hemolytic agent is introduced from the second introduction port 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 axially along the second flow path 20a together with the hemolyzing agent, and is introduced into the second flow path 20b from the valve V2b via the valve V2e. Here, by closing the valve V2b and opening the valve V2c and blowing air from the air introduction port A into the second flow path 20a, the blood cell component retained in the second flow path 20a is removed into the second flow path 20a. It can lead to the flow path 20b. The blood cell component introduced into the second flow path 20b can be circulated in the second circulation path including the second flow path 20b by closing the valve V2e and appropriately operating the pump unit P2. If necessary, reagents for blood cell analysis are introduced from inlet I2 and circulated through the second circulation pathway to mix with blood cell components. Alternatively, if necessary, a reagent for blood cell analysis is introduced from the inlet I3, and the third circulation pathway including the second flow paths 20b and 20c is circulated and 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 pathway or the third circulation pathway. The fluid circulating in the second circulation path or the third circulation path can be discharged to the waste liquid recovery unit W2 by the second discharge path or the third discharge path, if 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 methods of the present embodiment include (a) a step of introducing a blood sample into the first flow path from the first introduction port and causing the filter to capture blood cells in the blood sample, and (b) the first. It includes a hemolytic step of introducing a hemolytic agent from the introduction port or the second introduction port to hemolyze the blood cells in the filter.

The blood component separation method of the present embodiment can be carried out in the same manner as the method exemplified in "(Usage method)" described in the above "[Blood component separation device]".
As an example, the method of the present embodiment is after the step (a) and before the step (b), (c) the cleaning liquid is flowed from the first introduction port or the second introduction port to the first flow. A plasma removal step of introducing into the pathway and removing the plasma in the filter can be further included.

[Blood component analysis method]
In one embodiment, the present invention provides a method of analyzing a blood component using the blood component separation device of the above embodiment. The methods of the present embodiment include (a) a step of introducing a blood sample into the first flow path from the first introduction port 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). A step of introducing a hemolytic agent from the first introduction port or the second introduction port to hemolyze the blood cells in the filter, and (d) a step of analyzing the blood cells hemolyzed in the step (c). including.

The blood component analysis method of the present embodiment can be carried out in the same manner as the method exemplified in "(Usage method)" described in the above "[Blood component separation device]". When a blood component separation device including a blood cell analysis unit 60 is used in the method of the present embodiment, the blood cell component analysis in the step (d) can be performed by the blood cell analysis unit 60. When a blood component separation device not provided with the blood cell analysis unit 60 is used, the hemolyzed blood cell component may be collected from the blood component separation device and the collected blood cell component may be analyzed.

In the method of the present embodiment, in addition to the above steps (a) to (d), (d) the step of analyzing the plasma that has passed through the filter after the step (a) or the step (b) is further added. It may be included. When a blood component separation device including a plasma analysis unit 50 is used, plasma analysis can be performed by the plasma analysis unit 50. When a blood component separation device not provided with the plasma analysis unit 50 is used, hemolyzed plasma may be collected from the blood component separation device and the collected plasma may be analyzed.

[Hemoglobin A1c analysis method]
In one embodiment, the present invention provides a method of analyzing hemoglobin A1c. In the method of the present embodiment, (a) a step of causing a filter for separating blood cells and plasma to capture blood cells in a blood sample, and (b) after the step (a), a washing solution is passed through the filter to obtain the above-mentioned In the step of removing plasma in the filter, (c) after the step (b), a hemolytic agent is introduced into the filter to hemolyze the blood cells in the filter, and (d) the step (c). It comprises a step of analyzing hemoglobin A1c in the hemolyzed blood cells.

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

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

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

Although the embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and the design and the like within a range not deviating from the gist of the present invention are also included.

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 measured value HbA1c standard test reagents (HbA1c concentration 6.1%, HbA1c concentration 10%; hemoglobin A1c cutoff test certified standard substance, laboratory medical standard substance organization) 0 to 0 Human plasma was added to a concentration of 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 Rapidia (registered trademark) Auto HbA1c-L (Fujirebio). For each sample, the absorbance at 660 nm and the absorbance at 800 nm were measured, and the value of the absorbance at 660 nm −800 nm was determined.
The results are shown in Table 1 and FIG.

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

[Experimental Example 2] Measurement of Plasma Component in Blood Cell Fraction 1 μL of Cy5 was added to 1000 μL of whole blood of a rabbit to trace the plasma component and mixed to prepare whole blood of a Cy5-added rabbit.
The whole blood sample of the Cy5-added rabbit was centrifuged (centrifugal force (1000 × g), 5 minutes) to separate the blood cell fraction and the plasma fraction. Dilution series of blood cell fraction and plasma fraction were prepared respectively, and each dilution series was used to prepare a calibration curve for the amount of hemoglobin and the amount of Cy5.
Next, the blood cell separation of the Cy5-added rabbit whole blood sample was performed using the blood component separation device having the structure shown in FIG. 15 μL of Cy5-added rabbit whole blood sample was introduced into the first flow path from the introduction port (atmospheric pressure: 1 minute), and negative pressure was applied at 5 kPa for 2 minutes from the opposite side of the introduction port to filter the Cy5-containing rabbit whole blood sample. Passed through. Then, the ports b and d of the third flow path are closed, 100 μL of hypotonic solution (ultrapure water) is introduced from the port c of the second flow path, and the blood cell fraction captured by the filter is dissolved. A 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 blood cell fraction was collected. Hemoglobin absorbance and Cy5 fluorescence were measured for the collected blood cell fraction, 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 flow path 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)
In the same manner as in Experimental Example 1, a Cy5-added rabbit whole blood sample was prepared, and blood cells were separated from the Cy5-added rabbit whole blood sample using the blood component separation device having the structure shown in FIG. Pressure: 1 minute, 5 kPa negative pressure: 2 minutes). Next, 15 μL of PBS was introduced into the first flow path from the inlet (5 kPa negative pressure: 2 minutes), and the filter was washed. Then, 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 blood cell fraction was collected. Similar to Experimental Example 1, the plasma content in the collected blood cell fraction was calculated.

The results are shown in Figure 1 5. In FIG. 15 , “no washing” means that PBS washing was not performed, which is the same as the case where the blood cell fraction was collected in the second flow path of Experimental Example 1. “With washing” means that PBS washing was performed, and the plasma component content was significantly reduced as compared with “without washing”.

[Experimental Example 4] Evaluation of Filter Wash Solution Volume 1 μL of Cy5 was added to 9 μL of whole rabbit blood to trace plasma and mixed to prepare a Cy5-added rabbit whole blood sample.
Next, the blood cell separation of the Cy5-added rabbit whole blood sample was performed using the blood component separation device having the structure shown in FIG. 10 μL of a Cy5-added rabbit whole blood sample was introduced into the first flow path from the introduction port, and a negative pressure was applied at 10 kPa for 2 minutes from the opposite side of the introduction port, and the Cy5-containing rabbit whole blood sample was passed through a filter. Then, 20 μL, 25 μL, or 30 μL of PBS was introduced into the first flow path from the introduction port (10 kPa negative pressure: 2 minutes), and the filter was washed. After that, the port a of the second flow path and the port d of the third flow path are closed, 50 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 the port b of the third flow path. When most of the blood fraction was extruded, the introduction of hypotonic fluid was stopped.

FIG. 16 is a photograph of each blood component separation device after washing the filter with each PBS amount and collecting the blood cell fraction. Comparing the colors of the liquid immediately after passing through the filter (the elliptical portion of the dotted line), it was qualitatively shown that the larger the amount of the cleaning liquid, the lighter the blue component of Cy5. Assuming that the blue part 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 HbA1c measurement value (Example 1)
Using a blood component separation device with the structure shown in FIG. 1 3, human refrigerated blood (normal human whole blood · O type, Kohjin Bio) a blood cell separation of Been. 10 μL of human refrigerated blood was introduced into the first flow path from the introduction port, negative pressure was applied at 10 kPa for 2 minutes from the opposite side of the introduction port, and the human refrigerated blood was passed through a filter. Then, 25 μL, 25 μL, or 30 μL of PBS was introduced into the first flow path from the introduction port (10 kPa negative pressure: 2 minutes), and the filter was washed. After that, 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 the port b of the third flow path. When about 15 μL of the molten blood was extruded, the collection of the dissolved blood was completed.
Using 5 μL of the molten blood collected as described above, a latex agglutination method using Rapidia (registered trademark) Auto HbA1c-L was performed, and the absorbances at 660 nm and 800 nm were measured. From the measured absorbance, the HbAc1 concentration and the coefficient of variation (CV) were calculated.

(Comparative Example 1)
A latex agglutination inhibition method using 2.0 μL of human refrigerated blood was performed in a cartridge of Cobas® c101 (Roche) to calculate the HbAc1 concentration and the coefficient of variation (CV).

(Comparative Example 2)
1.5 μL of human refrigerated blood was hemolyzed in a cartridge of Affinion® HbAic (Alere). Then, the color development caused by HbA1c was measured by the boronic acid affinity method. The HbAc1 concentration and the coefficient of variation (CV) were calculated from the measured values of color development.

(result)
The results of Example 1, and Comparative Examples 1 and 2 shown in FIG 7. In Example 1, it was confirmed that the coefficient of variation (CV) was smaller and the measurement with high reproducibility was possible as compared with Comparative Examples 1 and 2.

[Experimental Example 6] Evaluation of Reproducibility of HbA1c Measurement Value Blood cell separation of human whole blood (normal human whole blood / O type, Kojinbio) was performed using a blood component separation device having the structure shown in FIG. 15 μL of human whole blood was introduced into the first flow path from the introduction port, negative pressure was applied at 10 kPa for 2 minutes from the opposite side of the introduction port, and the standard test reagent was passed through a filter. Next, 25 μL of PBS was introduced into the first flow path from the introduction port (10 kPa negative pressure: 2 minutes), and the filter was washed. After that, the port a of the second flow path and the port d of the third flow path are closed, 50 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 the port b of the third flow path. When about 50 μL of the molten blood was extruded, the collection of the dissolved blood was completed.
Using the molten blood collected as described above, a latex agglutination method using Rapidia (registered trademark) Auto HbA1c-L was performed, and the absorbances at 660 nm and 800 nm were measured. The coefficient of variation (CV) was calculated from the measured absorbance.
Further, a blood cell fraction of whole human blood was separated by a centrifugal separation method (centrifugal force (1000 × g), 5 minutes), and the absorbances at 660 nm and 800 nm were measured in the same manner.
When the HbAc1 concentration of whole human blood was measured using Affinion (registered trademark) HbAic (Alere), the HbAc1 concentration was 5.06%.

The results are shown in FIG. In FIG. 18, a standard sample (HbA1c concentration 6.1%, HbA1c concentration 10%; hemoglobin A1c cutoff test certified standard substance, laboratory medical standard substance organization) was measured using Rapidia (registered trademark) Auto HbA1c-L. The results are also shown. In the blood cell fraction separated from whole blood using the blood component separation device, an HbA1c measurement value equivalent to that of the blood cell fraction obtained by the centrifugation method was obtained. In addition, the coefficient of variation (CV) was smaller than that of the centrifugation method. Furthermore, the blood cell fractions separated using the blood component separation device had lower measured values by the latex agglutination method than the standard sample. Generally, the HbA1c concentration of normal human whole blood is 6% or less. Therefore, the reliability of the measured values by this method was shown by comparison with the measured values of the standard sample (HbA1c concentration 6.1%, HbA1c concentration 10%).

10, 10a, 10b first flow path,
11 Filter part 12 1st introduction port 20, 20a, 20b, 20c 2nd flow path 22 2nd introduction port 30 3rd flow path 40 4th flow path 42 4th introduction port 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, 311a, 311b, 311c Convex part 315 Flow path 320 Second substrate 330 Diaphragm member 331 Anchor part 340, 340a, 340b, 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 collection part A Air inlet

Claims (15)

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.
A second flow path connected to the filter portion of the first flow path and
Including
The second flow path has a second introduction port and intersects the first flow path at the filter portion of the first flow path .
Blood component separation device. The solution introduced from the first introduction port flows inside the filter portion in the axial direction of the first flow path, and flows.
The introduced solution from the second inlet, the flow in different directions inside the filter unit and the axial direction of the first flow path, the blood component separation device according to claim 1. 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.
A second flow path connected to the filter portion of the first flow path and
Including
El Bei plasma analysis portion connected to the first flow path,
Blood component separation device. 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.
A second flow path connected to the filter portion of the first flow path and
A third flow path connected to the filter portion of the first flow path is included.
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.
In the filter section
The connection portion with the second flow path is arranged at one end of the filter portion.
The connection portion with the third flow path is arranged at the other end of the filter portion .
Blood component separation device. The blood component separation device according to claim 4 , wherein the third flow path intersects the first flow path at the filter portion of the first flow path. 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.
A second flow path connected to the filter portion of the first flow path and
Including
El Bei hemocyte analyzer for analyzing the hemolyzed blood cells,
Blood component separation device. The blood cell analyzer is connected to the second passage,
The blood component separation device according to claim 6. Further including 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 cell analysis unit is connected to the second flow path or the third flow path.
The blood component separation device according to claim 6. The blood component separation device according to claim 6 or 7 , wherein the blood cell analysis unit is a hemoglobin A1c measuring unit that measures hemoglobin A1c. 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.
A second flow path connected to the filter portion of the first flow path and
Including
El Bei blood cell recovery unit,
Blood component separation device. 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.
Using a blood component separation device comprising a second flow path connected to the filter portion of the first flow path,
A method of separating blood components
(A) A solution containing blood cells and plasma is introduced into the first flow path from the first introduction port, the filter captures the blood cells in the solution, and the plasma in the solution is transferred to the first flow path. And the process of moving in
(B) A step of introducing a hemolytic agent from the first introduction port, hemolyzing the blood cells in the filter, and moving the blood cell component released by the hemolysis in the second flow path.
(C) prior to, after a is the step of the step (a) (b), the first inlet or al, introducing a cleaning liquid to the first flow path, the plasma is removed to remove the plasma in the filter Process and
Including methods. 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.
Includes a second flow path that connects to the filter section of the first flow path, has a second inlet, and intersects the first flow path at the filter section of the first flow path. Using a blood component separation device,
A method of separating blood components
(A) A solution containing blood cells and plasma is introduced into the first flow path from the first introduction port, the filter captures the blood cells in the solution, and the plasma in the solution is transferred to the first flow path. And the process of moving in
(B) A step of introducing a hemolytic agent from the first introduction port or the second introduction port, hemolyzing the blood cells in the filter, and moving the blood cell component released by hemolysis in the second flow path.
(C) After the step (a) and before the step (b), the washing liquid is introduced into the first flow path from the first introduction port or the second introduction port, and the plasma in the filter is introduced. and plasma removal step of removing,
Including methods. 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.
Using a blood component separation device comprising a second flow path connected to the filter portion of the first flow path,
A method of analyzing blood components
(A) A step of introducing a solution containing blood cells and plasma into the first flow path from the first introduction port and causing the filter to capture the blood cells in the solution.
(B) after the step (a), the first inlet or al, introducing a cleaning liquid to the first flow path, removing the plasma in the filter,
(C) after the step (b), introducing the first inlet or al hemolytic agent, a step of hemolyzing blood cells in the filter,
(D) A step of analyzing the blood cells hemolyzed in the step (c) and
Including methods. 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.
Includes a second flow path that connects to the filter section of the first flow path, has a second inlet, and intersects the first flow path at the filter section of the first flow path. Using a blood component separation device,
A method of analyzing blood components
(A) A step of introducing a solution containing blood cells and plasma into the first flow path from the first introduction port and causing the filter to capture the blood cells in the solution.
(B) After the step (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.
(C) After the step (b), a step of introducing a hemolytic agent from the first introduction port or the second introduction port to hemolyze the blood cells in the filter, and
(D) A step of analyzing the blood cells hemolyzed in the step (c) and
Including methods. (D) The step of analyzing the plasma that has passed through the filter after the step (a) or the step (b) is further included.
The method for analyzing a blood component according to claim 14.

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