Blood collection container

Abstract

Provided is a blood collection container that can suppress the release of exosomes from platelets and therefore suppress the incorporation of platelet-derived exosomes into plasma. [Solution] The blood collection container of the present invention is a blood collection container for collecting a predetermined amount of blood, and 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, wherein the aqueous solution contains an anticoagulant, and when a specific pH is measured, the pH of a specific mixed liquid (X) is 3.5 or more and 5.5 or less.

Description

[Technical field]

[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 acid, and a method for separating extracellular vesicles using the blood collection container. [Background technology]

[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, the following Patent Document 1 describes a blood collection container containing a plasma separation composition including a resin and an inorganic powder, and the following Patent Document 2 describes a blood collection container containing a plasma separation tool. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2010 / 053180A1 [Patent Document 2] WO2010 / 132783A1 Summary of the Invention [Problem to be solved by the invention]

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

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

[0007] An object of the present invention is to provide a blood collection container capable of suppressing the release of exosomes from platelets and thus suppressing the inclusion of platelet-derived exosomes in plasma. 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. [Means for solving the problem]

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

[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 in the blood collection container body, and an aqueous solution contained in 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] Measurement of pH: A solution with 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. The solution with a pH of 7.4 is collected in a blood collection container in an amount equal to a predetermined amount of blood to be collected in the blood collection container, and a mixed solution (X) is obtained in which the solution with a pH of 7.4 and the aqueous solution are mixed. 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 or succinic acid.

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

[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 6.0 or less.

[0018] Item 9. The blood collection container according to any one of Items 1 to 8, wherein the plasma separation material has a specific gravity at 25° C. of 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 composition for plasma separation.

[0020] Item 11. The blood collection container according to Item 10, wherein the plasma separation composition comprises an organic component having 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 resin, a cyclopentadiene resin, a polyester resin, or a (meth)acrylic 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 for separating 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 in 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 the 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. Effect of the Invention

[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 in the blood collection container body, and an aqueous solution contained in 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. Since 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 incorporation of platelet-derived exosomes into plasma. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a front cross-sectional view that shows a schematic diagram of a blood collection container according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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, the pH of the mixed liquid (X) is 3.5 or more and 5.5 or less when the pH is measured as described below.

[0032] Measurement of pH: A solution with 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. The solution with a pH of 7.4 is collected in a blood collection container in an amount equal to the amount of blood collected in the blood collection container, and a mixed solution (X) is obtained by mixing the solution with a pH of 7.4 and the aqueous solution. The pH of the resulting mixed solution (X) is measured.

[0033] Since the blood collection container according to the present invention has the above-mentioned configuration, it is possible to suppress the release of exosomes from platelets, and therefore to suppress the contamination of plasma with exosomes derived from platelets.

[0034] In general, it is difficult to suppress the inclusion of platelets in plasma in a blood collection container. Although a blood collection container equipped with a plasma separation material can suppress the inclusion of red blood cells and white blood cells in plasma to some extent, it is difficult to suppress the inclusion of platelets in plasma. This is because the specific gravity of the plasma separation material is approximately the same as the specific gravity of platelets. Therefore, in conventional blood collection containers, exosomes may be released from platelets mixed in plasma. The present inventors have intensively studied 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 the platelets is within a specific range.

[0035] The liquid having a pH of 7.4 in the pH measurement is a liquid having a pH similar to that of blood, and the mixed liquid (X) is a liquid having a pH similar to that of a mixed liquid of blood and the aqueous solution. When blood is collected in the blood collection container according to 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, in the blood collection container according to the present invention, even if the separated plasma is stored for several days, the release of exosomes from platelets mixed in 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 suppress the inclusion of red blood cells and white blood cells in the plasma.

[0037] The blood collection container of 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 can therefore reduce the amount of miRNA contaminating the plasma even if the separated plasma is stored for several days.

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

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

[0040] The pH of the mixed solution (X) and the solution having a pH of 7.4 are 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 equal to or more than the lower limit and equal to or less than the 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 contained in the blood collection container body. As the plasma separation material, a conventionally known plasma separation material can be used. As the plasma separation material, a plasma separation composition, a plasma separation tool, and the like can be mentioned. Since the plasma separation material is easily prepared, 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 equal to or more than the lower limit and equal to or less than the upper limit, the inclusion of white blood cells and red blood cells in the plasma can be more effectively prevented. Furthermore, when the specific gravity of the plasma separation material at 25°C is equal to or more than the lower limit and equal to or less than the upper limit, the inclusion of platelets in the plasma can be more effectively prevented. Furthermore, the plasma can be separated from the blood more effectively.

[0045] The location of the plasma separation material 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] <Composition for plasma separation> 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 is preferably thixotropic. 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 exerting the effects of the present invention more effectively, the plasma separation composition is preferably contained in the bottom of the blood collection container body.

[0047] As the above-mentioned 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 kinds.

[0049] Organic components that are flowable at 25°C: The above 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, and more preferably 200 Pa s or less. When the viscosity is equal to or more 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] The organic component includes a resin, and a mixture of a resin and an organic compound such as a plasticizer. Therefore, the organic component preferably includes the resin, and more preferably includes the resin and the organic compound. When the organic component is a mixture of the resin and the organic compound, the mixture (the organic component) may have fluidity, and the resin or the organic compound may not 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 of the resin and the organic compound may be used, or two or more of them may be used in combination.

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

[0054] The resin preferably contains a petroleum resin, a cyclopentadiene resin, a polyester resin, or a (meth)acrylic resin. In this case, the fluidity of the plasma separation composition is further increased, and the strength of the partition wall formed after centrifugation can be further increased.

[0055] An example of a commercially available product of the above petroleum resin is "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 the cyclopentadiene monomer and the copolymer of the cyclopentadiene monomer and an aromatic monomer may be an oligomer.

[0057] Examples of the cyclopentadiene 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 products of the dicyclopentadiene resin include "Scoretz SU500" and "Scoretz SU90" manufactured by Colon Co., Ltd.

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

[0061] The polyurethane resin may be 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 ester, (meth)acrylic acid polyalkylene glycol ester, (meth)acrylic acid alkoxyalkyl ester, (meth)acrylic acid hydroxyalkyl ester, (meth)acrylic acid glycidyl ester, (meth)acrylic acid dialkylamino alkyl ester, (meth)acrylic acid benzyl ester, (meth)acrylic acid phenoxy alkyl ester, (meth)acrylic acid cyclohexyl ester, (meth)acrylic acid isobornyl ester, and (meth)acrylic acid alkoxysilyl alkyl ester. When the (meth)acrylic acid ester monomer has an alkyl group, the number of carbon atoms of 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 a carbon number of 1 to 20. 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 derivative include phthalic acid ester, trimellitic acid ester, pyromellitic acid ester, etc. The benzene polycarboxylic acid alkyl ester derivative may be used alone or in combination of two or more kinds.

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

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

[0068] Commercially available products of the trimellitic acid ester 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] An example of a commercially available product of the pyromellitic acid ester is "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] In 100% by weight of the above-mentioned composition for plasma separation, the content of the above-mentioned organic component 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, fine glass 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, fine glass 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 fine silica. The inorganic fine powder may contain fine silica and an inorganic fine powder other than fine silica (second inorganic fine powder). The inorganic fine powder, the fine silica, and the second inorganic fine powder may each be used alone or in combination of two or more.

[0075] The fine silica powder includes natural silica and synthetic silica. The synthetic silica includes hydrophilic silica and hydrophobic silica. Hydrophilic silica, for example, imparts thixotropy to the plasma separation composition by hydrogen bonding between hydroxyl groups on the particle surface, and has the effect of adjusting the specific gravity. 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 higher 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 6 or less.

[0079] The average particle size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder is not particularly limited. The average particle size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder 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 diameters), and are the values ​​of the 50% median diameter (D50). The volume average particle diameter (D50) can be measured by a laser diffraction / scattering method, an image analysis method, the Coulter method, a centrifugal sedimentation method, and the like. The volume average particle diameter (D50) is preferably determined by a laser diffraction / scattering method or an image analysis method.

[0081] The specific surface area of ​​the fine silica powder is not particularly limited. The specific surface area of ​​the fine silica powder is 20 m 2 / g or more, 2 / g or more, 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] In 100% by weight of the plasma separation composition, the content of the hydrophilic silica 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, 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 a more suitable range.

[0084] In 100% by weight of the plasma separation composition, the content of the finely powdered silica is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, 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 a more suitable range.

[0085] In 100% by weight of the plasma separation composition, the content of the second inorganic fine powder is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, 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] In 100% by weight of the plasma separation composition, the content of the inorganic fine powder is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, 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 Ingredients: The plasma separation composition may contain other components in addition to the above-mentioned components, so 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, auxiliary solvents, 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 equal to or more than the lower limit and equal to or less than the upper limit, the inclusion of white blood cells and red blood cells in the plasma can be more effectively suppressed. When the specific gravity of the plasma separation composition at 25°C is equal to or more than the lower limit and equal to or less than the upper limit, the inclusion of platelets in the plasma can be more effectively suppressed. Furthermore, the plasma can be separated from the blood more effectively.

[0089] The specific gravity of the above-mentioned plasma separation composition at 25°C is measured by dropping one drop of the plasma separation composition into successively adjusted saline solutions at 25°C having specific gravities of 0.002 intervals, and observing the floating and sinking of the composition in the saline solution.

[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 more 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 above-mentioned 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 tool> 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, and is used for the purpose of preventing component migration between the plasma layer and the blood cell layer.

[0093] As the plasma separation tool, a conventionally known plasma separation tool can be used, for example, a mechanical separator (plasma separation tool) described in WO2010 / 132783A1 or the like.

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

[0095] (aqueous solution) The blood collection container includes an aqueous solution contained within the blood collection container body. The aqueous solution includes 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 enhance mixing with the blood and effectively suppress hemolysis.

[0096] <Anticoagulant> The aqueous solution contains an anticoagulant. As the anticoagulant, a conventionally known anticoagulant can be used. The anticoagulant may be used alone or in combination of two or more kinds.

[0097] Examples of the anticoagulant include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, and sodium citrate.

[0098] From the viewpoint of exerting 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. In 100% by weight of the aqueous solution, the content of the anticoagulant 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 well.

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

[0101] The organic acids include citric acid, succinic acid, formic acid, acetic acid, lactic acid, oxalic acid, gluconic acid, malonic acid, fumaric acid, malic acid, tartaric acid, etc. The inorganic acids include hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, boric acid, hydrofluoric acid, etc.

[0102] From the viewpoint of exerting the effects of the present invention more effectively and from the viewpoint of easily adjusting the pH of the mixed liquid (X), the aqueous solution preferably contains an organic acid.

[0103] 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 not less than the above lower limit and not more than the above upper limit, the effects of the present invention can be more effectively exhibited.

[0104] The organic acid is preferably a carboxylic acid, more preferably contains citric acid or succinic acid, and even more preferably contains citric acid. The aqueous solution preferably contains a carboxylic acid, more preferably contains citric acid or succinic acid, and even more preferably contains citric acid. In this case, the effect of the present invention can be more effectively exhibited. In addition, when the aqueous solution contains citric acid, hemolysis can be effectively suppressed.

[0105] The content of the organic acid and the content of the inorganic acid in the aqueous solution are not particularly limited as long as the pH of the mixed liquid (X) can be adjusted to the desired pH.

[0106] In the aqueous solution (100% by weight), the content of the organic acid 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 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.

[0107] In the aqueous solution (100% by weight), the content of citric acid is preferably 1% by weight or more, more preferably 2% by weight or more, even more preferably 2.5% by weight or more, 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 citric 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. In addition, when the content of citric acid is equal to or more than the lower limit and equal to or less than the upper limit, hemolysis can be more effectively suppressed.

[0108] In the aqueous solution (100% by weight), the content of succinic acid is preferably 1% by weight or more, more preferably 2% by weight or more, even more preferably 2.5% by weight or more, 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 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.

[0109] <Disaccharide> The aqueous solution preferably contains a disaccharide. In this case, hemolysis can be effectively suppressed. In addition, since a source of nutrition 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 disaccharide may be used alone or in combination of two or more kinds.

[0110] The disaccharides include sucrose, trehalose, maltose, and lactose.

[0111] The disaccharide preferably contains sucrose. The aqueous solution preferably contains sucrose. In this case, hemolysis can be more effectively suppressed. Also, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0112] In the aqueous solution (100% by weight), the content of the disaccharide 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 more than the lower limit and equal to or less than the upper limit, hemolysis can be more effectively suppressed. Also, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0113] In the aqueous solution (100% by weight), the content of sucrose 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 content of sucrose is equal to or more than the lower limit and equal to or less than the upper limit, hemolysis can be more effectively suppressed. Also, leakage of miRNA from red blood cells and white blood cells can be more effectively suppressed.

[0114] <Inorganic salts> 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.

[0115] 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.

[0116] The content of the inorganic salt in the aqueous solution (100% by weight) is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, and preferably 6% by weight or less, and 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.

[0117] <Water> The aqueous solution preferably contains water, in which the water serves as a solvent.

[0118] In the aqueous solution (100% by weight), 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, preferably 98% by weight or less, more preferably 95% by weight or less.

[0119] <Other ingredients> The aqueous solution may contain other components in addition to the above-mentioned components (anticoagulant, organic acid, inorganic acid, disaccharide, inorganic salt, and water). 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.

[0120] The aqueous solution may or may not contain a compound capable of releasing aldehyde. Examples of the compound capable of releasing 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.

[0121] <Other details of the aqueous solution> In the 100% by weight aqueous solution, the total content of the anticoagulant, the organic acid, and 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 90% by weight or more, and particularly preferably 95% by weight or more. When the total content is equal to or more than the lower limit, the effect of the present invention can be more effectively exhibited. In addition, in the 100% by weight 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.

[0122] In the 100% by weight aqueous solution, the total content of the anticoagulant, the organic acid, the disaccharide, and the water 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 more than the lower limit, the effect of the present invention can be more effectively exhibited. In addition, in the 100% by weight aqueous solution, the total content of the anticoagulant, the organic acid, the disaccharide, and the water may be 100% by weight or less, less than 100% by weight, or 99% by weight or less.

[0123] The pH of the aqueous solution is preferably 3.0 or more, more preferably 3.5 or more, and is preferably 6.0 or less, more preferably 5.5 or less, and even more preferably 5.0 or less. When the pH is equal to or more than the lower limit and equal to or less than the upper limit, the effects of the present invention can be more effectively exhibited.

[0124] The pH of the above aqueous solution is measured at 22° C. using a pH meter.

[0125] The amount of the aqueous solution contained in the blood collection container body is appropriately changed depending on the size of the blood collection container body, the amount of blood to be collected, etc. The amount of the 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, preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2.5 mL or less. When the amount of the aqueous solution is above the lower limit and below the upper limit, the blood is not excessively diluted, and the effect of the present invention can be more effectively exhibited.

[0126] (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 blood collection container body preferably has a closed bottom at the other end.

[0127] The material of the blood collection container body is not particularly limited. Examples of the material of 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 resins, epoxy resins, and epoxy-acrylate resins; 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. Only one type of material may be used for the blood collection container body, or two or more types may be used in combination.

[0128] (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 liquidtight manner. The stopper is preferably configured to be pierced by a blood collection needle.

[0129] 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.

[0130] The stopper may be a stopper including 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.

[0131] Examples of the material of the stopper (or the stopper main body) include synthetic resin, elastomer, rubber, metal foil, etc. Examples of the rubber include butyl rubber and halogenated butyl rubber, etc. Examples of the metal foil include aluminum foil, etc. From the viewpoint of improving the sealing performance, the material of the stopper is preferably butyl rubber. The stopper (or the stopper main body) is preferably a butyl rubber stopper.

[0132] (Other details of blood collection container) The blood collection container is a blood collection container in which a predetermined amount of blood is collected. The predetermined amount of blood is appropriately changed 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.

[0133] The blood collection container is preferably a blood collection container that collects 4 mL or more of blood per 1 mL of the aqueous solution contained in the blood collection container body, more preferably a blood collection container that collects 5 mL or more of blood, preferably a blood collection container that collects 9.5 mL or less of blood, and more preferably a blood collection container that collects 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 exhibited.

[0134] The blood collection container is preferably a blood collection tube. The blood collection container body is preferably a blood collection tube body.

[0135] The blood collection container is preferably used to separate plasma from blood. The blood collection container is preferably used to separate extracellular free nucleic acid or extracellular vesicles in blood, and is preferably used to isolate extracellular free nucleic acid or extracellular vesicles in blood. The extracellular free nucleic acid may be cell free DNA (cfDNA) or cell free RNA (cfRNA). The extracellular free nucleic acid is preferably cfRNA.

[0136] The blood collection container can be manufactured, for example, as follows.

[0137] An anticoagulant and an organic acid are dissolved in water to obtain an aqueous solution. If necessary, other components are also dissolved in water to obtain an aqueous solution. The obtained aqueous solution is added to the blood collection container body. Before or after the aqueous solution is added, a plasma separation material is placed in the blood collection container body.

[0138] FIG. 1 is a front cross-sectional view that shows a schematic diagram of a blood collection container according to one embodiment of the present invention.

[0139] 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 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 length direction of the blood collection container body 2, and the closed end 2b corresponds to the other end (bottom side) in the length direction of the blood collection container body 2. The plasma separation composition 3 is accommodated in the bottom part 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.

[0140] The aqueous solution 4 is disposed on the surface of the plasma separating composition 3, more specifically, on the upper surface (the surface on the open end 2a side of the blood collection container body 2) of the plasma separating composition 3. The aqueous solution 4 is disposed on the surface of the plasma separating composition 3 when the blood collection container 1 is in an upright position.

[0141] 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 on 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.

[0142] 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, the inside of which is evacuated and 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. In the case of a vacuum blood collection tube, a certain amount of blood can be easily collected regardless of the skill level of the blood collector.

[0143] 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.

[0144] (Method of separating plasma) The blood collection container can be used to separate plasma from blood. A method for separating plasma according to the present invention includes the steps of collecting blood in the blood collection container and centrifuging the blood collection container in which the blood has been collected.

[0145] In the plasma separation method according to the present invention, it is preferable to 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.

[0146] The centrifugation conditions in the centrifugation step are not particularly limited as long as the plasma separation material can form a partition 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.

[0147] (Method for isolating extracellular free nucleic acids and method for isolating extracellular vesicles) The method for isolating extracellular free nucleic acid according to the present invention comprises the steps of collecting blood in the blood collection container described above, centrifuging the blood collection container in which the blood has been collected to separate plasma from the blood, and separating extracellular free nucleic acid from the separated plasma.

[0148] The method for separating extracellular vesicles according to the present invention includes the steps of collecting blood in the blood collection container described above, centrifuging the blood collection container in which the blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.

[0149] In the method for separating extracellular free nucleic acids and the method for separating extracellular vesicles according to the present invention, it is preferable to include a step of mixing the collected blood with the aqueous solution between the step of collecting the blood and the step of centrifuging. Examples of a method for mixing the collected blood with the aqueous solution include mixing by inversion.

[0150] The centrifugation conditions in the centrifugation step are not particularly limited as long as the plasma separation material can form a partition 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.

[0151] In the step of separating the extracellular free nucleic acid, the extracellular free nucleic acid can be separated from the plasma using a conventionally known method. The extracellular free nucleic acid includes cell free DNA (cfDNA) and cell free RNA (cfRNA). The method of separating the extracellular free nucleic acid from the plasma includes a method using a commercially available nucleic acid purification kit. By using a commercially available nucleic acid purification kit, the extracellular free nucleic acid can be easily separated from the plasma. Examples of the commercially available nucleic acid purification kit include QIAamp Circulating Nucleic Acid Kit (QIAGEN), QIAamp MinElute ccfDNA Kits (QIAGEN), and MagMAX Cell-Free DNA Isolation Kit (Applied biosystems).

[0152] In the step of separating extracellular vesicles, extracellular vesicles can be separated from plasma using a conventionally known method.

[0153] (Method for inhibiting exosome release from platelets) The present specification also provides a method for suppressing exosome release from platelets, which comprises storing platelets in an environment having a pH of 3.5 or more and less than 7.0. The above "environment having a pH of 3.5 or more and less than 7.0" means that the pH of the liquid in contact with the platelets is 3.5 or more and less than 7.0.

[0154] In the above method, the platelets are preferably stored in an environment with a pH of 4.0 or higher, more preferably stored in an environment with a pH of 4.5 or higher, even more preferably stored in an environment with a pH of 5.0 or higher, preferably stored in an environment with a pH of 6.9 or lower, more preferably stored in an environment with a pH of 6.6 or lower, and even more preferably stored 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.

[0155] The above method is preferably the following method (1) or the following method (2).

[0156] The method (1) includes a step of mixing blood with an aqueous solution containing an anticoagulant to obtain a liquid (A), and a step of storing the liquid (A). In the method (1), the pH of the liquid (A) is 3.5 or more and less than 7.0. In the method (1), the liquid (A) is in contact with the platelets.

[0157] 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.

[0158] In the above method (1) and method (2), in the step of obtaining the liquid (A), blood, the aqueous solution, and other components (liquids, etc.) may be mixed. In addition, in the above method (1) and method (2), the aqueous solution (aqueous solution containing an anticoagulant) may be the aqueous solution described above.

[0159] In the above method (1) and method (2), the pH of the above liquid (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, even more preferably 6.4 or lower. When the pH of the above liquid (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.

[0160] In the above 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, even more preferably 6.4 or lower. When the pH of the platelet-containing plasma 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 inhibited.

[0161] The pH of the liquid (A) and the pH of the platelet-containing plasma are measured at 22° C. using a pH meter.

[0162] 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.

[0163] The following materials were prepared as the plasma separation composition.

[0164] (Materials made of organic components that are fluid at 25℃) (Meth)acrylic resin: 2-Ethylhexyl acrylate and butyl acrylate were radically polymerized in the presence of an azo-based polymerization initiator by solution polymerization to obtain a (meth)acrylate polymer having flowability at 25°C.

[0165] (Inorganic fine powder) Hydrophilic silica (fine silica powder, "200CF" manufactured by Nippon Aerosil Co., Ltd.)

[0166] (Other Ingredients) Silicone oil (Toray Dow Corning "SF8410") Organic gelling agent ("Gelall D" manufactured by New Japan Chemical Co., Ltd.) 1-Methyl-2-pyrrolidone (auxiliary solvent)

[0167] Preparation of plasma separation composition A: 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.

[0168] [Table 1]

[0169] A drop of each of the obtained compositions for plasma separation was dropped into saline at 25°C, the specific gravity of which had been adjusted stepwise at intervals of 0.002, and the specific gravity was measured by floating or sinking in the saline. The specific gravities of the obtained compositions for plasma separation at 25°C are shown in Tables 2 to 5.

[0170] The following materials were prepared as the aqueous solution materials. Granular disodium ethylenediaminetetraacetate (EDTA2Na) dihydrate (anticoagulant) was also prepared.

[0171] (Anticoagulant) Trisodium Citrate Dihydrate

[0172] Citric Acid Monohydrate Succinic acid sucrose Sodium chloride water

[0173] The following blood collection containers were prepared:

[0174] A PET bottomed tube (polyethylene terephthalate tube) with a length (distance between the open end and closed end) of 100 mm and an inside diameter of 14 mm at the open end.

[0175] Example 1 The components shown in Table 2 were dissolved in water (water for injection) to obtain an aqueous solution. The types and concentrations of the components in the obtained aqueous solution are shown in Table 2.

[0176] 1.2 g of the plasma separation composition A was placed in the bottom of the blood collection container body. 1.0 mL of the obtained aqueous solution was added onto the surface of the plasma separation composition A. The inside of the blood collection container was depressurized so that the blood collection volume was 8.5 mL, and the container was sealed with a butyl rubber stopper. In this way, the blood collection container was prepared.

[0177] (Examples 2 to 5, 8 to 10 and Comparative Examples 2 and 3) A blood collection container was produced in the same manner as in Example 1, except that the composition of the aqueous solution was changed as shown in Tables 2 to 5.

[0178] (Examples 6 and 7) The composition of the aqueous solution was changed as shown in Table 3. The inside of the blood collection container was depressurized so that the amount of blood collected was 5.0 mL (Example 6) or 9.5 mL (Example 7), and the container was sealed with a butyl rubber stopper. Except for this, the blood collection container was prepared in the same manner as in Example 1.

[0179] Comparative Example 1 10 mg of granular disodium ethylenediaminetetraacetate (EDTA2Na) dihydrate (anticoagulant) was placed in the bottom of the blood collection container. The inside of the blood collection container was depressurized so that the blood collection volume was 5.0 mL, and the container was sealed with a butyl rubber stopper. In this way, the blood collection container was prepared.

[0180] (evaluation) (1) pH of the aqueous solution The pH of the resulting aqueous solution was measured at 22° C. using a pH meter (HORIBA "F-52S").

[0181] (2) pH of the 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, and the pH was adjusted with hydrochloric acid (pH adjuster), and then water was added to make the solution 1 L, 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 ("F-52S" manufactured by HORIBA). The solution with a pH of 7.4 was collected in the obtained blood collection container. In Examples 1 to 5, 8 to 10 and Comparative Examples 2 and 3, 8.5 mL of the solution with a pH of 7.4 was collected in the blood collection container, 5.0 mL of the solution with a pH of 7.4 was collected in the blood collection container in Example 6, and 9.5 mL of the solution with a pH of 7.4 was collected in the blood collection container in Example 7. Next, the liquid with a pH of 7.4 and the aqueous solution were mixed by inversion to obtain a mixed liquid (X). In Comparative Example 1, 5.0 mL of the liquid with a pH of 7.4 was collected in a blood collection container, and then the liquid was inverted to obtain a mixed liquid (X) by dissolving a granular anticoagulant in the liquid with a pH of 7.4. The pH of the obtained mixed liquid (X) was measured at 22°C using a pH meter (HORIBA "F-52S").

[0182] (3) Blood evaluation Blood was collected from two blood donors in blood collection containers. In Examples 1-5, 8-10 and Comparative Examples 2 and 3, 8.5 mL of blood was collected in the blood collection container, in Example 6, 5.0 mL of blood was collected in the blood collection container, and in Example 7, 9.5 mL of blood was collected in the blood collection container. After collecting the blood, the blood was mixed by inversion to mix with the aqueous solution contained in the blood collection container. In Comparative Example 1, 5.0 mL of blood was collected in the blood collection container, and then the blood was mixed by inversion to dissolve the granular anticoagulant in the blood.

[0183] In the Examples and Comparative Examples other than Comparative Example 1, after mixing the blood with the above aqueous solution, the following (i) and (ii) were carried out.

[0184] (i) The blood collection container was centrifuged at 20° C. and 1900 G for 15 minutes. After centrifugation, the plasma was located above the partition formed by the plasma separation composition. A portion of the obtained plasma was collected. The collected plasma was transferred to another container, and the container was 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 as "plasma (A)".

[0185] (ii) The blood collection container after collecting a portion of the plasma in (i) above (the blood collection container in which the plasma is located above the partition formed by the plasma separation composition) was stored at 4°C for 3 days. Plasma was collected from the blood collection container after storage. The collected plasma was transferred to another container, and the container was centrifuged at 16000G at 4°C for 10 minutes. After centrifugation, the supernatant plasma was collected from the container, and the collected plasma was designated as "plasma (B)".

[0186] In Comparative Example 1, after dissolving a granular anticoagulant in blood, the following (a) and (b) were carried out.

[0187] (a): The blood collection container was centrifuged at 20° C. and 1900 G for 15 minutes to separate the blood cell layer and 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 as "plasma (A)".

[0188] (b): The blood collection container was stored at 4°C for 3 days. The stored blood collection container was centrifuged at 20°C and 1900G for 15 minutes to separate into a blood cell layer and a plasma layer. The plasma was collected from the plasma layer and transferred to another container, which was then centrifuged at 4°C and 16000G for 10 minutes. After centrifugation, the supernatant plasma was collected from the container, and the collected plasma was designated as "plasma (B)".

[0189] (3-1) Plasma pH The pH of the plasma (A) was measured at 22° C. using a pH meter (HORIBA "F-52S").

[0190] (3-2) Contamination of plasma with platelet-derived exosomes (3-2-1) Absorbance of CD9-positive exosomes (wavelength 450 nm) Plasma (A) and plasma (B) were each centrifuged through a 0.22 μm filter (Millipore Corporation, "0.22 μm GV DURAPORE"). The filtrate was diluted 20-fold with PBS(-), and the absorbance (wavelength 450 nm) was measured using a CD9-Capture human exosome ELISA kit (Wako Corporation, "Streptavidin HRP").

[0191] 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 incorporation of platelet-derived exosomes into plasma.

[0192] Increase in absorbance (%) = B / A x 100 A: Absorbance of plasma (A) B: Absorbance of plasma (B)

[0193] <Determination Criteria for Absorbance of CD9-Positive Exosomes> ○: The increase rate of absorbance is 350% or less △: The increase rate of absorbance exceeds 350% and is 600% or less ×: The increase rate of absorbance exceeds 600%

[0194] (3-2-2) Increase Rate of EV-miRNA (miR-126-3p) Concentration in Plasma For each of plasma (A) and plasma (B), the following tests were conducted. Using a purification kit (exoRNeasy Midi Kit, Qiagen), total EV-RNA was purified from 0.5 mL of plasma. Subsequently, reverse transcription reaction was performed using the following reagents and measuring instruments, and PCR for miR-126-3p was performed.

[0195] Reagents for reverse transcription reaction: miRCURY LNA RT Kit (Qiagen) Reagents for PCR: miRCURY LNA SYBR Green PCR Kit (Qiagen) Primer: miRCURY LNA miRNA PCR Assay (Qiagen, 339306), Primer #: YP00204227 Measuring instrument: C1000 Touch TM Thermal Cycler, CFX96 Deep Well TM Real-Time System (BioRad)

[0196] Also, the increase rate of EV-miRNA (miR-126-3p) concentration in plasma was determined by the following formula. The smaller the increase rate of EV-miRNA (miR-126-3p) concentration, the more the release of exosomes from platelets is suppressed. Therefore, it means that the contamination of platelet-derived exosomes into plasma is suppressed.

[0197] 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 with N=2 for each sample T The average value of

[0198] <Criteria for determining the increase rate of EV-miRNA (miR-126-3p) concentration in plasma> ○: Increase in EV-miRNA (miR-126-3p) concentration is less than 3-fold △: Increase in EV-miRNA (miR-126-3p) concentration is more than 3-fold and less than 6-fold ×: Increase in EV-miRNA (miR-126-3p) concentration exceeds 6-fold

[0199] (3-3) Contamination of plasma with exosomes derived from red blood cells (increase in EV-miRNA (miR-451a) concentration in plasma) The following tests were performed on 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, reverse transcription was performed using the following reagents and measuring equipment, and PCR of miR-451a was performed.

[0200] Reverse transcription reagent: miRCURY LNA RT Kit (Qiagen) PCR reagents: miRCURY LNA SYBR Green PCR Kit (Qiagen) Primer: miRCURY LNA miRNA PCR Assay (Qiagen, 339306), Primer#: YP02119305 Measuring equipment: C1000 Touch TM Thermal Cycler, CFX96 Deep Well TM Real-Time System (BioRad)

[0201] In addition, the increase rate of EV-miRNA (miR-451a) concentration in plasma was calculated using the following formula. The smaller the increase rate of EV-miRNA (miR-451a) concentration, the more suppressed the release of exosomes from red blood cells, and therefore the more suppressed the contamination of plasma with exosomes derived from red blood cells.

[0202] 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 for each sample when PCR was performed with N=2 for each sample T The average value of

[0203] <Criteria for determining the increase rate of EV-miRNA (miR-451a) concentration in plasma> ○: Increase in EV-miRNA (miR-451a) concentration is less than 3-fold △: Increase in EV-miRNA (miR-451a) concentration is more than 3-fold and less than 6-fold ×: Increase in EV-miRNA (miR-451a) concentration exceeds 6-fold

[0204] (3-4) Total EV-miRNA concentration in plasma The following tests were performed on 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 measuring device (Qubit microRNA Assay Kit (Thermo, Q32881)).

[0205] In addition, the rate of increase in total EV-miRNA concentration in plasma was calculated using the following formula.

[0206] Increase rate of total EV-miRNA concentration (%) = B / A × 100 A: Total EV-miRNA concentration in plasma (A) (ng / mL of plasma) B: Total EV-miRNA concentration in plasma (B) (ng / mL plasma)

[0207] <Criteria for determining total EV-miRNA concentration in plasma> ○: Increase in total EV-miRNA concentration is 150% or less △: Increase in total EV-miRNA concentration is more than 150% and less than 200% ×: Increase in total EV-miRNA concentration exceeds 200%

[0208] The compositions and results are shown in the following Tables 2 to 5. In the tables, the concentrations of the anticoagulant and the organic acid in the aqueous solution are the concentrations in an anhydrous state.

[0209] [Table 2]

[0210] [Table 3]

[0211] [Table 4]

[0212] [Table 5] [Explanation of symbols]

[0213] 1. Blood collection container 2…Blood collection container body 2a...Open end 2b...Closed end 3...Composition for plasma separation 4...Aqueous solution 5…Bolt

Claims

1. a blood collection container into which a predetermined amount of blood is collected; A blood collection container body; A plasma separation material contained in the blood collection container body; an aqueous solution contained in the blood collection container body; the aqueous solution comprises an anticoagulant; The pH of the aqueous solution is 3.0 or more and 5.5 or less, A blood collection container in which the pH of the mixed solution (X) is 3.5 or more and 5.5 or less when the pH is measured as described below. Measurement of pH: A solution having a pH of 7.4 is obtained, which contains 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. The solution having a pH of 7.4 is collected in a blood collection container in an amount equal to a predetermined amount of blood to be collected in the blood collection container, and a mixed solution (X) is obtained in which the solution having a pH of 7.4 and the aqueous solution are mixed. 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 of claim 1 , wherein the aqueous solution comprises an organic acid.

4. 4. The blood collection container of claim 3, wherein the organic acid has a molecular weight of 500 or less.

5. 4. The blood collection container according to claim 3, wherein the organic acid has a molecular weight of 100 or more and 250 or less.

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

7. 7. The blood collection container according to claim 3, 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.

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

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

10. 7. The blood collection container according to claim 1, wherein the anticoagulant is heparin, a metal salt of heparin, EDTA, a metal salt of EDTA, or sodium citrate.

11. 7. The blood collection container according to claim 1, wherein the plasma separation material has a specific gravity at 25° C. of 1.020 or more and 1.040 or less.

12. The blood collection container according to any one of claims 1 to 6, wherein the plasma separation material is a composition for plasma separation.

13. The plasma separation composition comprises an organic component having fluidity at 25° C. and an inorganic fine powder, the organic component comprises a resin; 13. The blood collection container of claim 12, wherein the fine inorganic powder comprises finely divided silica.

14. 14. The blood collection container of claim 13, wherein the resin comprises a petroleum resin, a cyclopentadiene-based resin, a polyester resin, or a (meth)acrylic resin.

15. The blood collection container according to any one of claims 1 to 6, 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.

16. The blood collection container according to any one of claims 1 to 6, which is used for separating extracellular free nucleic acids or extracellular vesicles in blood.

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