Classifications

• • 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
  • 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

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 acids, and a method for separating extracellular vesicles using the blood collection container.
• blood collection containers such as blood collection tubes are widely used to collect blood. After collecting blood in a blood collection container containing a plasma separation material, the blood can be separated into plasma and blood cells by centrifuging the blood collection container. At this time, the plasma is located above the plasma separation material, and the blood cells are located below.
• blood collection containers containing plasma separation material include blood collection containers containing a plasma separation composition containing a resin and an inorganic powder (e.g., Patent Document 1) and blood collection containers containing a plasma separation tool (e.g., Patent Document 2).
• the container In clinical settings, it is common for blood to be collected into a blood collection container and centrifuged on the same day the container is collected to separate the blood into plasma and blood cells. However, several days may pass between the time the blood is collected into the blood collection container and the time the container is centrifuged. For example, if blood is collected into a blood collection container at a hospital facility that does not have a centrifuge, the blood collection container must be transported to an external testing facility, so several days may pass between the time the blood is collected into the blood collection container and the time the container is centrifuged. Furthermore, if there is a large amount of specimens awaiting testing, several days may pass between the time the blood is collected into the blood collection container and the time the container is centrifuged.
• test results can fluctuate greatly depending on the DNA leaked from the white blood cells.
• the object of the present invention is to provide a blood collection container that can reduce the amount of leukocytes mixed into the plasma, even when the blood is centrifuged several days after collection.
• the present invention also aims to provide a method for separating plasma, a method for separating extracellular free nucleic acids, and a method for separating extracellular vesicles using the above-mentioned blood collection container.
• a blood collection container comprising a blood collection container body, a plasma separation material contained within the blood collection container body, and an aqueous solution contained within the blood collection container body, wherein the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant.
• Item 2 The blood collection container according to Item 1, wherein the content of the lithium salt is 1% by weight or more in 100% by weight of the aqueous solution.
• Item 3 The blood collection container according to Item 1 or 2, wherein the lithium salt includes lithium chloride, lithium acetate, lithium citrate, or lithium lactate.
• Item 5 The blood collection container according to any one of Items 1 to 4, wherein the specific gravity of the plasma separation material at 25°C is 1.027 or more and 1.060 or less.
• Item 6 The blood collection container according to any one of items 1 to 5, wherein the aqueous solution further contains ammonium sulfate.
• Item 7 The blood collection container according to any one of Items 1 to 6, wherein the aqueous solution further contains trehalose.
• Item 8 The blood collection container according to any one of items 1 to 7, wherein the aqueous solution further contains a water-soluble polymer compound.
• Item 9 The blood collection container according to any one of items 1 to 8, wherein the aqueous solution further contains propylene glycol.
• Item 10 The blood collection container according to any one of Items 1 to 9, wherein the plasma separation material is a plasma separation composition.
• Item 11 The blood collection container according to Item 10, wherein the plasma separation composition comprises an organic component that is fluid at 25°C and an inorganic fine powder, the organic component comprising a resin, and the inorganic fine powder comprising finely powdered silica.
• Item 12 The blood collection container according to Item 11, wherein the finely powdered silica contains hydrophilic silica and hydrophobic silica.
• Item 13 The blood collection container according to Item 11 or 12, wherein the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin.
• the resin comprises a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin.
• Item 14 A blood collection container according to any one of items 1 to 13, used to separate extracellular free nucleic acids or extracellular vesicles in blood.
• Item 17 A method for separating extracellular vesicles, comprising the steps of: collecting blood in the blood collection container described in any one of Items 1 to 14; centrifuging the blood collection container into which the blood has been collected to separate plasma from the blood; and separating extracellular vesicles from the separated plasma.
• the blood collection container of the present invention comprises a blood collection container body, a plasma separation material contained within the blood collection container body, and an aqueous solution contained within the blood collection container body.
• the aqueous solution contains an anticoagulant and also contains a lithium salt different from the anticoagulant. Because the blood collection container of the present invention is equipped with the above configuration, it is possible to reduce the amount of white blood cells contaminating the plasma, even when blood is centrifuged several days after collection.
• 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.
• the blood collection container comprises a blood collection container body, a plasma separation material contained in the blood collection container body, and an aqueous solution contained in the blood collection container body, wherein the aqueous solution contains an anticoagulant and a lithium salt different from the anticoagulant.
• the blood collection container of the present invention is equipped with the above-mentioned configuration, so the amount of white blood cells mixed into the plasma can be reduced even when the blood is centrifuged several days after collection.
• the blood collection container of the present invention can maintain a high level of effectiveness in separating plasma and white blood cells using the plasma separation material, even when the blood is centrifuged several days after collection.
• the blood is mixed with the aqueous solution.
• the inventors have discovered that the lithium salt contained in the aqueous solution can reduce the amount of leukocytes contaminating the plasma, even when the blood is centrifuged several days (e.g., two days) after collection.
• the blood collection container of the present invention can reduce the amount of leukocytes contaminating the plasma, and therefore the amount of leukocyte-derived DNA (genomic DNA) contaminating the plasma.
• (meth)acrylic refers to either or both of “acrylic” and “methacrylic.”
• 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.
• 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.
• the specific gravity of the plasma separation material at 25°C is preferably 1.027 or more, more preferably 1.029 or more, even more preferably 1.030 or more, particularly preferably 1.032 or more, and preferably 1.060 or less, more preferably 1.055 or less, and even more preferably 1.050 or less.
• the specific gravity of the plasma separation material at 25°C is above the above lower limit and below the above upper limit, plasma can be separated from blood more effectively, and the inclusion of white blood cells and red blood cells in the plasma can be more effectively suppressed.
• the location where the plasma separation material is contained is not particularly limited as long as it is within the blood collection container body.
• the plasma separation material may be placed at the bottom of the blood collection container body, or on the inner wall surface of the blood collection container 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.
• the plasma separation composition is used for the purpose of preventing component migration between the plasma layer and the blood cell layer after centrifugation.
• the plasma separation composition preferably has thixotropy.
• the plasma separation composition may be contained in the bottom of the blood collection container body, or may be disposed on the inner wall surface. From the viewpoint of more effectively exerting the effects of the present invention, the plasma separation composition is preferably contained in the bottom of the blood collection container body.
• a conventionally known plasma separation composition can be used as the plasma separation composition.
• the plasma separation composition preferably contains an organic component that is fluid at 25°C and an inorganic fine powder.
• the fluidity of the plasma separation composition is increased, and the strength of the partition wall formed by the centrifugation operation can be increased.
• the organic component that is fluid at 25°C and the inorganic fine powder may each be used alone, or two or more types 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, and preferably 200 Pa ⁇ s or less, more preferably 100 Pa ⁇ s or less.
• the viscosity is above the lower limit and below the upper limit, the fluidity of the plasma separation composition is increased, and the strength of the partition wall formed after centrifugation can be increased.
• the viscosity of the organic component at 25° C. is measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) under conditions of 25° C. and a shear rate of 1.0 sec ⁇ 1 .
• E-type viscometer for example, "TVE-35” manufactured by Toki Sangyo Co., Ltd.
• the organic component may be a resin, or 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, the mixture (the organic component) needs to have fluidity; the resin or the organic compound does not need to have fluidity. When the organic component is a mixture of the resin and the organic compound, the resin may be a solid at 25°C, for example. Only one of the resins and the organic compounds may be used, or two or more of them may be used in combination.
• the resins include petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, polyurethane-based resins, (meth)acrylic resins, silicone-based resins, ⁇ -olefin-fumaric acid ester copolymers, copolymers of sebacic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane-based resins, and polyether polyester-based resins.
• the above resins may be used alone or in combination of two or more.
• the above resin preferably contains a petroleum-based resin, a cyclopentadiene-based resin, a polyester-based resin, or a (meth)acrylic resin.
• the fluidity of the plasma separation composition is further improved, and the strength of the partition wall formed after centrifugation can be further increased.
• 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 resins may be hydrogenated.
• the polymers of cyclopentadiene-based monomers and copolymers of cyclopentadiene-based monomers and aromatic monomers may be oligomers.
• cyclopentadiene-based monomers examples include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.
• aromatic monomers examples include styrene, methylstyrene, indene, and methylindene.
• dicyclopentadiene resins include “Scoretz SU500” and “Scoretz SU90” manufactured by Colon Co., Ltd.
• the polyester resins mentioned above include polyalkylene terephthalate resins and polyalkylene naphthalate resins.
• Examples of the polyalkylene terephthalate resins mentioned above include polyethylene terephthalate, polybutylene terephthalate, and poly-1,4-cyclohexanedimethylene terephthalate.
• polyurethane resin examples include reaction products of polyol compounds and isocyanate compounds.
• the above-mentioned (meth)acrylic resins include resins obtained by polymerizing at least one type of (meth)acrylic acid ester monomer, and resins obtained by polymerizing at least one type of (meth)acrylic acid ester monomer and at least one type of monomer other than a (meth)acrylic acid ester monomer.
• 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.
• the alkyl group preferably has 1 or more carbon atoms 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 or more and 20 or less carbon atoms.
• the (meth)acrylic acid ester monomers may be used alone or in combination of two or more types.
• the organic compound may be a benzenepolycarboxylic acid alkyl ester derivative.
• the organic compound is preferably a benzenepolycarboxylic acid alkyl ester derivative. Therefore, the organic component is preferably a mixture of the resin and the benzenepolycarboxylic acid alkyl ester derivative.
• benzene polycarboxylic acid alkyl ester derivatives examples include phthalate esters, trimellitate esters, and pyromellitate esters.
• the benzene polycarboxylic acid alkyl ester derivatives may be used alone or in combination of two or more.
• trimellitic acid esters examples include trimellitic acid tri-n-octyl, triisooctyl, and triisodecyl trimellitic acid.
• pyromellitic acid esters examples include tetraisooctyl pyromellitic acid.
• pyromellitic acid esters include "Monocizer W-7010" manufactured by DIC Corporation.
• the above-mentioned benzenepolycarboxylic acid alkyl ester derivative is preferably a phthalate ester, a trimellitate ester, or a pyromellitate ester, and more preferably a trimellitate ester.
• the content of the organic component is preferably 80% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, particularly preferably 95% by weight or more, and preferably 98% by weight or less.
• Inorganic fine powder examples include fine silica powder, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, fine glass powder, talc powder, kaolin powder, bentonite powder, titania powder, and zirconium powder.
• the above-mentioned inorganic fine powder is preferably fine silica, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, or zirconium powder.
• the inorganic fine powder contain finely powdered silica.
• the inorganic fine powder When obtaining a plasma separation composition having a specific gravity of 1.050 or more at 25°C, it is more preferable that the inorganic fine powder contain finely powdered silica and an inorganic fine powder other than finely powdered silica (second inorganic fine powder).
• the specific gravity of the plasma separation composition at 25°C is 1.050 or more, the inorganic fine powder does not have to contain the second inorganic fine powder.
• 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.
• the finely powdered silica preferably contains hydrophilic silica, and more preferably contains both hydrophilic and hydrophobic silica.
• the second inorganic fine powder is preferably an inorganic fine powder with a higher specific gravity than fine silica powder, and more preferably an inorganic fine powder with a specific gravity of 3 or more, such as zinc oxide powder, titanium oxide powder, or alumina powder.
• 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. If the specific gravity is above the lower limit, the specific gravity of the plasma separation composition can be effectively increased.
• the specific gravity of the second inorganic fine powder may be 10 or less, or 6 or less.
• the average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are not particularly limited.
• the average particle diameters 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.
• the average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are average diameters measured on a volume basis (volume average particle diameter), and are the 50% median diameter (D50) values.
• the volume average particle diameter (D50) can be measured by laser diffraction/scattering method, image analysis method, Coulter method, centrifugal sedimentation method, etc. It is preferable to determine the volume average particle diameter (D50) by laser diffraction/scattering method or image analysis method.
• the specific surface area of the finely powdered silica is not particularly limited and 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.
• the specific surface area of the above-mentioned finely powdered silica is measured using the BET method.
• 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.
• content of the hydrophilic silica is above the above lower limit and below the above upper limit, both the specific gravity and thixotropy of the plasma separation composition can be maintained within even more suitable ranges.
• the content of the finely powdered silica is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, even more preferably 1.0% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 5% by weight or less, and particularly preferably 3% by weight or less.
• the content of the finely powdered silica is above the above lower limit and below the above upper limit, both the specific gravity and thixotropy of the plasma separation composition can be maintained within even more suitable ranges.
• the content of the second inorganic fine powder is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 4% by weight or less, and particularly preferably 2% by weight or less.
• the specific gravity of the plasma separation composition can be effectively increased.
• the content of the inorganic fine powder is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, even more preferably 1.0% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, even more preferably 5% by weight or less, and particularly preferably 3% by weight or less.
• the specific gravity of the plasma separation composition can be effectively increased.
• 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.
• 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.
• the specific gravity of the plasma separation composition at 25°C is preferably 1.027 or more, more preferably 1.029 or more, even more preferably 1.030 or more, particularly preferably 1.032 or more, and preferably 1.060 or less, more preferably 1.055 or less, and even more preferably 1.050 or less.
• 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 white blood cells can be effectively suppressed.
• 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 red blood cells can also be effectively suppressed.
• the specific gravity of the plasma separation composition at 25°C may be 1.050 or more, or may even exceed 1.050.
• 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.
• the viscosity is above the above lower limit and below the above upper limit, the effects of the present invention can be exerted even more effectively.
• the plasma separation device is a device that moves between the plasma layer and the blood cell layer during centrifugation to form a partition wall, and is used for the purpose of preventing component migration between the plasma layer and the blood cell layer.
• a conventionally known plasma separation tool can be used as the plasma separation tool.
• the plasma separation tool include the mechanical separator (plasma separation tool) described in WO2010/132783A1, etc.
• Examples of materials for the plasma separation device include elastomers.
• the blood collection container includes an aqueous solution contained within the blood collection container body.
• the aqueous solution includes an anticoagulant.
• the aqueous solution includes a lithium salt (hereinafter, sometimes referred to as "lithium salt A") different from the anticoagulant.
• the lithium salt A is different from the anticoagulant.
• the solutes contained in the aqueous solution include the anticoagulant and lithium salt A.
• the aqueous solution contains an anticoagulant.
• a conventionally known anticoagulant can be used as the anticoagulant. Only one type of anticoagulant can be used, or two or more types can be used in combination.
• anticoagulant examples include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, citric acid, and sodium salts of citric acid.
• the metal salts of heparin may be lithium salts of heparin.
• Examples of the sodium salts of citric acid include trisodium citrate.
• the anticoagulant is preferably EDTA, a metal salt of EDTA, heparin, a metal salt of heparin, or trisodium citrate.
• the concentration of the anticoagulant in the aqueous solution is not particularly limited, as long as it is a concentration at which anticoagulant performance is exhibited.
• 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.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• the concentration of the anticoagulant in the aqueous solution is preferably 50 mM or more, more preferably 70 mM or more, even more preferably 100 mM or more, preferably 300 mM or less, more preferably 200 mM or less, and even more preferably 140 mM or less.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• the concentration of the anticoagulant in the aqueous solution is preferably 50 IU/mL or more, more preferably 70 IU/mL or more, even more preferably 100 IU/mL or more, preferably 500 IU/mL or less, more preferably 450 IU/mL or less, and even more preferably 400 IU/mL or less.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• IU is an international unit.
• the content of the anticoagulant in the aqueous solution (100% by weight) is preferably 0.0003% by weight or more, more preferably 0.0004% by weight or more, even more preferably 0.0006% by weight or more, preferably 0.003% by weight or less, more preferably 0.0025% by weight or less, and even more preferably 0.002% by weight or less.
• the content of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• An amount of saline equal to the predetermined amount of blood 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.
• a mixed solution is obtained by mixing the saline and the aqueous solution.
• the concentration of the anticoagulant in the mixed solution satisfy the following conditions.
• the concentration of the anticoagulant in the mixed solution corresponds to the concentration of the anticoagulant in the mixed solution obtained by mixing the aqueous solution and the blood collected in the blood collection container.
• the concentration of the anticoagulant in the mixed solution is preferably 0.1 mM or more, more preferably 1 mM or more, even more preferably 4 mM or more, preferably 200 mM or less, more preferably 100 mM or less, even more preferably 50 mM or less, even more preferably 25 mM or less, even more preferably 10 mM or less, and particularly preferably 7 mM or less.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• the concentration of the anticoagulant in the mixed solution is preferably 5 mM or more, more preferably 7 mM or more, even more preferably 10 mM or more, preferably 30 mM or less, more preferably 20 mM or less, and even more preferably 14 mM or less.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• the concentration of the anticoagulant in the mixed solution is preferably 5 IU/mL or more, more preferably 7 IU/mL or more, even more preferably 10 IU/mL or more, preferably 50 IU/mL or less, more preferably 45 IU/mL or less, and even more preferably 40 IU/mL or less.
• concentration of the anticoagulant is above the above lower limit and below the above upper limit, good anticoagulant performance can be achieved.
• IU is an international unit.
• the aqueous solution contains a lithium salt A.
• the lithium salt A may be used alone or in combination of two or more kinds.
• lithium salt A examples include lithium chloride, lithium acetate, lithium citrate, and lithium lactate.
• the lithium salt A preferably contains lithium chloride, lithium acetate, lithium citrate, or lithium lactate, and more preferably contains lithium chloride, lithium acetate, or lithium lactate.
• the effect of the present invention of reducing the amount of leukocyte contamination in plasma is even more effectively achieved, even when blood is centrifuged several days after collection.
• leakage of genomic DNA from leukocytes can be effectively suppressed during the time from blood collection to centrifugation. That is, (1) the amount of leukocyte contamination in plasma can be reduced, thereby suppressing the contamination of leukocyte-derived DNA into plasma, and (2) the effect of effectively suppressing leakage of genomic DNA from leukocytes during the time from blood collection to centrifugation.
• the molecular weight of the lithium salt A is preferably 500 or less, more preferably 300 or less, even more preferably 200 or less, and particularly preferably 100 or less.
• the effect of the present invention that is, the amount of leukocytes contaminating the plasma can be reduced even when the blood is centrifuged several days after collection, is more effectively exerted.
• leakage of genomic DNA from leukocytes can be effectively suppressed during the period from blood collection to centrifugation.
• the molecular weight of the lithium salt A may be 30 or more, 40 or more, 50 or more, or 60 or more.
• the molecular weight of lithium salt A above refers to the molecular weight in an anhydrous state.
• the content of the lithium salt A is preferably 1% by weight or more, more preferably 3% by weight or more, even more preferably 5% by weight or more, preferably 20% by weight or less, more preferably 15% by weight or less, even more preferably 12% by weight or less, and particularly preferably 10% by weight or less.
• the content of the lithium salt A 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.
• a volume of saline equal to the predetermined amount of blood 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.
• a mixed solution is obtained by mixing the saline and the aqueous solution.
• the content of the lithium salt A in 100% by weight of the mixed solution is preferably 0.1% by weight or more, more preferably 0.3% by weight or more, even more preferably 0.5% by weight or more, preferably 1.9% by weight or less, more preferably 1.5% by weight or less, and even more preferably 1.3% by weight or less.
• the content of the lithium salt A in 100% by weight of the mixed solution corresponds to the content of the lithium salt A in 100% by weight of the mixed solution obtained by mixing the aqueous solution and the blood collected in the blood collection container.
• the content of the lithium salt A in 100% by weight of the aqueous solution and the content of the lithium salt A in 100% by weight of the mixed solution refer to the content of lithium salt A in an anhydrous state.
• the aqueous solution preferably contains ammonium sulfate.
• ammonium sulfate By using ammonium sulfate, the effects of the present invention can be more effectively achieved.
• leukocytes can be stabilized, and leakage of DNA from leukocytes can be suppressed.
• the content of ammonium sulfate in the above aqueous solution (100% by weight) is preferably 0.5% by weight or more, more preferably 1% by weight or more, even more preferably 2% by weight or more, preferably 20% by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less.
• the content of ammonium sulfate is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively exerted.
• the content of ammonium sulfate is above the above lower limit and below the above upper limit, leukocytes can be more effectively stabilized, and DNA leakage from leukocytes can be more effectively suppressed.
• a volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained.
• a mixture of the saline and the aqueous solution is obtained in a blood collection container capable of collecting 5 mL of blood.
• 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or other means to obtain a mixture.
• the ammonium sulfate content of the mixture (100% by weight) is preferably 0.05% by weight or more, more preferably 0.1% by weight or more, even more preferably 0.4% by weight or more, preferably 2.5% by weight or less, more preferably 1.9% by weight or less, and even more preferably 1.3% by weight or less.
• the ammonium sulfate content in 100% by weight of the above mixed solution corresponds to the ammonium sulfate content in 100% by weight of the solution obtained by mixing the above aqueous solution with blood collected in a blood collection container.
• the aqueous solution preferably contains trehalose, which effectively protects the cell membranes of leukocytes and stabilizes them, thereby further suppressing DNA leakage from leukocytes.
• the trehalose content in the above aqueous solution (100% by weight) is preferably 0.5% by weight or more, more preferably 1% by weight or more, even more preferably 2% by weight or more, preferably 10% by weight or less, more preferably 7% by weight or less, and even more preferably 5% by weight or less.
• leukocytes can be further stabilized and DNA leakage from leukocytes can be more effectively suppressed.
• 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.
• a mixed solution is obtained by mixing the saline and the aqueous solution.
• 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or other means to obtain a mixed solution.
• the trehalose content of the mixed solution (100% by weight) is preferably 0.06% by weight or more, more preferably 0.1% by weight or more, even more preferably 0.2% by weight or more, preferably 1.3% by weight or less, more preferably 0.9% by weight or less, and even more preferably 0.6% by weight or less.
• the trehalose content in 100% by weight of the above mixed solution corresponds to the trehalose content in 100% by weight of the solution obtained by mixing the above aqueous solution with blood collected in a blood collection container.
• the aqueous solution preferably contains a water-soluble polymer compound.
• the water-soluble polymer compound is different from both the anticoagulant and the lithium salt A.
• the effects of the present invention can be more effectively exhibited.
• leukocytes can be more stabilized and DNA leakage from leukocytes can be more effectively suppressed.
• water-soluble in the water-soluble polymer compound means that 0.1 g or more of the water-soluble polymer compound dissolves in 100 g of water at 25°C.
• the water-soluble polymer compound may be used alone or in combination of two or more types.
• the number-average molecular weight of the water-soluble polymer compound is preferably 300 or more, more preferably 1000 or more, even more preferably 1500 or more, even more preferably 2000 or more, particularly preferably 2500 or more, and preferably 180,000 or less, more preferably 170,000 or less, even more preferably 150,000 or less, even more preferably 120,000 or less, and particularly preferably 100,000 or less.
• the number-average molecular weight of the water-soluble polymer compound is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively exerted.
• leukocytes can be more effectively stabilized, and DNA leakage from leukocytes can be more effectively suppressed.
• the number average molecular weight of the above water-soluble polymer compound is the number average molecular weight calculated as standard polyethylene glycol, measured by gel permeation chromatography (GPC).
• water-soluble polymer compounds examples include dextran, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, methyl cellulose, hydroxyethyl cellulose, and corn starch.
• the water-soluble polymer compound is preferably dextran, polyethylene glycol, or polyvinylpyrrolidone, and more preferably dextran or polyethylene glycol.
• the effects of the present invention can be more effectively exerted.
• leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.
• the content of the water-soluble polymer compound 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.
• the content of the water-soluble polymer compound is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively exerted.
• leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.
• a volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained.
• a mixture of the saline and the aqueous solution is obtained in a blood collection container capable of collecting 5 mL of blood.
• 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or other means to obtain a mixture.
• the content of the water-soluble polymer compound in 100% by weight of the mixture is preferably 0.01% by weight or more, more preferably 0.06% by weight or more, even more preferably 0.1% by weight or more, preferably 2.5% by weight or less, more preferably 1.9% by weight or less, and even more preferably 1.3% by weight or less.
• the content of the water-soluble polymer compound is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively achieved. Furthermore, white blood cells can be more effectively stabilized, and DNA leakage from white blood cells can be more effectively suppressed.
• the content of the water-soluble polymer compound in 100% by weight of the mixed liquid corresponds to the content of the water-soluble polymer compound in 100% by weight of the mixed liquid obtained by mixing the aqueous solution and blood collected in the blood collection container.
• the aqueous solution preferably contains propylene glycol.
• propylene glycol By using propylene glycol, the effects of the present invention can be more effectively exhibited.
• leukocytes can be stabilized, and leakage of DNA from leukocytes can be suppressed.
• 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.
• the content of propylene glycol is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively exerted.
• leukocytes can be more effectively stabilized, and leakage of DNA from leukocytes can be more effectively suppressed.
• a volume of saline equal to the predetermined amount of blood to be collected in the blood collection container is collected into the blood collection container, and a mixture of the saline and the aqueous solution is obtained.
• a mixture of the saline and the aqueous solution is obtained in a blood collection container capable of collecting 5 mL of blood.
• 5 mL of saline is collected into the blood collection container, and the saline and the aqueous solution are mixed by inversion or other means to obtain a mixture.
• the propylene glycol content of the mixture (100% by weight) is preferably 0.1% by weight or more, more preferably 0.2% by weight or more, even more preferably 0.5% by weight or more, preferably 6% by weight or less, more preferably 5% by weight or less, and even more preferably 4% by weight or less.
• the propylene glycol content in 100% by weight of the above mixed solution corresponds to the propylene glycol content in 100% by weight of the solution obtained by mixing the above aqueous solution with blood collected in a blood collection container.
• the aqueous solution preferably contains water, which serves as a solvent.
• the water content is preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and preferably 95% by weight or less, more preferably 90% by weight or less.
• the aqueous solution may contain other components in addition to the above-mentioned components (anticoagulant, lithium salt A, ammonium sulfate, trehalose, water-soluble polymer compound, propylene glycol, and water).
• examples of the other components include salts (inorganic salts and organic salts other than those mentioned above), sugars, and sugar alcohols. Only one of the other components may be used, or two or more may be used in combination.
• salts examples include sodium salts such as sodium chloride and sodium hydrogen phosphate, and potassium salts such as potassium chloride, potassium acetate and potassium bicarbonate.
• sugar alcohols examples include D-mannitol and D-sorbitol.
• 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.
• the shape of the blood collection container body is not particularly limited.
• the blood collection container body is preferably a tubular container with a bottom.
• the blood collection container body preferably has an open end at one end and a closed end at the other end.
• the open end of the blood collection container body is one end in the length direction of the blood collection container body, and the closed end of the blood collection container body is the other end in the length direction of the blood collection container body.
• the blood collection container body preferably has a closed bottom at the other end.
• 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 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, soda-lime glass, phosphosilicate glass, and borosilicate glass, and quartz glass. Only one type of material may be used for the blood collection container body, or two or more types may be used in combination.
• stopper examples include a stopper shaped to fit into the open end of the blood collection container body, a sheet-shaped seal stopper, etc.
• the stopper may comprise a stopper body such as a rubber stopper and a cap member made of plastic or the like.
• a stopper body such as a rubber stopper and a cap member made of plastic or the like.
• the material of the stopper may be, for example, synthetic resin, elastomer, rubber, metal foil, etc.
• the rubber include butyl rubber and halogenated butyl rubber.
• the metal foil include aluminum foil, etc. From the perspective of improving sealing performance, the material of the stopper is preferably butyl rubber.
• the stopper (or the stopper main body) is preferably a butyl rubber stopper.
• the blood collection container is a blood collection container for collecting a predetermined amount of blood.
• the blood collection container is used by collecting a predetermined amount of blood.
• the predetermined amount of blood is changed as appropriate depending on the size and internal pressure of the blood collection container.
• the predetermined amount of blood may be 1 mL or more, 2 mL or more, 4 mL or more, 12 mL or less, 11 mL or less, or 10 mL or less.
• 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.
• a mixed solution is obtained by mixing the saline and the aqueous solution.
• the osmotic pressure of the mixed solution obtained by mixing the saline and the aqueous solution is preferably 320 mOsm/L or more, more preferably 350 mOsm/L or more, even more preferably 400 mOsm/L or more, preferably 2000 mOsm/L or less, more preferably 1000 mOsm/L or less, and even more preferably 800 mOsm/L or less.
• the osmotic pressure of the mixed solution is above the above lower limit and below the above upper limit, the effects of the present invention can be more effectively achieved.
• the osmotic pressure of the above mixture is measured by the freezing point depression method using an osmometer (e.g., Arkray's "OM-6060").
• the blood collection container is preferably one that can collect 3 mL or more of blood per 1 mL of the aqueous solution contained in the blood collection container body, more preferably one that can collect 4 mL or more of blood, preferably one that can collect 11 mL or less of blood, and even more preferably one that can collect 10 mL or less of blood.
• the blood will not be excessively diluted, and the effects of the present invention can be achieved even more effectively.
• the blood collection container is preferably a blood collection tube.
• the blood collection container body is preferably a blood collection tube body.
• the blood collection container is preferably used to separate plasma from blood.
• the blood collection container is also preferably used to separate extracellular free nucleic acids or extracellular vesicles in blood, and is preferably used to isolate extracellular free nucleic acids or extracellular vesicles in blood.
• the extracellular free nucleic acids may be cell-free DNA (cfDNA) or cell-free RNA (cfRNA).
• the extracellular free nucleic acids are preferably cfDNA.
• the above-mentioned blood collection container can be manufactured, for example, as follows.
• An anticoagulant and lithium salt A are dissolved in water to obtain an aqueous solution. If necessary, other components are also dissolved in water to obtain an aqueous solution. The resulting aqueous solution is added to the blood collection container body. Before or after adding the aqueous solution, the plasma separator is placed in the blood collection container body.
• 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.
• 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 plasma separation composition 3 and the aqueous solution 4 are each contained within the blood collection container body 2.
• the blood collection container body 2 has an open end 2a and a closed end 2b.
• the open end 2a corresponds to one end in the lengthwise direction of the blood collection container body 2
• the closed end 2b corresponds to the other end (bottom side) in the lengthwise direction of the blood collection container body 2.
• the plasma separation composition 3 is contained at the bottom of the blood collection container body 2.
• the aqueous solution 4 contains an anticoagulant and lithium salt A.
• the stopper 5 is inserted into the open end 2a of the blood collection container body 2.
• the aqueous solution 4 is disposed on the surface of the plasma separation composition 3; more specifically, when the blood collection container 1 is in an upright position, it is disposed on the upper surface of the plasma separation composition 3 (the surface on the open end 2a side of the blood collection container body 2). When the blood collection container 1 is in an upright position, the aqueous solution 4 is disposed on the surface of the plasma separation composition 3.
• 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.
• the plasma separation tool may be used instead of the plasma separation composition.
• 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, with the interior evacuated and sealed with the stopper.
• the internal pressure of the vacuum blood collection tube is reduced so that a predetermined amount of blood can be collected.
• a vacuum blood collection tube When using a vacuum blood collection tube, a fixed amount of blood can be easily collected regardless of the skill level of the blood collector.
• the inside of the blood collection container be sterilized in accordance with ISO or JIS standards.
• the blood collection container can be used to separate plasma from blood.
• the method for separating plasma according to the present invention preferably includes a step of centrifuging the blood collection container in which blood has been collected. More preferably, the method for separating plasma according to the present invention includes a step of collecting blood in the blood collection container described above, and a step of centrifuging the blood collection container in which the blood has been collected.
• the plasma separation method according to the present invention preferably includes a step of mixing the collected blood with the aqueous solution between the blood collection step and the centrifugation step.
• methods for mixing the collected blood with the aqueous solution include mixing by inversion.
• centrifugation conditions for the centrifugation step are not particularly limited, as long as a partition can be formed using the plasma separation material, separating plasma from blood cells.
• centrifugation conditions include centrifugation at 400 G or higher and 4000 G or lower for 10 minutes or longer and 120 minutes or shorter.
• the method for separating extracellular free nucleic acids according to the present invention preferably comprises the steps of centrifuging the blood collection container containing collected blood to separate plasma from the blood, and separating extracellular free nucleic acids from the separated plasma. More preferably, the method for separating extracellular free nucleic acids according to the present invention comprises the steps of collecting blood into the blood collection container described above, centrifuging the blood collection container containing collected blood to separate plasma from the blood, and separating extracellular free nucleic acids from the separated plasma.
• the method for separating extracellular vesicles according to the present invention preferably comprises the steps of centrifuging the blood collection container into which blood has been collected to separate plasma from the blood, and separating extracellular vesicles from the separated plasma.
• the method for separating extracellular vesicles according to the present invention more preferably comprises the steps of collecting blood in the blood collection container 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.
• 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.
• methods for mixing the collected blood with the aqueous solution include mixing by inversion.
• centrifugation conditions for the centrifugation step are not particularly limited, as long as a partition can be formed using the plasma separation material, separating plasma from blood cells.
• centrifugation conditions include centrifugation at 400 G or higher and 4000 G or lower for 10 minutes or longer and 120 minutes or shorter.
• nucleic acid purification kits include the QIAamp Circulating Nucleic Acid Kit (manufactured by QIAGEN), QIAamp MinElute ccfDNA Kits (manufactured by QIAGEN), and MagMAX Cell-Free DNA Isolation Kit (manufactured by Applied biosystems).
• the extracellular vesicles can be separated from the plasma using conventional methods.
• (Meth)acrylic resin 2-Ethylhexyl acrylate and butyl acrylate were radically polymerized by solution polymerization in the presence of an azo-based polymerization initiator to obtain a (meth)acrylate polymer having fluidity at 25°C.
• Trimellitic acid ester (benzenepolycarboxylic acid alkyl ester derivative, "Monocizer W700” manufactured by DIC Corporation)
• 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 (co-solvent)
• compositions A and B for plasma separation were prepared by mixing an organic component having fluidity at 25°C, an inorganic fine powder, and other components in the proportions shown in Table 1.
• Preparation of plasma separation composition C The materials for the organic component having fluidity at 25° C. listed in Table 1 were blended, heated to 130° C. 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.
• One drop of the obtained plasma separation composition was dropped into saline solution at 25°C, the specific gravity of which had been adjusted in increments of 0.002, and the specific gravity was measured by floating or sinking in the saline.
• the specific gravities of the obtained plasma separation compositions at 25°C are shown in the table below.
• Lithium Salt A Lithium chloride monohydrate (molecular weight in anhydrous state: 42) Lithium acetate dihydrate (molecular weight in anhydrous state: 66) Lithium citrate (trilithium citrate tetrahydrate, molecular weight in anhydrous state: 210) Lithium L-lactate (molecular weight in anhydrous state: 96)
• a PET (polyethylene terephthalate) tube with a bottom, measuring 100 mm in length (distance between the open and closed ends) and an inner diameter of 14 mm at the open end.
• Example 1 The components shown in Table 2 were dissolved in water to obtain aqueous solutions. The types and concentrations of the components in the resulting aqueous solutions are shown in Table 2.
• 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.
• Examples 2 to 12 and Comparative Examples 2 to 6 The type of plasma separation composition and the composition of the aqueous solution were changed as shown in the table below. Except for these, blood collection containers were produced in the same manner as in Example 1.
• a mixed solution was obtained by dissolving 24 parts by weight of dipotassium ethylenediaminetetraacetate dihydrate in 76 parts by weight of water.
• 1.2 g of plasma separation composition A was placed in the bottom of a blood collection container body.
• 60 mg of the mixed solution was applied to the inner wall surface of the blood collection container body and allowed to dry.
• the pressure inside the blood collection container was reduced so that the blood collection volume was 8 mL, and the container was sealed with a butyl rubber stopper. In this manner, a blood collection container was prepared.
• Osmotic Pressure of Mixed Solution 8 mL of saline was collected in a blood collection container. After collecting the saline, the container was inverted to mix the saline and the aqueous solution contained in the blood collection container (Examples 1 to 12 and Comparative Examples 2 to 6), 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.).
• the DNA contained in the collected plasma was purified using a cfDNA purification kit (QIAGEN's "QIAamp Circulating Nucleic Acid Kit”). The DNA purification procedure was performed on the day the plasma was collected from the blood collection container.
• the DNA concentration in the purified extract was measured using the Qubit dsDNA HS Assay kit (Invitrogen).
• the cfDNA concentration (cfDNA content per mL of plasma) was then calculated using the following formula, and designated as the "cfDNA concentration (A)."
• cfDNA concentration [A] x [B]/[C]
• the concentration of the anticoagulant is the concentration of EDTA2K, not the concentration of EDTA2K ⁇ 2H 2 O, and the concentration of lithium salt A is the concentration in an anhydrous state.
• cfDNA concentration (2) indicates the amount of DNA in the plasma that increased due to leakage of genomic DNA from white blood cells contaminating the plasma during the five-day storage period of the sample after centrifugation.
• the aqueous solution contains an anticoagulant and lithium salt A, the amount of white blood cells contaminating the plasma can be reduced even when the blood is centrifuged two days after collection, and it can be seen that this reduces the contamination of genomic DNA into the plasma during subsequent sample storage.
• “Increase in cfDNA concentration (3)” indicates the increase in cfDNA from the day of blood collection, and is the sum of the genomic DNA that leaked from white blood cells over the two days from blood collection to centrifugation, and the genomic DNA that leaked from white blood cells that mixed into the plasma when the blood was centrifuged two days after collection and then during sample storage.
• the aqueous solution contains specific lithium salt A, these effects are exerted synergistically, and it can be seen that the increase in cfDNA from the day of blood collection is further suppressed.
• Blood collection container 2 Blood collection container body 2a
• Open end 2b Opend end 3
• Composition for plasma separation 4 ...
• Aqueous solution 5 ... Stopper

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