Blood collection container, method for separating plasma, method for separating extracellular free nucleic acid, and method for separating extracellular vesicle

Abstract

Provided is a blood collection container which can suppress the release of exosomes from platelets, and therefore, can suppress the contamination of plasma by exosomes derived from platelets.　A blood collection container according to the present invention is configured so as to collect a predetermined amount of blood, and comprises: a blood collection container body; a plasma separation material stored in the blood collection container body; and an aqueous solution stored in the blood collection container body. The aqueous solution contains an anticoagulant. When a specific pH is measured, the pH of a specific mixed liquid (X) is 3.5-5.5.

Description

Blood collection container, plasma separation method, extracellular free nucleic acid separation method, and extracellular vesicle separation method

[0001] The present invention relates to a blood collection container. The present invention also relates to a method for separating plasma, a method for separating extracellular free nucleic acids, and a method for separating extracellular vesicles using the blood collection container.

[0002] In clinical testing, blood collection containers containing anticoagulants are widely used. After blood is collected in the blood collection container containing the anticoagulant, the blood can be separated into plasma and blood cells by centrifuging the blood collection container.

[0003] Also known are blood collection containers containing a plasma separation material, as shown in the following Patent Documents 1 and 2. For example, Patent Document 1 describes a blood collection container containing a plasma separation composition containing a resin and an inorganic powder, and Patent Document 2 describes a blood collection container containing a plasma separation tool.

[0004] WO2010 / 053180A1 WO2010 / 132783A1

[0005] In clinical settings, after blood collected in a blood collection container is centrifuged to separate plasma and blood cells, several days may pass before the plasma is available for testing. With conventional blood collection containers, exosomes may be released from platelets mixed in the plasma before the plasma is available for testing.

[0006] Platelet-derived exosomes contain miRNA (microRNA). Therefore, when exosomes are released from platelets, they can affect test results. For example, in tests that detect extracellular free nucleic acids (e.g., cell-free RNA) in plasma, the test results can vary depending on the miRNA present in the plasma.

[0007] An object of the present invention is to provide a blood collection container that can suppress the release of exosomes from platelets and therefore suppress the contamination of plasma with platelet-derived exosomes. Another object of the present invention is to provide a method for separating plasma, a method for separating extracellular free nucleic acids, and a method for separating extracellular vesicles using the blood collection container.

[0008] This specification discloses the following blood collection container, plasma separation method, extracellular free nucleic acid separation method, and extracellular vesicle separation method.

[0009] Item 1. A blood collection container for collecting a predetermined amount of blood, comprising a blood collection container body, a plasma separation material contained within the blood collection container body, and an aqueous solution contained within the blood collection container body, wherein the aqueous solution contains an anticoagulant, and when the pH is measured as described below, the pH of the mixed solution (X) is 3.5 or more and 5.5 or less.

[0010] pH measurement: A solution having a pH of 7.4 containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 0.24 g / L potassium dihydrogen phosphate, and water is obtained. A volume of the solution having a pH of 7.4 equal to the predetermined volume of blood to be collected in the blood collection container is collected into the blood collection container, and a mixed solution (X) is obtained by mixing the solution having a pH of 7.4 with the aqueous solution. The pH of the resulting mixed solution (X) is measured.

[0011] Item 2. The blood collection container according to Item 1, wherein the aqueous solution contains an organic acid or an inorganic acid.

[0012] Item 3. The blood collection container according to Item 1 or 2, wherein the aqueous solution contains an organic acid.

[0013] Item 4. The blood collection container according to Item 3, wherein the organic acid includes citric acid, succinic acid, oxalic acid, or acetic acid.

[0014] Item 5. The blood collection container according to Item 3 or 4, wherein the content of the organic acid in 100% by weight of the aqueous solution is 1% by weight or more and 10% by weight or less.

[0015] Item 6. The blood collection container according to any one of Items 1 to 5, wherein the aqueous solution contains a disaccharide, and the disaccharide contains sucrose.

[0016] Item 7. The blood collection container according to any one of Items 1 to 6, wherein the aqueous solution contains an inorganic salt.

[0017] Item 8. The blood collection container according to any one of Items 1 to 7, wherein the aqueous solution has a pH of 3.0 or more and 5.5 or less.

[0018] Item 9. The blood collection container according to any one of Items 1 to 8, wherein the specific gravity of the plasma separation material at 25°C is 1.020 or more and 1.040 or less.

[0019] Item 10. The blood collection container according to any one of Items 1 to 9, wherein the plasma separation material is a plasma separation composition.

[0020] Item 11. The blood collection container according to Item 10, wherein the plasma separation composition comprises an organic component that has fluidity at 25°C and an inorganic fine powder, the organic component comprising a resin, and the inorganic fine powder comprising finely powdered silica.

[0021] Item 12. The blood collection container according to Item 11, wherein the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic-based resin.

[0022] Item 13. The blood collection container according to any one of Items 1 to 12, wherein 4 mL to 9.5 mL of blood is collected per 1 mL of the aqueous solution contained in the blood collection container body.

[0023] Item 14. The blood collection container according to any one of Items 1 to 13, which is used to separate extracellular free nucleic acids or extracellular vesicles in blood.

[0024] Item 15. A method for separating plasma, comprising the steps of collecting blood in the blood collection container according to any one of Items 1 to 14, and centrifuging the blood collection container into which the blood has been collected.

[0025] Item 16. A method for separating extracellular free nucleic acid, comprising the steps of: collecting blood in the blood collection container according to any one of Items 1 to 14; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; and separating extracellular free nucleic acid from the separated plasma.

[0026] Item 17. A method for separating extracellular vesicles, comprising the steps of collecting blood in the blood collection container according to any one of Items 1 to 14, centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.

[0027] The blood collection container according to the present invention is a blood collection container from which a predetermined amount of blood is collected. The blood collection container according to the present invention comprises a blood collection container body, a plasma separation material contained within the blood collection container body, and an aqueous solution contained within the blood collection container body, the aqueous solution containing an anticoagulant. In the blood collection container according to the present invention, when a specific pH is measured, the pH of the specific mixed solution (X) is 3.5 or more and 5.5 or less. Because the blood collection container according to the present invention has the above configuration, it is possible to suppress the release of exosomes from platelets, and therefore to suppress the contamination of plasma with platelet-derived exosomes.

[0028] FIG. 1 is a front cross-sectional view showing a schematic diagram of a blood collection container according to one embodiment of the present invention.

[0029] The present invention will be described in detail below.

[0030] (Blood collection container) The blood collection container according to the present invention is a blood collection container for collecting a predetermined amount of blood. The blood collection container according to the present invention comprises a blood collection container body, a plasma separation material contained in the blood collection container body, and an aqueous solution contained in the blood collection container body, the aqueous solution containing an anticoagulant.

[0031] In the blood collection container according to the present invention, when the pH is measured as described below, the pH of the mixed solution (X) is 3.5 or more and 5.5 or less.

[0032] pH measurement: A solution having a pH of 7.4 containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 0.24 g / L potassium dihydrogen phosphate, and water is obtained. An amount of the solution having a pH of 7.4 equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixed solution (X) is obtained by mixing the solution having a pH of 7.4 with the aqueous solution. The pH of the resulting mixed solution (X) is measured.

[0033] The blood collection container according to the present invention has the above-described configuration, and therefore can suppress the release of exosomes from platelets, thereby suppressing the contamination of plasma with platelet-derived exosomes.

[0034] In general, it is difficult to prevent platelets from contaminating plasma in blood collection containers. While blood collection containers equipped with plasma separators can prevent red blood cells and white blood cells from contaminating plasma to some extent, it is difficult to prevent platelets from contaminating plasma. This is because the specific gravity of the plasma separator is similar to that of platelets. Therefore, conventional blood collection containers can release exosomes from platelets contaminated in plasma. The present inventors have intensively investigated a configuration for effectively suppressing the release of exosomes from platelets. As a result, the present inventors have found that the release of exosomes from platelets can be effectively suppressed when the pH of the liquid in contact with platelets is within a specific range.

[0035] The liquid with a pH of 7.4 is a liquid having a pH similar to that of blood (a liquid simulating the pH of blood). The mixed liquid (X) is a liquid having a pH similar to that of a mixed liquid of blood and the aqueous solution (a liquid simulating the pH of a mixed liquid of blood and the aqueous solution). When blood is collected in the blood collection container of the present invention, the mixed liquid of blood and the aqueous solution becomes weakly acidic, and the plasma separated by centrifugation also becomes weakly acidic. Therefore, with the blood collection container of the present invention, even if the separated plasma is stored for several days, the release of exosomes from platelets contaminating the plasma is effectively suppressed, and as a result, the incorporation of platelet-derived exosomes into the plasma can be suppressed.

[0036] Furthermore, since the blood collection container according to the present invention is provided with a plasma separation material, by controlling the specific gravity of the plasma separation material, it is possible to prevent red blood cells and white blood cells from being mixed into the plasma.

[0037] The blood collection container according to the present invention can suppress the contamination of plasma with platelet-derived exosomes, as well as the contamination of plasma with red blood cells and white blood cells, and therefore can reduce the amount of miRNA contaminating the plasma even if the separated plasma is stored for several days.

[0038] The pH of the mixed solution (X) is measured as follows.

[0039] A solution having a pH of 7.4 containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 0.24 g / L potassium dihydrogen phosphate, and water is prepared. The composition of the solution having a pH of 7.4 is 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, and 0.24 g / L potassium dihydrogen phosphate, and the solvent is water. The solution having a pH of 7.4 can be obtained, for example, by mixing 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 900 mL of water, adjusting the pH with a pH adjuster (e.g., hydrochloric acid) as needed, and then adding water to make the solution 1 L. The liquid with a pH of 7.4 is collected into the blood collection container in an amount equal to the predetermined amount of blood to be collected in the blood collection container, and a mixed liquid (X) is obtained by mixing the liquid with a pH of 7.4 and the aqueous solution. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of the liquid with a pH of 7.4 is collected into the blood collection container, and the liquid with a pH of 7.4 and the aqueous solution are mixed by inversion or the like to obtain a mixed liquid (X). The pH of the resulting mixed liquid (X) is measured.

[0040] The pH of the mixed solution (X) and the solution having a pH of 7.4 is measured at 22°C using a pH meter.

[0041] From the viewpoint of exerting the effects of the present invention, the pH of the mixed solution (X) is 3.5 or more and 5.5 or less. The pH of the mixed solution (X) is preferably 3.8 or more, more preferably 4.0 or more, even more preferably 4.2 or more, preferably 5.2 or less, more preferably 5.0 or less, even more preferably 4.8 or less, and particularly preferably 4.6 or less. When the pH of the mixed solution (X) is not less than the above lower limit and not more than the above upper limit, the effects of the present invention can be exerted more effectively.

[0042] The blood collection container according to the present invention will be described in detail below. In this specification, "(meth)acrylic" means either or both of "acrylic" and "methacrylic".

[0043] (Plasma separation material) The blood collection container includes a plasma separation material accommodated in the blood collection container body. A conventionally known plasma separation material can be used as the plasma separation material. Examples of the plasma separation material include a plasma separation composition and a plasma separation tool. Because the plasma separation material is easy to prepare, the plasma separation material is preferably the plasma separation composition.

[0044] The specific gravity of the plasma separation material at 25°C is preferably 1.020 or more, more preferably 1.022 or more, even more preferably 1.025 or more, particularly preferably 1.028 or more, preferably 1.040 or less, more preferably 1.038 or less, and even more preferably 1.036 or less. When the specific gravity of the plasma separation material at 25°C is above the above lower limit and below the above upper limit, contamination of plasma with white blood cells and red blood cells can be more effectively suppressed. Furthermore, when the specific gravity of the plasma separation material at 25°C is above the above lower limit and below the above upper limit, contamination of plasma with platelets can be more effectively suppressed. Furthermore, plasma can be separated from blood more effectively.

[0045] The location where the plasma separation material is accommodated is not particularly limited as long as it is within the blood collection container body. The plasma separation material may be disposed at the bottom of the blood collection container body or on the inner wall surface of the blood collection container body.

[0046] <Plasma Separation Composition> The plasma separation composition is a composition that migrates between the plasma layer and the blood cell layer during centrifugation to form a partition. The plasma separation composition is also used for the purpose of preventing component migration between the plasma layer and the blood cell layer after centrifugation. The plasma separation composition preferably has thixotropy. The plasma separation composition may be contained in the bottom of the blood collection container body or may be disposed on the inner wall surface. From the viewpoint of more effectively exerting the effects of the present invention, the plasma separation composition is preferably contained in the bottom of the blood collection container body.

[0047] As the plasma separation composition, a conventionally known plasma separation composition can be used.

[0048] The plasma separation composition preferably contains an organic component having fluidity at 25° C. and an inorganic fine powder. In this case, the fluidity of the plasma separation composition is increased, and the strength of the partition wall formed after centrifugation can be increased. The organic component having fluidity at 25° C. and the inorganic fine powder may each be used alone or in combination of two or more types.

[0049] Organic component having fluidity at 25°C: The phrase "having fluidity at 25°C" means that the viscosity at 25°C is 500 Pa·s or less.

[0050] The viscosity of the organic component at 25° C. is preferably 10 Pa s or more, more preferably 30 Pa s or more, and preferably 300 Pa s or less, more preferably 200 Pa s or less. When the viscosity is equal to or greater than the lower limit and equal to or less than the upper limit, the fluidity of the composition for plasma separation is increased, and the strength of the partition wall formed after centrifugation can be increased.

[0051] The viscosity of the organic component at 25°C was measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 sec. －１ It is measured under the following conditions.

[0052] Examples of the organic component include a resin and a mixture of a resin and an organic compound such as a plasticizer. Therefore, the organic component preferably contains the resin, and more preferably contains the resin and the organic compound. When the organic component is a mixture of the resin and the organic compound, it is sufficient that the mixture (the organic component) has fluidity, and the resin or the organic compound does not have to have fluidity. When the organic component is a mixture of the resin and the organic compound, the resin may be, for example, a resin that is solid at 25°C. Only one type of the resin and the organic compound may be used, or two or more types may be used in combination.

[0053] Examples of the resin include petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, polyurethane-based resins, (meth)acrylic resins, silicone-based resins, α-olefin-fumaric acid ester copolymers, copolymers of sebacic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane-based resins, polyether polyester-based resins, etc. Only one type of the resins may be used, or two or more types may be used in combination.

[0054] The resin preferably includes a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin, and more preferably includes a (meth)acrylic resin, which further enhances the fluidity of the plasma separation composition and further enhances the strength of the partition wall formed after centrifugation.

[0055] Commercially available petroleum-based resins include "Regalite S5090" manufactured by Eastman Chemical Company.

[0056] Examples of the cyclopentadiene resin include a polymer of a cyclopentadiene monomer, a copolymer of a cyclopentadiene monomer and an aromatic monomer, and a dicyclopentadiene resin. The cyclopentadiene resin may be hydrogenated. The polymer of a cyclopentadiene monomer and the copolymer of a cyclopentadiene monomer and an aromatic monomer may be an oligomer.

[0057] Examples of the cyclopentadiene-based monomer include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0058] Examples of the aromatic monomer include styrene, methylstyrene, indene, and methylindene.

[0059] Commercially available dicyclopentadiene resins include "Scoretz SU500" and "Scoretz SU90" manufactured by Colon Co., Ltd.

[0060] Examples of the polyester resin include polyalkylene terephthalate resins and polyalkylene naphthalate resins, etc. Examples of the polyalkylene terephthalate resin include polyethylene terephthalate, polybutylene terephthalate, and poly-1,4-cyclohexanedimethylene terephthalate.

[0061] Examples of the polyurethane resin include a reaction product of a polyol compound and an isocyanate compound.

[0062] Examples of the (meth)acrylic resin include a resin obtained by polymerizing at least one type of (meth)acrylic acid ester monomer, and a resin obtained by polymerizing at least one type of (meth)acrylic acid ester monomer and at least one type of monomer other than a (meth)acrylic acid ester monomer.

[0063] Examples of the (meth)acrylic acid ester monomer include (meth)acrylic acid alkyl esters, (meth)acrylic acid polyalkylene glycol esters, (meth)acrylic acid alkoxyalkyl esters, (meth)acrylic acid hydroxyalkyl esters, (meth)acrylic acid glycidyl esters, (meth)acrylic acid dialkylaminoalkyl esters, (meth)acrylic acid benzyl esters, (meth)acrylic acid phenoxyalkyl esters, (meth)acrylic acid cyclohexyl esters, (meth)acrylic acid isobornyl esters, and (meth)acrylic acid alkoxysilylalkyl esters. When the (meth)acrylic acid ester monomer has an alkyl group, the number of carbon atoms in the alkyl group is preferably 1 or more and preferably 20 or less. The (meth)acrylic acid alkyl ester is preferably a (meth)acrylic acid alkyl ester having an alkyl group having 1 to 20 carbon atoms. The (meth)acrylic acid ester monomer may be used alone or in combination of two or more.

[0064] The organic compound may be a benzene polycarboxylic acid alkyl ester derivative. The organic compound is preferably a benzene polycarboxylic acid alkyl ester derivative. Therefore, the organic component is preferably a mixture of the resin and the benzene polycarboxylic acid alkyl ester derivative.

[0065] Examples of the benzene polycarboxylic acid alkyl ester derivatives include phthalates, trimellitates, pyromellitates, etc. The benzene polycarboxylic acid alkyl ester derivatives may be used alone or in combination of two or more.

[0066] Examples of the trimellitic acid ester include trimellitic acid tri-n-octyl, triisooctyl, and triisodecyl trimellitate.

[0067] Examples of the pyromellitic acid ester include tetraisooctyl pyromellitic acid.

[0068] Commercially available trimellitic acid esters include "Monocizer W700" and "Monocizer W-750" manufactured by DIC Corporation, and "Sansocizer TOTM" and "Sansocizer TITM" manufactured by New Japan Chemical Co., Ltd.

[0069] Commercially available pyromellitic acid esters include "Monocizer W-7010" manufactured by DIC Corporation.

[0070] The benzenepolycarboxylic acid alkyl ester derivative is preferably a phthalate ester, a trimellitate ester, or a pyromellitate ester, and more preferably a trimellitate ester.

[0071] The content of the organic component in 100% by weight of the plasma separation composition is preferably 75% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, and preferably 97% by weight or less.

[0072] Inorganic fine powder: Examples of the inorganic fine powder include fine silica powder, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, and zirconium powder.

[0073] The inorganic fine powder is preferably fine silica, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, or zirconium powder.

[0074] From the viewpoint of maintaining both the specific gravity and thixotropy of the plasma separation composition within a suitable range, the inorganic fine powder preferably contains finely powdered silica. The inorganic fine powder may contain finely powdered silica and an inorganic fine powder other than finely powdered silica (a second inorganic fine powder). The inorganic fine powder, the finely powdered silica, and the second inorganic fine powder may each be used alone or in combination of two or more.

[0075] The finely powdered silica includes natural silica and synthetic silica. Synthetic silica includes hydrophilic silica and hydrophobic silica. Hydrophilic silica imparts thixotropy to the plasma separation composition and adjusts the specific gravity by, for example, hydrogen bonding between hydroxyl groups on the particle surface. On the other hand, hydrophobic silica has a smaller effect of imparting thixotropy than hydrophilic silica.

[0076] From the viewpoint of maintaining both the specific gravity and thixotropy of the plasma separation composition within a suitable range, the finely powdered silica preferably contains hydrophilic silica, and more preferably contains hydrophilic silica and hydrophobic silica.

[0077] The second inorganic fine powder is preferably an inorganic fine powder having a larger specific gravity than fine silica powder, and more preferably an inorganic fine powder having a specific gravity of 3 or more, such as zinc oxide powder, titanium oxide powder, or alumina powder.

[0078] The specific gravity of the second inorganic fine powder is preferably 3 or more, more preferably 3.5 or more, and even more preferably 4 or more. The higher the specific gravity of the second inorganic fine powder, the better. When the specific gravity is equal to or higher than the lower limit, the specific gravity of the plasma separation composition can be effectively increased. The specific gravity of the second inorganic fine powder may be 10 or less, or may be 6 or less.

[0079] The average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are not particularly limited, and may be 1 nm or more, 10 nm or more, 500 nm or less, or 100 nm or less.

[0080] The average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are average diameters measured on a volume basis (volume average particle diameter), and are the 50% median diameter (D50) values. The volume average particle diameter (D50) can be measured by laser diffraction / scattering method, image analysis method, Coulter method, centrifugal sedimentation method, etc. The volume average particle diameter (D50) is preferably determined by laser diffraction / scattering method or image analysis method.

[0081] The specific surface area of ​​the finely powdered silica is not particularly limited. ２ / g or more, and ２ / g or more, and ２ / g or less, and ２ / g or less.

[0082] The specific surface area of ​​the finely powdered silica is measured by the BET method.

[0083] The content of the hydrophilic silica in 100% by weight of the plasma separation composition is preferably 0.01% by weight or more, more preferably 0.10% by weight or more, even more preferably 0.30% by weight or more, and preferably 2.50% by weight or less, more preferably 2.00% by weight or less. When the content of the hydrophilic silica is equal to or more than the lower limit and equal to or less than the upper limit, both the specific gravity and thixotropy of the plasma separation composition can be maintained within more suitable ranges.

[0084] The content of the finely powdered silica in 100% by weight of the plasma separation composition is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, even more preferably 0.4% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the finely powdered silica is equal to or more than the lower limit and equal to or less than the upper limit, both the specific gravity and thixotropy of the plasma separation composition can be maintained within more suitable ranges.

[0085] The content of the second inorganic fine powder in 100% by weight of the plasma separation composition is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the second inorganic fine powder is equal to or more than the lower limit and equal to or less than the upper limit, the specific gravity of the plasma separation composition can be effectively increased.

[0086] The content of the inorganic fine powder in 100% by weight of the plasma separation composition is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the inorganic fine powder is equal to or more than the lower limit and equal to or less than the upper limit, the specific gravity of the plasma separation composition can be effectively increased.

[0087] Other Components: The plasma separation composition may contain other components in addition to the components described above, as long as the effects of the present invention are not impaired. Examples of the other components include organic gelling agents, thermoplastic elastomers, polyalkylene glycols, silicone oils, cosolvents, antioxidants, colorants, and water. Each of the other components may be used alone or in combination of two or more.

[0088] The specific gravity of the plasma separation composition at 25°C is preferably 1.020 or more, more preferably 1.022 or more, even more preferably 1.025 or more, particularly preferably 1.028 or more, preferably 1.040 or less, more preferably 1.038 or less, and even more preferably 1.036 or less. When the specific gravity of the plasma separation composition at 25°C is above the above lower limit and below the above upper limit, contamination of plasma with white blood cells and red blood cells can be more effectively suppressed. When the specific gravity of the plasma separation composition at 25°C is above the above lower limit and below the above upper limit, contamination of plasma with platelets can be more effectively suppressed. Furthermore, plasma can be separated from blood more effectively.

[0089] The specific gravity of the plasma separation composition at 25°C is measured by dropping one drop of the plasma separation composition into saline solutions at 25°C, each having a specific gravity adjusted in increments of 0.002, and observing the floating and sinking of the composition in the saline solution. The specific gravity of the saline solution at 25°C is measured using a hydrometer (for example, "DA-130N" manufactured by Kyoto Electronics Manufacturing Co., Ltd.).

[0090] The viscosity of the plasma separation composition at 25° C. is preferably 50 Pa s or more, more preferably 70 Pa s or more, and preferably 500 Pa s or less, more preferably 400 Pa s or less. When the viscosity is equal to or greater than the lower limit and equal to or less than the upper limit, the fluidity of the plasma separation composition is increased, and the strength of the partition wall formed after centrifugation can be increased.

[0091] The viscosity of the composition for plasma separation at 25°C was measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 sec. －１ It is measured under the following conditions.

[0092] <Plasma Separation Device> The plasma separation device is a device that moves between the plasma layer and the blood cell layer during centrifugation to form a partition wall. The plasma separation device is used for the purpose of preventing component migration between the plasma layer and the blood cell layer.

[0093] The plasma separation device may be a conventionally known plasma separation device, such as the mechanical separator (plasma separation device) described in WO 2010 / 132783 A1.

[0094] Examples of materials for the plasma separation device include elastomers.

[0095] (Aqueous Solution) The blood collection container includes an aqueous solution contained within the blood collection container body. The aqueous solution contains an anticoagulant. By dissolving components that dissolve in blood, such as the anticoagulant, in the aqueous solution contained within the blood collection container body, it is possible to improve the mixing property with the blood and effectively suppress hemolysis.

[0096] <Anticoagulant> The aqueous solution contains an anticoagulant. A conventionally known anticoagulant can be used as the anticoagulant. Only one type of anticoagulant may be used, or two or more types may be used in combination.

[0097] Examples of the anticoagulant include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, sodium citrate, etc. The anticoagulant is an anticoagulant different from citric acid.

[0098] From the viewpoint of exhibiting good anticoagulant performance, the anticoagulant is preferably heparin, a metal salt of heparin, EDTA, a metal salt of EDTA, or sodium citrate.

[0099] The content of the anticoagulant in the aqueous solution is not particularly limited as long as it is a concentration at which anticoagulant performance is exhibited. The content of the anticoagulant in 100% by weight of the aqueous solution is preferably 0.5% by weight or more, more preferably 1% by weight or more, even more preferably 2% by weight or more, preferably 10% by weight or less, more preferably 6% by weight or less, and even more preferably 4% by weight or less. When the content of the anticoagulant is equal to or more than the lower limit and equal to or less than the upper limit, the anticoagulant performance can be exhibited satisfactorily.

[0100] <Organic Acid or Inorganic Acid> The aqueous solution preferably contains an organic acid or an inorganic acid. In this case, the aqueous solution may contain an organic acid, an inorganic acid, or both an organic acid and an inorganic acid. When the aqueous solution contains an organic acid or an inorganic acid, the pH of the mixed solution (X) can be easily adjusted to a specific range. The organic acid and the inorganic acid serve as pH adjusters. Only one type of organic acid may be used, or two or more types may be used in combination. Only one type of inorganic acid may be used, or two or more types may be used in combination.

[0101] Examples of the organic acid include citric acid, succinic acid, formic acid, acetic acid, lactic acid, oxalic acid, gluconic acid, malonic acid, fumaric acid, malic acid, and tartaric acid. The organic acid is different from ethylenediaminetetraacetic acid (EDTA).

[0102] Examples of the inorganic acid include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, boric acid, and hydrofluoric acid.

[0103] From the viewpoint of more effectively exerting the effects of the present invention and from the viewpoint of easily adjusting the pH of the mixed liquid (X), the aqueous solution preferably contains an organic acid.

[0104] The molecular weight of the organic acid is preferably 20 or more, more preferably 100 or more, and preferably 500 or less, more preferably 300 or less, and even more preferably 250 or less. When the molecular weight of the organic acid is equal to or more than the above lower limit and equal to or less than the above upper limit, the effects of the present invention can be more effectively exhibited.

[0105] The organic acid is preferably a carboxylic acid (a carboxylic acid different from EDTA), more preferably citric acid, succinic acid, oxalic acid, or acetic acid, even more preferably citric acid or succinic acid, and particularly preferably citric acid. The aqueous solution preferably contains a carboxylic acid (a carboxylic acid different from EDTA), more preferably citric acid, succinic acid, oxalic acid, or acetic acid, even more preferably citric acid or succinic acid, and particularly preferably citric acid. In this case, the effects of the present invention can be more effectively exerted. Furthermore, when the aqueous solution contains citric acid, hemolysis can also be effectively suppressed.

[0106] The contents of the organic acid and the inorganic acid in the aqueous solution are not particularly limited as long as they allow the mixed solution (X) to have a desired pH.

[0107] The content of the organic acid in 100% by weight of the aqueous solution is preferably 1% by weight or more, more preferably 2% by weight or more, even more preferably 2.5% by weight or more, and preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 4% by weight or less. When the content of the organic acid is equal to or more than the above lower limit and equal to or less than the above upper limit, the pH of the mixed solution (X) can be easily adjusted to the desired pH, and the effects of the present invention can be more effectively exhibited.

[0108] The content of citric acid in the aqueous solution (100 wt %) is preferably 1 wt % or more, more preferably 2 wt % or more, even more preferably 2.5 wt % or more, and preferably 10 wt % or less, more preferably 5 wt % or less, and even more preferably 4 wt % or less. When the content of citric acid is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the pH of the mixed solution (X) can be easily adjusted to the desired pH, and the effects of the present invention can be more effectively exhibited. Furthermore, when the content of citric acid is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, hemolysis can be more effectively suppressed.

[0109] The content of succinic acid in 100% by weight of the aqueous solution is preferably 1% by weight or more, more preferably 2% by weight or more, even more preferably 2.5% by weight or more, and preferably 10% by weight or less, more preferably 8% by weight or less, and even more preferably 6% by weight or less. When the content of succinic acid is equal to or more than the above lower limit and equal to or less than the above upper limit, the pH of the mixed solution (X) can be easily adjusted to a desired pH, and the effects of the present invention can be more effectively exhibited.

[0110] The content of oxalic acid in the aqueous solution (100% by weight) is preferably 0.25% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, and preferably 7% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less. When the content of oxalic acid is equal to or more than the lower limit and equal to or less than the upper limit, the pH of the mixed solution (X) can be easily adjusted to a desired pH, and the effects of the present invention can be more effectively exhibited.

[0111] The content of acetic acid in 100% by weight of the aqueous solution is preferably 0.5% by weight or more, more preferably 1% by weight or more, even more preferably 2% by weight or more, and preferably 5% by weight or less, more preferably 4% by weight or less, and even more preferably 3.5% by weight or less. When the content of acetic acid is equal to or more than the above lower limit and equal to or less than the above upper limit, the pH of the mixed solution (X) can be easily adjusted to a desired pH, and the effects of the present invention can be more effectively exhibited.

[0112] <Disaccharides> The aqueous solution preferably contains disaccharides. In this case, hemolysis can be effectively suppressed. Furthermore, since a source of nutrients is supplied to red blood cells and white blood cells, the survival rate of red blood cells and white blood cells can be increased, and as a result, leakage of miRNA from red blood cells and white blood cells can be further suppressed. The disaccharides may be used alone or in combination of two or more.

[0113] The disaccharides include sucrose, trehalose, maltose, and lactose.

[0114] The disaccharide preferably contains sucrose. The aqueous solution preferably contains sucrose. In this case, hemolysis can be more effectively suppressed. Furthermore, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0115] The content of the disaccharide in the aqueous solution (100% by weight) is preferably 0.2% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, preferably 10% by weight or less, more preferably 6% by weight or less, and even more preferably 4% by weight or less. When the content of the disaccharide is equal to or greater than the lower limit and equal to or less than the upper limit, hemolysis can be more effectively suppressed. Furthermore, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0116] The sucrose content in the aqueous solution (100% by weight) is preferably 0.2% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, even more preferably 1.5% by weight or more, even more preferably 2% by weight or more, preferably 10% by weight or less, more preferably 6% by weight or less, and even more preferably 4% by weight or less. When the sucrose content is equal to or greater than the lower limit and equal to or less than the upper limit, hemolysis can be more effectively suppressed. Furthermore, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0117] <Inorganic Salt> The aqueous solution preferably contains an inorganic salt. In this case, it is easy to adjust the osmotic pressure of the mixture of blood and the aqueous solution. The inorganic salt may be used alone or in combination of two or more.

[0118] Examples of the inorganic salt include sodium salts such as sodium chloride and sodium hydrogen phosphate, and potassium salts such as potassium chloride and potassium hydrogen carbonate.

[0119] The content of the inorganic salt in 100% by weight of the aqueous solution is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, and preferably 6% by weight or less, more preferably 3% by weight or less. When the content of the inorganic salt is equal to or more than the lower limit and equal to or less than the upper limit, the osmotic pressure of the mixture of blood and the aqueous solution is easily adjusted to a suitable range.

[0120] <Water> The aqueous solution preferably contains water, which serves as a solvent.

[0121] In 100% by weight of the aqueous solution, the content of the water is preferably 70% by weight or more, more preferably 80% by weight or more, even more preferably 85% by weight or more, even more preferably 88% by weight or more, particularly preferably 90% by weight or more, and preferably 98% by weight or less, more preferably 95% by weight or less.

[0122] <Other Components> The aqueous solution may contain components other than the above-mentioned components (anticoagulant, organic acid, inorganic acid, disaccharide, inorganic salt, and water). Examples of the other components include monosaccharides, polysaccharides, and sugar alcohols. Each of the other components may be used alone or in combination of two or more.

[0123] The aqueous solution may or may not contain a compound capable of releasing an aldehyde. Examples of the compound capable of releasing an aldehyde include diazolidinyl urea, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, oxazolidine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, quaternium-15, DMDM ​​hydantoin, 2-bromo-2-nitropropane-1,3-diol, 5-bromo-5-nitro-1,3-dioxane, tris(hydroxymethyl)nitromethane, hydroxymethylglycinate, and polyquaternium.

[0124] <Other details of the aqueous solution> The total content of the anticoagulant, the organic acid, and the water in 100% by weight of the aqueous solution is preferably 70% by weight or more, more preferably 80% by weight or more, even more preferably 85% by weight or more, even more preferably 90% by weight or more, and particularly preferably 95% by weight or more. When the total content is equal to or greater than the lower limit, the effects of the present invention can be more effectively exhibited. Note that, in 100% by weight of the aqueous solution, the total content of the anticoagulant, the organic acid, and the water may be 100% by weight or less, less than 100% by weight, or 99% by weight or less.

[0125] The total content of the anticoagulant, the organic acid, the disaccharide, and the water in 100% by weight of the aqueous solution is preferably 75% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, even more preferably 95% by weight or more, particularly preferably 98% by weight or more, and most preferably 99% by weight or more. When the total content is equal to or greater than the lower limit, the effects of the present invention can be more effectively exhibited. Note that the total content of the anticoagulant, the organic acid, the disaccharide, and the water in 100% by weight of the aqueous solution may be 100% by weight or less, less than 100% by weight, or 99% by weight or less.

[0126] The pH of the aqueous solution is preferably 3.0 or higher, more preferably 3.5 or higher, and preferably 6.0 or lower, more preferably 5.5 or lower, even more preferably 5.2 or lower, still more preferably 5.0 or lower, and particularly preferably 4.5 or lower. When the pH is equal to or higher than the lower limit and equal to or lower than the upper limit, the effects of the present invention can be more effectively exhibited.

[0127] The pH of the aqueous solution is measured at 22°C using a pH meter.

[0128] The amount of aqueous solution contained in the blood collection container body can be varied as appropriate depending on the size of the blood collection container body, the amount of blood to be collected, etc. The amount of aqueous solution contained in the blood collection container body is preferably 0.1 mL or more, more preferably 0.5 mL or more, even more preferably 0.7 mL or more, and preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2.5 mL or less. When the amount of aqueous solution is above the above-mentioned lower limit and below the above-mentioned upper limit, the blood is not excessively diluted, and the effects of the present invention can be more effectively achieved.

[0129] (Blood collection container body) The shape of the blood collection container body is not particularly limited. The blood collection container body is preferably a tubular container with a bottom. The blood collection container body preferably has an open end at one end and a closed end at the other end. The open end of the blood collection container body is one end in the length direction of the blood collection container body, and the closed end of the blood collection container body is the other end in the length direction of the blood collection container body. The blood collection container body preferably has a closed bottom at the other end.

[0130] The material of the blood collection container body is not particularly limited. Examples of materials for the blood collection container body include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; thermosetting resins such as unsaturated polyester resin, epoxy resin, and epoxy-acrylate resin; modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, and ethyl chitin; and glass such as silicate glass, such as soda-lime glass, phosphosilicate glass, and borosilicate glass, and quartz glass. The blood collection container body may be made of one material or two or more materials.

[0131] (Stopper) The blood collection container preferably includes a stopper. The stopper is preferably attached to the open end of the blood collection container body. A conventionally known stopper can be used as the stopper. The stopper is preferably made of a material and has a shape that allows it to be attached to the open end of the blood collection container body in an airtight and liquid-tight manner. The stopper is preferably configured so that it can be pierced by a blood collection needle.

[0132] Examples of the stopper include a stopper having a shape that fits into the open end of the blood collection container body, a sheet-like seal stopper, and the like.

[0133] The stopper may also comprise a stopper body such as a rubber stopper and a cap member made of plastic, etc. In this case, the risk of blood coming into contact with the human body can be reduced when the stopper body is pulled out from the open end of the blood collection container body after blood collection.

[0134] Examples of materials for the stopper (or the stopper main body) include synthetic resin, elastomer, rubber, and metal foil. Examples of the rubber include butyl rubber and halogenated butyl rubber. Examples of the metal foil include aluminum foil. From the viewpoint of improving sealing performance, the stopper is preferably made of butyl rubber. The stopper (or the stopper main body) is preferably a butyl rubber stopper.

[0135] (Other Details of the Blood Collection Container) The blood collection container is a blood collection container from which a predetermined amount of blood is collected. The blood collection container is used by collecting a predetermined amount of blood. The predetermined amount of blood is changed as appropriate depending on the size and internal pressure of the blood collection container. The predetermined amount of blood may be 1 mL or more, 2 mL or more, 4 mL or more, 12 mL or less, 11 mL or less, or 10 mL or less.

[0136] The blood collection container is preferably a blood collection container that can collect 4 mL or more of blood per mL of the aqueous solution contained in the blood collection container body, more preferably a blood collection container that can collect 5 mL or more of blood, preferably a blood collection container that can collect 9.5 mL or less of blood, and even more preferably a blood collection container that can collect 9 mL or less of blood. In this case, the blood is not excessively diluted, and the effects of the present invention can be more effectively achieved.

[0137] The blood collection container is preferably a blood collection tube, and the blood collection container body is preferably a blood collection tube body.

[0138] The blood collection container is preferably used to separate plasma from blood. The blood collection container is also preferably used to separate extracellular free nucleic acids or extracellular vesicles in blood, and is preferably used to isolate extracellular free nucleic acids or extracellular vesicles in blood. The extracellular free nucleic acids may be cell-free DNA (cfDNA) or cell-free RNA (cfRNA). The extracellular free nucleic acids are preferably cfRNA.

[0139] The blood collection container can be manufactured, for example, as follows.

[0140] An anticoagulant and an organic acid are dissolved in water to obtain an aqueous solution. If necessary, other components may also be dissolved in water to obtain an aqueous solution. The resulting aqueous solution is added to the blood collection container body. Before or after adding the aqueous solution, a plasma separator is placed in the blood collection container body.

[0141] FIG. 1 is a front cross-sectional view showing a schematic diagram of a blood collection container according to one embodiment of the present invention.

[0142] The blood collection container 1 shown in FIG. 1 comprises a blood collection container body 2, a plasma separation composition 3, an aqueous solution 4, and a stopper 5. The plasma separation composition 3 and the aqueous solution 4 are each contained within the blood collection container body 2. The blood collection container body 2 has an open end 2a and a closed end 2b. The open end 2a corresponds to one end in the lengthwise direction of the blood collection container body 2, and the closed end 2b corresponds to the other end (bottom side) in the lengthwise direction of the blood collection container body 2. The plasma separation composition 3 is contained in the bottom portion of the blood collection container body 2. The aqueous solution 4 contains an anticoagulant. The stopper 5 is inserted into the open end 2a of the blood collection container body 2.

[0143] The aqueous solution 4 is disposed on the surface of the plasma separating composition 3, more specifically, on the upper surface of the plasma separating composition 3 (the surface on the side of the open end 2a of the blood collecting container body 2) when the blood collecting container 1 is in an upright position. The aqueous solution 4 is disposed on the surface of the plasma separating composition 3 when the blood collecting container 1 is in an upright position.

[0144] In the blood collection container according to the present invention, the plasma separation composition may be disposed on the side wall surface of the blood collection container body, and the aqueous solution may be disposed at the bottom of the blood collection container body when the blood collection container is in an upright position. Also, the plasma separation tool may be used instead of the plasma separation composition.

[0145] The internal pressure of the blood collection container is not particularly limited. The blood collection container can also be used as a vacuum blood collection tube, which is evacuated and then sealed with the stopper. The internal pressure of the vacuum blood collection tube is reduced so that a predetermined amount of blood can be collected. When using a vacuum blood collection tube, a predetermined amount of blood can be easily collected regardless of the skill level of the blood collector.

[0146] From the viewpoint of preventing bacterial infection, it is preferable that the inside of the blood collection container is sterilized in accordance with ISO or JIS standards.

[0147] (Method for separating plasma) Plasma can be separated from blood using the above-mentioned blood collection container. The method for separating plasma according to the present invention preferably includes a step of centrifuging the blood collection container in which blood has been collected. More preferably, the method for separating plasma according to the present invention includes a step of collecting blood in the above-mentioned blood collection container, and a step of centrifuging the blood collection container in which the blood has been collected.

[0148] In the method for separating plasma according to the present invention, it is preferable to further include a step of mixing the collected blood with the aqueous solution between the step of collecting the blood and the step of centrifuging the blood. Examples of a method for mixing the collected blood with the aqueous solution include mixing by inversion.

[0149] The centrifugation conditions in the centrifugation step are not particularly limited as long as a partition can be formed using the plasma separation material to separate plasma from blood cells. Examples of the centrifugation conditions include centrifugation at 400 G or more and 4000 G or less for 10 minutes or more and 120 minutes or less.

[0150] (Method for separating extracellular free nucleic acid and method for separating extracellular vesicles) The method for separating extracellular free nucleic acid according to the present invention preferably comprises the steps of centrifuging the blood collection container into which blood has been collected to separate plasma from the blood, and separating extracellular free nucleic acid from the separated plasma. More preferably, the method for separating extracellular free nucleic acid according to the present invention comprises the steps of collecting blood into the blood collection container described above, centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood, and separating extracellular free nucleic acid from the separated plasma.

[0151] The method for separating extracellular vesicles according to the present invention preferably comprises the steps of centrifuging the blood collection container into which blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.The method for separating extracellular vesicles according to the present invention more preferably comprises the steps of collecting blood in the blood collection container, centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.

[0152] The method for separating extracellular free nucleic acids and the method for separating extracellular vesicles according to the present invention preferably include a step of mixing the collected blood with the aqueous solution between the step of collecting the blood and the step of centrifuging the blood. Examples of a method for mixing the collected blood with the aqueous solution include mixing by inversion.

[0153] The centrifugation conditions in the centrifugation step are not particularly limited as long as a partition can be formed using the plasma separation material to separate plasma from blood cells. Examples of the centrifugation conditions include centrifugation at 400 G or more and 4000 G or less for 10 minutes or more and 120 minutes or less.

[0154] In the step of isolating extracellular free nucleic acids, the extracellular free nucleic acids can be isolated from plasma using a conventionally known method. Examples of the extracellular free nucleic acids include cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Examples of methods for isolating extracellular free nucleic acids from plasma include methods using commercially available nucleic acid purification kits. By using commercially available nucleic acid purification kits, extracellular free nucleic acids can be easily isolated from plasma. Examples of commercially available nucleic acid purification kits include QIAamp Circulating Nucleic Acid Kit (manufactured by QIAGEN), QIAamp MinElute ccfDNA Kits (manufactured by QIAGEN), and MagMAX Cell-Free DNA Isolation Kit (manufactured by Applied biosystems).

[0155] In the step of separating extracellular vesicles, the extracellular vesicles can be separated from the plasma using a conventionally known method.

[0156] (Method for inhibiting exosome release from platelets) The present specification also provides a method for inhibiting exosome release from platelets, which comprises storing platelets in an environment with a pH of 3.5 or higher and lower than 7.0. The term "an environment with a pH of 3.5 or higher and lower than 7.0" means that the pH of the liquid in contact with the platelets is 3.5 or higher and lower than 7.0.

[0157] In the above method, platelets are preferably stored in an environment with a pH of 4.0 or higher, more preferably in an environment with a pH of 4.5 or higher, even more preferably in an environment with a pH of 5.0 or higher, preferably in an environment with a pH of 6.9 or lower, more preferably in an environment with a pH of 6.6 or lower, and even more preferably in an environment with a pH of 6.4 or lower. When the pH is equal to or higher than the above lower limit and equal to or lower than the above upper limit, the release of exosomes from platelets can be more effectively suppressed.

[0158] The above method is preferably the following method (1) or the following method (2).

[0159] The method (1) includes the steps of mixing blood with an aqueous solution containing an anticoagulant to obtain a liquid (A) and storing the liquid (A). In the method (1), the pH of the liquid (A) is equal to or greater than 3.5 and less than 7.0. In the method (1), the liquid (A) is in contact with the platelets.

[0160] The method (2) includes the steps of: mixing blood with an aqueous solution containing an anticoagulant to obtain a liquid (A); separating platelet-containing plasma from the liquid (A); and storing the platelet-containing plasma. In the method (2), the platelet-containing plasma is a liquid in contact with the platelets.

[0161] In the method (1) and the method (2), in the step of obtaining the liquid (A), blood, the aqueous solution, and other components (liquids, etc.) may be mixed. In the method (1) and the method (2), the aqueous solution (aqueous solution containing an anticoagulant) described above can be used.

[0162] In the above methods (1) and (2), the pH of the solution (A) is preferably 4.0 or higher, more preferably 4.5 or higher, even more preferably 5.0 or higher, and preferably 6.9 or lower, more preferably 6.6 or lower, and even more preferably 6.4 or lower. When the pH of the solution (A) is equal to or higher than the above lower limit and equal to or lower than the above upper limit, the release of exosomes from platelets can be more effectively suppressed.

[0163] In the method (2), the pH of the platelet-containing plasma is preferably 4.0 or higher, more preferably 4.5 or higher, even more preferably 5.0 or higher, and preferably 6.9 or lower, more preferably 6.6 or lower, and even more preferably 6.4 or lower. When the pH of the platelet-containing plasma is equal to or higher than the lower limit and equal to or lower than the upper limit, the release of exosomes from platelets can be more effectively suppressed.

[0164] The pH of the liquid (A) and the pH of the platelet-containing plasma are measured at 22°C using a pH meter.

[0165] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0166] The following materials were prepared as the plasma separation composition.

[0167] (Organic component materials having fluidity at 25°C) (Meth)acrylic resin: 2-Ethylhexyl acrylate and butyl acrylate were radically polymerized by solution polymerization in the presence of an azo-based polymerization initiator to obtain a (meth)acrylic acid ester polymer having fluidity at 25°C.

[0168] (Inorganic fine powder) Hydrophilic silica (fine powder silica, "200CF" manufactured by Nippon Aerosil Co., Ltd.)

[0169] (Other ingredients) Silicone oil ("SF8410" manufactured by Toray Dow Corning Co., Ltd.) Organic gelling agent ("Gelall D" manufactured by New Japan Chemical Co., Ltd.) 1-methyl-2-pyrrolidone (auxiliary solvent)

[0170] Preparation of composition A for plasma separation: An organic component having fluidity at 25° C., inorganic fine powder, and other components were mixed in the blending ratios shown in Table 1 to prepare composition A for plasma separation.

[0171]

[0172] One drop of the obtained plasma separation composition was successively dropped into saline solutions at 25°C, each having a specific gravity adjusted in increments of 0.002, and the specific gravity was measured by floating or sinking in the saline solution. The specific gravities of the obtained plasma separation compositions at 25°C are shown in the table below.

[0173] The following materials were prepared for the aqueous solution: Granular disodium ethylenediaminetetraacetic acid (EDTA2Na) dihydrate (anticoagulant) was also prepared.

[0174] (Anticoagulant) Trisodium Citrate Dihydrate

[0175] Citric acid monohydrate, succinic acid, oxalic acid, acetic acid, sucrose, sodium chloride, water

[0176] The following blood collection containers were prepared:

[0177] A PET bottomed tube (polyethylene terephthalate tube) with a length (distance between the open end and the closed end) of 100 mm and an inner diameter of the open end of 14 mm

[0178] (Example 1) Aqueous solutions were obtained by dissolving the components shown in Table 2 in water (water for injection). The types and concentrations of the components in the obtained aqueous solutions are shown in Table 2.

[0179] 1.2 g of plasma separation composition A was placed in the bottom of the blood collection container body. 1.0 mL of the resulting aqueous solution was added to the surface of plasma separation composition A. The pressure inside the blood collection container was reduced so that the blood collection volume was 8.5 mL, and the container was sealed with a butyl rubber stopper. In this way, a blood collection container was prepared.

[0180] Examples 2 to 5, 8 to 13 and Comparative Examples 2 and 3 Blood collection containers were produced in the same manner as in Example 1, except that the composition of the aqueous solution was changed as shown in the table below.

[0181] (Examples 6 and 7) The composition of the aqueous solution was changed as shown in the table below. The pressure inside the blood collection container was reduced so that the blood collection volume was 5.0 mL (Example 6) or 9.5 mL (Example 7), and the container was sealed with a butyl rubber stopper. Other than these, blood collection containers were prepared in the same manner as in Example 1.

[0182] Comparative Example 1: 10 mg of granular disodium ethylenediaminetetraacetate (EDTA2Na) dihydrate (anticoagulant) was placed in the bottom of a blood collection container. The pressure inside the blood collection container was reduced so that the blood collection volume was 5.0 mL, and the container was sealed with a butyl rubber stopper. In this manner, a blood collection container was prepared.

[0183] (Evaluation) (1) pH of Aqueous Solution The pH of the resulting aqueous solution was measured at 22° C. using a pH meter ("F-52S" manufactured by HORIBA).

[0184] (2) pH of Mixed Solution (X): 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 900 mL of water were mixed, the pH was adjusted with hydrochloric acid (pH adjuster), and then the mixture was diluted to 1 L with water to obtain a solution with a pH of 7.4. The pH of the solution with a pH of 7.4 was confirmed by measuring it at 22°C using a pH meter (HORIBA "F-52S"). The solution with a pH of 7.4 was collected into the prepared blood collection container. In Examples 1 to 5, 8 to 13, and Comparative Examples 2 and 3, 8.5 mL of the solution with a pH of 7.4 was collected into the blood collection container; in Example 6, 5.0 mL of the solution with a pH of 7.4 was collected into the blood collection container; and in Example 7, 9.5 mL of the solution with a pH of 7.4 was collected into the blood collection container. Next, the solution with a pH of 7.4 and the aqueous solution were mixed by inversion to obtain a mixed solution (X). In Comparative Example 1, 5.0 mL of the solution with a pH of 7.4 was collected in a blood collection container, and then the solution was inverted to obtain a mixed solution (X) by dissolving a granular anticoagulant in the solution with a pH of 7.4. The pH of the obtained mixed solution (X) was measured at 22°C using a pH meter ("F-52S" manufactured by HORIBA).

[0185] (3) Blood Evaluation Blood was collected from two subjects using the prepared blood collection containers. In Examples 1 to 5, 8 to 13 and Comparative Examples 2 and 3, 8.5 mL of blood was collected in the blood collection container, 5.0 mL of blood was collected in Example 6, and 9.5 mL of blood was collected in Example 7. After collecting the blood, the blood was mixed with the aqueous solution contained in the blood collection container by inversion. In Comparative Example 1, 5.0 mL of blood was collected in the blood collection container, and then the container was inverted to mix, dissolving the granular anticoagulant in the blood.

[0186] In Examples 1 to 13 and Comparative Examples 2 and 3, after mixing the blood with the aqueous solution, the following steps (i) and (ii) were carried out.

[0187] (i) The blood collection container was centrifuged at 20°C and 1900G for 15 minutes. After centrifugation, the plasma was located above the partition formed by the plasma separation composition. A portion of the resulting plasma was collected. The collected plasma was transferred to another container, and the container was centrifuged at 16000G for 10 minutes at 4°C. After centrifugation, the supernatant plasma was collected from the container, and the collected plasma was designated "plasma (A)."

[0188] (ii) After collecting a portion of the plasma in (i) above, the blood collection container (in which the plasma was located above the partition formed by the plasma separation composition) was stored at 4°C for 3 days. The plasma was recovered from the blood collection container after storage. The recovered plasma was transferred to another container, and the container was centrifuged at 16,000 G for 10 minutes at 4°C. After centrifugation, the supernatant plasma was recovered from the container, and the recovered plasma was designated "plasma (B)."

[0189] In Comparative Example 1, after dissolving a granular anticoagulant in blood, the following (a) and (b) were carried out.

[0190] (a): The blood collection container was centrifuged at 1900 G for 15 minutes at 20°C to separate the blood cell layer from the plasma layer. The plasma was collected from the plasma layer and transferred to another container, which was then centrifuged at 16000 G for 10 minutes at 4°C. After centrifugation, the supernatant plasma was collected from the container, and the collected plasma was designated "plasma (A)."

[0191] (b): The blood collection container was stored at 4°C for 3 days. After storage, the blood collection container was centrifuged at 1900G for 15 minutes at 20°C to separate the blood cell layer and plasma layer. The plasma was recovered from the plasma layer and transferred to another container, which was then centrifuged at 16000G for 10 minutes at 4°C. After centrifugation, the supernatant plasma was recovered from the container, and the recovered plasma was designated "plasma (B)."

[0192] (3-1) pH of Plasma The pH of plasma (A) was measured at 22°C using a pH meter ("F-52S" manufactured by HORIBA).

[0193] (3-2) Contamination of platelet-derived exosomes in plasma (3-2-1) Absorbance of CD9-positive exosomes (wavelength 450 nm) Plasma (A) and plasma (B) were each centrifuged using a 0.22 μm pore size filter (Millipore "0.22 μm GV DURAPORE"). The filtrate was diluted 20-fold with PBS(-), and then the absorbance (wavelength 450 nm) was measured using a CD9-Capture human exosome ELISA kit (Wako "Streptavidin HRP").

[0194] The rate of increase in absorbance was calculated using the following formula: The smaller the rate of increase in absorbance, the more suppressed the release of exosomes from platelets, and therefore the more suppressed the contamination of plasma with platelet-derived exosomes.

[0195] Increase rate of absorbance (%) = B / A x 100 A: absorbance of plasma (A) B: absorbance of plasma (B)

[0196] <Criteria for determining absorbance of CD9-positive exosomes> ○: Increase in absorbance is 350% or less △: Increase in absorbance is more than 350% but not more than 600% ×: Increase in absorbance is more than 600%

[0197] (3-2-2) Rate of increase in EV-miRNA (miR-126-3p) concentration in plasma The following test was performed for each of plasma (A) and plasma (B). Total EV-RNA was purified from 0.5 mL of plasma using a purification kit (exoRNeasy Midi Kit, Qiagen). Next, a reverse transcription reaction was performed using the following reagents and measuring equipment, followed by PCR of miR-126-3p.

[0198] Reverse transcription reagents: miRCURY LNA RT Kit (Qiagen) PCR reagents: miRCURY LNA SYBR Green PCR Kit (Qiagen) Primers: miRCURY LNA miRNA PCR Assay (Qiagen, 339306), Primer #: YP00204227 Measurement equipment: C1000 Touch TM Thermal Cycler, CFX96 Deep WellTM Real-Time System (BioRad)

[0199] The rate of increase in EV-miRNA (miR-126-3p) concentration in plasma was calculated using the following formula: The smaller the rate of increase in EV-miRNA (miR-126-3p) concentration, the more suppressed the release of exosomes from platelets, and therefore the more suppressed the contamination of plasma with platelet-derived exosomes.

[0200] R ３Ｐ =2 （－ΔＣＴ） R ３Ｐ : Increase rate of EV-miRNA (miR-126-3p) concentration ΔCT: Mean C of plasma (B) Ｔ - Mean C of plasma (A) Ｔ average C Ｔ : C of each sample when PCR was performed on each sample with N = 2 Ｔ Average value of C Ｔ : The cycle number at which the fluorescent signal of the PCR reaction crosses the threshold line

[0201] <Criteria for determining the rate of increase in EV-miRNA (miR-126-3p) concentration in plasma> ○: The rate of increase in EV-miRNA (miR-126-3p) concentration is 3-fold or less △: The rate of increase in EV-miRNA (miR-126-3p) concentration is more than 3-fold but not more than 6-fold ×: The rate of increase in EV-miRNA (miR-126-3p) concentration is more than 6-fold

[0202] (3-3) Contamination of erythrocyte-derived exosomes in plasma (rate of increase in EV-miRNA (miR-451a) concentration in plasma) The following test was performed on each of plasma (A) and plasma (B). Total EV-RNA was purified from 0.5 mL of plasma using a purification kit (exoRNeasy Midi Kit, Qiagen). Next, a reverse transcription reaction was performed using the following reagents and measuring equipment, followed by PCR of miR-451a.

[0203] Reverse transcription reagents: miRCURY LNA RT Kit (Qiagen) PCR reagents: miRCURY LNA SYBR Green PCR Kit (Qiagen) Primers: miRCURY LNA miRNA PCR Assay (Qiagen, 339306), Primer #: YP02119305 Measurement equipment: C1000 Touch TM Thermal Cycler, CFX96 Deep Well TM Real-Time System (BioRad)

[0204] The rate of increase in EV-miRNA (miR-451a) concentration in plasma was calculated using the following formula. The smaller the rate of increase in EV-miRNA (miR-451a) concentration, the more suppressed the release of exosomes from erythrocytes, and therefore the more suppressed the contamination of plasma with exosomes derived from erythrocytes.

[0205] R ４５１ａ =2 （－ΔＣＴ） R ４５１ａ : Increase rate of EV-miRNA (miR-451a) concentration ΔCT: Mean C of plasma (B) Ｔ - Mean C of plasma (A) Ｔ average C Ｔ : C of each sample when PCR was performed on each sample with N = 2 Ｔ Average value of C Ｔ : The cycle number at which the fluorescent signal of the PCR reaction crosses the threshold line

[0206] <Criteria for determining the rate of increase in EV-miRNA (miR-451a) concentration in plasma> ○: The rate of increase in EV-miRNA (miR-451a) concentration is 3-fold or less △: The rate of increase in EV-miRNA (miR-451a) concentration is more than 3-fold and 6-fold or less ×: The rate of increase in EV-miRNA (miR-451a) concentration is more than 6-fold

[0207] (3-4) Total EV-miRNA Concentration in Plasma The following test was performed on each of plasma (A) and plasma (B). Total EV-RNA was purified from 0.5 mL of plasma using a purification kit (exoRNeasy Midi Kit, Qiagen). The total EV-miRNA concentration was then measured using a measurement device (Qubit microRNA Assay Kit (Thermo, Q32881)).

[0208] In addition, the rate of increase in total EV-miRNA concentration in plasma was calculated using the following formula.

[0209] Increase rate (%) of total EV-miRNA concentration = B / A × 100 A: Total EV-miRNA concentration in plasma (A) (ng / 1 mL of plasma) B: Total EV-miRNA concentration in plasma (B) (ng / 1 mL of plasma)

[0210] <Criteria for determining total EV-miRNA concentration in plasma> ○: The increase in total EV-miRNA concentration is 150% or less. △: The increase in total EV-miRNA concentration is more than 150% and 200% or less. ×: The increase in total EV-miRNA concentration is more than 200%.

[0211] The compositions and results are shown in the following Tables 2 to 13. In the tables, the concentrations of the anticoagulant and the organic acid in the aqueous solution are concentrations in an anhydrous state.

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224] 1. Blood collection container 2. Blood collection container body 2a. Open end 2b. Closed end 3. Plasma separation composition 4. Aqueous solution 5. Plug

Claims

1. A blood collection container for collecting a predetermined amount of blood, comprising: a blood collection container body; a plasma separator contained within the blood collection container body; and an aqueous solution contained within the blood collection container body, the aqueous solution containing an anticoagulant, wherein the pH of the resulting mixed solution (X) is 3.5 or greater and 5.5 or less when measured as follows: pH measurement: A solution having a pH of 7.4 is obtained containing 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 0.24 g / L potassium dihydrogen phosphate, and water. An amount of the solution having a pH of 7.4 equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and the solution having a pH of 7.4 and the aqueous solution are mixed to obtain a mixed solution (X). The pH of the resulting mixed solution (X) is measured.

2. The blood collection container of claim 1, wherein the aqueous solution comprises an organic or inorganic acid.

3. The blood collection container according to claim 1 or 2, wherein the aqueous solution contains an organic acid.

4. The blood collection container of claim 3, wherein the organic acid comprises citric acid, succinic acid, oxalic acid, or acetic acid.

5. A blood collection container according to claim 3 or 4, wherein the content of said organic acid in 100% by weight of said aqueous solution is 1% by weight or more and 10% by weight or less.

6. The blood collection container according to any one of claims 1 to 5, wherein the aqueous solution comprises a disaccharide, and the disaccharide comprises sucrose.

7. A blood collection container according to any one of claims 1 to 6, wherein the aqueous solution contains an inorganic salt.

8. A blood collection container according to any one of claims 1 to 7, wherein the pH of the aqueous solution is 3.0 or more and 5.5 or less.

9. A blood collection container according to any one of claims 1 to 8, wherein the specific gravity of the plasma separation material at 25°C is 1.020 or more and 1.040 or less.

10. A blood collection container according to any one of claims 1 to 9, wherein the plasma separation material is a composition for plasma separation.

11. The blood collection container according to claim 10, wherein the plasma separation composition comprises an organic component that is fluid at 25°C and an inorganic fine powder, the organic component comprising a resin, and the inorganic fine powder comprising finely powdered silica.

12. The blood collection container according to claim 11, wherein the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic-based resin.

13. A blood collection container according to any one of claims 1 to 12, wherein 4 mL to 9.5 mL of blood is collected per 1 mL of the aqueous solution contained in the blood collection container body.

14. The blood collection container according to any one of claims 1 to 13, which is used to separate extracellular free nucleic acids or extracellular vesicles in blood.

15. A method for separating plasma, comprising the steps of: collecting blood in a blood collection container according to any one of claims 1 to 14; and centrifuging the blood collection container into which the blood has been collected.

16. A method for separating free extracellular nucleic acids, comprising the steps of: collecting blood in a blood collection container according to any one of claims 1 to 14; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; and separating free extracellular nucleic acids from the separated plasma.

17. A method for separating extracellular vesicles, comprising the steps of: collecting blood in a blood collection container according to any one of claims 1 to 14; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; and separating extracellular vesicles from the separated plasma.

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