WO2023145137A1 - 血液採取容器、血漿の分離方法、細胞外遊離核酸の分離方法及び細胞外小胞の分離方法 - Google Patents

血液採取容器、血漿の分離方法、細胞外遊離核酸の分離方法及び細胞外小胞の分離方法 Download PDF

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Publication number
WO2023145137A1
WO2023145137A1 PCT/JP2022/036449 JP2022036449W WO2023145137A1 WO 2023145137 A1 WO2023145137 A1 WO 2023145137A1 JP 2022036449 W JP2022036449 W JP 2022036449W WO 2023145137 A1 WO2023145137 A1 WO 2023145137A1
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Prior art keywords
collection container
blood collection
blood
plasma
aqueous solution
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PCT/JP2022/036449
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English (en)
French (fr)
Japanese (ja)
Inventor
邦哉 駒井
エムディ シャハダット ホセイン
智雅 井上
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Sekisui Medical Co Ltd
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Sekisui Medical Co Ltd
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Application filed by Sekisui Medical Co Ltd filed Critical Sekisui Medical Co Ltd
Priority to CN202280075520.1A priority Critical patent/CN118235042A/zh
Priority to KR1020247019557A priority patent/KR20240142406A/ko
Priority to CA3250108A priority patent/CA3250108A1/en
Priority to JP2023576612A priority patent/JPWO2023145137A1/ja
Priority to EP22923995.9A priority patent/EP4471401A4/en
Priority to US18/727,494 priority patent/US20250073703A1/en
Publication of WO2023145137A1 publication Critical patent/WO2023145137A1/ja
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter

Definitions

  • the present invention relates to a blood collection container.
  • the present invention also relates to a method for separating plasma, a method for separating extracellular free nucleic acid, and a method for separating extracellular vesicles using the blood collection container.
  • Blood collection containers such as blood collection tubes are widely used in clinical examinations 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 positioned above the plasma separation material, and the blood cells are positioned below.
  • the blood collection container containing the plasma separation material include a blood collection container containing a composition for plasma separation containing resin and inorganic powder (for example, Patent Document 1) and a blood collection container containing a jig for plasma separation. A container (for example, Patent Document 2) is known.
  • white blood cells may be mixed in the separated plasma.
  • the separated plasma is stored for a certain period of time without freezing until it is used for testing.
  • conventional blood collection containers such as those described in Patent Documents 1 and 2
  • leukocytes mixed in the plasma are destroyed over time due to death or the like, and the DNA in the leukocytes may leak into the plasma. Leakage of DNA in white blood cells into plasma can affect test results.
  • test results will vary greatly depending on the DNA leaked from the leukocytes.
  • a blood collection container main body a plasma separation material contained in the blood collection container main body, and an aqueous solution contained in the blood collection container main body, Specific gravity 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 or ammonium sulfate having a number average molecular weight of 500 or more and 180,000 or less.
  • a blood collection container is provided.
  • the aqueous solution contains a water-soluble polymer compound with a number average molecular weight of 500 or more and 180,000 or less.
  • the aqueous solution contains the ammonium sulfate.
  • 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.
  • 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.
  • 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 having a number average molecular weight of 2,000 or more and 150,000 or less. is a compound.
  • the aqueous solution further contains trehalose.
  • the aqueous solution further contains glucose, adenine or inositol.
  • the aqueous solution further contains an apoptosis inhibitor.
  • the blood collection container is a blood collection container from which a predetermined amount of blood is collected, and an amount of physiological fluid equivalent to the predetermined amount of blood collected in the blood collection container.
  • the mixture has an osmotic pressure of 300 mOsm/L or more.
  • the aqueous solution further contains propylene glycol.
  • the plasma separation material is a composition for plasma separation.
  • the composition for plasma separation contains an organic component having fluidity at 25° C. and an inorganic fine powder, the organic component contains a resin, and the The inorganic fine powder includes finely divided silica.
  • the finely divided silica contains hydrophilic silica.
  • the hydrophilic silica content is 0.01% by weight or more and 2.50% by weight or less in 100% by weight of the plasma separation composition.
  • the finely divided silica contains hydrophilic silica and hydrophobic silica.
  • the plasma separation composition has a specific gravity of 1.050 or more at 25° C., and the inorganic fine powder has a higher specific gravity than the fine powder silica. Contains fine powder.
  • the resin includes petroleum resin, cyclopentadiene resin, polyester resin, or (meth)acrylic resin.
  • the blood collection container is used to separate extracellular free nucleic acids or extracellular vesicles in blood.
  • a plasma separation method comprising the steps of: collecting blood in the blood collection container described above; and centrifuging the blood collection container containing the blood. .
  • a blood collection container comprises a blood collection container main body, a plasma separation material contained in the blood collection container main body, and an aqueous solution contained in the blood collection container main body.
  • the specific gravity at 25°C is 1.027 or more and 1.060 or less.
  • 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. Since the blood collection container according to the present invention has the above configuration, it is possible to suppress the contamination of plasma with leukocyte-derived DNA during sample storage.
  • FIG. 1 is a front cross-sectional view schematically showing a blood collection container according to one embodiment of the present invention.
  • a blood collection container comprises a blood collection container main body, a plasma separation material contained in the blood collection container main body, and an aqueous solution contained in the blood collection container main body.
  • the specific gravity at 25°C is 1.027 or more and 1.060 or less.
  • 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.
  • the blood collection container according to the present invention has the above configuration, it is possible to suppress contamination of plasma with leukocyte-derived DNA during sample storage.
  • the blood and the aqueous solution are mixed.
  • the present inventors have found that the specific water-soluble polymer compound or ammonium sulfate contained in the aqueous solution can suppress the contamination of the separated plasma with leukocytes. It was also found that the specific water-soluble polymer compound or ammonium sulfate contained in the aqueous solution can stabilize leukocytes contaminating the separated plasma. Therefore, in the blood collection container according to the present invention, leakage of DNA from leukocytes due to destruction of leukocytes over time can be effectively suppressed, and an increase in the amount of DNA in plasma during specimen storage can be suppressed. In addition, in the blood collection container according to the present invention, since the destruction of leukocytes mixed in plasma over time can be effectively suppressed, leakage of contents such as proteins from the leukocytes can also be effectively suppressed. .
  • (meth)acryl means one or both of “acryl” and “methacryl”.
  • the blood collection container includes a plasma separation material housed within the blood collection container main body.
  • a conventionally known plasma separation material can be used as the plasma separation material.
  • the plasma separation material include plasma separation compositions and plasma separation jigs.
  • the plasma separation material is preferably the plasma separation composition, because the plasma separation material can be easily produced.
  • the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less.
  • the specific gravity of the plasma separation material at 25°C is preferably 1.029 or more, more preferably 1.030 or more, still more preferably 1.032 or more, preferably 1.055 or less, and more preferably 1.050 or less. be.
  • the specific gravity of the plasma separation material at 25°C is equal to or higher than the lower limit and equal to or lower than the upper limit, plasma can be separated from blood more effectively, and leukocytes and erythrocytes are more effectively prevented from being mixed into plasma. can be effectively suppressed.
  • the storage location of the plasma separation material is not particularly limited as long as it is inside the blood collection container main body.
  • the plasma separation material may be arranged at the bottom of the blood collection container main body, or may be arranged on the inner wall surface of the blood collection container main body.
  • the plasma separation composition is a composition that migrates between the plasma layer and the blood cell layer during centrifugation to form a partition wall. Moreover, the composition for plasma separation 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 thixotropic properties.
  • the plasma separation composition may be contained in the bottom of the blood collection container main body, or may be arranged on the inner wall surface. From the viewpoint of exhibiting the effects of the present invention more effectively, the plasma separation composition is preferably contained in the bottom portion of the blood collection container main body.
  • a conventionally known composition for plasma separation can be used as the composition for plasma separation.
  • the composition for plasma separation preferably contains an organic component having fluidity at 25°C and an inorganic fine powder.
  • Each of the organic component having fluidity at 25° C. and the inorganic fine powder may be used alone, or two or more thereof may be used in combination.
  • the viscosity of the organic component at 25°C is preferably 10 Pa ⁇ s or more, more preferably 30 Pa ⁇ s or more, preferably 200 Pa ⁇ s or less, more preferably 100 Pa ⁇ s or less.
  • the viscosity is equal to or higher than the lower limit and equal to or lower than the upper limit, the fluidity of the composition for plasma separation is enhanced, and the strength of partition walls formed by centrifugation can be enhanced.
  • the viscosity of the above organic component at 25°C is measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) under the conditions of 25°C and a shear rate of 1.0 sec -1 . .
  • the organic component preferably contains the resin, and more preferably contains the resin and the organic compound.
  • 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 has fluidity. It doesn't have to be.
  • the resin may be, for example, a solid resin at 25°C.
  • Each of the above resin and the above organic compound may be used alone, or two or more thereof may be used in combination.
  • Examples of the above resins include petroleum resins, cyclopentadiene resins, polyester resins, polyurethane resins, (meth)acrylic resins, silicone resins, ⁇ -olefin-fumarate ester copolymers, sebacic acid and 2,2-dimethyl-1. ,3-propanediol and 1,2-propanediol copolymers, polyether polyurethane resins, and polyether polyester resins. Only one kind of the above resin may be used, or two or more kinds thereof may be used in combination.
  • the resin preferably contains petroleum resin, cyclopentadiene resin, polyester resin, or (meth)acrylic resin.
  • Examples of the cyclopentadiene-based resin include polymers of cyclopentadiene-based monomers, copolymers of cyclopentadiene-based monomers and aromatic monomers, and dicyclopentadiene resins.
  • the cyclopentadiene-based resin may be hydrogenated.
  • the polymer of the cyclopentadiene monomer and the copolymer of the cyclopentadiene monomer and the aromatic monomer may be oligomers.
  • cyclopentadiene-based monomers examples include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.
  • Styrene, methylstyrene, indene, methylindene and the like are mentioned as the aromatic monomers.
  • polyester resins include polyalkylene terephthalate resins and polyalkylene naphthalate resins.
  • polyalkylene terephthalate resin examples include polyethylene terephthalate, polybutylene terephthalate and poly-1,4-cyclohexanedimethylene terephthalate.
  • polyurethane resin examples include a reaction product of a polyol compound and an isocyanate compound.
  • the (meth)acrylic resin includes a resin obtained by polymerizing at least one (meth)acrylic acid ester monomer, and at least one (meth)acrylic acid ester monomer and at least one and a resin obtained by polymerizing a monomer other than the (meth)acrylic acid ester monomer.
  • Examples of the (meth)acrylic acid ester monomers 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 ester, (meth)acrylic acid dialkylaminoalkyl ester, (meth)acrylic acid benzyl ester, (meth)acrylic acid phenoxyalkyl ester, (meth)acrylic acid cyclohexyl ester, (meth)acrylic acid isobornyl ester esters, and (meth)acrylic acid alkoxysilylalkyl esters, and the like.
  • 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 from 1 to 20 carbon atoms. Only one kind of the (meth)acrylic acid ester monomer may be used, or two or more kinds thereof may be used in combination.
  • Examples of the above organic compounds include benzene polycarboxylic acid alkyl ester derivatives.
  • 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.
  • benzene polycarboxylic acid alkyl ester derivatives examples include phthalates, trimellitates, pyromellitic esters, and the like. Only one kind of the benzene polycarboxylic acid alkyl ester derivative may be used, or two or more kinds thereof may be used in combination.
  • trimellitate examples include tri-n-octyl trimellitate, triisooctyl trimellitate, and triisodecyl trimellitate.
  • Examples of the pyromellitic acid ester include tetraisooctyl pyromellitic acid.
  • trimellitate esters include “Monocizer W700” and “Monocizer W-750” manufactured by DIC, and “Sansocizer TOTM” and “Sansocizer TITM” manufactured by Shin Nippon Rika.
  • the benzene polycarboxylic acid alkyl ester derivative is preferably a phthalate, a trimellitate, or a pyromellitate, more preferably a trimellitate.
  • the content of the organic component in 100% by weight of the composition for plasma separation is preferably 80% by weight or more, more preferably 85% by weight or more, still more preferably 90% by weight or more, and preferably 97% by weight or less. .
  • Inorganic fine powder examples include fine powder silica, 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.
  • the inorganic fine powder preferably contains fine powder silica.
  • the inorganic fine powder includes finely powdered silica and an inorganic fine powder other than finely powdered silica (second inorganic fine powder). and more preferably.
  • the specific gravity of the plasma separation composition at 25° C. is 1.050 or higher
  • the inorganic fine powder may not contain the second inorganic fine powder.
  • the specific gravity of the composition for plasma separation 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 fine silica powder, and the second inorganic fine powder may be used alone or in combination of two or more.
  • the finely divided silica includes natural silica and synthetic silica.
  • Synthetic silica includes hydrophilic silica and hydrophobic silica.
  • Hydrophilic silica has the effect of imparting thixotropy to the composition for plasma separation and adjusting the specific gravity, for example, by hydrogen bonding between hydroxyl groups on the particle surfaces.
  • hydrophobic silica has a smaller effect of imparting thixotropy than hydrophilic silica.
  • the finely divided silica preferably contains hydrophilic silica, and includes hydrophilic silica and hydrophobic silica. is more preferable.
  • the second inorganic fine powder is preferably an inorganic fine powder having a higher specific gravity than finely divided silica, and is an inorganic fine powder having a specific gravity of 3 or more, such as zinc oxide powder, titanium oxide powder, and alumina powder. is more preferred.
  • 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 average particle size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder is not particularly limited.
  • the average particle size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder may be 1 nm or more, 10 nm or more, 500 nm or less, or 100 nm or less. There may be.
  • the average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are the average diameters measured on a volume basis, and are the values of the median diameter (D50) at 50%.
  • the volume average particle size (D50) can be measured by a laser diffraction/scattering method, an image analysis method, a Coulter method, a centrifugal sedimentation method, or the like.
  • the volume average particle size (D50) is preferably obtained by measurement by a laser diffraction/scattering method or an image analysis method.
  • the specific surface area of the finely divided silica is not particularly limited.
  • the specific surface area of the finely divided silica may be 20 m 2 /g or more, 100 m 2 /g or more, 500 m 2 /g or less, or 300 m 2 /g or less. good too.
  • the specific surface area of the finely divided silica is measured by the BET method.
  • the content of the hydrophilic silica in 100% by weight of the composition for plasma separation is preferably 0.01% by weight or more, more preferably 0.10% by weight or more, and still more preferably 0.30% by weight or more. is 2.50% by weight or less, more preferably 2.00% by weight or less.
  • the content of the hydrophilic silica is equal to or higher than the lower limit and equal to or lower than the upper limit, both the specific gravity and thixotropy of the composition for plasma separation can be maintained within a more suitable range.
  • the content of the finely divided silica in 100% by weight of the composition for plasma separation is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, preferably 10% by weight or less, more preferably 7% by weight. % by weight or less.
  • content of the finely divided silica is above the lower limit and below the upper limit, both the specific gravity and thixotropy of the composition for plasma separation can be maintained within a more suitable range.
  • the content of the second inorganic fine powder is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 10% by weight or less. Preferably, it is 7% by weight or less.
  • the content of the second inorganic fine powder is not less than the lower limit and not more than the upper limit, the specific gravity of the plasma separation composition can be effectively increased.
  • the content of the inorganic fine powder in 100% by weight of the composition for plasma separation is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, preferably 10% by weight or less, and more preferably 7% by weight. % by weight or less.
  • the specific gravity of the plasma separation composition can be effectively increased.
  • composition for plasma separation may contain other ingredients than those mentioned above as long as the effects of the present invention are not impaired.
  • other components include organic gelling agents, thermoplastic elastomers, polyalkylene glycols, silicone oils, co-solvents, antioxidants, colorants and water.
  • organic gelling agents include organic gelling agents, thermoplastic elastomers, polyalkylene glycols, silicone oils, co-solvents, antioxidants, colorants and water.
  • the specific gravity of the plasma separation composition at 25° C. is preferably 1.027 or higher, more preferably 1.029 or higher, still more preferably 1.030 or higher, particularly preferably 1.032 or higher, and preferably 1.060. Below, more preferably 1.055 or less, still more preferably 1.050 or less.
  • the specific gravity of the composition for plasma separation at 25°C is equal to or higher than the lower limit and equal to or lower than the upper limit, plasma can be well separated from blood, and contamination of plasma with leukocytes can be effectively suppressed. can be done.
  • the specific gravity of the plasma separation composition at 25° C. is equal to or higher than the lower limit and equal to or lower than the upper limit, contamination of plasma with red blood cells can be effectively suppressed.
  • the specific gravity at 25°C of the composition for plasma separation may be 1.050 or more, or may exceed 1.050.
  • the specific gravity of the composition for plasma separation at 25° C. was obtained by dropping one drop of the composition for plasma separation sequentially into saline at 25° C. whose specific gravity was adjusted stepwise at intervals of 0.002, and floating and sinking in the saline. measured by
  • the viscosity of the plasma separation composition at 25°C is preferably 50 Pa ⁇ s or more, more preferably 70 Pa ⁇ s or more, preferably 500 Pa ⁇ s or less, more preferably 400 Pa ⁇ s or less.
  • the viscosity is at least the lower limit and at most the upper limit, the effects of the present invention can be exhibited more effectively.
  • the viscosity of the composition for plasma separation at 25° C. was measured using an E-type viscometer (eg, “TVE-35” manufactured by Toki Sangyo Co., Ltd.) under conditions of 25° C. and a shear rate of 1.0 sec ⁇ 1 . measured.
  • E-type viscometer eg, “TVE-35” manufactured by Toki Sangyo Co., Ltd.
  • the jig for plasma separation is a jig that moves between the plasma layer and the blood cell layer during centrifugation to form a partition wall. Moreover, the jig for plasma separation is used for the purpose of preventing component transfer between the plasma layer and the blood cell layer.
  • a conventionally known plasma separation jig can be used as the plasma separation jig.
  • Examples of the plasma separation jig include a mechanical separator (plasma separation jig) described in WO2010/132783A1 and the like.
  • Examples of materials for the plasma separation jig include elastomers.
  • the blood collection container includes an aqueous solution contained within 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 or more and 180,000 or less (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 contains the water-soluble polymer compound (X) or ammonium sulfate.
  • the aqueous solution may contain the water-soluble polymer compound (X) and ammonium sulfate.
  • the aqueous solution contains an anticoagulant.
  • Conventionally known anticoagulants can be used as the anticoagulant. Only one kind of the anticoagulant may be used, or two or more kinds thereof may be used in combination.
  • anticoagulant examples include heparin, heparin metal salts, ethylenediaminetetraacetic acid (EDTA), EDTA metal salts, citric acid and sodium citrate.
  • the anticoagulant is preferably EDTA, a metal salt of EDTA, heparin, a metal salt of heparin, or sodium citrate.
  • the concentration of the anticoagulant in the aqueous solution is preferably 2 mM or more, more preferably 5 mM or more, still more preferably 10 mM or more, preferably 2000 mM or less, more preferably 1000 mM or less, even more preferably 500 mM or less, still more preferably 250 mM. Below, even more preferably 100 mM or less, particularly preferably 50 mM or less.
  • concentration of the anticoagulant is equal to or higher than the lower limit and equal to or lower than the upper limit, good anticoagulant performance can be exhibited.
  • 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 contamination of the separated plasma with leukocytes can be suppressed by using the water-soluble polymer compound (X) is that the water-soluble polymer compound (X) keeps the viscosity of the blood good. It is presumed that this is because leukocytes move more smoothly during centrifugation, but the present invention is not limited to this.
  • water-soluble polymer compound (X) suppresses DNA leakage from leukocytes contaminating blood plasma. It is presumed that this is for stabilizing leukocytes, but is not limited to this.
  • water-soluble in the water-soluble polymer compound (X) means that 0.1 g or more is dissolved in 100 g of water at 25°C.
  • the water-soluble polymer compound (X) only one type may be used, or two or more types may be used in combination.
  • the number average molecular weight of the water-soluble polymer compound (X) is 500 or more and 180,000 or less.
  • the number average molecular weight of the water-soluble polymer compound (X) is preferably 1,000 or more, more preferably 1,500 or more, still more preferably 2,000 or more, particularly preferably 2,500 or more, preferably 170,000. 150,000 or less, more preferably 120,000 or less, and particularly preferably 100,000 or less.
  • the number average molecular weight of the water-soluble polymer compound (X) is at least the above lower limit and below the above upper limit, leukocyte contamination in the separated plasma can be further suppressed, and leukocytes can be further stabilized. and the effect of the present invention can be exhibited more effectively.
  • the number average molecular weight of the water-soluble polymer compound (X) is the number average molecular weight in terms of standard polyethylene glycol measured by gel permeation chromatography (GPC).
  • water-soluble polymer compounds (X) include dextran, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, methylcellulose, hydroxyethylcellulose and corn starch.
  • the water-soluble polymer compound (X) is preferably dextran, polyethylene glycol, or polyvinylpyrrolidone.
  • leukocytes can be further stabilized, and the effects of the present invention can be exhibited more effectively.
  • each water-soluble polymer compound (X) is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, still more preferably 1% by weight or more, preferably It is 15% by weight or less, more preferably 10% by weight or less, and still more preferably 7% by weight or less.
  • each content of the water-soluble polymer compound (X) is equal to or more than the above lower limit and equal to or less than the above upper limit, leukocytes can be further stabilized, and the effects of the present invention can be exhibited more effectively. .
  • each content of the 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).
  • each content of the 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).
  • the aqueous solution contains two or more types of water-soluble polymer compounds (X), it means the content of each water-soluble polymer compound (X).
  • 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, still more preferably 1% by weight or more, and preferably 20% by weight. % by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less.
  • the content of the water-soluble polymer compound (X) is at least the above lower limit and below the above upper limit, leukocytes can be further stabilized, and the effects of the present invention can be exhibited more effectively.
  • the content of the water-soluble polymer compound (X) is equal to or higher 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. The amount of leukocyte contamination can be further reduced.
  • the aqueous solution preferably contains ammonium sulfate.
  • the present inventors have found that the use of ammonium sulfate can further suppress the contamination of the separated plasma with leukocytes, and that the leakage of DNA from the leukocytes that are contamination with the plasma can be suppressed. Found it.
  • the aqueous solution contains the water-soluble polymer compound (X) and ammonium sulfate, the effects of the present invention are exhibited more effectively.
  • the content of ammonium sulfate in 100% by weight of the aqueous solution is preferably 0.5% by weight or more, more preferably 1% by weight or more, still more preferably 4% by weight or more, preferably 20% by weight or less, and more preferably 15% by weight. % or less, more preferably 10% by weight or less.
  • ammonium sulfate content is equal to or higher than the lower limit and equal to or lower than the upper limit, contamination of the separated plasma with leukocytes can be further suppressed, and leukocytes can be further stabilized. The effects of the invention can be exhibited more effectively.
  • the osmotic pressure of the mixed liquid obtained by mixing the collected blood and the aqueous solution can be effectively increased, so that the amount of leukocytes mixed into the plasma can be reduced. can be much less.
  • the aqueous solution preferably contains trehalose.
  • the solute contained in the aqueous solution preferably contains trehalose.
  • the content of trehalose in 100% by weight of the aqueous solution is preferably 0.5% by weight or more, more preferably 1% by weight or more, still more preferably 2% by weight or more, preferably 10% by weight or less, and more preferably 7% by weight. % or less, more preferably 5 wt % or less.
  • content of trehalose is equal to or higher than the lower limit and equal to or lower than the upper limit, leukocytes can be further stabilized, and the effects of the present invention can be exhibited more effectively.
  • the osmotic pressure of the mixed liquid in which the collected blood and the aqueous solution are mixed can be effectively increased, so that the amount of leukocytes mixed into the plasma can be reduced. can be much less.
  • the aqueous solution preferably contains glucose, adenine, or inositol.
  • the solute contained in the aqueous solution preferably contains glucose, adenine or inositol.
  • the aqueous solution may contain only one of glucose, adenine and inositol, or may contain two or more of them.
  • the content of glucose in 100% by weight of the aqueous solution is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, and more preferably 10% by weight or less. is 5% by weight or less.
  • the content of glucose is equal to or more than the lower limit and equal to or less than the upper limit, the effect of the present invention can be exhibited more effectively.
  • the glucose content is equal to or higher than the lower limit, the osmotic pressure of the mixed liquid in which the collected blood and the aqueous solution are mixed can be effectively increased. can be much less.
  • the content of adenine in 100% by weight of the aqueous solution is preferably 0.01% by weight or more, more preferably 0.05% by weight or more, and more preferably 2% by weight or less. is 1% by weight or less.
  • the adenine content is at least the lower limit and at most the upper limit, the effects of the present invention can be exhibited more effectively.
  • the osmotic pressure of the mixed liquid obtained by mixing the collected blood and the aqueous solution can be effectively increased, so that the amount of leukocytes mixed into plasma can be reduced. can be much less.
  • 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, and more preferably 2% by weight or less. is 1% by weight or less.
  • the inositol content is at least the lower limit and at most the upper limit, the effects of the present invention can be exhibited more effectively.
  • the inositol content is equal to or higher than the lower limit, the osmotic pressure of the mixed liquid in which the collected blood and the aqueous solution are mixed can be effectively increased, so that the amount of leukocytes mixed into the plasma can be reduced. can be much less.
  • the aqueous solution preferably contains an apoptosis inhibitor.
  • the solute contained in the aqueous solution preferably contains an apoptosis inhibitor.
  • 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 plasma can be further suppressed. Only one kind of the apoptosis inhibitor may be used, or two or more kinds thereof may be used in combination.
  • Examples of the apoptosis inhibitor include Q-VD-Oph and Z-VAD-FMK.
  • the apoptosis inhibitor is preferably a caspase inhibitor, more preferably Q-VD-Oph.
  • the aqueous solution preferably contains a caspase inhibitor, more preferably Q-VD-Oph.
  • the concentration of the apoptosis inhibitor in the aqueous solution is preferably 1 nM or higher, more preferably 20 nM or higher, preferably 10 ⁇ M or lower, more preferably 5 ⁇ M or lower.
  • concentration of the apoptosis inhibitor is equal to or higher than the lower limit and equal to or lower than the upper limit, the effects of the present invention can be exhibited more effectively.
  • the aqueous solution preferably contains propylene glycol.
  • the solute contained in the aqueous solution preferably contains propylene glycol.
  • the content of propylene glycol in 100% by weight of the aqueous solution is preferably 1% by weight or more, more preferably 5% by weight or more, preferably 50% by weight or less, and more preferably 30% by weight or less.
  • the aqueous solution may contain components other than the components described above (anticoagulant, water-soluble polymer compound (X), ammonium sulfate, trehalose, glucose, adenine, inositol, apoptosis inhibitor, and propylene glycol).
  • Inorganic salts, sugars, sugar alcohols, and the like are mentioned as the above-mentioned other ingredients. Only one of the other components may be used, or two or more thereof may be used in combination.
  • inorganic salts examples include sodium salts such as sodium chloride and sodium hydrogen phosphate, and potassium salts such as potassium chloride and potassium hydrogen carbonate.
  • saccharides examples include dihydroxyacetone, fructose, galactose, sucrose, maltose, lactulose, and polysaccharides that do not correspond to the water-soluble polymer compound (X).
  • sugar alcohols examples include D-mannitol and D-sorbitol.
  • the amount of the aqueous solution contained in the blood collection container main body is appropriately changed depending on the size of the blood collection container main body, the amount of blood to be collected, and the like.
  • the amount of the aqueous solution contained in the blood collection container body is preferably 0.1 mL or more, more preferably 0.5 mL or more, still more preferably 0.7 mL or more, preferably 5 mL or less, more preferably 3 mL or less, More preferably, it is 2.5 mL or less.
  • the amount of the aqueous solution 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 exhibited more effectively without excessive dilution of blood.
  • the shape of the blood collection container main body is not particularly limited.
  • the blood collection container main body is preferably a bottomed tubular container.
  • the material of the blood collection container body is not particularly limited.
  • 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 resins, epoxy resins, and epoxy-acrylate resins; Modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose and ethyl chitin; silicate glasses such as soda lime glass, phosphosilicate glass and borosilicate glass; and glasses such as quartz glass. Only one kind of material may be used for the blood collection container main body, or two or more kinds may be used in combination.
  • the blood collection container preferably has a stopper.
  • the stopper is preferably attached to the opening of the blood collection container main body.
  • a conventionally known plug can be used as the plug. It is preferable that the stopper is made of a material and has a shape that can be attached to the opening of the blood collection container main body in an airtight and liquid-tight manner.
  • the plug is preferably configured so that a blood collection needle can be pierced therethrough.
  • the plug examples include a plug having a shape that fits into the opening of the blood collection container main body, a sheet-like sealing plug, and the like.
  • the plug body may be a plug body including a plug body such as a rubber plug and a cap member made of plastic or the like. In this case, it is possible to reduce the risk of blood coming into contact with the human body when the plug is pulled out from the opening of the blood collection container main body after blood collection.
  • the plug body examples include synthetic resin, elastomer, rubber, and metal foil.
  • the rubber examples include butyl rubber and halogenated butyl rubber. Aluminum foil etc. are mentioned as said metal foil.
  • the material of the stopper is preferably butyl rubber.
  • the stopper (or the stopper body) is preferably a butyl rubber stopper.
  • the blood collection container is a blood collection container from which a predetermined amount of blood is collected.
  • the predetermined amount of blood is appropriately changed depending on the size and internal pressure of the blood collection container.
  • the predetermined amount of the 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. may be
  • a physiological saline solution in an amount equal to a predetermined amount of blood to be collected in the blood collection container is collected in the blood collection container to obtain a mixed liquid in which the physiological saline solution and the aqueous solution are mixed.
  • a mixed liquid in which the physiological saline solution and the aqueous solution are mixed For example, in a blood collection container from which 5 mL of blood is collected, 5 mL of physiological saline is collected into the blood collection container and mixed by inversion or the like to mix the physiological saline and the aqueous solution.
  • the osmotic pressure of the mixed solution obtained by mixing the physiological saline and the aqueous solution is preferably 300 mOsm/L or more, more preferably 330 mOsm/L or more, still more preferably 350 mOsm/L or more, and preferably 1300 mOsm/L or less. It is more preferably 1000 mOsm/L or less, still more preferably 800 mOsm/L or less. If the osmotic pressure of the mixed liquid is equal to or higher than the lower limit, when blood is collected in the blood collection container, the blood and the aqueous solution are mixed to increase the osmotic pressure of the blood.
  • the water content in white blood cells and the water content in red blood cells move out of the blood cells, and the specific gravities of white blood cells and red blood cells increase.
  • the white blood cells and red blood cells with increased specific gravity move well below the plasma separation material.
  • contamination of white blood cells and red blood cells into plasma can be effectively suppressed.
  • the osmotic pressure of the mixed solution is equal to or higher than the lower limit and equal to or lower than the upper limit, excessive stress on leukocytes can be suppressed, and leukocytes can be further stabilized, so that the effects of the present invention are further enhanced. can be effectively demonstrated.
  • the osmotic pressure of the above mixed solution is measured by the freezing point depression method using an osmometer (for example, "OM-6060” manufactured by ARKRAY).
  • 3 mL or more of blood is preferably collected, more preferably 4 mL or more, preferably 11 mL or less, and 10 mL or less is collected from 1 mL of the aqueous solution contained in the blood collection container. more preferably.
  • the effects of the present invention can be exhibited more effectively without excessive dilution of blood.
  • the blood collection container is preferably a blood collection tube.
  • the blood collection container main body is preferably a blood collection tube main body.
  • the blood collection container is preferably used for separating plasma from blood.
  • the blood collection container is preferably used for isolating extracellular free nucleic acids or extracellular vesicles in blood. preferably.
  • the blood collection container can be manufactured as follows.
  • An aqueous solution is obtained by dissolving an anticoagulant and a water-soluble polymer compound (X) or ammonium sulfate in water. If necessary, components other than these (trehalose, glucose, adenine, inositol, apoptosis inhibitor, propylene glycol, or other components mentioned above) are also dissolved in water to obtain an aqueous solution. The resulting aqueous solution is added into the body of the blood collection container. Before or after adding the aqueous solution, the plasma separation material is accommodated in the blood collection container main body.
  • FIG. 1 is a front cross-sectional view schematically showing a blood collection container according to one embodiment of the present invention.
  • the blood collection container main body 2 has an opening at one end and a closed bottom at the other end.
  • the plasma separation composition 3 is housed 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 main body 2 .
  • the aqueous solution 4 is placed on the surface of the plasma separation composition 3, more specifically, on the upper surface (one end side surface) of the plasma separation composition 3.
  • the aqueous solution 4 is placed on the surface of the plasma separation composition 3 when the blood collection container 1 is in an upright state.
  • the composition for plasma separation is arranged on the side wall surface of the blood collection container main body, and the aqueous solution is applied to the blood collection container when the blood collection container is placed in an upright state. It may be arranged at the bottom of the container body. Moreover, the jig for plasma separation may be used instead of the composition for plasma separation.
  • 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 whose inside is evacuated and then sealed with the stopper. In the case of a vacuum blood collection tube, a fixed amount of blood can be collected easily regardless of the skill of the blood collector.
  • the inside of the blood collection container is sterilized according to ISO or JIS standards.
  • the blood collection container can be used to separate plasma from blood.
  • a plasma separation method according to the present invention comprises the steps of collecting blood in the above-described blood collection container and centrifuging the blood collection container containing the blood.
  • the plasma separation method according to the present invention preferably includes a step of mixing the collected blood and the aqueous solution between the step of collecting the blood and the step of centrifuging.
  • a method for mixing the collected blood and the aqueous solution inversion mixing and the like can be mentioned.
  • the centrifugation conditions in the centrifugation step are not particularly limited as long as the plasma separation material can form partition walls to separate plasma and blood cells.
  • Examples of the centrifugation conditions include conditions for centrifugation at 400 G or more and 4000 G or less for 10 minutes or more and 120 minutes or less.
  • the method for separating extracellular free nucleic acids according to the present invention includes the steps of collecting blood in the above-described blood collection container, and centrifuging the blood collection container containing the blood to separate plasma from the blood. and separating extracellular free nucleic acids from the separated plasma.
  • a method for separating extracellular vesicles according to the present invention includes the steps of collecting blood in the blood collection container described above, and centrifuging the blood collection container containing the blood to separate plasma from the blood. and separating extracellular vesicles from the separated plasma.
  • the collected blood and the aqueous solution are mixed between the step of collecting the blood and the step of centrifuging. It is preferable to include steps.
  • steps As a method for mixing the collected blood and the aqueous solution, inversion mixing and the like can be mentioned.
  • the centrifugation conditions in the centrifugation step are not particularly limited as long as the plasma separation material can form partition walls to separate plasma and blood cells.
  • Examples of the centrifugation conditions include conditions for centrifugation at 400 G or more and 4000 G or less for 10 minutes or more and 120 minutes or less.
  • the extracellular free nucleic acid can be separated from plasma using a conventionally known method.
  • the extracellular free nucleic acid include cell free DNA (cfDNA), cell free RNA (cfRNA), and the like.
  • the method for isolating the extracellularly released nucleic acid from the plasma include a method using a commercially available nucleic acid purification kit.
  • Extracellular free nucleic acids can be easily separated from plasma by using a commercially available nucleic acid purification kit.
  • 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 (Applied biosystem ms company) and the like.
  • extracellular vesicles can be separated from plasma using a conventionally known method.
  • the following materials were prepared for the plasma separation composition.
  • (Material of organic component having fluidity at 25°C) (Meth) acrylic resin 1: 2-Ethylhexyl acrylate and butyl acrylate are radically polymerized by a solution polymerization method in the presence of an azo polymerization initiator to form a (meth)acrylic acid ester polymer ((meth)acrylic ester polymer having fluidity at 25°C). A system resin 1) was obtained. The specific gravity of the (meth)acrylic resin 1 at 25° C. was 1.034.
  • (Meth) acrylic resin 2 A (meth)acrylic acid ester-based fluid at 25° C. was prepared in the same manner as in (meth)acrylic resin 1, except that the blending ratio of 2-ethylhexyl acrylate and butyl acrylate was changed. A polymer ((meth)acrylic resin 2) was obtained. The specific gravity of the (meth)acrylic resin 2 at 25° C. was 1.015.
  • Trimellitate ester (benzene polycarboxylic acid alkyl ester derivative, "Monocizer W700" manufactured by DIC)
  • 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 Co., Ltd.)
  • Silicone oil (“SF8410” manufactured by Dow Corning Toray Co., Ltd.)
  • Organic gelling agent (“Gelol D” manufactured by Shin Nippon Rika Co., Ltd.) 1-methyl-2-pyrrolidone (co-solvent)
  • Preparation of plasma separation compositions A, B, and D An organic component, an inorganic fine powder, and other components having flowability at 25° C. were mixed at the mixing ratios shown in Table 1 to prepare compositions A, B, and D for plasma separation.
  • Preparation of plasma separation composition C The materials of the organic component having fluidity at 25°C shown in Table 1 were blended, heated and dissolved at 130°C, and mixed to prepare an organic component having fluidity at 25°C. Next, the organic component, the inorganic fine powder, and the other components, which are fluid at 25°C, were mixed at the mixing ratio shown in Table 1 to prepare a composition C for plasma separation.
  • PET bottomed tube polyethylene terephthalate tube with a length of 100 mm and an inner diameter of 14 mm at the opening
  • Example 1 An aqueous solution was obtained by dissolving the components shown in Table 2 in water. Table 2 shows the types and concentrations of the components in the resulting aqueous solution.
  • 1.2 g of plasma separation composition A was accommodated in the bottom of the blood collection container main body. Also, 1 mL of the obtained aqueous solution was added onto the surface of the composition A for plasma separation. The inside of the blood collection container was evacuated so that the blood collection volume was 8 mL, and the container was sealed with a butyl rubber stopper. Thus, a blood collection container was produced.
  • Examples 2 to 12 and Comparative Examples 2 and 3 The type of composition for plasma separation and the composition of the aqueous solution were changed as shown in Tables 2-5.
  • a blood collection container was produced in the same manner as in Example 1 except for these.
  • composition A for plasma separation was accommodated in the bottom of the blood collection container main body. Also, 60 mg of the obtained mixture was applied to the inner wall surface of the blood collection container main body and dried. The inside of the blood collection container was evacuated so that the blood collection volume was 8 mL, and the container was sealed with a butyl rubber plug. Thus, a blood collection container was produced.
  • the number of white blood cells in the plasma was measured by analyzing the collected plasma using a multiitem automatic blood cell analyzer ("XE5000" manufactured by Sysmex Corporation).
  • the prepared blood (whole blood sample) was similarly measured for the white blood cell count in the whole blood sample.
  • the above white blood cell count is the average value of the results obtained by evaluating the prepared blood of three persons.
  • the residual ratio of leukocytes was calculated by the following formula.
  • Leukocyte survival rate (%) (number of leukocytes contained in separated plasma (cells)) / (number of leukocytes contained in whole blood sample (cells)) x 100
  • Leukocyte residual rate is less than 10%
  • Leukocyte residual rate is 10% or more
  • DNA contained in the collected plasma was purified using a cfDNA purification kit ("QIAamp Circulating Nucleic Acid Kit” manufactured by QIAGEN). The DNA purification operation was performed on the day plasma was collected from the blood collection container.
  • the DNA concentration in the extract after purification was measured using the Qubit dsDNA HS Assay kit (manufactured by Invitrogen). Then, the cfDNA concentration (the content of cfDNA contained per 1 mL of plasma) was calculated according to the following formula.
  • cfDNA concentration [A] x [B]/[C]
  • the average concentration of cfDNA collected from plasma on the day of blood collection is defined as “cfDNA concentration (on the day of collection)", and the average concentration of cfDNA collected from plasma after storage for 7 days is defined as “cfDNA concentration (storage for 7 days)”. and in addition, the difference between the cfDNA concentration (stored for 7 days) and the cfDNA concentration (on the day of collection) was defined as "increased amount of cfDNA concentration”.
  • the average value of the cfDNA concentration is the average value of the results obtained using the blood of three persons.
  • the recovered amount of cfDNA was determined according to the following criteria. During storage, the more leukocytes contaminating the plasma are destroyed, the greater the amount of increase in the cfDNA concentration. Therefore, the smaller the increase in the cfDNA concentration, the more the leakage of DNA from leukocytes contaminating plasma is suppressed, and the contamination of leukocyte-derived DNA into plasma during specimen storage is suppressed.
  • the concentration of anticoagulant means the concentration of EDTA2K, not the concentration of EDTA2K ⁇ 2H 2 O.

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