JPWO2004083852A1 - Blood cell separation membrane and blood holding device using the same - Google Patents

Abstract

A blood cell separation membrane for preventing hemolysis of blood cells and separating serum or plasma and blood cells is provided. A porous membrane that separates blood cells from the supplied blood contains a solution containing hydrophobic aminocarboxylic acid, silk-derived protein, Tris, TES, ε-aminohexanoic acid, tranexamic acid, or heparin, and is then dried. A separation membrane is used.

Description

  The present invention relates to a blood cell separation membrane for separating blood cells in blood and serum or plasma, and a blood holding device used for holding and testing blood.

When testing blood, a sheet-like blood test tool (also called a test piece) that is used up for each specimen is widely used. Examples of the test tool include a type that holds blood, extracts blood from the blood, and performs a test, and a test tool that contains a reagent or the like in advance. With the latter tool, for example, blood can be reacted with a reagent by dropping blood onto the test tool, and the reaction can be measured by an optical technique or an electrochemical technique.
Such blood test tools are not only widely used in general clinical tests and the like, but are also actually used in, for example, remote clinical test systems. This remote clinical test system is a system in which a patient collects blood at home, and the blood is stored in a blood test tool, dried, and mailed to a test organization such as a hospital for testing. Patients who have sent blood by mail can know the test result by mail, etc., or can go directly to a hospital or the like to know the test result.
When the test item is a component in serum or plasma, such as a blood glucose level, it is necessary to separate blood into blood cells and serum or plasma in a blood test tool. For this reason, in a conventional blood test tool, it has been common to incorporate a blood cell separator such as a glass filter into the blood test tool. According to such a blood test tool, for example, blood is separated into blood cells and serum / plasma by a blood cell separator, and the serum / plasma that has passed through the blood cell separator is further developed by capillary action on a deployment part. Serum and plasma samples can be prepared. In addition, a blood test tool using an asymmetric porous membrane whose pore diameter changes in the thickness direction has recently been developed (for example, Japanese Patent Publication No. 11-505327). When blood is supplied from the surface with the larger pore diameter of the asymmetric porous membrane, blood cells are separated from the blood in the process of blood permeating in the thickness direction, and serum and plasma come out on the opposite side of the supply surface. What is necessary is just to collect this. A blood test tool using such an asymmetric porous membrane has the advantage that clogging of blood cells is prevented.
However, in such a conventional blood test tool, the blood cells may be hemolyzed with blood cell separation, and the components in the blood cells may be mixed into serum or the like. It is necessary to contain in (for example, Unexamined-Japanese-Patent No. 9-196908). However, in this case, although hemolysis is prevented, the hematocrit (Ht) value in the serum and plasma obtained by the influence of the additive is greatly reduced, and it is difficult to accurately measure the serum and plasma components. There is a problem that there is.

Therefore, an object of the present invention is to provide a blood cell separation membrane capable of separating blood cells and serum / plasma while preventing hemolysis of blood cells accompanying blood cell separation, and a blood holding device using the same.
In order to achieve the above object, the blood cell separation membrane of the present invention includes a porous membrane that separates serum or plasma in blood and blood cells, and the porous membrane is a hydrophobic aminocarboxylic acid, a protein derived from silk. And at least one hemolysis inhibitor selected from the group consisting of Tris, TES, ε-aminohexanoic acid, tranexamic acid and heparin.
Thus, the blood cell separation membrane of the present invention can prevent hemolysis of blood cells by containing the type of hemolysis inhibitor as described above. For this reason, the serum or plasma sample separated after passing through the blood cell separation membrane does not contain the components in the blood cells, and can measure components such as serum with excellent accuracy. In addition, since the hemoglobin pigment is prevented from being mixed due to hemolysis, the separated sample can be directly measured by optical means or visual observation.
Moreover, if a serum / plasma sample is prepared using the blood cell separation membrane of the present invention, various components can be measured with high accuracy. The inventors of the present invention have conducted intensive research on a decrease in hematocrit (Ht) value when using the conventional blood cell separation membrane as described above and a decrease in measurement accuracy in various analysis items. As a result, the additive substance conventionally used for hemolysis prevention is glycine, which is usually contained at a high concentration of about 100 mg to 200 mg per volume (cm ³ ) of the blood cell separation membrane. I found out. In particular, for analysis items such as GGT, it was found that not only the dilution effect by glycine but also the measurement accuracy was lowered as a result of changes in serum or plasma components by glycine. And if it contains not the glycine but the above-mentioned hemolysis inhibitor like the blood cell separation membrane of this invention, it does not need to be high concentration like glycine, for example. Therefore, it has been found that the influence on the Ht value is also reduced, and various analysis items such as GGT can be measured with high accuracy. As described above, by using the blood cell separation membrane of the present invention, blood cells and serum / plasma can be separated while preventing hemolysis of blood cells accompanying blood cell separation, and serum or plasma samples that do not affect various analyzes Can be prepared.
Next, the blood holding device of the present invention has a blood cell separation unit that separates blood cells in the blood, and a development unit that develops serum or plasma in the blood by capillary action, and the blood cell separation unit comprises the book It is a blood cell separation membrane of invention. Since the blood holding device of the present invention has a blood cell separation membrane having the above-described effects, it can be said that it is particularly useful for the above-described remote clinical examination system, for example. The specimen holding tool of the present invention can also be used as a tool for holding a blood specimen from which blood cells and serum / plasma are separated, for example, for mailing, etc. It can also be used.

FIG. 1 is a cross-sectional view showing an embodiment of the blood holding device of the present invention.
FIG. 2 is a cross-sectional view showing another embodiment of the blood holding device of the present invention.
FIG. 3 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 4 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 5 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 6 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 7 is a cross-sectional view showing still another embodiment of the blood retention tool of the present invention.
FIG. 8 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 9 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.
FIG. 10 is a cross-sectional view showing still another embodiment of the blood holding device of the present invention.

As described above, the blood cell separation membrane of the present invention includes a porous membrane that separates serum or plasma in blood and blood cells, and the porous membrane comprises a hydrophobic aminocarboxylic acid, a silk-derived protein, Tris, It contains at least one hemolysis inhibitor selected from the group consisting of TES, ε-aminohexanoic acid, tranexamic acid and heparin.
As the hydrophobic aminocarboxylic acid, for example, a hydrophobic amino acid can be used, and specifically, alanine, valine, leucine, isoleucine and the like can be used. The protein derived from silk refers to a hydrolyzate of fibroin (hereinafter also referred to as “silk essence”).
Among the hemolysis inhibitors, valine, leucine, ε-aminohexanoic acid, tranexamic acid, and silk essence are preferable. This is because when these are used, in addition to the effect of preventing hemolysis as described above, the effect of separating serum / plasma and blood cells with excellent efficiency is also achieved. Although the mechanism is unknown, the penetration rate of blood cells in the blood cell separation membrane is suppressed by containing these anti-hemolytic agents in the blood cell separation membrane, while the blood and blood plasma penetration rates are promoted. . Further, even when blood cells pass through the blood cell separation membrane, for example, the development speed of blood cells in the development part is suppressed, and the development speed of serum or plasma is promoted. Therefore, in the development part, the development distance of blood cells can be shortened, and the development distance of serum or plasma can be lengthened. That is, since both contradictory effects of suppressing the development of blood cells and promoting the development of serum and plasma can be realized, the amount of development of only serum or plasma can be increased. Therefore, according to the blood cell separation membrane containing the hemolysis inhibitor as described above, blood cells and serum or plasma can be easily separated. Furthermore, even when blood cells pass through the blood cell separation membrane, the influence on the serum / plasma recovery rate due to the development of blood cells can be suppressed, and therefore the recovery rate can be improved. For this reason, the blood volume to supply can also be reduced, for example, the burden of the patient at the time of blood collection can also be reduced.
Furthermore, among them, valine, tranexamic acid, and ε-aminohexanoic acid are particularly preferable. When these are used, for example, compared with the above-mentioned glycine, a sufficient effect can be obtained with a content (weight) of about 1/5 to 1/10 of glycine.
Moreover, any one type of the hemolysis inhibitor may be used, or two or more types may be combined. Examples of the combination include a combination of valine and heparin, tranexamic acid and heparin, and the like.
In the present invention, the content (weight) of the hemolysis inhibitor is, for example, the volume of the porous membrane (cm ³ ) Is preferably in the range of 1 mg to 50 mg, more preferably in the range of 5 mg to 40 mg, and particularly preferably in the range of 10 mg to 30 mg. The content of the hemolysis inhibitor is the volume of the porous membrane (cm ³ If it is 1 mg or more per unit, hemolysis can be sufficiently prevented. Here, the “volume of the porous membrane” refers to the volume including the pores of the porous membrane.
Specifically, when the hemolysis inhibitor is valine, the content (weight) is, for example, the volume of the porous membrane (cm ³ ) Is preferably in the range of 5 mg to 50 mg, more preferably in the range of 15 mg to 45 mg, and particularly preferably in the range of 30 mg to 40 mg.
When the hemolysis inhibitor is leucine, its content (weight) is, for example, the volume of the porous membrane (cm ³ ) Is preferably in the range of 1 mg to 30 mg, more preferably in the range of 5 mg to 25 mg, and particularly preferably in the range of 10 mg to 20 mg.
When the hemolysis inhibitor is tranexamic acid, its content (weight) is, for example, the volume of the porous membrane (cm ³ ) Is preferably in the range of 5 mg to 50 mg, more preferably in the range of 15 mg to 45 mg, and particularly preferably in the range of 30 mg to 40 mg.
When the hemolysis inhibitor is ε-aminohexanoic acid, its content (weight) is, for example, the volume of the porous membrane (cm ³ ) Is preferably in the range of 5 mg to 50 mg, more preferably in the range of 10 mg to 45 mg, and particularly preferably in the range of 30 mg to 40 mg.
The blood separation membrane of the present invention may further contain additives such as pullulan and BSA in addition to the hemolysis inhibitor. By using these and the hemolysis inhibitor in combination, the hemolysis prevention effect can be further improved.
The blood cell separation membrane preferably contains the hemolysis inhibitor in its entirety. For example, the hemolysis agent is contained only on at least one surface of the blood cell separation membrane, particularly the surface to which blood is supplied. It may be.
In general, it is desirable that the porous membrane is difficult for blood cells to pass through the inside thereof. For example, the porous membrane preferably has pores having a diameter that prevents blood cells from passing therethrough. In the present invention, “a hole having a pore diameter through which blood cells cannot pass” does not mean that the pore diameter is smaller than that of blood cells, and any mechanism may be used as long as it does not allow blood cells to pass through. . Therefore, the pore diameter through which blood cells cannot pass includes pore diameters larger than the blood cell diameter. Further, according to the blood cell separation membrane of the present invention, as described above, for example, even when blood cells pass through the separation membrane, the hemolysis inhibitor accelerates the development rate of serum or plasma, It is also possible to suppress the deployment speed. In this case, the porous membrane does not completely hold the blood cells inside the porous membrane and does not hold the blood cells. For example, the porous membrane is a porous membrane through which blood cells partially pass. May be.
Specifically, the porous membrane preferably has pores having a pore size of 0.1 μm to 20 μm, more preferably 1 μm to 10 μm, and particularly preferably 2 μm to 8 μm.
The porous membrane is not particularly limited, and various materials conventionally used for blood cell separation can be used. Specifically, for example, a glass filter or an asymmetric porous membrane having a pore size distribution in which the average pore size continuously or discontinuously decreases in the thickness direction can be used.
As the glass filter, for example, one having a low fiber density is preferable, and a commercially available glass filter, for example, trade name AP25 manufactured by Millipore Corporation may be used.
In addition, when an asymmetric porous membrane is used as the porous membrane, the pore diameter changes in the thickness direction. For example, blood can gradually pass through the porous membrane as blood advances in the thickness direction. The blood cells are retained at the site of the pore diameter where the blood cells cannot pass. Therefore, clogging hardly occurs and blood cells can be separated quickly and easily. In the present invention, “the average pore diameter becomes discontinuously smaller” means, for example, that it becomes smaller stepwise.
The pore diameter of the asymmetric porous membrane is preferably such that the maximum pore diameter is in the range of 10 μm to 300 μm and the minimum pore diameter is in the range of 0.1 μm to 30 μm, more preferably the maximum pore diameter is in the range of 100 μm to 200 μm. The pore diameter is in the range of 1 μm to 10 μm, particularly preferably the maximum pore diameter is in the range of 150 μm to 200 μm and the minimum pore diameter is in the range of 1 μm to 5 μm.
The material of the asymmetric porous membrane is not particularly limited, and examples thereof include resins such as polyester, polysulfone, polyethersulfone, polycarbonate, cellulose acetate, polyamide, polyimide, and polystyrene. These porous membranes may be used alone or in combination of two or more thereof. Among these, preferred are, for example, polysulfone and polyethersulfone, and particularly preferred is polyethersulfone.
Further, the asymmetric porous membrane may be produced using various resins as described above, or a commercially available asymmetric porous membrane such as U.S.A. The product name BST-SP manufactured by S Filter, the product name S / G manufactured by Primecare, etc. may be used.
The size of the blood cell separation membrane can be appropriately determined according to, for example, the amount of blood to be supplied. Specifically, when the blood volume to be supplied is 120 μL, for example, the length is 4 mm × width 1.5 mm × thickness 50 μm to length 50 mm × width 20 mm × thickness 2000 μm, preferably length 5 mm × width 3 mm × thickness. It is 75 μm to 30 mm long × 15 mm wide × 1250 μm thick, more preferably 10 mm long × 5 mm wide × 90 μm thick to 20 mm long × 12 mm wide × 1100 μm thick. The length refers to the length in the longitudinal direction of the blood cell separation membrane, and the width refers to the length in the width direction, and so on.
When a glass filter is used as the blood cell separation membrane, the thickness thereof is, for example, in the range of 200 μm to 2000 μm, and preferably in the range of 500 μm to 1000 μm. When the amount of blood to be supplied is 120 μL, the size is, for example, length 4 mm × width 1.5 mm × thickness 100 μm to length 50 mm × width 20 mm × thickness 2000 μm, preferably length 5 mm × width 3 mm. X thickness 200 μm to length 30 mm × width 15 mm × thickness 1250 μm, more preferably length 10 mm × width 5 mm × thickness 250 μm to length 20 mm × width 12 mm × thickness 1100 μm.
When an asymmetric porous membrane is used as the blood cell separation membrane, the thickness thereof is, for example, in the range of 50 μm to 400 μm, and preferably in the range of 100 μm to 350 μm. In addition, when the amount of blood to be supplied is 40 μL, the size is, for example, 4 mm long × 1.5 mm wide × 50 μm thick to 50 mm long × 20 mm wide × 400 μm thick, preferably 5 mm long × 3 mm wide X thickness 100 μm to length 30 mm × width 15 mm × thickness 350 μm, more preferably length 10 mm × width 5 mm × thickness 200 μm to length 20 mm × width 12 mm × thickness 300 μm.
The method for producing the blood cell separation membrane of the present invention is not particularly limited, and for example, the porous membrane is immersed in a dispersion or solution of the hemolysis inhibitor and dried, or the dispersion or the like is applied to the porous membrane. There is a method of drying after dropping and infiltrating.
The concentration of the hemolysis inhibitor in the dispersion or solution is, for example, in the range of 0.1 wt% to 5 wt%, preferably in the range of 0.5 wt% to 4 wt%, more preferably 2 wt%. It is in the range of ˜3% by weight.
Moreover, since the porous membrane can rapidly permeate whole blood, before retaining the hemolysis inhibitor, for example, hydroxypropylcellulose (HPC), polyvinyl alcohol (PVA), carboxymethylcellulose (CMC) in advance. Hydrophilic treatment may be performed by immersing in a treatment solution such as a hydrophilic polymer. The concentration of the treatment solution is, for example, in the range of 0.1 wt% to 50 wt%, and the treatment time is, for example, in the range of 0.1 hour to 24 hours. Examples of the solvent for the solution include water and various organic solvents, and examples of the organic solvent include alcohols such as ethanol.
Next, as described above, the blood holding device of the present invention includes a blood cell separation unit that separates blood cells in blood, and a deployment unit that develops serum or plasma in blood by capillary action, and the blood cell separation The part is the blood cell separation membrane of the present invention.
As the blood holding tool of the present invention, for example, the laminated embodiment A-1 in which the development part and the blood cell separation part are laminated, the asymmetric porous membrane includes the development part and the blood cell separation part. There are single layer type embodiments (A-2 and A-3) and single layer type embodiments in which the blood separation membrane comprises a blood cell separation part and a development part. The forms of A-1, A-2 and A-3 will be specifically described below.
(Embodiment A-1)
In this embodiment, the blood cell separation part is the blood cell separation membrane of the present invention, the development part is composed of a porous membrane, and the blood cell separation film is laminated on the development part (a porous membrane for development). This is an example. In this embodiment, the surface of the blood cell separation membrane serves as a blood supply unit.
An example of the blood holding tool is shown in the sectional view of FIG. As shown in the figure, the blood holding device 10 has a structure in which a blood cell separating unit 12 is laminated on one end of the developing unit 11, and the surface of the blood cell separating unit 12 is connected to the blood supply unit 13. It has become. In the same figure, an arrow A indicates the development direction of serum / plasma in blood (the same applies to FIGS. 2 to 10).
For example, a filter paper, a cellulose acetate film, a porous film, or a glass fiber film can be used as the developing porous film constituting the developing unit 11. Examples of the material of the porous membrane include resins such as polyester, polysulfone, polyethersulfone, polycarbonate, cellulose acetate, polyamide, polyimide, polystyrene, and the like, and preferably polyethersulfone and polycarbonate. Also, the asymmetric porous membrane as described above can be used. If the asymmetric porous membrane is used as the developing portion, blood cells can be further separated in the developing portion by the pore structure of the asymmetric porous membrane even when blood cells pass through the blood cell separating portion. These porous membranes may be used alone or in combination of two or more thereof. Among these, for example, filter paper, cellulose acetate membrane, nitrocellulose membrane, polysulfone porous membrane, polyester porous membrane, polycarbonate porous membrane, particularly preferably filter paper, polysulfone porous membrane, It is a porous film made of polyester. Moreover, since the development of serum or plasma can be further promoted similarly to the aforementioned blood cell separation membrane, a hydrophilization treatment may be performed.
The average pore diameter of the porous membrane for development is not particularly limited as long as, for example, serum or plasma can be developed by capillary action, but is preferably in the range of 0.1 μm to 300 μm, more preferably 1 μm to 100 μm, and particularly preferably 2 μm. ~ 50 μm. The size of the porous membrane for development can be appropriately determined according to, for example, the amount of blood to be supplied. For example, when the amount of blood to be supplied is 40 μL, the length is 4 mm × width 1.5 mm × thickness 50 μm Length 50 mm × width 20 mm × thickness 400 μm, preferably length 5 mm × width 3 mm × thickness 100 μm to length 30 mm × width 15 mm × thickness 350 μm, more preferably length 10 mm × width 5 mm × thickness 200 μm to length It is 20 mm × width 12 mm × thickness 300 μm.
Moreover, the hemolysis inhibitor may be contained not only in the blood cell separation membrane but also in the development porous membrane.
The combination of the blood cell separation membrane and the development porous membrane is not particularly limited. For example, a combination using a glass filter as the blood cell separation membrane and an asymmetric porous membrane as the development porous membrane, or a blood cell separation membrane A combination of using an asymmetric porous membrane and a nitrocellulose membrane as a developing porous membrane is preferred.
In the blood holding device of the present embodiment, for example, the blood cell separation membrane may be disposed and stacked on the development porous membrane, or the positioning of the development porous membrane and the blood cell separation membrane may be performed. It can manufacture by adhering or crimping | bonding the both ends of said both.
Moreover, it is preferable that the said development | deployment porous membrane is supported by the support body. This is because a blood holding device having a sufficient strength can be obtained regardless of the strength of the developing porous membrane, and the handling becomes simple. As the material for forming the support, for example, plastics such as polystyrene, polyethylene terephthalate (PET), polyvinyl chloride, acrylic resin, acrylonitrile-butadiene-styrene copolymer (ABS) can be used. Any one kind of the material may be used, or two or more kinds may be used in combination. In the case of directly measuring by an optical method or the like using a blood holding tool as described later, the support is preferably light transmissive, for example, a support made of polystyrene, PET, acrylic resin, or the like. The body can be used.
Next, an example of preparing a serum or plasma sample by adding blood to the blood holding device 10 will be described with reference to FIG.
First, blood is dropped onto the blood supply unit 13 on the surface of the blood cell separation unit 12. The blood is separated into blood cells and serum / plasma while moving in the blood cell separator 12 in the thickness direction. Then, the separated serum / plasma reaches the development part 11 and develops in the development part 11 in the surface direction (in the direction of arrow A in the figure) by capillary action. At this time, according to the blood holding device of the present invention, the blood cell separation unit 12 contains the hemolysis inhibitor, so that the hemolysis that occurs when the blood cells are separated is prevented, and the serum or plasma in which the mixing of blood cell components is suppressed. A sample is obtained.
When collecting the developed serum or plasma sample, the blood holding tool 10 is dried by air drying or natural drying. Thereafter, the developing part 11 is peeled off from the blood holding tool 10 and only the developing part of serum or plasma is cut out from the developing part 11.
Then, a section obtained by cutting or the like is placed in, for example, a test tube, an extraction solution is added thereto, and the serum or plasma is extracted and collected by allowing to stand. The extraction solution is not particularly limited as long as it can extract serum or plasma and does not affect the detection of the analysis target component in the serum or plasma. For example, it may be a buffer solution, physiological saline, purified water, protein solution, or a mixture thereof. Examples of the buffer solution include various buffer solutions containing phosphoric acid, citric acid, hydrochloric acid, acetic acid and the like, and the pH is, for example, in the range of pH 6-8. The addition amount of the extraction solution is not particularly limited, but can be determined as appropriate depending on the size of the section, and is, for example, in the range of 1 to 1000 times the section volume. Further, the extraction processing time is not particularly limited, and is, for example, in the range of 1 minute to 300 minutes.
Moreover, after developing serum or plasma, it can also be recovered by cutting out the developed part and directly centrifuging it.
Using this recovered solution, the component to be analyzed in serum or plasma can be measured.
On the other hand, in this blood holding tool 10, the serum or plasma held in the development part 11 can be analyzed as it is without collecting it with the extract. In this case, the reagent part may be formed in advance by containing the analysis reagent in the developing part 11. Examples of the method for arranging the reagent in the developing unit 11 include a printing method, an impregnation method, and a spraying method.
The analysis reagent is not particularly limited, and can be appropriately determined depending on the type of the target analysis target component. Examples of the components of the reagent include various enzymes, buffer substances such as phosphates and carbonates, color formers, and the like. Specifically, when analyzing glucose, for example, glucokinase, glucose-6-phosphate dehydrogenase, β-NADP, ATP, a buffer solution and the like may be contained.
Further, if the developing unit 11 contains a reagent in parallel with the direction of movement of serum or plasma, it is possible to perform multi-item analysis using the same blood holding device. In this case, it is preferable to provide a boundary layer between the reagent parts by, for example, adding a hydrophobic resin solution so that the reagent of many items is not mixed.
According to such a blood holding device 10, when blood is supplied and serum or plasma is developed as described above, various components to be analyzed react with the detection reagents in the development unit 11. If color development or the like due to this reaction is detected by an electrochemical technique or an optical technique (including visual observation), analysis can be easily performed.
(Embodiment A-2)
In this embodiment, the blood cell separation portion is an asymmetric porous membrane, the asymmetric porous membrane further has the development portion, and in the asymmetric porous membrane, the blood cell separation portion, the development portion, Is located in the surface direction, the blood cell supplying side is the blood cell separating unit, and the downstream side is the developing unit along the developing direction of the supplied blood. That is, there is a blood cell separation portion and a development portion in the plane direction of one asymmetric porous membrane, and a configuration in which blood cell separation and development of serum or plasma can be performed in one asymmetric porous membrane. It is.
An example of the blood holding tool is shown in the sectional view of FIG. As shown in the figure, the blood holding device 20 is composed of an asymmetric porous membrane having a pore size distribution in which the average pore size continuously or discontinuously decreases in the thickness direction. The upstream side in the direction of arrow A is the blood separation unit 22, and the downstream side is the development unit 21. The surface of the blood separation unit 22 becomes the blood supply unit 23.
As for the pore size of the asymmetric porous membrane, the maximum pore size is, for example, in the range of 10 μm to 300 μm, preferably the range of 50 μm to 150 μm, and the minimum pore size is, for example, in the range of 0.1 μm to 30 μm, preferably It is in the range of 1 μm to 10 μm.
The size of the blood holding tool 20 is not particularly limited as described above, and can be appropriately determined according to the amount of blood sample to be supplied. When the whole blood to be supplied is about 100 μL, the size of the entire blood holding device 20 is, for example, length 4 mm × width 1.5 mm × thickness 50 μm to length 60 mm × width 20 mm × thickness 400 μm, preferably length 5 mm × width 3 mm × thickness 100 μm to length 30 mm × width 15 mm × thickness 350 μm, more preferably length 10 mm × width 5 mm × thickness 200 μm to length 20 mm × width 12 mm × thickness 300 μm. The length refers to the length in the longitudinal direction of the blood retention tool, and the width refers to the length in the width direction.
In such a blood holding device 20, for example, the blood separating unit 22 and the developing unit 21 do not need to be strictly separated by a boundary line. In the blood holding device 20, when blood is dropped on the blood supply unit 23, the blood moves in the thickness direction and separates into blood cells and serum or plasma, and in the surface direction (in the direction of arrow A in the figure). Further expanded. The size of the blood supply unit 23 is, for example, length 2 mm × width 1.5 mm to length 15 mm × width 20 mm, preferably length 4 mm × width 3 mm to length 10 mm × width 15 mm, more preferably Length 4 mm x width 5 mm to length 6 mm x width 12 mm. The region in the thickness direction of the blood supply unit 23 normally corresponds to the blood cell separation unit 22.
The blood holding device 20 is composed of a single layer asymmetric porous membrane as described above. For this reason, for example, the solution containing the hemolysis inhibitor may be contained only in the portion to be the blood cell separation part 22, or the asymmetric porous membrane is immersed in the solution to impregnate the whole. You may let them.
Next, an example of preparing a serum or plasma sample by adding blood to the blood holding device 20 will be described with reference to FIG.
First, blood is dropped into the blood supply unit 23. The blood separates blood cells while moving inside the blood separation unit 22 in the thickness direction. Then, the serum or plasma moves in the surface direction (the direction of arrow A in the figure), and is developed on the development part 21 by capillary action. Thereafter, serum or plasma may be collected from the development part 21 as in the embodiment A-1.
(Embodiment A-3)
In order to further improve the efficiency of blood cell separation, a blood holding device composed of an asymmetric porous membrane having both a blood separation unit and a development unit as shown in Embodiment A-2, for example, the blood cell separation unit and It is preferable that a blood cell blocking part is further provided at the boundary with the development part. Specifically, a blood cell blocking portion composed only of pores having a diameter that cannot pass blood cells is formed in the width direction of the asymmetric porous membrane between the blood cell separating portion and the developing portion. And when blood is supplied, along the direction of blood deployment, the downstream side of the blood cell blocking part is a developing part, and the blood cell blocking part and the upstream side of the blood cell blocking part are a blood separating part and It is the structure which becomes. With such a structure, for example, even when the blood cells in the blood move not only in the thickness direction but also in the surface direction, the blood cell blocking portion reliably prevents the blood cells from passing through the deployment portion. In addition, it becomes possible to deploy only serum or plasma to the development part. Further, the blood cell blocking portion is, for example, a portion in which a groove is formed in the width direction of the asymmetric porous membrane and between the bottom surface of the groove and the asymmetric porous membrane surface corresponding to the bottom surface. Is preferred. Unless otherwise indicated, the blood holding device of the present embodiment has the same conditions (size, material, etc.) as those of Embodiment A-2.
An example of the blood holding tool is shown in the sectional view of FIG. As shown in the figure, the blood holding tool 30 is composed of a single-layered asymmetric porous membrane, similarly to the blood holding tool 20 of the embodiment A-2, and further has a groove in its width direction. Is formed. A portion between the bottom surface of the groove and the asymmetric porous membrane surface corresponding to the bottom surface is a blood cell blocking portion 32. The blood cell blocking part 32 and the upstream side in the direction of arrow A serve as a blood cell separation part, and the downstream side of the blood cell blocking part 32 serves as a deployment part 31. In addition, the blood supply part 33 is a surface having a large pore diameter of the blood cell separation part.
In the asymmetric porous membrane, the pore diameter of the blood cell blocking part 32 is, for example, in the range of 0.1 μm to 50 μm, and preferably in the range of 1 μm to 30 μm.
The size of the blood holding tool 30 is not particularly limited as described above, and can be appropriately determined according to the amount of blood sample to be supplied. When the whole blood to be supplied is about 100 μL, the size of the blood cell blocker 32 is, for example, length 0.1 mm × width 1.5 mm × thickness 10 μm to length 10 mm × width 20 mm × thickness 400 μm, preferably long The thickness is 0.2 mm × width 3 mm × thickness 15 μm to length 8 mm × width 15 mm × thickness 350 μm, and more preferably length 0.3 mm × width 5 mm × thickness 20 μm to length 6 mm × width 12 mm × thickness 300 μm.
The groove can be formed, for example, by compressing a part of the surface of the asymmetric porous membrane. For example, it can be formed by rolling and compressing a disk-shaped roller, or by compressing it to such an extent that the cutting edge cannot be cut with a dull blade. As described above, when the groove is formed by compression, the blood cell blocking portion includes a hole having a large pore diameter of the asymmetric porous membrane. However, since the hole is deformed or crushed by the compression, the blood cell Never pass.
Moreover, you may form the said groove | channel by cutting off a part of said asymmetric porous film, for example using cutters, such as a cutter. The size of the groove at this time is the same as described above.
Next, as a specific example of the blood retention tool of the present invention, an embodiment that can be used as a blood test tool will be described with reference to FIGS. The blood holding device of the present invention is not limited to these.
(Embodiment B-1)
The first embodiment (B-1) of the blood retention tool is shown in the sectional view of FIG. As shown in the figure, the blood holding device 40 includes a blood separation membrane 46 and a support 44, and a reagent portion 45 containing an analysis reagent is formed at one end of the blood separation membrane 46. A support 44 is laminated on one surface of the blood separation membrane 46 so that one end (the side opposite to the reagent part 45) is exposed, and this exposed part becomes the blood supply part 43.
According to such a blood holding device 40, for example, when blood is dropped on the blood supply unit 43, the dropped blood is separated into blood cells and serum while moving in the blood separation membrane 46 in the direction of arrow A. The Since the blood separation membrane 46 contains the hemolysis inhibitor as described above, blood cells and serum are separated while preventing hemolysis of the blood cells. Then, the separated serum moves through the blood separation membrane 46 and is developed in the reagent part 45, where the analysis reagent reacts with the analyte in the serum. This reaction may be detected by, for example, an electrochemical technique or an optical technique (including visual observation).
(Embodiment B-2)
The second embodiment (B-2) of the blood retention tool is shown in the sectional view of FIG. Unless otherwise indicated, this embodiment is the same as the first embodiment (B-1), and the same applies to the third to sixth embodiments (B-3 to B-6) shown below.
As shown in the figure, the blood holding device 50 includes a blood separation membrane 56, a reagent layer 55 containing an analysis reagent, and a support 54. A support 54 is stacked on one surface of the blood separation membrane 56 so that one end of the blood separation membrane 56 is exposed. Further, the reagent layer 55 is laminated on the other surface of the blood separation membrane 56 (the surface opposite to the support laminate surface) and on the end opposite to the blood supply unit 53.
According to such a blood holding device 50, for example, the serum separated in the blood separation membrane 56 is developed on the reagent layer 55 laminated on the blood separation membrane 56, and the analysis reagent in the reagent layer 55 and the serum in the serum are separated. Reacts with the analyte.
(Embodiment B-3)
The third embodiment (B-3) of the blood retention tool is shown in the sectional view of FIG. As shown in the figure, the blood holding device 60 includes a blood separation membrane 66 and supports 64 and 67. A support 67 is laminated on one surface of the blood separation membrane 66, and a support 64 is laminated on the other surface of the blood separation membrane 66 so that one end thereof is exposed. This exposed portion becomes the blood supply unit 63. The blood separation membrane 66 has three reagent parts containing an analysis reagent, and these first reagent part 651, second reagent part 652 and third reagent part 653 are arranged along the direction of arrow A in the figure. It is formed in order. The first reagent part 651, the second reagent part 652, and the third reagent part 653 contain the first analysis reagent, the second analysis reagent, and the third analysis reagent, respectively.
According to such a blood holding device 60, for example, the serum separated in the blood separation membrane 66 is first developed in the first reagent unit 651, and the analyte in the serum reacts with the first analysis reagent. To do. This reaction solution is further developed in the second reagent portion 652 and reacts with the second analysis reagent. And this reaction liquid is finally expand | deployed by the 3rd reagent part 653, and reacts with a 3rd analysis reagent. This reaction may be detected as described above.
Such a blood holding device 60 is suitable for the case where the measurement of an analysis target is performed using a multistage reaction, and according to the reaction sequence, the first reagent unit 651, the second reagent unit 652, and the third reagent. Reagents corresponding to the portions 653 may be disposed.
(Embodiment B-4)
The fourth embodiment (B-4) of the blood retention tool is shown in the sectional view of FIG. As shown in the figure, the blood retention tool 70 includes a blood separation membrane 76, three reagent layers (751, 752 and 753) containing analysis reagents, and supports 74 and 77. A support body 74 is laminated on one surface of the blood separation membrane 76 so that one end of the blood separation membrane 76 is exposed. In addition, a first reagent layer 751, a second reagent layer 752, and a third reagent layer 753 are sequentially stacked on the other surface of the blood separation membrane 76 along the direction of arrow A in FIG. A support 77 is further laminated through the layers. The first reagent layer 751, the second reagent layer 752, and the third reagent layer 753 contain the first analysis reagent, the second analysis reagent, and the third analysis reagent, respectively.
According to such a blood holding device 70, for example, the serum separated in the blood separation membrane 76 is obtained by stacking the first reagent portion 751, the second reagent layer 752, and the third reagent layer 753 stacked on the blood separation membrane 76. Respectively. Then, the analyte in the serum reacts with each analysis reagent in each reagent layer (751, 752 and 753). Therefore, for example, if a reagent corresponding to an analysis item is arranged in each reagent layer, a multi-item analysis can be performed on the same sample with the same tool.
(Embodiment B-5)
The fifth embodiment (B-5) of the blood retention tool is shown in the sectional view of FIG.
As shown in the figure, the blood holding tool 80 includes a blood separation membrane 86, a reagent layer 85 containing an analysis reagent, and supports 84 and 87. A support body 84 is laminated on one surface of the blood separation membrane 86 so that one end of the blood separation membrane 86 is exposed. Further, a support 87 having a through hole is laminated on the other surface of the blood separation membrane 86, and a reagent layer 85 is laminated on a portion corresponding to the through hole.
Further, as shown in the cross-sectional view of FIG. 9, three through holes of the support 97 are provided in order along the direction of the arrow A, and reagent layers (851, 852, and 85) are provided on the surface of the blood separation membrane 86 corresponding to each through hole. 853) may be provided. This blood retention tool 90 is suitable for the multi-item inspection, as in the fourth mode (B-4). In addition, the same code | symbol is attached | subjected to the same location as FIG.
(Embodiment B-6)
The sixth embodiment (B-6) of the blood retention tool is shown in the sectional view of FIG.
As shown in the figure, the blood holding device 100 includes a blood separation membrane 102, a development layer 101, and supports 104 and 107. A support 107 is laminated on one surface of the development layer 101, and a support 104 is laminated on the other surface so that one end thereof is exposed, and a blood separation membrane is formed on the exposed portion. 102 are stacked. The surface of the blood separation membrane 102 becomes the blood supply unit 103. In the development layer 101, a first reagent part 151 and a second reagent part 152 containing an analysis reagent are sequentially formed along the direction of arrow A in the figure. The second reagent unit 152 also serves as a first detection unit, and the second detection unit 153 is located downstream of the second reagent unit (first detection unit) 152 in the direction of arrow A.
Hereinafter, an example of a method for analyzing serum components by antigen-antibody reaction using the blood retention tool 100 will be described. In the first reagent part 151, a labeled first antibody for the analyte (antigen) and a labeled third antibody for the second antibody described later are arranged. An unlabeled second antibody against the antigen is immobilized on the second reagent part 152. For the labeled first antibody and the labeled third antibody, colored latex particles can be used as labels, respectively.
First, when blood is dropped into the blood supply unit 103, serum is separated by passing through the blood separation membrane 102, and the separated serum moves in the direction of arrow A in the development layer 101. The serum is first developed in the first reagent unit 151, and the antigen in the serum and the labeled first antibody form a complex by an antigen-antibody reaction. This complex, serum containing unlabeled labeled first antibody and labeled third antibody is further developed in the second reagent part 152. Here, the complex and the labeled third antibody bind to the unlabeled second antibody immobilized on the second reagent unit 152 by an antigen-antibody reaction and are captured by the second reagent unit 152. Then, a serum containing the labeled first antibody that has not been bound to the antigen, the complex that has not been captured by the first detection unit (second reagent unit) 152, and the labeled third antibody is further added to the second detection unit 153. Expanded to
And the measurement is performed as follows. First, the first detection unit (second reagent unit) 152 detects a complex of the captured antigen and the labeled first antibody. Further, the first detection unit 152 detects the captured labeled third antibody, and the second detection unit 153 detects the labeled third antibody that has not been captured by the second reagent unit 152. These detections can be performed by measuring the absorbance at a wavelength specific to each label (colored latex) of the labeled first antibody and labeled third antibody. And the capture efficiency in the 2nd reagent part 152 is calculated | required from the detection result of a labeled 3rd antibody. The true complex amount (antigen amount) is calculated from the capture efficiency and the apparent detection result of the complex. By measuring together the labeled third antibody in this way, the capture efficiency can be used for correction. Therefore, according to the present embodiment, the analysis system for the analysis target (antigen) can be improved.

前記表１に示すように、比較例１では溶血が見られたが、同じ濃度もしくはそれ以下の濃度の溶血防止剤溶液を含有させた実施例１（１−１〜１−８）では溶血が防止された。
（実施例２、比較例２）
溶血防止剤として、下記表２に示す各種溶血防止剤を含有させた以外は、実施例１と同様にして血液保持用具を作製し、これを用い血球の展開を確認した。
（血球展開距離の測定）
血球展開距離は、血漿が展開して展開部の先端（図１において、展開部１１の右端部）に達した時点において、展開用多孔質膜の血液を点着した側の端から血球の展開した先端（図１において、展開部１１の左端部）までの距離を測定した。

前記表１および表２に示すように、５％グリシン（比較例２）では溶血が生じたのに対し、溶血防止剤を同量若しくはそれ以下の量を含有させた実施例２（２−１〜２−６）では、溶血防止はもちろんのこと、血球の展開距離も比較例２より抑えることができた。このように、前記溶血防止剤を含有させた本実施例の血液保持用具によれば、血球展開の抑制と血漿展開の促進とにより血球と血漿とが十分に分離されるため、血漿の展開量が増加し、血漿回収率を向上できることがわかった。
（実施例３、比較例３）
以下に示す展開用多孔質膜（ろ紙、酢酸セルロース膜）および血球分離用多孔質膜（グラスフィルター）を使用し、溶血防止剤としてバリンを用いた以外は、前記実施例１と同様にして血液保持用具を作製した（実施例３−１、３−２）。なお、比較例３としては、血球分離用多孔質膜にバリンを含有させない以外は、前記実施例３−１もしくは３−２と同様にして血液保持用具を作製した（比較例３−１、３−２）。そして、これらを用いて実施例１および２と同様に血球分離を行い、溶血の有無、および血球の展開を確認した。これらの結果を下記表３に示す。
（ろ紙）
商品名３ＭＭｃｈｒ（ワットマン社製）
材質：セルロース製
厚み：３４０μｍ
重量：１８５ｇ／ｍｍ^２
空孔率（ｐｏｒｏｓｉｔｙ）：２０ｓｅｃ／１００ｍＬ／ｉｎｃｈ^２
（酢酸セルロース膜）
商品名Ｃ３００Ａ２９３Ｃ（東洋ろ紙社製）
材質：酢酸セルロース
厚み：１８５μｍ
重量：４０ｇ／ｍｍ^２
（血球分離用多孔質膜）
厚み１２００μｍのグラスフィルター（商品名ＡＰ２５：ミリポア社製）を使用した。

前記表３に示すように、グラスフィルターにバリンを含有させていない比較例３−１、３−２では溶血が生じたのに対して、実施例３−１、３−２では共に溶血が起こらなかった。また、実施例３−１は、比較例３−１よりも短い血球展開距離を示し、実施例３−２も、比較例３−２より短い血球展開距離を示した。このことから、前記実施例２と同様に、本実施例の血液保持用具によれば、血球の展開を抑制できるため、血漿のみの展開を増加でき、その回収率を向上できるといえる。なお、前記実施例３−１と３−２とにおいて、血球展開距離が異なるが、これは展開用多孔質膜の種類に起因するものである。
（実施例４、比較例４）
溶血防止剤として、各種濃度（５％、２％、１％）のバリン溶液、２％のロイシン溶液を含有させた以外は、実施例１と同様にして血液保持用具をそれぞれ１０個ずつ作製し、これを用いて血漿を回収し、血漿成分の測定を行った（実施例４）。
比較例４としては、所定濃度（２０％、５％、０％）のグリシン溶液をグラスフィルターに含有させた以外は、前記実施例１と同様にして血液保持用具それぞれ１０個ずつを作製した。
各血液保持用具に全血検体を滴下し、血球分離および血漿展開を行った。血漿の展開後、展開用多孔質膜のうち血漿のみが展開された部分を１０ｍｍ×６ｍｍの大きさに切り出した。この切片を、各溶血防止剤について１０枚集め、これらを直接遠心分離機にかけて（１５，０００ｇ、１０分間）、血漿を回収した。この血漿を試料として各成分量の測定を行った。なお、コントロールとしては、前記全血検体を遠心分離（３０００ｇ、１０分間）して得られた血漿を使用した。
（成分量の測定）
各成分量は、以下に示す市販キットを使用し、それらの使用方法に準じて、自動分析器（商品名：日立７０７０、日立製作所社製）により測定した。各種測定において、ブランクとして精製水を用いた。
１． 高比重リポ蛋白コレステロール（ＨＤＬ−Ｃ）
：商品名 リキテック ＨＤＬ−Ｃ（ロシュ社製）
２． アミラーゼ（ＡＭＹ）
：商品名 ＡＭＹ−ＮＰ（デンカ生研社製）
３． アルブミン（ＡＬＢ）
：商品名 クリンメイトアルブミン（第一化学薬品社製）
４． グルタミック−オキサロアセティックトランスアミナーゼ（ＧＯＴ）
：商品名 リキテック ＧＯＴ ＩＦＣＣ（ロシュ社製）
５． ガンマグルタミルトランスペプチダーゼ（ＧＧＴ）
：商品名 γ−ＧＴＰ−Ｊ−ＨＡワコー（和光純薬社製）
６． グルタミック−ピルビックトランスアミナーゼ（ＧＰＴ）
：商品名 リキテック ＧＰＴ ＩＦＣＣ（ロシュ社製）
７． トリグリセライド（ＴＧ）
：商品名 リキテック ＴＧ（ロシュ社製）
８． ラクテートデヒドロゲナーゼ（ＬＤＨ）
：商品名 ＬＤＨ ＩＩ−ＨＡワコー（和光純薬社製）
９． 総タンパク（ＴＰ）
：商品名 ＲＤ総タンパク（ロシュ社製）
１０． 総ビリルビン（Ｔ−Ｂｉｌ）
：商品名 Ｔ−Ｂｉｌ−Ｖ５（アズウェル社製）
１１． クレアチニン（ＣＲＥ）
：商品名 リキテック クレアチニン ＰＡＰ（ロシュ社製）
１２． 総コレステロール（ＴＣ）
：商品名 Ｔ−Ｃｈｏ−ＫＬ（国際試薬社製）
１３． アルカリホスファターゼ（ＡＬＰ）
：商品名 アルカリ性ホスファＩＩ−ＨＲテストワコー（和光純薬社製）
１４． 尿素窒素（ＢＵＮ）
：商品名 尿素窒素ＩＩ−ＨＡテストワコー（和光純薬社製）
１５． 尿酸（ＵＡ）
：商品名 ウリカラー・リキッド尿酸（小野薬品社製）
１６． フルクトサミン（ＦＲＡ）
：商品名 リキテックフルクトサミン（ロシュ社製）
１７． グルコース（Ｇｌｕ）
：商品名 リキテックグルコース（ロシュ社製）
１８． クレアチニンホスホキナーゼ（ＣＰＫ）
：商品名 ＣＫ Ｅ−ＨＡテストワコー（和光純薬社製）
これらの実施例４および比較例４の結果について、コントロールの測定値を１００％とした場合の相対値（平均％）を下記表４に示す。

前記表４に示すように、グリシン添加の比較例によると、各成分の測定精度にバラツキが見られた。特にＧＧＴについてはコントロールに対する相対値が５０％前後という結果になった。これに対して、バリンまたはロイシンを添加した血液保持用具によれば、各成分ともに優れた測定結果が得られ、特にＧＧＴについても同様に優れた結果をなった。また、実施例４は全て溶血を防止できたが、比較例４においては、５％のグリシン溶液を含有させた血液保持用具について溶血が確認された。また、２０％のグリシン溶液を含有させた比較例４の血液保持用具については、すべての成分項目において、低い相対値を示す結果となった。これは、高グリシン濃度が原因となって血球が萎縮し、その分血漿成分の体積増加が起こり、その結果血漿成分の希釈が生じたためと考えられる。以上の結果から、本発明の血液保持用具によれば、溶血を防止し、かつ、各種成分測定に影響を与えることのないサンプルを調製できるといえる。(Example 1, Comparative Example 1)
A blood retention tool shown in FIG. 1 in the embodiment A-1 was prepared, and blood cells were separated using this to confirm the presence or absence of hemolysis. Hereinafter, unless otherwise indicated, the solution concentration (%) of the hemolysis inhibitor indicates the weight (unit: g) of the hemolysis inhibitor in the volume of the solvent (100 mL) (unit: (w / v)%). .
(Porous membrane for deployment)
A commercially available asymmetric porous membrane made of polyether sulfonic acid (trade name Prime Care S / G: manufactured by Spectral Co.) was washed under the following conditions and used as a porous membrane for development. The porous membrane has a thickness of 290 μm, a length of 20 mm, and a width of 6 mm, a maximum pore diameter of 200 μm, and a minimum pore diameter of 2 μm. In the washing, first, the asymmetric porous membrane was immersed in purified water and allowed to stand for 10 minutes, then replaced with new purified water, and this operation was performed 5 times in total. Then, after the asymmetric porous material was air-dried for half a day, it was further dried for half a day in a desiccator as the porous membrane for development.
(Blood separation membrane)
Each hemolysis inhibitor shown in Table 1 below was prepared by dissolving the hemolysis inhibitor in a solvent (distilled water) in advance. Subsequently, 30 μL of the hemolysis inhibitor solution having various concentrations shown in Table 1 below was dropped onto a commercially available glass filter (trade name AP25: manufactured by Millipore) having a thickness of 1200 μm, a length of 12 mm, and a width of 6 mm and dried. The glass filter after drying was used as a blood separation membrane. The following silk essence was used as a brand name silk essence manufactured by Fukui Silk Corporation.
(Method for producing blood holding device)
A PET film having a length of 50 mm, a width of 6 mm, and a thickness of 180 μm is prepared as a support, and the development porous membrane is adhered to the PET film with an adhesive. Further, as shown in FIG. The glass filter was placed on one end of the glass. Then, PET for covering (thickness: 100 μm) was disposed on the surface of the developing porous membrane where the glass filter was not disposed. This was used as a blood retention tool (Examples 1-1 to 1-8).
On the other hand, as Comparative Example 1, a blood retention tool was produced in the same manner as in Example 1 except that a glass filter contained a 5% glycine solution.
(Hemolysis of blood cells)
100 μL of whole blood (hematocrit 38%) was spotted on the surface of the glass filter of each produced blood holding tool, and the blood cells and plasma were separated to develop the plasma. After 2 minutes, the presence or absence of hemolysis of blood cells was confirmed. The plasma was developed until it completely penetrated into the tip of the development part (the right end part of the development part 11 in FIG. 1). Hemolysis of blood cells was judged by whether or not the developed plasma was colored red by Hb in the blood cells.

As shown in Table 1, hemolysis was observed in Comparative Example 1, but hemolysis was observed in Example 1 (1-1 to 1-8) containing a hemolysis inhibitor solution having the same or lower concentration. Prevented.
(Example 2, comparative example 2)
A blood retention tool was prepared in the same manner as in Example 1 except that various hemolysis inhibitors shown in Table 2 below were included as the hemolysis inhibitor, and the development of blood cells was confirmed using this.
(Measurement of blood cell deployment distance)
The blood cell deployment distance is determined by the expansion of the blood cell from the end of the porous membrane for spreading on the side where the blood is spotted when the plasma spreads and reaches the tip of the deployment portion (the right end portion of the deployment portion 11 in FIG. 1). The distance to the tip (the left end of the developed portion 11 in FIG. 1) was measured.

As shown in Table 1 and Table 2 above, hemolysis occurred in 5% glycine (Comparative Example 2), whereas Example 2 (2-1) containing the same or less amount of the hemolysis inhibitor was used. In 2-6), the development distance of blood cells as well as the prevention of hemolysis could be suppressed as compared with Comparative Example 2. Thus, according to the blood holding device of the present example containing the hemolysis inhibitor, blood cells and plasma are sufficiently separated by suppressing blood cell development and promoting plasma development. It has been found that the plasma recovery can be improved.
(Example 3, Comparative Example 3)
Blood was prepared in the same manner as in Example 1 except that the following development porous membrane (filter paper, cellulose acetate membrane) and blood cell separation porous membrane (glass filter) were used, and valine was used as a hemolysis inhibitor. A holding tool was produced (Examples 3-1, 3-2). In Comparative Example 3, a blood holding device was prepared in the same manner as in Example 3-1 or 3-2 except that valine was not contained in the blood cell separation porous membrane (Comparative Examples 3-1, 3). -2). Using these, blood cells were separated in the same manner as in Examples 1 and 2, and the presence or absence of hemolysis and the development of blood cells were confirmed. These results are shown in Table 3 below.
(Filter paper)
Product name 3MMchr (manufactured by Whatman)
Material: Cellulose Thickness: 340 μm
Weight: 185 g / mm ²
Porosity: 20 sec / 100 mL / inch ²
(Cellulose acetate membrane)
Product name C300A293C (manufactured by Toyo Roshi)
Material: Cellulose acetate Thickness: 185μm
Weight: 40 g / mm ²
(Porous membrane for blood cell separation)
A glass filter having a thickness of 1200 μm (trade name AP25: manufactured by Millipore) was used.

As shown in Table 3, hemolysis occurred in Comparative Examples 3-1 and 3-2 where the glass filter did not contain valine, whereas hemolysis occurred in Examples 3-1 and 3-2. There wasn't. In addition, Example 3-1 showed a shorter blood cell deployment distance than Comparative Example 3-1, and Example 3-2 also showed a shorter blood cell deployment distance than Comparative Example 3-2. From this, it can be said that, as in the case of Example 2, according to the blood holding device of this example, the development of blood cells can be suppressed, the development of only plasma can be increased, and the recovery rate can be improved. In addition, although the blood cell expansion | deployment distance differs in the said Examples 3-1 and 3-2, this originates in the kind of porous membrane for expansion | deployment.
(Example 4, comparative example 4)
Ten blood-holding devices were prepared in the same manner as in Example 1 except that various concentrations (5%, 2%, 1%) of valine solution and 2% leucine solution were included as hemolysis inhibitors. Using this, plasma was collected, and plasma components were measured (Example 4).
As Comparative Example 4, ten blood retention tools were prepared in the same manner as in Example 1 except that a glycine solution having a predetermined concentration (20%, 5%, 0%) was contained in a glass filter.
A whole blood sample was dropped into each blood holding device, and blood cell separation and plasma expansion were performed. After the development of the plasma, a portion of the development porous membrane where only the plasma was developed was cut out to a size of 10 mm × 6 mm. Ten pieces of these sections were collected for each hemolysis inhibitor, and these were directly centrifuged (15,000 g, 10 minutes) to collect plasma. Using this plasma as a sample, the amount of each component was measured. As a control, plasma obtained by centrifuging the whole blood sample (3000 g, 10 minutes) was used.
(Measurement of amount of ingredients)
The amount of each component was measured with an automatic analyzer (trade name: Hitachi 7070, manufactured by Hitachi, Ltd.) according to the method of using the following commercially available kits. In various measurements, purified water was used as a blank.
1. High density lipoprotein cholesterol (HDL-C)
: Product name Rikitech HDL-C (Roche)
2. Amylase (AMY)
: Product name AMY-NP (manufactured by Denka Seken)
3. Albumin (ALB)
: Product name Klimate albumin (Daiichi Chemical Co., Ltd.)
4). Glutamic-oxaloacetic transaminase (GOT)
: Product name Rikitech GOT IFCC (Roche)
5). Gamma glutamyl transpeptidase (GGT)
: Product name γ-GTP-J-HA Wako (manufactured by Wako Pure Chemical Industries, Ltd.)
6). Glutamic-Pyrvic transaminase (GPT)
: Product name Rikitec GPT IFCC (Roche)
7). Triglyceride (TG)
: Product name LIQUITECH TG (Roche)
8). Lactate dehydrogenase (LDH)
: Product name LDH II-HA Wako (Wako Pure Chemical Industries, Ltd.)
9. Total protein (TP)
: Product name RD total protein (Roche)
10. Total bilirubin (T-Bil)
: Product name T-Bil-V5 (manufactured by Aswell)
11. Creatinine (CRE)
: Product name Liquitec Creatinine PAP (Roche)
12 Total cholesterol (TC)
: Product name T-Cho-KL (made by Kokusai Reagent Co., Ltd.)
13. Alkaline phosphatase (ALP)
: Product name Alkaline Phospha II-HR Test Wako (Wako Pure Chemical Industries, Ltd.)
14 Urea nitrogen (BUN)
: Product name Urea Nitrogen II-HA Test Wako (Wako Pure Chemical Industries, Ltd.)
15. Uric acid (UA)
: Product name Uricolor liquid uric acid (manufactured by Ono Pharmaceutical)
16. Fructosamine (FRA)
: Product name Liquitec fructosamine (Roche)
17. Glucose (Glu)
: Product name Liquitec Glucose (Roche)
18. Creatinine phosphokinase (CPK)
: Product name CK E-HA Test Wako (Wako Pure Chemical Industries, Ltd.)
Regarding the results of Example 4 and Comparative Example 4, the relative values (average%) when the measured value of the control is 100% are shown in Table 4 below.

As shown in Table 4, according to the comparative example in which glycine was added, the measurement accuracy of each component varied. Especially for GGT, the value relative to the control was about 50%. On the other hand, according to the blood holding device to which valine or leucine was added, excellent measurement results were obtained for each component, and particularly excellent results were obtained for GGT as well. Further, all of Example 4 could prevent hemolysis, but in Comparative Example 4, hemolysis was confirmed for a blood holding device containing a 5% glycine solution. Moreover, about the blood holding tool of the comparative example 4 containing a 20% glycine solution, it became a result which shows a low relative value in all the component items. This is presumably because blood cells were atrophied due to the high glycine concentration, and the volume of the plasma component increased accordingly, resulting in dilution of the plasma component. From the above results, it can be said that according to the blood holding device of the present invention, it is possible to prepare a sample that prevents hemolysis and does not affect the measurement of various components.

  As described above, according to the blood cell separation membrane of the present invention, blood cells and serum or plasma can be separated while preventing hemolysis of blood cells, so that a serum or plasma sample can be efficiently prepared. Moreover, since the sample prepared by this has little influence on the measurement system of various components, a highly accurate measurement is attained. For this reason, the blood cell separation membrane and blood holding device of the present invention are useful, for example, in the fields of clinical medicine as described above.

Claims (20)

Including a porous membrane that separates blood cells from serum or plasma in blood,
The blood cell separation membrane, wherein the porous membrane contains at least one hemolysis inhibitor selected from the group consisting of hydrophobic aminocarboxylic acid, silk-derived protein, Tris, TES, ε-aminohexanoic acid, tranexamic acid and heparin . The blood cell separation membrane according to claim 1, wherein the hydrophobic aminocarboxylic acid is a hydrophobic amino acid. The blood cell separation membrane according to claim 2, wherein the hydrophobic amino acid is at least one amino acid selected from the group consisting of alanine, valine, leucine and isoleucine. The blood cell separation membrane according to claim 1, wherein the porous membrane contains at least one hemolysis inhibitor selected from the group consisting of valine, leucine, ε-aminohexanoic acid, tranexamic acid, and silk essence. The blood cell separation membrane according to any one of claims 1 to 4, wherein the content of the hemolysis inhibitor is in the range of 1 mg to 50 mg per volume (cm ³ ) of the porous membrane. 6. The porous membrane according to claim 1, wherein the porous membrane is at least one of a glass filter and an asymmetric porous membrane having a pore size distribution in which an average pore size continuously or discontinuously decreases in a thickness direction. 2. The blood cell separation membrane according to 1. The blood cell separation membrane according to claim 6, wherein the material of the asymmetric porous membrane is at least one resin selected from the group consisting of polysulfone, polyethersulfone, polyamide, polyimide, polycarbonate, polystyrene, and polyarylhydrazide. The blood cell separation membrane according to claim 6 or 7, wherein the asymmetric porous membrane has a maximum pore size in the range of 30 to 300 µm and a minimum pore size in the range of 1 to 30 µm. The blood cell separation membrane according to any one of claims 1 to 8, wherein the hemolysis inhibitor is contained on at least one surface side of the blood cell separation membrane. A blood cell according to any one of claims 1 to 9, further comprising: a blood cell separation unit that separates blood cells in blood; and a development unit that develops serum or plasma in blood. A blood retention tool that is a separation membrane. The blood holding tool according to claim 10, wherein the blood cell separation part is laminated on the development part. The blood retention tool according to any one of claims 10 to 11, wherein the development portion is a porous membrane. The blood holding device according to claim 10, wherein the blood separation membrane comprises a blood cell separation part and a development part. The blood retention tool according to claim 12, wherein the material of the porous membrane is at least one resin selected from the group consisting of polysulfone, polyethersulfone, polyamide, polyimide, polycarbonate, polystyrene, and polyarylhydrazide. The blood cell separation portion is an asymmetric porous membrane, the asymmetric porous membrane further has the development portion, and in the asymmetric porous membrane, the blood cell separation portion and the development portion are positioned in a plane direction. The blood holding device according to claim 10, wherein the blood supply side is the blood cell separation part, and the downstream side is a development part along a development direction of the supplied blood. The blood cell separation portion is an asymmetric porous membrane, the asymmetric porous membrane further has the development portion, and in the asymmetric porous membrane, the blood cell separation portion and the development portion are positioned in a plane direction. 16. A blood cell blocking portion comprising only pores having a pore diameter through which blood cells cannot pass is formed in the width direction of the asymmetric porous membrane between the blood cell separating portion and the developing portion. Blood retention tool. 17. The asymmetric porous membrane according to claim 16, wherein a groove is formed in the width direction, and a portion between the bottom surface of the groove and the asymmetric porous membrane surface corresponding to the bottom surface is a blood cell blocking portion. Blood retention tool. The development part further contains at least one hemolysis inhibitor selected from the group consisting of hydrophobic aminocarboxylic acid, silk-derived protein, Tris, TES, ε-aminohexanoic acid, tranexamic acid and heparin. The blood holding tool according to any one of 10 to 17. The blood retention tool according to any one of claims 10 to 18, wherein a reagent part containing an analysis reagent is formed in the development part. Furthermore, the blood retention tool as described in any one of Claims 10-19 provided with the reagent layer containing a reagent.

Images