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

Abstract

Provided is a blood collection container which makes it possible to suppress the introduction of leukocyte-derived DNA into plasma during the storage of a specimen.　The blood collection container according to the present invention comprises: a blood collection container body; a plasma separating material accommodated in the blood collection container body; and an aqueous solution accommodated in the blood collection container body, wherein the specific gravity of the plasma separating material at 25 °C is 1.027-1.060, and the aqueous solution contains an anticoagulant and also contains a water-soluble polymer compound or ammonium sulfate having a number average molecular weight of 500-180,000.

Description

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

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

[0002] In clinical testing, blood collection containers such as blood collection tubes are widely used to collect blood. After blood is collected 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 including 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).

[0003] WO2010 / 053180A1 WO2010 / 132783A1

[0004] When plasma is separated from blood using a blood collection container containing a plasma separator, the separated plasma may contain leukocytes.

[0005] The separated plasma is stored for a certain period of time without freezing until it is used for testing. In conventional blood collection containers such as those described in Patent Documents 1 and 2, white blood cells contaminating the plasma are destroyed over time due to death or other reasons, and DNA in the white blood cells may leak into the plasma. If DNA in the white blood cells leaks into the plasma, it may affect the test results.

[0006] For example, in a test for detecting extracellular free nucleic acids (e.g., cell-free DNA) in plasma, test results vary greatly due to DNA leaked from white blood cells.

[0007] An object of the present invention is to provide a blood collection container that can prevent leukocyte-derived DNA from being mixed into plasma during sample storage. Another object of the present invention is to provide a method for separating plasma, a method for separating extracellular free nucleic acids, and a method for separating extracellular vesicles using the blood collection container.

[0008] According to a broad aspect of the present invention, there is provided 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 specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less, and the aqueous solution contains an anticoagulant, and also contains a water-soluble polymer compound or ammonium sulfate having a number average molecular weight of 500 or more and 180,000 or less.

[0009] In a specific aspect of the blood collection container according to the present invention, the aqueous solution contains the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less.

[0010] In a specific aspect of the blood collection container according to the present invention, the aqueous solution contains ammonium sulfate.

[0011] In a specific aspect of the blood collection container according to the present invention, the aqueous solution contains the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less, and the ammonium sulfate.

[0012] In a specific aspect of the blood collection container according to the present invention, the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less is dextran, polyethylene glycol, or polyvinylpyrrolidone.

[0013] In a specific aspect of the blood collection container according to the present invention, the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less is a water-soluble polymer compound having a number average molecular weight of 2,000 or more and 150,000 or less.

[0014] In a specific aspect of the blood collection container according to the present invention, the aqueous solution further contains trehalose.

[0015] In a specific aspect of the blood collection container according to the present invention, the aqueous solution further contains glucose, adenine, or inositol.

[0016] In a specific aspect of the blood collection container according to the present invention, the aqueous solution further contains an apoptosis inhibitor.

[0017] In a specific aspect of the blood collection container according to the present invention, the blood collection container is a blood collection container for collecting a predetermined amount of blood, and when an amount 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 mixed solution is obtained by mixing the saline and the aqueous solution, the osmotic pressure of the mixed solution is 300 mOsm / L or more.

[0018] In a specific aspect of the blood collection container according to the present invention, the aqueous solution further contains propylene glycol.

[0019] In a specific aspect of the blood collection container according to the present invention, the plasma separation material is a composition for plasma separation.

[0020] In a specific aspect of the blood collection container according to the present invention, 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.

[0021] In a specific aspect of the blood collection container according to the present invention, the finely powdered silica includes hydrophilic silica.

[0022] In a specific aspect of the blood collection container according to the present invention, the content of the hydrophilic silica in 100% by weight of the plasma separation composition is 0.01% by weight or more and 2.50% by weight or less.

[0023] In a specific aspect of the blood collection container according to the present invention, the finely powdered silica includes hydrophilic silica and hydrophobic silica.

[0024] In a specific aspect of the blood collection container according to the present invention, the specific gravity of the plasma separation composition at 25°C is 1.050 or more, and the inorganic fine powder contains an inorganic fine powder having a higher specific gravity than the fine silica powder.

[0025] In a specific aspect of the blood collection container according to the present invention, the resin includes a petroleum resin, a cyclopentadiene-based resin, a polyester resin, or a (meth)acrylic resin.

[0026] In a particular aspect of the blood collection container according to the present invention, the blood collection container is used to separate extracellular free nucleic acids or extracellular vesicles in blood.

[0027] According to a broad aspect of the present invention, there is provided a method for separating plasma, comprising the steps of collecting blood in the blood collection container described above and centrifuging the blood collection container with the collected blood.

[0028] According to a broad aspect of the present invention, there is provided a method for separating extracellular free nucleic acids, comprising the steps of: collecting blood in the above-described blood collection container; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; and separating extracellular free nucleic acids from the separated plasma.

[0029] According to a broad aspect of the present invention, there is provided a method for separating extracellular vesicles, comprising the steps of collecting blood in the above-described 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.

[0030] 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, wherein the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less. In the blood collection container according to the present invention, the aqueous solution contains an anticoagulant, and also contains a water-soluble polymer compound or ammonium sulfate having a number average molecular weight of 500 or more and 180,000 or less. Because the blood collection container according to the present invention has the above configuration, it is possible to prevent leukocyte-derived DNA from being mixed into plasma during sample storage.

[0031] FIG. 1 is a front cross-sectional view showing a schematic diagram of a blood collection container according to one embodiment of the present invention.

[0032] The present invention will be described in detail below.

[0033] 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 specific gravity of the plasma separation material at 25° C. is 1.027 or more and 1.060 or less. In the blood collection container according to the present invention, the aqueous solution contains an anticoagulant, and also contains a water-soluble polymer compound or ammonium sulfate having a number average molecular weight of 500 or more and 180,000 or less.

[0034] The blood collection container according to the present invention has the above-described configuration, and therefore can prevent leukocyte-derived DNA from being mixed into plasma during sample storage.

[0035] When blood is collected in the blood collection container of the present invention, the blood and the aqueous solution are mixed. The inventors have found that the specific water-soluble polymer compound or ammonium sulfate contained in the aqueous solution can suppress the contamination of leukocytes with separated plasma. They have also found that the specific water-soluble polymer compound or ammonium sulfate contained in the aqueous solution can stabilize leukocytes contaminating separated plasma. Therefore, the blood collection container of the present invention effectively suppresses DNA leakage from leukocytes due to destruction of leukocytes over time, thereby suppressing an increase in the amount of DNA in plasma during sample storage. Furthermore, the blood collection container of the present invention effectively suppresses destruction of leukocytes contaminating plasma over time, thereby effectively suppressing leakage of contents such as proteins from the leukocytes.

[0036] 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".

[0037] (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.

[0038] From the viewpoint of separating plasma from blood well, the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less.

[0039] The specific gravity of the plasma separation material at 25° C. is preferably 1.029 or more, more preferably 1.030 or more, even more preferably 1.032 or more, and preferably 1.055 or less, more preferably 1.050 or less. When the specific gravity of the plasma separation material at 25° C. is equal to or more than the above lower limit and equal to or less 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.

[0040] 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.

[0041] <Plasma Separation Composition> The plasma separation composition is a composition that migrates between the plasma layer and the blood cell layer during centrifugation to form a partition. The plasma separation composition is also used for the purpose of preventing component migration between the plasma layer and the blood cell layer after centrifugation. The plasma separation composition preferably has thixotropy. The plasma separation composition may be contained in the bottom of the blood collection container body or may be disposed on the inner wall surface. From the viewpoint of more effectively exerting the effects of the present invention, the plasma separation composition is preferably contained in the bottom of the blood collection container body.

[0042] As the plasma separation composition, a conventionally known plasma separation composition can be used.

[0043] The composition for plasma separation preferably contains an organic component having fluidity at 25° C. and an inorganic fine powder. 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.

[0044] Organic component having fluidity at 25°C: The phrase "having fluidity at 25°C" means that the viscosity at 25°C is 500 Pa·s or less.

[0045] 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 by centrifugation can be increased.

[0046] 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.

[0047] Examples of the organic component include a resin and a mixture of a resin and an organic compound such as a plasticizer. Therefore, the organic component preferably contains the resin, and more preferably contains the resin and the organic compound. When the organic component is a mixture of the resin and the organic compound, it is sufficient that the mixture (the organic component) has fluidity, and the resin or the organic compound does not have to have fluidity. When the organic component is a mixture of the resin and the organic compound, the resin may be, for example, a resin that is solid at 25°C. Only one type of the resin and the organic compound may be used, or two or more types may be used in combination.

[0048] Examples of the resin include petroleum resins, cyclopentadiene resins, polyester resins, polyurethane resins, (meth)acrylic resins, silicone resins, α-olefin-fumaric acid ester copolymers, copolymers of sebacic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane resins, polyether polyester resins, etc. Only one of the resins may be used, or two or more of them may be used in combination.

[0049] The resin preferably includes a petroleum resin, a cyclopentadiene-based resin, a polyester resin, or a (meth)acrylic resin.

[0050] Commercially available petroleum resins include "Regalite S5090" manufactured by Eastman Chemical Company.

[0051] 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.

[0052] Examples of the cyclopentadiene-based monomer include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0053] Examples of the aromatic monomer include styrene, methylstyrene, indene, and methylindene.

[0054] Commercially available dicyclopentadiene resins include "Scoretz SU500" and "Scoretz SU90" manufactured by Colon Co., Ltd.

[0055] 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.

[0056] Examples of the polyurethane resin include a reaction product of a polyol compound and an isocyanate compound.

[0057] 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 the (meth)acrylic acid ester monomer.

[0058] Examples of the (meth)acrylic acid ester monomer include (meth)acrylic acid alkyl esters, (meth)acrylic acid polyalkylene glycol esters, (meth)acrylic acid alkoxyalkyl esters, (meth)acrylic acid hydroxyalkyl esters, (meth)acrylic acid glycidyl esters, (meth)acrylic acid dialkylaminoalkyl esters, (meth)acrylic acid benzyl esters, (meth)acrylic acid phenoxyalkyl esters, (meth)acrylic acid cyclohexyl esters, (meth)acrylic acid isobornyl esters, and (meth)acrylic acid alkoxysilylalkyl esters. When the (meth)acrylic acid ester monomer has an alkyl group, the number of carbon atoms in the alkyl group is preferably 1 or more and preferably 20 or less. The (meth)acrylic acid alkyl ester is preferably a (meth)acrylic acid alkyl ester having an alkyl group having 1 to 20 carbon atoms. The (meth)acrylic acid ester monomer may be used alone or in combination of two or more.

[0059] 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.

[0060] 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.

[0061] Examples of the trimellitic acid ester include trimellitic acid tri-n-octyl, triisooctyl, and triisodecyl trimellitate.

[0062] Examples of the pyromellitic acid ester include tetraisooctyl pyromellitic acid.

[0063] Commercially available trimellitic acid esters include "Monocizer W700" and "Monocizer W-750" manufactured by DIC Corporation, and "Sansocizer TOTM" and "Sansocizer TITM" manufactured by New Japan Chemical Co., Ltd.

[0064] Commercially available pyromellitic acid esters include "Monocizer W-7010" manufactured by DIC Corporation.

[0065] The benzenepolycarboxylic acid alkyl ester derivative is preferably a phthalate ester, a trimellitate ester, or a pyromellitate ester, and more preferably a trimellitate ester.

[0066] The content of the organic component in 100% by weight of the plasma separation composition is preferably 80% 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.

[0067] Inorganic fine powder: Examples of the inorganic fine powder include fine silica powder, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, and zirconium powder.

[0068] 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. To obtain 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 necessarily contain the second inorganic fine powder. Furthermore, 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. The inorganic fine powder, the finely powdered silica, and the second inorganic fine powder may each be used alone or in combination of two or more.

[0069] 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.

[0070] 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.

[0071] The second inorganic fine powder is preferably an inorganic fine powder having a specific gravity greater than that of 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.

[0072] 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.

[0073] 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.

[0074] The average particle diameters of the inorganic fine powder, the fine silica, and the second inorganic fine powder are average diameters measured on a volume basis, 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 measurement using laser diffraction / scattering method or image analysis method.

[0075] 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.

[0076] The specific surface area of ​​the finely powdered silica is measured by the BET method.

[0077] 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.

[0078] 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, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the finely powdered silica is equal to or more than the lower limit and equal to or less than the upper limit, both the specific gravity and thixotropy of the plasma separation composition can be maintained within more suitable ranges.

[0079] The content of the second inorganic fine powder in 100% by weight of the plasma separation composition is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the second inorganic fine powder is equal to or more than the lower limit and equal to or less than the upper limit, the specific gravity of the plasma separation composition can be effectively increased.

[0080] The content of the inorganic fine powder in 100% by weight of the plasma separation composition is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the inorganic fine powder is equal to or more than the lower limit and equal to or less than the upper limit, the specific gravity of the plasma separation composition can be effectively increased.

[0081] Other Components: The plasma separation composition may contain other components in addition to the components described above, as long as the effects of the present invention are not impaired. Examples of the other components include organic gelling agents, thermoplastic elastomers, polyalkylene glycols, silicone oils, cosolvents, antioxidants, colorants, and water. Each of the other components may be used alone or in combination of two or more.

[0082] 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, 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 lower limit and below the above 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 lower limit and below the above 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.

[0083] 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 saline solutions at 25°C whose specific gravities have been adjusted in steps of 0.002, and observing the floating and sinking of the composition in the saline solution.

[0084] 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.

[0085] The viscosity of the composition for plasma separation at 25°C was measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 sec. －１ It is measured under the following conditions.

[0086] <Plasma Separation Device> The plasma separation device is a device that moves between the plasma layer and the blood cell layer during centrifugation to form a partition wall. The plasma separation device is used for the purpose of preventing component migration between the plasma layer and the blood cell layer.

[0087] The plasma separation device may be a conventionally known plasma separation device, such as the mechanical separator (plasma separation device) described in WO 2010 / 132783 A1.

[0088] Examples of materials for the plasma separation device include elastomers.

[0089] (Aqueous Solution) The blood collection container includes an aqueous solution accommodated in the blood collection container body. The aqueous solution preferably contains an anticoagulant and water. The aqueous solution contains a water-soluble polymer compound having a number-average molecular weight of 500 to 180,000 (hereinafter, sometimes referred to as water-soluble polymer compound (X)) or ammonium sulfate. The solute contained in the aqueous solution includes an anticoagulant. The solute contained in the aqueous solution includes the water-soluble polymer compound (X) or ammonium sulfate. The aqueous solution may contain the water-soluble polymer compound (X) and ammonium sulfate.

[0090] <Anticoagulant> The aqueous solution contains an anticoagulant. A conventionally known anticoagulant can be used as the anticoagulant. Only one type of anticoagulant may be used, or two or more types may be used in combination.

[0091] Examples of the anticoagulant include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, citric acid, and sodium citrate.

[0092] 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 sodium citrate.

[0093] 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 not less than the above lower limit and not more than the above upper limit, the anticoagulant can exhibit good anticoagulant performance.

[0094] <Water-soluble polymer compound (water-soluble polymer compound (X)) having a number-average molecular weight of 500 to 180,000> The aqueous solution preferably contains a water-soluble polymer compound (water-soluble polymer compound (X)) having a number-average molecular weight of 500 to 180,000 (500 to 180,000). The reason why the use of the water-soluble polymer compound (X) can suppress the contamination of separated plasma with leukocytes is presumed to be, but not limited to, that the water-soluble polymer compound (X) maintains a good viscosity of the blood, thereby allowing the leukocytes to move more smoothly during centrifugation. The reason why the use of the water-soluble polymer compound (X) can suppress the leakage of DNA from leukocytes contaminating plasma is presumed to be, but not limited to, that the water-soluble polymer compound (X) effectively protects the cell membranes of leukocytes, thereby stabilizing the leukocytes. Note that "water solubility" in the water-soluble polymer compound (X) means that 0.1 g or more of the compound dissolves in 100 g of water at 25°C. The water-soluble polymer compound (X) may be used alone or in combination of two or more kinds.

[0095] In order to exert the effects of the present invention, the number average molecular weight of the water-soluble polymer compound (X) is 500 or more and 180,000 or less.

[0096] The number-average molecular weight of the water-soluble polymer compound (X) is preferably 1,000 or more, more preferably 1,500 or more, even more preferably 2,000 or more, particularly preferably 2,500 or more, and preferably 170,000 or less, 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 (X) is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, contamination of the separated plasma with leukocytes can be further suppressed, and leukocytes can be further stabilized, allowing the effects of the present invention to be exerted more effectively.

[0097] The number average molecular weight of the water-soluble polymer compound (X) is a number average molecular weight calculated as a standard polyethylene glycol, measured by gel permeation chromatography (GPC).

[0098] Examples of the water-soluble polymer compound (X) include dextran, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, and corn starch.

[0099] The water-soluble polymer compound (X) is preferably dextran, polyethylene glycol, or polyvinylpyrrolidone, which can further stabilize leukocytes and more effectively exert the effects of the present invention.

[0100] The content of each water-soluble polymer compound (X) in 100% by weight of the aqueous solution 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 15% by weight or less, more preferably 10% by weight or less, and even more preferably 7% by weight or less. When the content of each water-soluble polymer compound (X) is above the above-mentioned lower limit and below the above-mentioned upper limit, leukocytes can be further stabilized, and the effects of the present invention can be more effectively exhibited. Furthermore, when the content of each water-soluble polymer compound (X) is above the above-mentioned lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood and the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes mixed into the plasma. Note that the "content of each water-soluble polymer compound (X)" refers to the content of the water-soluble polymer compound (X) when the aqueous solution contains one type of water-soluble polymer compound (X). When the aqueous solution contains two or more types of water-soluble polymer compounds (X), it refers to the content of each water-soluble polymer compound (X).

[0101] The content of the water-soluble polymer compound (X) in 100% by weight of the aqueous solution 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 (X) 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 further stabilized, and the effects of the present invention can be more effectively exhibited. Furthermore, when the content of the water-soluble polymer compound (X) is equal to or greater than the above-mentioned lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood with the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes mixed into the plasma.

[0102] <Ammonium sulfate> The aqueous solution preferably contains ammonium sulfate. The present inventors have found that the use of ammonium sulfate can further suppress the contamination of separated plasma with leukocytes and also suppress the leakage of DNA from leukocytes contaminating the plasma. When the aqueous solution contains a water-soluble polymer compound (X) and ammonium sulfate, the effects of the present invention are even more effectively exhibited.

[0103] 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 4% 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 ammonium sulfate is above the lower limit and below the upper limit, contamination of leukocytes into the separated plasma can be further suppressed, and leukocytes can be further stabilized, allowing the effects of the present invention to be more effectively exerted. Furthermore, when the content of ammonium sulfate is above the lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood with the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes contaminating the plasma.

[0104] <Trehalose> From the viewpoint of more effectively exerting the effects of the present invention, the aqueous solution preferably contains trehalose. The solute contained in the aqueous solution preferably contains trehalose. By using trehalose, the cell membranes of leukocytes can be effectively protected and the leukocytes can be stabilized, thereby further suppressing leakage of DNA from leukocytes mixed into plasma.

[0105] 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 above the above lower limit and below the above upper limit, leukocytes can be further stabilized, and the effects of the present invention can be more effectively exerted. Furthermore, when the trehalose content is above the above lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood and the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes contaminating the plasma.

[0106] <Glucose, adenine, inositol> From the viewpoint of more effectively exerting the effects of the present invention, the aqueous solution preferably contains glucose, adenine, or inositol. The solute contained in the aqueous solution preferably contains glucose, adenine, or inositol. In this case, the aqueous solution may contain only one of glucose, adenine, and inositol, or may contain two or more of them. By using these compounds, a nutrient source is supplied to leukocytes, thereby increasing the survival rate of leukocytes and, as a result, further suppressing leakage of DNA from leukocytes mixed in plasma.

[0107] When the aqueous solution contains glucose, the glucose content is preferably 0.1 wt % or more, more preferably 0.3 wt % or more, preferably 10 wt % or less, and more preferably 5 wt % or less, based on 100 wt % of the aqueous solution. When the glucose content 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. Furthermore, when the glucose content is equal to or greater than the lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood and the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes mixed into the plasma.

[0108] When the aqueous solution contains adenine, the adenine content is preferably 0.01 wt % or more, more preferably 0.05 wt % or more, and preferably 2 wt % or less, more preferably 1 wt % or less, based on 100 wt % of the aqueous solution. When the adenine content 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. Furthermore, when the adenine content is equal to or greater than the lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood and the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes mixed into the plasma.

[0109] When the aqueous solution contains inositol, the content of inositol in 100% by weight of the aqueous solution is preferably 0.01% by weight or more, more preferably 0.05% by weight or more, preferably 2% by weight or less, and more preferably 1% by weight or less. When the inositol content is equal to or greater 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. Furthermore, when the inositol content is equal to or greater than the above lower limit, the osmotic pressure of the mixture obtained by mixing the collected blood and the aqueous solution can be effectively increased, thereby further reducing the amount of leukocytes mixed into the plasma.

[0110] <Apoptosis inhibitor> From the viewpoint of more effectively exerting the effects of the present invention, the aqueous solution preferably contains an apoptosis inhibitor. The solute contained in the aqueous solution preferably contains an apoptosis inhibitor. By using an apoptosis inhibitor, cell death of leukocytes during sample storage can be effectively suppressed, and as a result, leakage of DNA from leukocytes contaminating the plasma can be further suppressed. The apoptosis inhibitor may be used alone or in combination of two or more types.

[0111] Examples of the apoptosis inhibitor include Q-VD-Oph and Z-VAD-FMK.

[0112] From the viewpoint of more effectively exerting the effects of the present invention, the apoptosis inhibitor is preferably a caspase inhibitor, more preferably Q-VD-Oph, and the aqueous solution preferably contains a caspase inhibitor, more preferably Q-VD-Oph.

[0113] The concentration of the apoptosis inhibitor in the aqueous solution is preferably 1 nM or more, more preferably 20 nM or more, and preferably 10 μM or less, more preferably 5 μM or less. When the concentration of the apoptosis inhibitor 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.

[0114] <Propylene Glycol> From the viewpoint of more effectively exhibiting the effects of the present invention, the aqueous solution preferably contains propylene glycol. The solute contained in the aqueous solution preferably contains propylene glycol.

[0115] 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.

[0116] <Other Components> The aqueous solution may contain components other than the above-mentioned components (anticoagulant, water-soluble polymer compound (X), ammonium sulfate, trehalose, glucose, adenine, inositol, apoptosis inhibitor, and propylene glycol).

[0117] Examples of the other components include inorganic salts, sugars, sugar alcohols, etc. The other components may be used alone or in combination of two or more.

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

[0119] Examples of the sugars include dihydroxyacetone, fructose, galactose, sucrose, maltose, lactulose, and polysaccharides that do not correspond to the water-soluble polymer compound (X).

[0120] Examples of the sugar alcohol include D-mannitol and D-sorbitol.

[0121] The amount of aqueous solution contained in the blood collection container body can be varied as appropriate depending on the size of the blood collection container body, the amount of blood to be collected, etc. The amount of aqueous solution contained in the blood collection container body is preferably 0.1 mL or more, more preferably 0.5 mL or more, even more preferably 0.7 mL or more, and preferably 5 mL or less, more preferably 3 mL or less, and even more preferably 2.5 mL or less. When the amount of aqueous solution is above the above-mentioned lower limit and below the above-mentioned upper limit, the blood is not excessively diluted, and the effects of the present invention can be more effectively achieved.

[0122] (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.

[0123] 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.

[0124] (Stopper) The blood collection container preferably includes a stopper. The stopper is preferably attached to the opening 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 opening 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.

[0125] Examples of the stopper include a stopper having a shape that fits into the opening of the blood collection container body, a sheet-like sealing stopper, and the like.

[0126] 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 opening of the blood collection container body after blood collection.

[0127] 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.

[0128] (Other Details of the Blood Collection Container) The blood collection container is a blood collection container into which a predetermined amount of blood is collected. 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.

[0129] 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, resulting in a mixture of the saline solution and the aqueous solution. 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 other means to obtain a mixture. The osmotic pressure of the mixture of the saline solution and the aqueous solution is preferably 300 mOsm / L or higher, more preferably 330 mOsm / L or higher, even more preferably 350 mOsm / L or higher, preferably 1300 mOsm / L or lower, more preferably 1000 mOsm / L or lower, and even more preferably 800 mOsm / L or lower. If the osmotic pressure of the mixture is above the lower limit, the blood and the aqueous solution will mix when collected in the blood collection container, increasing the osmotic pressure of the blood. As a result, the water content in the white blood cells and the water content in the red blood cells move out of the blood cells, increasing the specific gravity of the white blood cells and the red blood cells. The white blood cells and the red blood cells with increased specific gravity move smoothly below the plasma separation material when the blood collection container is centrifuged. As a result, contamination of the white blood cells and the red blood cells with the plasma can be effectively suppressed. Furthermore, when the osmotic pressure of the mixed solution is above the lower limit and below the upper limit, excessive stress on the white blood cells is suppressed, and the white blood cells can be further stabilized, thereby more effectively achieving the effects of the present invention.

[0130] 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).

[0131] In the blood collection container, preferably 3 mL or more, more preferably 4 mL or more, preferably 11 mL or less, and more preferably 10 mL or less of blood is collected per mL of the aqueous solution contained therein. In this case, the blood is not excessively diluted, and the effects of the present invention can be more effectively exhibited.

[0132] The blood collection container is preferably a blood collection tube, and the blood collection container body is preferably a blood collection tube body.

[0133] The blood collection container is preferably used for separating plasma from blood, and is also preferably used for separating extracellular free nucleic acids or extracellular vesicles in blood, and is preferably used for isolating extracellular free nucleic acids or extracellular vesicles in blood.

[0134] The blood collection container can be manufactured, for example, as follows.

[0135] An anticoagulant and a water-soluble polymer compound (X) or ammonium sulfate are dissolved in water to obtain an aqueous solution. If necessary, other components (trehalose, glucose, adenine, inositol, an apoptosis inhibitor, propylene glycol, or other components described above) 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.

[0136] FIG. 1 is a front cross-sectional view showing a schematic diagram of a blood collection container according to one embodiment of the present invention.

[0137] The blood collection container 1 shown in Figure 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 opening at one end and a closed bottom at the other end. The plasma separation composition 3 is contained in the bottom of the blood collection container body 2. The aqueous solution 4 contains an anticoagulant, and also contains a water-soluble polymer compound (X) or ammonium sulfate. The stopper 5 is inserted into the opening of the blood collection container body 2.

[0138] The aqueous solution 4 is disposed on the surface of the plasma separation composition 3, more specifically, on the upper surface (surface on one end side) of the plasma separation composition 3. 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.

[0139] 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.

[0140] 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. When used as a vacuum blood collection tube, a fixed amount of blood can be easily collected regardless of the skill level of the blood collection person.

[0141] 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.

[0142] (Plasma Separation Method) Plasma can be separated from blood using the above-mentioned blood collection container. The plasma separation method according to the present invention includes the steps of collecting blood in the above-mentioned blood collection container and centrifuging the blood collection container containing the collected blood.

[0143] 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.

[0144] 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.

[0145] (Method for separating extracellular free nucleic acids and method for separating extracellular vesicles) The method for separating extracellular free nucleic acids according to the present invention comprises the steps of collecting blood in the blood collection container described above, centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood, and separating extracellular free nucleic acids from the separated plasma.

[0146] The method for separating extracellular vesicles according to the present invention comprises the steps of collecting blood in the blood collection container described above, centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.

[0147] 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.

[0148] 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.

[0149] In the step of isolating extracellular free nucleic acids, the extracellular free nucleic acids can be isolated from plasma using a conventionally known method. Examples of the extracellular free nucleic acids include cell-free DNA (cfDNA) and cell-free RNA (cfRNA). Examples of methods for isolating extracellular free nucleic acids from plasma include methods using commercially available nucleic acid purification kits. By using commercially available nucleic acid purification kits, extracellular free nucleic acids can be easily isolated from plasma. Examples of commercially available nucleic acid purification kits include QIAamp Circulating Nucleic Acid Kit (manufactured by QIAGEN), QIAamp MinElute ccfDNA Kits (manufactured by QIAGEN), and MagMAX Cell-Free DNA Isolation Kit (manufactured by Applied biosystems).

[0150] In the step of separating extracellular vesicles, the extracellular vesicles can be separated from the plasma using a conventionally known method.

[0151] 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.

[0152] The following materials were prepared as the plasma separation composition.

[0153] (Organic component materials having fluidity at 25°C) (Meth)acrylic resin 1: 2-Ethylhexyl acrylate and butyl acrylate were radically polymerized by solution polymerization in the presence of an azo polymerization initiator to obtain a (meth)acrylic acid ester polymer ((meth)acrylic resin 1) having fluidity at 25°C. The specific gravity of (meth)acrylic resin 1 at 25°C was 1.034.

[0154] (Meth)acrylic resin 2: A (meth)acrylic acid ester polymer ((meth)acrylic resin 2) having fluidity at 25°C was obtained in the same manner as in the preparation of (meth)acrylic resin 1, except that the compounding ratio of 2-ethylhexyl acrylate and butyl acrylate was changed. The specific gravity of (meth)acrylic resin 2 at 25°C was 1.015.

[0155] Other resins: Petroleum resin (Eastman Chemical Company's "Rigalite S5090") Dicyclopentadiene resin 1 (Colon Co.'s "Scoretz SU500") Dicyclopentadiene resin 2 (Colon Co.'s "Scoretz SU90")

[0156] Organic compound: Trimellitic acid ester (benzenepolycarboxylic acid alkyl ester derivative, "Monocizer W700" manufactured by DIC Corporation)

[0157] (Inorganic fine powder) Hydrophilic silica (fine powder silica, "200CF" manufactured by Nippon Aerosil Co., Ltd.) Hydrophobic silica (fine powder silica, "R974" manufactured by Nippon Aerosil Co., Ltd.) Titanium oxide powder ("A-100" manufactured by Ishihara Sangyo Kaisha, Ltd.)

[0158] (Other ingredients) Silicone oil ("SF8410" manufactured by Toray Dow Corning Co., Ltd.) Organic gelling agent ("Gelall D" manufactured by New Japan Chemical Co., Ltd.) 1-methyl-2-pyrrolidone (auxiliary solvent)

[0159] Preparation of compositions A, B, and D for plasma separation: Organic components having fluidity at 25°C, inorganic fine powder, and other components were mixed in the proportions shown in Table 1 to prepare compositions A, B, and D for plasma separation.

[0160] Preparation of composition C for plasma separation: The organic component materials having fluidity at 25° C. listed in Table 1 were blended, heated to 130° C. to dissolve, and mixed to prepare 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 prepare composition C for plasma separation.

[0161]

[0162] One drop of each of the obtained compositions for plasma separation was dropped successively 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 Tables 2 to 5.

[0163] The following solutes were prepared for the aqueous solutions:

[0164] (Anticoagulant) Dipotassium ethylenediaminetetraacetate dihydrate (EDTA2K.2H ２ O)

[0165] (Water-soluble polymer compound (X)) Dextran (number average molecular weight: 40,000) Dextran (number average molecular weight: 70,000) Polyethylene glycol (number average molecular weight: 3,000) Polyethylene glycol (number average molecular weight: 20,000) Polyvinylpyrrolidone (number average molecular weight: 10,000) Polyvinylpyrrolidone (number average molecular weight: 58,000)

[0166] (Water-soluble polymer compounds not corresponding to the water-soluble polymer compound (X)) Dextran (number average molecular weight: 200,000) Polyethylene glycol (number average molecular weight: 500,000)

[0167] Ammonium sulfate

[0168] Trehalose

[0169] Glucose Adenine myo-inositol

[0170] Apoptosis inhibitor (Q-VD-Oph)

[0171] Propylene glycol

[0172] (Other ingredients) Sodium chloride (osmolality adjuster)

[0173] The following blood collection containers were prepared:

[0174] A PET tube with a bottom (polyethylene terephthalate tube) having a length of 100 mm and an inner diameter of 14 mm at the opening

[0175] 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 obtained aqueous solution are shown in Table 2.

[0176] 1.2 g of plasma separation composition A was placed in the bottom of the blood collection container body. 1 mL of the resulting aqueous solution was added to the surface of plasma separation composition A. The pressure inside the blood collection container was reduced so that the blood collection volume was 8 mL, and the container was sealed with a butyl rubber stopper. In this way, a blood collection container was prepared.

[0177] (Examples 2 to 12 and Comparative Examples 2 and 3) The type of plasma separation composition and the composition of the aqueous solution were changed as shown in Tables 2 to 5. Other than these, blood collection containers were produced in the same manner as in Example 1.

[0178] Comparative Example 1: 24 parts by weight of anticoagulant was dissolved in 76 parts by weight of water to obtain a mixed solution. 1.2 g of plasma separation composition A was placed in the bottom of a blood collection container body. 60 mg of the resulting 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 manner, a blood collection container was produced.

[0179] (Evaluation) (1) Osmotic Pressure of Mixed Solution 8 mL of saline was collected into the obtained blood collection container. After collecting the saline, the container was inverted to mix the saline with the aqueous solution (Examples 1 to 12 and Comparative Examples 2 and 3) or water-soluble component (Comparative Example 1) contained in the blood collection container, thereby obtaining a mixed solution. The osmotic pressure of the obtained mixed solution was measured by the freezing point depression method using an osmometer ("OM-6060" manufactured by Arkray, Inc.).

[0180] (2) Residual White Blood Cell Quantity Blood was prepared from three individuals, and the following procedure was performed on each. One blood collection container was prepared, and 8 mL of blood was collected into each. After collection, the blood was mixed by inversion to mix the blood with the aqueous solution (Examples 1 to 12 and Comparative Examples 2 and 3) or aqueous component (Comparative Example 1) contained in the blood collection container. The blood collection container was then centrifuged at 1500 G 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, and the remaining blood cells deposited on the partition were suspended, and the plasma was then recovered. The recovered plasma was analyzed using a multi-parameter automated blood cell analyzer (Sysmex Corporation, "XE5000") to measure the white blood cell count in the plasma. The white blood cell count in the prepared blood (whole blood sample) was also measured in the same manner. The white blood cell count is the average value of the results obtained by evaluating the blood of three people. The residual rate of white blood cells was calculated using the following formula.

[0181] Leukocyte survival rate (%) = (number of leukocytes (cells) contained in separated plasma) / (number of leukocytes (cells) contained in whole blood sample) × 100

[0182] <Criteria for determining residual white blood cell volume> ◯: residual white blood cell rate is less than 10% ×: residual white blood cell rate is 10% or more

[0183] (3) Amount of cfDNA recovered Blood was prepared from three individuals, and the following procedure was performed on each. Two blood collection containers were prepared, and 8 mL of blood was collected into each. After collecting the blood, the blood was mixed by inversion to mix with the aqueous solution (Examples 1 to 12 and Comparative Examples 2 and 3) or water-soluble component (Comparative Example 1) contained in the blood collection container. The blood collection container was then centrifuged at 1500 G for 15 minutes. After centrifugation, the plasma was located above the partition formed by the plasma separation composition.

[0184] For one of the two blood collection containers after plasma separation, plasma was collected from the blood collection container on the same day as the blood was collected. For the remaining one of the two blood collection containers after plasma separation, the blood collection container with the plasma separated was stored at room temperature (25°C) for 7 days, after which the plasma was collected from the blood collection container.

[0185] DNA contained in the collected plasma was purified using a cfDNA purification kit (QIAGEN "QIAamp Circulating Nucleic Acid Kit"). The DNA purification procedure was performed on the day the plasma was collected from the blood collection container.

[0186] The DNA concentration in the purified extract was measured using a Qubit dsDNA HS Assay kit (Invitrogen). Then, the cfDNA concentration (the amount of cfDNA contained per mL of plasma) was calculated according to the following formula.

[0187] cfDNA concentration (ng / 1 mL of plasma) = [A] x [B] / [C]

[0188] [A]: Measured DNA concentration in the extract after purification (ng / mL) [B]: Total volume of the extract after purification (mL) [C]: Volume of plasma used for DNA purification (mL)

[0189] The average concentration of cfDNA recovered from plasma on the day of blood collection was designated as the "cfDNA concentration (day of collection)," and the average concentration of cfDNA recovered from plasma after 7 days of storage was designated as the "cfDNA concentration (storage for 7 days)." The difference between the cfDNA concentration (storage for 7 days) and the cfDNA concentration (day of collection) was designated as the "increase in cfDNA concentration." The average cfDNA concentration is the average value of the results obtained using the blood of three individuals.

[0190] The amount of cfDNA recovered was determined according to the following criteria. Note that the more leukocytes contaminating the plasma are destroyed during storage, the greater the increase in cfDNA concentration. Therefore, the smaller the increase in cfDNA concentration, the more suppressed DNA leakage from leukocytes contaminating the plasma, and the less leukocyte-derived DNA is contaminated into the plasma during sample storage.

[0191] <Criteria for determining the amount of recovered cfDNA> XX: The increase in cfDNA concentration is less than 10 ng / 1 mL of plasma. ○: The increase in cfDNA concentration is 10 ng / 1 mL or more and less than 70 ng / 1 mL of plasma. ×: The increase in cfDNA concentration is 70 ng / 1 mL or more of plasma.

[0192] The composition and results are shown in Tables 2 to 5 below. In the tables, the concentration of the anticoagulant is EDTA 2K / 2H. ２ This refers to the concentration of EDTA2K, not the concentration of EDTA2K.

[0193]

[0194]

[0195]

[0196]

[0197] 1...Blood collection container 2...Blood collection container main body 3...Composition for plasma separation 4...Aqueous solution 5...Blug body

Claims

1. 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 specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less, and the aqueous solution contains an anticoagulant and a water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less or ammonium sulfate, a blood collection container.

2. The blood collection container according to claim 1, wherein the aqueous solution contains the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less.

3. The blood collection container according to claim 1, wherein the aqueous solution contains the ammonium sulfate.

4. The blood collection container according to claim 1, wherein the aqueous solution contains the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less and the ammonium sulfate.

5. The blood collection container according to any one of claims 1 to 4, wherein the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less is dextran, polyethylene glycol, or polyvinylpyrrolidone.

6. The blood collection container according to any one of claims 1 to 4, wherein the water-soluble polymer compound having a number average molecular weight of 500 or more and 180,000 or less is a water-soluble polymer compound having a number average molecular weight of 2,000 or more and 150,000 or less.

7. The blood collection container according to any one of claims 1 to 4, wherein the aqueous solution further contains trehalose.

8. The blood collection container according to any one of claims 1 to 4, wherein the aqueous solution further contains glucose, adenine, or inositol.

9. The blood collection container according to any one of claims 1 to 4, wherein the aqueous solution further contains an apoptosis inhibitor.

10. A blood collection container in which a predetermined amount of blood is collected, when a physiological saline equivalent to the predetermined amount of blood collected in the blood collection container is collected into the blood collection container to obtain a mixed solution in which the physiological saline and the aqueous solution are mixed, the osmotic pressure of the mixed solution is 300 mOsm / L or more, the blood collection container according to any one of claims 1 to 4.

11. The blood collection container according to any one of claims 1 to 4, wherein the aqueous solution further contains propylene glycol.

12. The blood collection container according to any one of claims 1 to 4, wherein the plasma separation material is a plasma separation composition.

13. The composition for plasma separation contains an organic component having fluidity at 25°C and inorganic fine powder, the organic component contains a resin, The blood collection container according to claim 12, wherein the inorganic fine powder contains fine silica powder.

14. The blood collection container according to claim 13, wherein the fine silica powder contains hydrophilic silica.

15. The blood collection container according to claim 14, wherein the content of the hydrophilic silica in 100% by weight of the composition for plasma separation is 0.01% by weight or more and 2.50% by weight or less.

16. The blood collection container according to claim 13, wherein the fine silica powder contains hydrophilic silica and hydrophobic silica.

17. The specific gravity of the composition for plasma separation at 25°C is 1.050 or more, The blood collection container according to claim 13, wherein the inorganic fine powder contains inorganic fine powder having a specific gravity greater than that of the fine silica powder.

18. The blood collection container according to claim 13, wherein the resin contains a petroleum resin, a cyclopentadiene-based resin, a polyester resin, or a (meth)acrylic-based resin.

19. The blood collection container according to any one of claims 1 to 4, which is used for separating extracellular free nucleic acids or extracellular vesicles in blood.

20. A method for separating plasma, comprising a step of collecting blood in the blood collection container according to any one of claims 1 to 4, and a step of centrifuging the blood collection container in which the blood has been collected.

21. A method for separating extracellular free nucleic acids, comprising a step of collecting blood in the blood collection container according to any one of claims 1 to 4, a step of centrifuging the blood collection container in which the blood has been collected to separate plasma from the blood, and a step of separating extracellular free nucleic acids from the separated plasma.

22. A method for separating extracellular vesicles, comprising a step of collecting blood in the blood collection container according to any one of claims 1 to 4, a step of centrifuging the blood collection container in which the blood has been collected to separate plasma from the blood, and a step of separating extracellular vesicles from the separated plasma.

23. A method for separating extracellular vesicles, comprising a step of collecting blood in the blood collection container according to any one of claims 1 to 4, a step of centrifuging the blood collection container in which the blood has been collected to separate plasma from the blood, and a step of separating extracellular vesicles from the separated plasma. ​ ​ ​