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

Abstract

To provide a blood collection container capable of reducing the amount of leukocytes mixed into plasma even when blood is centrifuged several days after collection. 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, wherein the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant.

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

[0002] In clinical testing, blood collection containers such as blood collection tubes are widely used to collect blood. After collecting blood in a blood collection container containing a plasma separation material, the blood can be separated into plasma and blood cells by centrifuging the blood collection container. At this time, the plasma is located above the plasma separation material, and the blood cells are located below. Known blood collection containers containing a plasma separation material include a blood collection container containing a plasma separation composition containing a resin and an inorganic powder (e.g., Patent Document 1) and a blood collection container containing a plasma separation tool (e.g., Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2010 / 053180A1 [Patent Document 2] WO2010 / 132783A1 Summary of the Invention [Problem to be solved by the invention]

[0004] In clinical settings, it is common to separate blood into plasma and blood cells by centrifuging the blood collection container on the same day the blood is collected. However, several days may pass between the time the blood is collected into the blood collection container and the time the blood collection container is centrifuged. For example, if blood is collected into a blood collection container at a hospital facility that does not have a centrifuge, the blood collection container must be transported to an external testing facility, and several days may pass between the time the blood is collected into the blood collection container and the time the blood collection container is centrifuged. Furthermore, if there are a large number of samples waiting to be tested, several days may pass between the time the blood is collected into the blood collection container and the time the blood collection container is centrifuged.

[0005] When conventional blood collection containers are centrifuged on the same day blood is collected, the amount of leukocytes mixed into the separated plasma can be relatively small. However, when conventional blood collection containers are centrifuged several days after blood collection, the amount of leukocytes mixed into the separated plasma can be large.

[0006] When the amount of white blood cells contaminating plasma is high, the amount of DNA leaked from the white blood cells into the plasma due to death and other reasons also increases. DNA leaked from white blood cells into the plasma can affect test results. For example, in tests that detect extracellular free nucleic acids (e.g., cell-free DNA) in plasma, test results can vary significantly depending on the DNA leaked from white blood cells.

[0007] An object of the present invention is to provide a blood collection container that can reduce the amount of leukocytes mixed into plasma even when blood is centrifuged several days after collection. 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 above blood collection container. [Means for solving the problem]

[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 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 a lithium salt different from the anticoagulant.

[0010] Item 2. The blood collection container according to Item 1, wherein the content of the lithium salt in 100% by weight of the aqueous solution is 1% by weight or more.

[0011] Item 3. The blood collection container according to Item 1 or 2, wherein the lithium salt comprises lithium chloride, lithium acetate, lithium citrate, or lithium lactate.

[0012] Item 4. The blood collection container according to any one of Items 1 to 3, wherein the lithium salt has a molecular weight of 100 or less.

[0013] Item 5. The blood collection container according to any one of Items 1 to 4, wherein the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less.

[0014] Item 6. The blood collection container according to any one of Items 1 to 5, wherein the aqueous solution further contains ammonium sulfate.

[0015] Item 7. The blood collection container according to any one of Items 1 to 6, wherein the aqueous solution further contains trehalose.

[0016] Item 8. The blood collection container according to any one of Items 1 to 7, wherein the aqueous solution further contains a water-soluble polymer compound.

[0017] Item 9. The blood collection container according to any one of Items 1 to 8, wherein the aqueous solution further contains propylene glycol.

[0018] 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.

[0019] Item 11. The blood collection container according to Item 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.

[0020] Item 12. The blood collection container according to Item 11, wherein the finely powdered silica comprises hydrophilic silica and hydrophobic silica.

[0021] Item 13. The blood collection container according to Item 11 or 12, wherein the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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. [Effects of the Invention]

[0026] 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. In the blood collection container according to the present invention, the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant. Because the blood collection container according to the present invention has the above configuration, it is possible to reduce the amount of leukocytes contaminating the plasma even when blood is centrifuged several days after collection. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a front cross-sectional view that schematically shows a blood collection container according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention will be described in detail below.

[0029] (Blood collection container) 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, wherein the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant.

[0030] The blood collection container according to the present invention is provided with the above-described configuration, so that the amount of leukocytes mixed into the plasma can be reduced even when the blood is centrifuged several days after collection. The blood collection container according to the present invention can maintain a high degree of separation effect between plasma and leukocytes by the plasma separator, even when the blood is centrifuged several days after collection.

[0031] When blood is collected in the blood collection container of the present invention, the blood is mixed with the aqueous solution. The inventors have found that the lithium salt contained in the aqueous solution can reduce the amount of leukocytes contaminating the plasma, even when the blood is centrifuged several days (e.g., two days) after collection. The blood collection container of the present invention can reduce the amount of leukocytes contaminating the plasma, and therefore can also reduce the amount of leukocyte-derived DNA (genomic DNA) contaminating the plasma.

[0032] 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".

[0033] (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.

[0034] The specific gravity of the plasma separation material at 25°C is preferably 1.027 or more, more preferably 1.029 or more, even more preferably 1.030 or more, particularly preferably 1.032 or more, and preferably 1.060 or less, more preferably 1.055 or less, and even more preferably 1.050 or less. When the specific gravity of the plasma separation material at 25°C is not less than the above lower limit and not more than the above upper limit, plasma can be separated from blood more effectively, and contamination of plasma with white blood cells and red blood cells can be more effectively suppressed.

[0035] 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.

[0036] <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 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.

[0037] As the plasma separation composition, a conventionally known plasma separation composition can be used.

[0038] The composition for plasma separation preferably contains an organic component having fluidity at 25°C and an inorganic fine powder. In this case, the fluidity of the composition for plasma separation is increased, and the strength of the partition wall formed by 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.

[0039] 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.

[0040] 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 200 Pa s or less, more preferably 100 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.

[0041] 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.

[0042] 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; 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 of the resin and the organic compound may be used, or two or more of them may be used in combination.

[0043] 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 of the above resins may be used, or two or more of them may be used in combination.

[0044] The resin preferably contains a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin, which further enhances the fluidity of the plasma separation composition and further increases the strength of the partition wall formed after centrifugation.

[0045] Commercially available petroleum-based resins include "Regalite S5090" manufactured by Eastman Chemical Company.

[0046] 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.

[0047] Examples of the cyclopentadiene-based monomer include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0048] Examples of the aromatic monomer include styrene, methylstyrene, indene, and methylindene.

[0049] Commercially available dicyclopentadiene resins include "Scoretz SU500" and "Scoretz SU90" manufactured by Colon Co., Ltd.

[0050] 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.

[0051] Examples of the polyurethane resin include a reaction product of a polyol compound and an isocyanate compound.

[0052] 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.

[0053] 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 monomers may be used alone or in combination of two or more.

[0054] 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.

[0055] 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.

[0056] Examples of the trimellitic acid ester include tri-n-octyl trimellitate, triisooctyl trimellitate, and triisodecyl trimellitate.

[0057] Examples of the pyromellitic acid ester include tetraisooctyl pyromellitic acid.

[0058] Commercially available trimellitic acid esters include "Monocizer W700" and "Monocizer W-750" manufactured by DIC Corporation, and "Sansocizer TO™" and "Sansocizer TI™" manufactured by New Japan Chemical Co., Ltd.

[0059] Commercially available pyromellitic acid esters include "Monocizer W-7010" manufactured by DIC Corporation.

[0060] The benzenepolycarboxylic acid alkyl ester derivative is preferably a phthalate ester, a trimellitate ester, or a pyromellitate ester, and more preferably a trimellitate ester.

[0061] In 100% by weight of the above-mentioned composition for plasma separation, the content of the above-mentioned organic components is preferably 80% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, particularly preferably 95% by weight or more, and preferably 98% by weight or less.

[0062] 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.

[0063] 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.

[0064] 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. When obtaining a plasma separation composition having a specific gravity of 1.050 or more at 25°C, the inorganic fine powder more preferably contains finely powdered silica and an inorganic fine powder other than finely powdered silica (second inorganic fine powder). However, even when the specific gravity of the plasma separation composition at 25°C is 1.050 or more, the inorganic fine powder does not have to contain the second inorganic fine powder. Moreover, even when the specific gravity of the plasma separation composition at 25°C is less than 1.050, the inorganic fine powder may contain the second inorganic fine powder. Each of the inorganic fine powder, the finely powdered silica, and the second inorganic fine powder may be used alone or in combination of two or more.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 composition for plasma separation can be effectively increased. The specific gravity of the second inorganic fine powder may be 10 or less, or 6 or less.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The specific surface area of ​​the finely powdered silica is measured by the BET method.

[0073] 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.

[0074] 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 0.5% by weight or more, even more preferably 1.0% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 5% by weight or less, and particularly preferably 3% 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.

[0075] 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, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 4% by weight or less, and particularly preferably 2% 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.

[0076] 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, even more preferably 1.0% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 5% by weight or less, and particularly preferably 3% 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.

[0077] Other Ingredients: 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.

[0078] The specific gravity of the plasma separation composition at 25°C is preferably 1.027 or more, more preferably 1.029 or more, even more preferably 1.030 or more, particularly preferably 1.032 or more, and preferably 1.060 or less, more preferably 1.055 or less, and even more preferably 1.050 or less. When the specific gravity of the plasma separation composition at 25°C is above the above-mentioned lower limit and below the above-mentioned upper limit, plasma can be separated well from blood and contamination of plasma with leukocytes can be effectively suppressed. Furthermore, when the specific gravity of the plasma separation composition at 25°C is above the above-mentioned lower limit and below the above-mentioned upper limit, contamination of plasma with erythrocytes can also be effectively suppressed. However, the specific gravity of the plasma separation composition at 25°C may be 1.050 or more, or may exceed 1.050.

[0079] The specific gravity of the plasma separation composition at 25°C is measured by dropping one drop of the 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 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.).

[0080] 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 effects of the present invention can be more effectively exhibited.

[0081] 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 -1It is measured under the following conditions.

[0082] <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.

[0083] The plasma separation device can be a conventionally known plasma separation device, such as the mechanical separator (plasma separation device) described in WO2010 / 132783A1.

[0084] Examples of materials for the plasma separation device include elastomers.

[0085] (aqueous solution) The blood collection container includes an aqueous solution contained within the blood collection container body. The aqueous solution contains an anticoagulant. The aqueous solution contains a lithium salt (hereinafter, sometimes referred to as "lithium salt A") different from the anticoagulant. The lithium salt A is different from the anticoagulant. The solutes contained in the aqueous solution include the anticoagulant and lithium salt A. By containing the anticoagulant and lithium salt A dissolved in the aqueous solution within the blood collection container body, it is possible to improve the mixability with blood and effectively suppress hemolysis.

[0086] <Anticoagulant> The aqueous solution contains an anticoagulant. A conventionally known anticoagulant can be used as the anticoagulant. Only one type of anticoagulant can be used, or two or more types can be used in combination.

[0087] Examples of the anticoagulant include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, citric acid, and sodium salts of citric acid. The metal salts of heparin may be lithium salts of heparin. Examples of the sodium salts of citric acid include trisodium citrate.

[0088] From the viewpoint of exhibiting good anticoagulant performance, the anticoagulant is preferably EDTA, a metal salt of EDTA, heparin, a metal salt of heparin, or trisodium citrate.

[0089] The concentration of the anticoagulant in the aqueous solution is not particularly limited as long as it is a concentration at which anticoagulant performance is exhibited.

[0090] When the anticoagulant is EDTA or a metal salt of EDTA, the concentration of the anticoagulant in the aqueous solution is preferably 2 mM or more, more preferably 5 mM or more, even more preferably 10 mM or more, preferably 2000 mM or less, more preferably 1000 mM or less, even more preferably 500 mM or less, even more preferably 250 mM or less, even more preferably 100 mM or less, and particularly preferably 50 mM or less. When the concentration of the anticoagulant is above the lower limit and below the upper limit, the anticoagulant can exhibit good anticoagulant performance.

[0091] When the anticoagulant is trisodium citrate, the concentration of the anticoagulant in the aqueous solution is preferably 50 mM or more, more preferably 70 mM or more, even more preferably 100 mM or more, preferably 300 mM or less, more preferably 200 mM or less, and even more preferably 140 mM or less. When the concentration of the anticoagulant is equal to or more than the lower limit and equal to or less than the upper limit, the anticoagulant can exhibit good anticoagulant performance.

[0092] When the anticoagulant is heparin or a metal salt of heparin, the concentration of the anticoagulant in the aqueous solution is preferably 50 IU / mL or more, more preferably 70 IU / mL or more, even more preferably 100 IU / mL or more, preferably 500 IU / mL or less, more preferably 450 IU / mL or less, and even more preferably 400 IU / mL or less. When the concentration of the anticoagulant is above the lower limit and below the upper limit, the anticoagulant performance can be satisfactorily exhibited. Here, IU is an international unit.

[0093] When the anticoagulant is heparin or a metal salt of heparin, the content of the anticoagulant in the aqueous solution (100% by weight) is preferably 0.0003% by weight or more, more preferably 0.0004% by weight or more, even more preferably 0.0006% by weight or more, preferably 0.003% by weight or less, more preferably 0.0025% by weight or less, and even more preferably 0.002% 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 can exhibit good anticoagulant performance.

[0094] A volume of saline solution equivalent to the predetermined volume of blood collected in the blood collection container is collected into the blood collection container, and a mixture of the saline solution and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline solution is collected into the blood collection container, and the saline solution and the aqueous solution are mixed by inversion or the like to obtain a mixture. In this case, it is preferable that the concentration of the anticoagulant in the mixture satisfy the following conditions. The concentration of the anticoagulant in the mixture corresponds to the concentration of the anticoagulant in the mixture obtained by mixing the aqueous solution and the blood collected in the blood collection container.

[0095] When the anticoagulant is EDTA or a metal salt of EDTA, the concentration of the anticoagulant in the mixed solution is preferably 0.1 mM or more, more preferably 1 mM or more, even more preferably 4 mM or more, preferably 200 mM or less, more preferably 100 mM or less, even more preferably 50 mM or less, even more preferably 25 mM or less, even more preferably 10 mM or less, and particularly preferably 7 mM or less. When the concentration of the anticoagulant is above the lower limit and below the upper limit, the anticoagulant can exhibit good anticoagulant performance.

[0096] When the anticoagulant is trisodium citrate, the concentration of the anticoagulant in the mixed solution is preferably 5 mM or more, more preferably 7 mM or more, even more preferably 10 mM or more, preferably 30 mM or less, more preferably 20 mM or less, and even more preferably 14 mM or less. When the concentration of the anticoagulant is equal to or more than the lower limit and equal to or less than the upper limit, the anticoagulant can exhibit good anticoagulant performance.

[0097] When the anticoagulant is heparin or a metal salt of heparin, the concentration of the anticoagulant in the mixed solution is preferably 5 IU / mL or more, more preferably 7 IU / mL or more, even more preferably 10 IU / mL or more, preferably 50 IU / mL or less, more preferably 45 IU / mL or less, and even more preferably 40 IU / mL or less. When the concentration of the anticoagulant is above the lower limit and below the upper limit, the anticoagulant performance can be satisfactorily exhibited. Here, IU is an international unit.

[0098] <Lithium salt A (a lithium salt different from anticoagulants)> The aqueous solution contains a lithium salt A. The lithium salt A may be used alone or in combination of two or more kinds.

[0099] Examples of the lithium salt A include lithium chloride, lithium acetate, lithium citrate, and lithium lactate.

[0100] The lithium salt A preferably contains lithium chloride, lithium acetate, lithium citrate, or lithium lactate, and more preferably contains lithium chloride, lithium acetate, or lithium lactate. In this case, the effect of the present invention of reducing the amount of leukocyte contamination in plasma is more effectively exhibited, even when blood is centrifuged several days after collection. Furthermore, even when blood is centrifuged several days after collection, leakage of genomic DNA from leukocytes can be effectively suppressed during the period from blood collection to centrifugation. Specifically, (1) the effect of reducing the amount of leukocyte contamination in plasma, thereby suppressing leukocyte-derived DNA contamination in plasma, and (2) the effect of effectively suppressing genomic DNA leakage from leukocytes during the period from blood collection to centrifugation are exhibited. These two effects significantly reduce the amount of leukocyte-derived DNA contamination in plasma, even when blood is centrifuged several days after collection.

[0101] The molecular weight of the lithium salt A is preferably 500 or less, more preferably 300 or less, even more preferably 200 or less, and particularly preferably 100 or less. When the molecular weight is equal to or less than the upper limit, the effect of the present invention, that is, the amount of leukocytes contaminating plasma can be reduced even when blood is centrifuged several days after collection, is more effectively exhibited. Furthermore, even when blood is centrifuged several days after collection, leakage of genomic DNA from leukocytes can be effectively suppressed during the period from blood collection to centrifugation. That is, due to the effects (1) and (2) above, the amount of leukocyte-derived DNA contaminating plasma can be significantly reduced even when blood is centrifuged several days after collection. The molecular weight of the lithium salt A may be 30 or more, 40 or more, 50 or more, or 60 or more.

[0102] The molecular weight of the lithium salt A means the molecular weight in an anhydrous state.

[0103] The content of the lithium salt A in 100% by weight of the aqueous solution is preferably 1% by weight or more, more preferably 3% by weight or more, even more preferably 5% by weight or more, and preferably 20% by weight or less, more preferably 15% by weight or less, even more preferably 12% by weight or less, and particularly preferably 10% by weight or less. When the content of the lithium salt A 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.

[0104] A volume of physiological saline equal to the predetermined amount of blood collected in the blood collection container is collected into the blood collection container, and a mixture of the physiological saline and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of physiological saline is collected into the blood collection container, and the physiological saline and the aqueous solution are mixed by inversion or the like to obtain a mixture. The content of the lithium salt A in 100 wt% of the mixture is preferably 0.1 wt% or more, more preferably 0.3 wt% or more, even more preferably 0.5 wt% or more, preferably 1.9 wt% or less, more preferably 1.5 wt% or less, and even more preferably 1.3 wt% or less. When the content of the lithium salt A is above the above-mentioned lower limit and below the above-mentioned upper limit, the effects of the present invention can be more effectively achieved. The content of the lithium salt A in 100 wt% of the mixture corresponds to the content of the lithium salt A in 100 wt% of the mixture obtained by mixing the aqueous solution and the blood collected in the blood collection container.

[0105] The content of the lithium salt A in 100% by weight of the aqueous solution and the content of the lithium salt A in 100% by weight of the mixed solution refer to the content of lithium salt A in an anhydrous state.

[0106] <Ammonium sulfate> The aqueous solution preferably contains ammonium sulfate. The use of ammonium sulfate can further enhance the effects of the present invention. It can also stabilize leukocytes and suppress DNA leakage from leukocytes.

[0107] The content of ammonium sulfate in the aqueous solution (100% by weight) is preferably 0.5% by weight or more, more preferably 1% by weight or more, even more preferably 2% by weight or more, preferably 20% by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less. When the content of ammonium sulfate is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. When the content of ammonium sulfate is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, leukocytes can be more effectively stabilized and DNA leakage from leukocytes can be more effectively suppressed.

[0108] A volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or the like to obtain a mixture. The content of ammonium sulfate in the mixture (100 wt%) is preferably 0.05 wt% or more, more preferably 0.1 wt% or more, even more preferably 0.4 wt% or more, preferably 2.5 wt% or less, more preferably 1.9 wt% or less, and even more preferably 1.3 wt% or less. When the content of ammonium sulfate is above the above-mentioned lower limit and below the above-mentioned upper limit, the effects of the present invention can be more effectively achieved. When the content of ammonium sulfate is above the above-mentioned lower limit and below the above-mentioned upper limit, leukocytes can be more effectively stabilized and DNA leakage from leukocytes can be more effectively suppressed. The content of ammonium sulfate in 100% by weight of the above mixed solution corresponds to the content of ammonium sulfate in 100% by weight of a solution obtained by mixing the above aqueous solution with blood collected in a blood collection container.

[0109] <Trehalose> The aqueous solution preferably contains trehalose, which effectively protects the cell membranes of leukocytes and stabilizes them, thereby further suppressing DNA leakage from leukocytes.

[0110] The trehalose content in the aqueous solution (100% by weight) 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 10% by weight or less, more preferably 7% by weight or less, and even more preferably 5% by weight or less. When the trehalose content is equal to or greater than the above lower limit and equal to or less than the above upper limit, leukocytes can be further stabilized and DNA leakage from leukocytes can be more effectively suppressed.

[0111] A volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or other means to obtain a mixture. The trehalose content in the mixture (100 wt%) is preferably 0.06 wt% or more, more preferably 0.1 wt% or more, even more preferably 0.2 wt% or more, preferably 1.3 wt% or less, more preferably 0.9 wt% or less, and even more preferably 0.6 wt% or less. A trehalose content above the above lower limit and below the above upper limit can further stabilize leukocytes and more effectively suppress DNA leakage from leukocytes. The trehalose content in the mixture (100 wt%) corresponds to the trehalose content in the mixture (100 wt%) obtained by mixing the aqueous solution and the blood collected in the blood collection container.

[0112] <Water-soluble polymer compounds> The aqueous solution preferably contains a water-soluble polymer compound. The water-soluble polymer compound is different from both the anticoagulant and the lithium salt A. By using the water-soluble polymer compound, the effects of the present invention can be more effectively exhibited. Furthermore, by using the water-soluble polymer compound, leukocytes can be more stabilized and DNA leakage from leukocytes can be more effectively suppressed. Note that "water-soluble" in the water-soluble polymer compound means that 0.1 g or more of the water-soluble polymer compound dissolves in 100 g of water at 25°C. The water-soluble polymer compound may be used alone or in combination of two or more.

[0113] The number-average molecular weight of the water-soluble polymer compound is preferably 300 or more, more preferably 1000 or more, even more preferably 1500 or more, even more preferably 2000 or more, particularly preferably 2500 or more, and preferably 180,000 or less, more preferably 170,000 or less, even more preferably 150,000 or less, even more preferably 120,000 or less, and particularly preferably 100,000 or less. When the number-average molecular weight of the water-soluble polymer compound is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. In addition, leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.

[0114] The number average molecular weight of the water-soluble polymer compound is a number average molecular weight measured by gel permeation chromatography (GPC) in terms of standard polyethylene glycol.

[0115] Examples of the water-soluble polymer compound include dextran, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, and corn starch.

[0116] The water-soluble polymer compound is preferably dextran, polyethylene glycol, or polyvinylpyrrolidone, and more preferably dextran or polyethylene glycol. In this case, the effects of the present invention can be more effectively exhibited. In addition, leukocytes can be more effectively stabilized, and DNA leakage from leukocytes can be more effectively suppressed.

[0117] The content of the water-soluble polymer compound in the aqueous solution (100% by weight) is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, even more preferably 1% by weight or more, and preferably 20% by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less. When the content of the water-soluble polymer compound is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. In addition, leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.

[0118] A volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or the like to obtain a mixture. The content of the water-soluble polymer compound in 100% by weight of the mixture is preferably 0.01% by weight or more, more preferably 0.06% by weight or more, even more preferably 0.1% by weight or more, preferably 2.5% by weight or less, more preferably 1.9% by weight or less, and even more preferably 1.3% by weight or less. When the content of the water-soluble polymer compound is above the above-mentioned lower limit and below the above-mentioned upper limit, the effects of the present invention can be more effectively exerted. Furthermore, leukocytes can be more effectively stabilized, and DNA leakage from leukocytes can be more effectively suppressed. The content of the water-soluble polymer compound in 100% by weight of the mixed liquid corresponds to the content of the water-soluble polymer compound in 100% by weight of the liquid obtained by mixing the aqueous solution and blood collected in a blood collection container.

[0119] <Propylene glycol> The aqueous solution preferably contains propylene glycol. By using propylene glycol, the effects of the present invention can be more effectively achieved. In addition, leukocytes can be stabilized, and leakage of DNA from leukocytes can be suppressed.

[0120] The content of propylene glycol in the aqueous solution (100% by weight) is preferably 1% by weight or more, more preferably 5% by weight or more, and preferably 50% by weight or less, more preferably 30% by weight or less. When the content of propylene glycol 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. In addition, leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.

[0121] A volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or the like to obtain a mixture. The content of propylene glycol in the mixture (100% by weight) is preferably 0.1% by weight or more, more preferably 0.2% by weight or more, even more preferably 0.5% by weight or more, preferably 6% by weight or less, more preferably 5% by weight or less, and even more preferably 4% by weight or less. When the content of propylene glycol is above the above-mentioned lower limit and below the above-mentioned upper limit, the effects of the present invention can be more effectively achieved. Furthermore, leukocytes can be more effectively stabilized, and DNA leakage from leukocytes can be more effectively suppressed. The content of propylene glycol in 100% by weight of the above-mentioned mixed liquid corresponds to the content of propylene glycol in 100% by weight of the liquid obtained by mixing the above-mentioned aqueous solution and blood collected in the blood collection container.

[0122] <Water> The aqueous solution preferably contains water, which serves as a solvent.

[0123] The water content in the aqueous solution (100% by weight) is preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and preferably 95% by weight or less, more preferably 90% by weight or less.

[0124] <Other ingredients> The aqueous solution may contain other components in addition to the above-mentioned components (anticoagulant, lithium salt A, ammonium sulfate, trehalose, water-soluble polymer compound, propylene glycol, and water). Examples of the other components include salts (inorganic salts and organic salts not corresponding to the above-mentioned components), sugars, and sugar alcohols. One or more of the other components may be used alone or in combination.

[0125] Examples of the salt include sodium salts such as sodium chloride and sodium hydrogen phosphate, and potassium salts such as potassium chloride, potassium acetate and potassium hydrogen carbonate.

[0126] Examples of the sugars include dihydroxyacetone, fructose, galactose, sucrose, maltose, and lactulose.

[0127] Examples of the sugar alcohol include D-mannitol and D-sorbitol.

[0128] <Other details of aqueous solution> The amount of aqueous solution contained in the blood collection container body varies 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, 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 lower limit and below the above 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 in combination.

[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 to be pierceable 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 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] A volume of saline solution equivalent to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline solution and the aqueous solution is obtained. For example, in a blood collection container capable of collecting 5 mL of blood, 5 mL of saline solution is collected into the blood collection container, and the saline solution and the aqueous solution are mixed by inversion or the like to obtain a mixture. The osmotic pressure of the mixture of the saline solution and the aqueous solution is preferably 320 mOsm / L or more, more preferably 350 mOsm / L or more, even more preferably 400 mOsm / L or more, preferably 2000 mOsm / L or less, more preferably 1000 mOsm / L or less, and even more preferably 800 mOsm / L or less. When the osmotic pressure of the mixture is above the above-mentioned lower limit and below the above-mentioned upper limit, the effects of the present invention can be more effectively achieved.

[0137] The osmotic pressure of the mixed solution is measured by the freezing point depression method using an osmometer (for example, "OM-6060" manufactured by Arkray).

[0138] The blood collection container is preferably a blood collection container that can collect 3 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 4 mL or more of blood, preferably a blood collection container that can collect 11 mL or less of blood, and more preferably a blood collection container that can collect 10 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.

[0139] The blood collection container is preferably a blood collection tube, and the blood collection container body is preferably a blood collection tube body.

[0140] 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 cfDNA.

[0141] The blood collection container can be manufactured, for example, as follows.

[0142] An anticoagulant and lithium salt A are dissolved in water to obtain an aqueous solution. If necessary, other components are also 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.

[0143] FIG. 1 is a front cross-sectional view that schematically shows a blood collection container according to one embodiment of the present invention.

[0144] 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 part of the blood collection container body 2. The aqueous solution 4 contains an anticoagulant and lithium salt A. The stopper 5 is inserted into the open end 2a of the blood collection container body 2.

[0145] The aqueous solution 4 is disposed on the surface of the plasma separation composition 3, more specifically, on the upper surface of the plasma separation composition 3 (the surface on the side of the open end 2a of the blood collection container body 2) when the blood collection container 1 is in an upright position. The aqueous solution 4 is disposed on the surface of the plasma separation composition 3 when the blood collection container 1 is in an upright position.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] (Plasma Separation Method) The blood collection container can be used to separate plasma from blood. 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 blood collection container described above, and a step of centrifuging the blood collection container in which the blood has been collected.

[0150] 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.

[0151] 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.

[0152] (Method for isolating extracellular free nucleic acids and method for isolating extracellular vesicles) The method for separating extracellular free nucleic acids according to the present invention preferably comprises the steps of centrifuging the blood collection container containing collected blood to separate plasma from the blood, and separating extracellular free nucleic acids from the separated plasma. More preferably, the method for separating extracellular free nucleic acids according to the present invention comprises the steps of collecting blood into the blood collection container described above, centrifuging the blood collection container containing collected blood to separate plasma from the blood, and separating extracellular free nucleic acids from the separated plasma.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In the step of separating the extracellular free nucleic acids, the extracellular free nucleic acids can be separated 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 separating the extracellular free nucleic acids from plasma include methods using commercially available nucleic acid purification kits. By using commercially available nucleic acid purification kits, the extracellular free nucleic acids can be easily separated from plasma. Examples of commercially available nucleic acid purification kits include the 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).

[0157] In the step of separating extracellular vesicles, the extracellular vesicles can be separated from the plasma using a conventionally known method.

[0158] 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.

[0159] The following materials were prepared as the plasma separation composition.

[0160] (Organic material that is fluid 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)acrylate polymer having fluidity at 25°C.

[0161] Other resins: Petroleum-based resin (Eastman Chemical Company's "Rigalite S5090") Dicyclopentadiene resin 1 (Colon "Scolez SU500") Dicyclopentadiene resin 2 (Colon "Scolez SU90")

[0162] Organic compounds: Trimellitic acid ester (benzenepolycarboxylic acid alkyl ester derivative, DIC "Monocizer W700")

[0163] (Inorganic fine powder) Hydrophilic silica (fine powder silica, Nippon Aerosil "200CF") Hydrophobic silica (fine powder silica, Nippon Aerosil "R974") Titanium oxide powder (Ishihara Sangyo Kaisha "A-100", specific gravity: 4)

[0164] (Other ingredients) Silicone oil (Toray Dow Corning "SF8410") Organic gelling agent ("Gelall D" manufactured by New Japan Chemical Co., Ltd.) 1-Methyl-2-pyrrolidone (co-solvent)

[0165] Preparation of plasma separation compositions A and B: 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 compositions A and B for plasma separation.

[0166] Preparation of plasma separation composition C: The materials for the organic component having fluidity at 25°C listed in Table 1 were blended, heated to 130°C, dissolved, and mixed to produce an organic component having fluidity at 25°C. Next, the organic component having fluidity at 25°C, inorganic fine powder, and other components were mixed in the blending ratios listed in Table 1 to produce composition C for plasma separation.

[0167] [Table 1]

[0168] A drop of each of the obtained compositions for plasma separation was dropped into saline solutions at 25°C, each having a specific gravity adjusted in increments of 0.002, and the specific gravities were measured by measuring whether the composition floated or sank in the saline solution. The specific gravities of the obtained compositions for plasma separation at 25°C are shown in the table below.

[0169] The following materials were prepared for the aqueous solution:

[0170] (anticoagulant) Ethylenediaminetetraacetic acid dipotassium dihydrate (EDTA2K·2H2O) Lithium heparin

[0171] (Lithium salt A) Lithium chloride monohydrate (molecular weight in anhydrous state: 42) Lithium acetate dihydrate (molecular weight in anhydrous state: 66) Lithium citrate (trilithium citrate tetrahydrate, molecular weight in anhydrous state: 210) Lithium L-lactate (molecular weight in anhydrous state: 96)

[0172] (Water-soluble polymer compound) Polyethylene glycol (number average molecular weight: 3000) Dextran (number average molecular weight: 70,000)

[0173] Sodium chloride potassium acetate Ammonium sulfate Trehalose Propylene glycol

[0174] water

[0175] The following blood collection containers were prepared:

[0176] A PET bottomed tube (polyethylene terephthalate tube) with a length (distance between the open end and closed end) of 100 mm and an inner diameter of 14 mm at the open end.

[0177] Example 1 An aqueous solution was obtained by dissolving the components shown in Table 2 in water. The types and concentrations of the components in the resulting aqueous solution are shown in Table 2.

[0178] 1.2 g of plasma separation composition A was placed in the bottom of the blood collection container body. 1 mL of the obtained 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 mL, and the container was sealed with a butyl rubber stopper. In this way, a blood collection container was prepared.

[0179] (Examples 2 to 12 and Comparative Examples 2 to 6) The type of composition for plasma separation and the composition of the aqueous solution were changed as shown in the table below. Except for these, blood collection containers were produced in the same manner as in Example 1.

[0180] (Comparative Example 1) A mixed solution was obtained by dissolving 24 parts by weight of dipotassium ethylenediaminetetraacetate dihydrate in 76 parts by weight of water. 1.2 g of plasma separation composition A was placed in the bottom of a blood collection container body. 60 mg of the mixed solution was applied to the inner wall surface of the blood collection container body and allowed to dry. The pressure inside the blood collection container was reduced so that the blood collection volume was 8 mL, and the container was sealed with a butyl rubber stopper. In this way, a blood collection container was produced.

[0181] (evaluation) (1) Osmotic pressure of the mixed solution 8 mL of saline was collected in a blood collection container. After collecting the saline, the container was inverted to mix the saline with the aqueous solution contained in the blood collection container (Examples 1 to 12 and Comparative Examples 2 to 6), yielding a mixture. The osmotic pressure of the resulting mixture was measured by the freezing point depression method using an osmometer ("OM-6060" manufactured by Arkray).

[0182] (2) Evaluation on the day blood was collected 8 mL of blood was collected in a blood collection container. After collection, the blood was mixed by inversion with the aqueous solution (Examples 1 to 12 and Comparative Examples 2 to 6) or aqueous component (Comparative Example 1) contained in the blood collection container. 2 mL of the resulting liquid was collected and added to a Spitz tube. The Spitz tube was then centrifuged at 1500 G for 15 minutes. After centrifugation, plasma was collected.

[0183] The DNA contained in the collected plasma was purified using a cfDNA purification kit (QIAGEN's "QIAamp Circulating Nucleic Acid Kit") on the same day the plasma was collected from the blood collection container.

[0184] The DNA concentration in the purified extract was measured using the Qubit dsDNA HS Assay kit (Invitrogen). The cfDNA concentration (cfDNA content per mL of plasma) was calculated using the following formula:

[0185] cfDNA concentration (ng / 1mL plasma)=[A]×[B] / [C]

[0186] [A]: Measured DNA concentration in the purified extract (ng / mL) [B]: Total volume of purified extract (mL) [C]: Volume of plasma used for DNA purification (mL)

[0187] (3) Evaluation of blood collected by centrifugation two days after collection (3-1) Percentage of leukocytes in plasma After collecting 2 mL of the liquid in the above-mentioned "Evaluation on the day of blood collection", the blood collection container was stored at 25°C for 2 days. After the 2-day storage, the blood collection container was centrifuged at 1500G for 15 minutes. After centrifugation, the plasma was located above the partition formed by the plasma separation composition. The plasma located above the partition was stirred by pipetting to suspend the blood cells deposited and remaining on the partition, and then 2 mL of the plasma was collected. The white blood cell count in the collected plasma was measured by analyzing the collected plasma using a multi-item automatic blood cell analyzer ("XE5000" manufactured by Sysmex Corporation). Also, for the prepared blood (whole blood sample), the white blood cell count in the whole blood sample was measured in the same manner.

[0188] Mixing ratio of white blood cells (%) = (number of white blood cells (cells) contained in the separated plasma) / (number of white blood cells (cells) contained in the whole blood sample) × 100

[0189] <Judgment criteria for the mixing ratio of white blood cells into plasma> 〇: Mixing ratio of white blood cells is less than 10% ×: Mixing ratio of white blood cells is 10% or more

[0190] (3-2) Increase amount (I) of cfDNA concentration Also, the plasma collected in the above-mentioned "(3-1) Mixing ratio of white blood cells into plasma" was centrifuged at 1500G for 15 minutes to precipitate the suspended white blood cells. For the supernatant plasma, the cfDNA concentration (content of cfDNA per 1 mL of plasma) was calculated by the method described in the column of the above-mentioned "(2) Evaluation on the day of blood collection" and designated as "cfDNA concentration (B)". Also, the increase amount (I) of cfDNA concentration was calculated by the following formula.

[0191] Increase amount (I) of cfDNA concentration = [cfDNA concentration (B)] - [cfDNA concentration (A)]

[0192] <Judgment criteria for the increase amount (I) of cfDNA concentration> ○○○: Increase amount (I) of cfDNA concentration is less than 10 ng / 1 mL of plasma ○○: Increase amount (1) of cfDNA concentration is 10 ng / plasma 1 mL or more and less than 25 ng / plasma 1 mL 〇: Increase amount (1) of cfDNA concentration is 25 ng / plasma 1 mL or more and less than 50 ng / plasma 1 mL ×: Increase amount (1) of cfDNA concentration is 50 ng / plasma 1 mL or more

[0193] (4) Evaluation when plasma is stored for 5 days (7 days after blood collection) (4-1) Increase amount (2) of cfDNA concentration After collecting 2 mL of plasma at the "Mixing ratio of white blood cells into plasma in (3-1)", the blood collection container was stored at 25°C for 5 days (7 days have passed since blood collection). For the plasma after 5-day storage, the cfDNA concentration (content of cfDNA per 1 mL of plasma) was calculated by the method described in the column of "(2) Evaluation on the day of blood collection", and designated as "cfDNA concentration (C)". Also, the increase amount (2) of cfDNA concentration and the increase amount (3) of cfDNA concentration were calculated by the following formulae.

[0194] Increase amount (2) of cfDNA concentration = [cfDNA concentration (C)] - [cfDNA concentration (B)] Increase amount (3) of cfDNA concentration = [cfDNA concentration (C)] - [cfDNA concentration (A)]

[0195] <Judgment criteria for increase amount (2) of cfDNA concentration> ○○○: Increase amount (2) of cfDNA concentration is less than 10 ng / plasma 1 mL ○○: Increase amount (2) of cfDNA concentration is 10 ng / plasma 1 mL or more and less than 25 ng / plasma 1 mL 〇: Increase amount (2) of cfDNA concentration is 25 ng / plasma 1 mL or more and less than 50 ng / plasma 1 mL ×: Increase amount (2) of cfDNA concentration is 50 ng / plasma 1 mL or more

[0196] <Judgment criteria for increase amount (3) of cfDNA concentration> ○○○: Increase amount (3) of cfDNA concentration is less than 10 ng / plasma 1 mL ○○: Increase in cfDNA concentration (3) is 10 ng / 1 mL or more and less than 25 ng / 1 mL of plasma 〇: Increase in cfDNA concentration (3) is 25ng / 1mL or more and less than 50ng / 1mL of plasma ×: Increase in cfDNA concentration (3) is 50 ng / 1 mL of plasma or more

[0197] The compositions and results are shown in Tables 2 to 13 below. In the tables, the concentrations of the anticoagulants are those of EDTA2K, not EDTA2K·2H2O, and the concentrations of lithium salt A are those in an anhydrous state.

[0198] [Table 2]

[0199] [Table 3]

[0200] [Table 4]

[0201] [Table 5]

[0202] [Table 6]

[0203] [Table 7]

[0204] [Table 8]

[0205] [Table 9]

[0206] [Table 10]

[0207] [Table 11]

[0208] [Table 12]

[0209] [Table 13]

[0210] From the results of "proportion of leukocytes in plasma" in the table above, it can be seen that when the aqueous solution contains an anticoagulant and lithium salt A, the amount of leukocytes in plasma can be reduced even when the blood is centrifuged two days after collection.

[0211] Furthermore, the "increase in cfDNA concentration (1)" increases the more genomic DNA leaks from white blood cells during the two days between blood collection and centrifugation. The results for "increase in cfDNA concentration (1)" indicate that when the aqueous solution contains an anticoagulant and lithium salt A, leakage of genomic DNA from white blood cells is effectively suppressed, even if two days have passed between blood collection and centrifugation.

[0212] Additionally, "increase in cfDNA concentration (2)" indicates the amount of DNA in plasma that increased due to leakage of genomic DNA from leukocytes contaminating the plasma during the five-day storage period of the sample after centrifugation. When the aqueous solution contains an anticoagulant and lithium salt A, the amount of leukocytes contaminating the plasma can be reduced even when the blood is centrifuged two days after collection, which indicates that the contamination of plasma with genomic DNA during subsequent sample storage is suppressed.

[0213] Furthermore, "increase in cfDNA concentration (3)" indicates the increase in cfDNA from the day of blood collection, and is the sum of the genomic DNA leaked from white blood cells during the two days from blood collection to centrifugation, and the genomic DNA leaked from white blood cells mixed into the plasma when the blood was centrifuged two days after collection and during subsequent sample storage. When the aqueous solution contains a specific lithium salt A, these effects are exerted synergistically, and it can be seen that the increase in cfDNA from the day of blood collection is further suppressed. [Explanation of symbols]

[0214] 1...Blood collection container 2...Blood collection container body 2a...Open end 2b...Closed end 3...Composition for plasma separation 4...Aqueous solution 5…Bung body

Claims

1. A blood collection container body; a plasma separator housed in the blood collection container body; an aqueous solution contained in the blood collection container body, the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant; A blood collection container, wherein the content of the lithium salt in 100% by weight of the aqueous solution is 1% by weight or more.

2. 10. The blood collection container of claim 1, wherein the lithium salt comprises lithium chloride, lithium acetate, lithium citrate, or lithium lactate.

3. 3. The blood collection container according to claim 1, wherein the lithium salt has a molecular weight of 100 or less.

4. 3. The blood collection container according to claim 1, wherein the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less.

5. 3. The blood collection container of claim 1, wherein the aqueous solution further comprises ammonium sulfate.

6. 3. The blood collection container of claim 1, wherein the aqueous solution further comprises trehalose.

7. The blood collection container according to claim 1 or 2, wherein the aqueous solution further contains a water-soluble polymer compound.

8. 3. The blood collection container of claim 1, wherein the aqueous solution further comprises propylene glycol.

9. 3. The blood collection container according to claim 1, wherein the plasma separation material is a plasma separation composition.

10. The plasma separation composition comprises an organic component having fluidity at 25°C and an inorganic fine powder, the organic component comprises a resin; 10. The blood collection container of claim 9, wherein the fine inorganic powder comprises finely powdered silica.

11. 11. The blood collection container of claim 10, wherein the finely divided silica comprises hydrophilic silica and hydrophobic silica.

12. The blood collection container according to claim 10 , wherein the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic-based resin.

13. The blood collection container according to claim 1 or 2, which is used to separate extracellular free nucleic acids or extracellular vesicles in blood.

14. collecting blood in the blood collection container according to claim 1 or 2; and centrifuging the blood collection container in which the blood has been collected.

15. collecting blood in the blood collection container according to claim 1 or 2; 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.

16. collecting blood in the blood collection container according to claim 1 or 2; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; A method for separating extracellular vesicles, comprising a step of separating extracellular vesicles from the separated plasma.

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