Mononuclear cell-containing plasma separation composition and blood collection container

Abstract

Provided is a mononuclear cell-containing plasma separation composition which is capable of suppressing the flow of a mononuclear cell-containing plasma separation composition during storage, is capable of satisfactorily forming partition walls during centrifugal separation, and is capable of obtaining mononuclear cell-containing plasma in which a small amount of blood cell components other than mononuclear cells are mixed. The mononuclear cell-containing composition for plasma separation according to the present invention contains an organic component having fluidity at 25 DEG C and two or more inorganic fine powders, and has a specific gravity of 1.060-1.080 (inclusive) at 25 DEG C.

Description

[0001] This application is a divisional application of the invention patent application with the application number 202080007051.0, the application date of May 19, 2020, and the invention title of "Composition for Separating Plasma Containing Mononuclear Cells and Blood Collection Container". Technical Field

[0002] The present invention relates to a composition for separating plasma containing mononuclear cells for separating plasma containing mononuclear cells from blood. In addition, the present invention relates to a blood collection container in which the composition for separating plasma containing mononuclear cells is accommodated in a blood collection container body. Background Art

[0003] In clinical examinations, blood collection containers are widely used to collect blood samples. In a blood collection container containing a composition for separating serum or plasma, by appropriately adjusting the specific gravity of the separating composition, centrifugal separation can be performed using the specific gravity difference to separate serum or plasma from whole blood.

[0004] The following Patent Document 1 discloses a composition for separating serum or plasma, which contains a liquid organic compound, a thixotropy-imparting component, and a thermoplastic elastomer. In addition, in all the examples of Patent Document 1, a composition for separating serum or plasma with a specific gravity of 1.043 to 1.045 at 25°C was prepared. The composition for separating serum or plasma of Patent Document 1 has thixotropy. Therefore, for example, the composition for separating serum or plasma accommodated at the bottom of the blood collection container body can be prevented from flowing easily to the opening end side of the blood collection container body.

[0005] In addition, a separating composition capable of separating specific blood cell components from blood is known.

[0006] The following Patent Document 2 discloses a method for separating platelets, lymphocytes, and mononuclear large white blood cells (monocytes) from anticoagulated blood, which includes the following steps (a), (b), and (c). (a) A water-insoluble thixotropic gel-like substance (separating composition) having a specific gravity of 1.065 to 1.077 g / cc and being chemically inactive with respect to blood components is added to an anticoagulated blood sample. (b) The gel-like substance and the blood sample are centrifuged at a force of at least 1200G for a sufficient time to form a barrier between the gel-like substance and the plasma, platelets, lymphocytes, mononuclear large white blood cells (monocytes), and heavier blood cells. (c) The plasma, platelets, lymphocytes, and mononuclear large white blood cells are recovered from the barrier. In addition, in Patent Document 2, silica powder is used as the inorganic fine powder contained in the water-insoluble thixotropic gel-like substance (separating composition).

[0007] Prior art documents

[0008] Patent documents

[0009] Patent document 1: WO2016 / 199851A1

[0010] Patent document 2: Japanese Patent Laid-Open No. 02-111374 Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] In clinical examinations, examinations using mononuclear cells (lymphocytes and monocytes) are being carried out. However, when using a sample solution containing blood cell components other than mononuclear cells (for example, red blood cells and granulocytes, etc.) to perform an examination using mononuclear cells, it sometimes affects the examination results.

[0013] In the conventional plasma separation composition as described in Patent Document 1, it was not intended to separate mononuclear cells and blood cell components other than mononuclear cells. Therefore, when using the conventional plasma separation composition to separate plasma, the number of mononuclear cells contained in the obtained plasma is extremely small, and it is difficult to use this plasma as a sample solution for an examination using mononuclear cells.

[0014] On the other hand, in the case of the water-insoluble thixotropic gel-like substance (separation composition) described in Patent Document 2, it is possible to separate platelets, lymphocytes, and mononuclear large white blood cells (monocytes) from blood. However, in the case of using only silica powder as the inorganic fine powder to increase the specific gravity of the separation composition as described in Patent Document 2, the viscosity and thixotropic index of this separation composition become too high, and this separation composition cannot exhibit sufficient fluidity during centrifugation, and it is difficult to form a partition wall well. It should be noted that in Patent Document 2, by arranging the water-insoluble thixotropic gel-like substance (separation composition) on the liquid surface of the collected blood after blood collection, the formability of the partition wall can be improved to a certain extent, but the operation of opening the blood collection container after blood collection and arranging the separation composition on the liquid surface of the blood is not practical in clinical examinations.

[0015] In addition, in the conventional separation composition, the separation composition sometimes flows during storage. For example, in the conventional separation composition, the separation composition accommodated at the bottom of the blood collection container main body sometimes flows to the opening end side of the blood collection container main body. When the separation composition flows, the blood collection needle is sometimes blocked by the separation composition during blood collection, resulting in poor blood collection.

[0016] An object of the present invention is to provide a composition for mononuclear cell-containing plasma separation that can suppress the flow of the composition for mononuclear cell-containing plasma separation during storage, can form a partition wall well during centrifugation, and can obtain a mononuclear cell-containing plasma with a small amount of contamination of blood cell components other than mononuclear cells. In addition, an object of the present invention is to provide a blood collection container containing the composition for mononuclear cell-containing plasma separation.

[0017] Technical means for solving the problem

[0018] According to a broad aspect of the present invention, there is provided a composition for mononuclear cell-containing plasma separation, which comprises: an organic component having fluidity at 25°C and two or more kinds of inorganic fine powders, and the specific gravity of the composition at 25°C is 1.060 or more and 1.080 or less.

[0019] In a specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the organic component having fluidity at 25°C contains a resin.

[0020] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the organic component having fluidity at 25°C is a mixture of the resin and an alkyl benzenepolycarboxylate derivative.

[0021] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the resin contains a petroleum resin, a cyclopentadiene resin or a polyester resin.

[0022] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the resin contains a petroleum resin or a cyclopentadiene resin.

[0023] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the inorganic fine powder contains silica fine powder.

[0024] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the inorganic fine powder contains silica fine powder and an inorganic fine powder having a specific gravity of 3 or more.

[0025] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the silica fine powder contains hydrophilic silica.

[0026] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the silica fine powder contains hydrophilic silica and hydrophobic silica.

[0027] In another specific aspect of the composition for mononuclear cell-containing plasma separation of the present invention, the composition for mononuclear cell-containing plasma separation contains an organic gelling agent.

[0028] According to a broad aspect of the present invention, there is provided a blood collection container including a blood collection container main body and the composition for separating mononuclear cell-containing plasma, and the composition for separating mononuclear cell-containing plasma is accommodated in the blood collection container main body.

[0029] Effects of the Invention

[0030] The composition for separating mononuclear cell-containing plasma of the present invention includes: an organic component having fluidity at 25°C and two or more kinds of inorganic fine powders, and the specific gravity of the composition at 25°C is 1.060 or more and 1.080 or less. Since the composition for separating mononuclear cell-containing plasma of the present invention has the above-described configuration, it is possible to suppress the flow of the composition for separating mononuclear cell-containing plasma during storage, to form a partition wall well during centrifugation, and to obtain mononuclear cell-containing plasma with a small amount of contamination of blood cell components other than mononuclear cells. Detailed Embodiments

[0031] Hereinafter, the present invention will be described in detail.

[0032] The composition for separating mononuclear cell-containing plasma of the present invention includes an organic component having fluidity at 25°C and two or more kinds of inorganic fine powders. The specific gravity of the composition for separating mononuclear cell-containing plasma of the present invention at 25°C is 1.060 or more and 1.080 or less.

[0033] In the case of the composition for separating mononuclear cell-containing plasma of the present invention, since it has the above-described configuration, it is possible to suppress the flow of the composition for separating mononuclear cell-containing plasma during storage, to form a partition wall well during centrifugation, and to obtain mononuclear cell-containing plasma with a small amount of contamination of blood cell components other than mononuclear cells.

[0034] In the case of the composition for separating mononuclear cell-containing plasma of the present invention, by collecting blood into a blood collection container containing the composition for separating mononuclear cell-containing plasma and performing centrifugation, a partition wall can be formed by the composition for separating mononuclear cell-containing plasma, and the mononuclear cell-containing plasma can be separated above the partition wall. The composition for separating mononuclear cell-containing plasma of the present invention can simply and rapidly obtain plasma containing mononuclear cells (lymphocytes and monocytes) from blood. In the present invention, since mononuclear cells can be separated from blood by simple centrifugation, damage to mononuclear cells can be suppressed.

[0035] In the case of the composition for mononuclear cell-containing plasma separation of the present invention, the specific gravity at 25°C is in the range of 1.060 or more and 1.080 or less, and the viscosity and thixotropic index of the composition for mononuclear cell-containing plasma separation can be improved. Therefore, the flow of the composition for mononuclear cell-containing plasma separation during storage can be suppressed. In addition, even when the composition for mononuclear cell-containing plasma separation is accommodated at the bottom of a blood collection main body, for example, the composition for mononuclear cell-containing plasma separation can exhibit good fluidity during centrifugation and can form a partition wall well.

[0036] In the case of the composition for mononuclear cell-containing plasma separation of the present invention, using the obtained mononuclear cell-containing plasma, mononuclear cells can be analyzed with good accuracy.

[0037] The specific gravity at 25°C of the composition for mononuclear cell-containing plasma separation is 1.060 or more and 1.080 or less. When the specific gravity is less than 1.060 or exceeds 1.080, it may not be possible to separate mononuclear cell-containing plasma well from blood, or the amount of contamination of blood cell components other than mononuclear cells may increase, or the recovery rate of mononuclear cells may decrease.

[0038] The specific gravity at 25°C of the composition for mononuclear cell-containing plasma separation is preferably more than 1.060, more preferably 1.065 or more, preferably less than 1.080, more preferably 1.077 or less, and further preferably 1.070 or less. When the specific gravity satisfies the above preferred range, mononuclear cell-containing plasma can be separated well from blood, and plasma with a small amount of contamination of blood cell components other than mononuclear cells and a large amount of mononuclear cells can be obtained. In particular, when the specific gravity is 1.065 or more, the recovery rate of mononuclear cells can reach 70% or more. In addition, in particular, when the specific gravity is 1.070 or less, mononuclear cell-containing plasma with an extremely small amount of contamination of granulocytes can be appropriately obtained. For example, when the specific gravity is 1.070 or less, the removal rate of granulocytes can reach 90% or more. In addition, when the specific gravity satisfies the above preferred range, a partition wall with good strength can be formed even at low temperatures or under low centrifugal forces. In addition, when the specific gravity is at least the lower limit, the recovery rate of mononuclear cells can be increased.

[0039] The specific gravity at 25°C of the composition for mononuclear cell-containing plasma separation is measured by sequentially dropping one drop of the composition for mononuclear cell-containing plasma into 25°C saline with the specific gravity adjusted in a gradient of 0.002 and observing the floating and sinking in the saline.

[0040] The viscosity of the composition for mononuclear cell-containing plasma separation at 25°C is preferably 100 Pa·s or more, more preferably 150 Pa·s or more, preferably 400 Pa·s or less, and more preferably 300 Pa·s or less. When the viscosity is above the lower limit and below the upper limit, the effects of the present invention can be further effectively exerted.

[0041] The viscosity of the composition for mononuclear cell-containing plasma separation at 25°C is measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 second -1 under the conditions.

[0042] The thixotropic index of the composition for mononuclear cell-containing plasma separation at 25°C is preferably 1.1 or more, more preferably 1.2 or more, preferably 1.5 or less, and more preferably 1.3 or less. When the thixotropic index is above the lower limit and below the upper limit, the flow of the composition for mononuclear cell-containing plasma separation during storage can be further effectively suppressed. In addition, even when the composition for mononuclear cell-containing plasma separation is accommodated at the bottom of the blood collection body, for example, the composition for mononuclear cell-containing plasma separation can exhibit good fluidity during centrifugation and further form a partition wall well.

[0043] The thixotropic index of the composition for mononuclear cell-containing plasma separation at 25°C is obtained as follows. The viscosity value of the composition for mononuclear cell-containing plasma separation measured using an E-type viscometer at 25°C and a shear rate of 1.0 second -1 under the conditions is set as the first viscosity value. The viscosity value of the composition for mononuclear cell-containing plasma separation measured using an E-type viscometer at 25°C and a shear rate of 2.0 second -1 under the conditions is set as the second viscosity value. The ratio of the first viscosity value to the second viscosity value (first viscosity value / second viscosity value) is set as the thixotropic index of the composition for mononuclear cell-containing plasma separation at 25°C. It should be noted that as the E-type viscometer, for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd. can be cited.

[0044] When separating mononuclear cell-containing plasma from blood using the composition for mononuclear cell-containing plasma separation, the ratio of the number of red blood cells contained in the separated mononuclear cell-containing plasma to the number of red blood cells contained in the blood before separation (number of red blood cells contained in the separated mononuclear cell-containing plasma / number of red blood cells contained in the blood before separation × 100) is set as ratio (1). Ratio (1) is preferably 10% or less, more preferably 5% or less, and further preferably 1% or less.

[0045] When separating mononuclear cell-containing plasma from blood using the composition for separating mononuclear cell-containing plasma, the ratio of the number of granulocytes contained in the separated mononuclear cell-containing plasma to the number of granulocytes contained in the blood before separation (the number of granulocytes contained in the separated mononuclear cell-containing plasma / the number of granulocytes contained in the blood before separation × 100) is defined as ratio (2). Ratio (2) is preferably 30% or less, more preferably 20% or less, and still more preferably 10% or less.

[0046] When separating mononuclear cell-containing plasma from blood using the composition for separating mononuclear cell-containing plasma, the ratio of the number of mononuclear cells contained in the separated mononuclear cell-containing plasma to the number of mononuclear cells contained in the blood before separation (the number of mononuclear cells contained in the separated mononuclear cell-containing plasma / the number of mononuclear cells contained in the blood before separation × 100) is defined as ratio (3). Ratio (3) is preferably 50% or more, more preferably 60% or more, and still more preferably 70% or more.

[0047] Hereinafter, details of the components contained in the composition for separating mononuclear cell-containing plasma of the present invention will be described.

[0048] <Organic component having fluidity at 25°C>

[0049] The composition for separating mononuclear cell-containing plasma of the present invention contains an organic component having fluidity at 25°C (hereinafter, sometimes simply referred to as "organic component"). As the organic component, one kind can be used alone, or two or more kinds can be used in combination.

[0050] The "having fluidity at 25°C" means that the viscosity at 25°C is 500 Pa·s or less.

[0051] The viscosity of the organic component at 25°C is preferably 30 Pa·s or more, more preferably 50 Pa·s or more, preferably 200 Pa·s or less, and more preferably 100 Pa·s or less. When the viscosity is above the lower limit and below the upper limit, the fluidity of the organic component can be improved, the fluidity of the composition for separating mononuclear cell-containing plasma can be increased, and the strength of the partition wall can be enhanced.

[0052] 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 the conditions of 25°C and a shear rate of 1.0 second -1 conditions.

[0053] Examples of the organic component include resins and mixtures of organic compounds such as resins and plasticizers. When the organic component is a mixture of the resin and the organic compound, it is sufficient for the mixture (the organic component) to have fluidity, and the resin or the organic compound may not have fluidity. When the organic component is a mixture of the resin and the organic compound, the resin can be, for example, a solid resin at 25°C. The resin and the organic compound can each be used alone or in combination of two or more.

[0054] From the viewpoint of further effectively exerting the effects of the present invention, the organic component preferably contains the resin, and more preferably is a mixture of the resin and the organic compound. When the organic component contains two or more components (including the case where the organic component contains a solid component and a liquid component), these components can be separately mixed in the manufacturing step of the composition for mononuclear cell plasma separation.

[0055] Examples of the resin include petroleum resins, cyclopentadiene resins, polyester resins, polyurethane resins, (meth)acrylic resins, silicone resins, α-olefin-fumarate copolymers, copolymers of sebacic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane resins, and polyether polyester resins. The resin can be used alone or in combination of two or more.

[0056] Examples of the petroleum resin include resins obtained by steam cracking of petroleum. Examples of the petroleum resin include homopolymers or copolymers of compounds (such as cyclopentadiene, isoprene, piperylene, 2-methylbutene-1, and 2-methylbutene-2) contained in the C 5 fraction; homopolymers or copolymers of compounds (such as styrene, vinyltoluene, α-methylstyrene, indene, and coumarone) contained in the C 9 fraction; copolymers formed from compounds contained in the C 5 fraction and compounds contained in the C 9 fraction, etc. The petroleum resin can be an unhydrogenated resin, a partially hydrogenated resin, or a fully hydrogenated resin.

[0057] Examples of commercially available products of the petroleum resin include "RIGARAITOS5090" manufactured by EASTMAN CHEMICALS.

[0058] Examples of the cyclopentadiene resin include polymers of cyclopentadiene monomers, copolymers of cyclopentadiene monomers and aromatic monomers, and dicyclopentadiene resins. The cyclopentadiene resin may or may not be hydrogenated. The polymers of cyclopentadiene monomers and the copolymers of cyclopentadiene monomers and aromatic monomers may be oligomers.

[0059] Examples of the cyclopentadiene monomer include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0060] Examples of the aromatic monomer include styrene, methylstyrene, indene, and methylindene.

[0061] Examples of commercially available products of the polymers of cyclopentadiene monomers include "ESCOREZ5380", "ESCOREZ5300", "ESCOREZ5320", "ESCOREZ5340", "ESCOREZ5400", and "ESCOREZ ECR251" manufactured by EXXON MOBIL Corporation.

[0062] Examples of commercially available products of the copolymers of cyclopentadiene monomers and aromatic monomers include "ESCOREZ ECR227E", "ESCOREZ ECR235E", "ESCOREZ ECR231C", "ESCOREZ5690", and "ESCOREZ5600" manufactured by EX XONMOBIL Corporation.

[0063] Examples of commercially available products of the dicyclopentadiene resins include "SUKORET TSU (Sukoretsu) SU500" and "SUKORETTSU SU90" manufactured by COLON Corporation.

[0064] Examples of the polyester resin include polyalkylene terephthalate resins and polyalkylene naphthalate resins. Examples of the polyalkylene terephthalate resin include polyethylene terephthalate, polybutylene terephthalate, and poly-1,4-cyclohexanedimethylene terephthalate.

[0065] Examples of the polyurethane resin include reaction products of polyol compounds and isocyanate compounds.

[0066] Examples of the (meth)acrylic resin include resins obtained by polymerizing at least one (meth)acrylate monomer, and resins obtained by polymerizing at least one (meth)acrylate monomer and at least one monomer other than the (meth)acrylate monomer.

[0067] Examples of the (meth)acrylate monomer include alkyl (meth)acrylates having an alkyl group with 1 to 20 carbon atoms, polyalkylene glycol (meth)acrylates, alkoxyalkyl (meth)acrylates, hydroxyalkyl (meth)acrylates, glycidyl (meth)acrylates, dialkylaminoalkyl (meth)acrylates, benzyl (meth)acrylates, phenoxyalkyl (meth)acrylates, cyclohexyl (meth)acrylates, isobornyl (meth)acrylates, and alkoxysilylalkyl (meth)acrylates. As the (meth)acrylate monomer, only one kind may be used, or two or more kinds may be used in combination.

[0068] From the viewpoint of further effectively exerting the effects of the present invention, the resin preferably contains a petroleum resin, a cyclopentadiene resin, a polyester resin, or a polyurethane resin, more preferably contains a petroleum resin, a cyclopentadiene resin, or a polyester resin, and further preferably contains a petroleum resin or a cyclopentadiene resin.

[0069] From the viewpoint of further effectively exerting the effects of the present invention, the organic compound is preferably an alkyl benzenepolycarboxylate derivative. Therefore, the organic component is preferably a mixture of the resin and the alkyl benzenepolycarboxylate derivative.

[0070] Examples of the alkyl benzenepolycarboxylate derivative include phthalates, trimellitates, and pyromellitates. As the alkyl benzenepolycarboxylate derivative, only one kind may be used, or two or more kinds may be used in combination.

[0071] Examples of the trimellitate include tri-n-octyl trimellitate, tri-iso-octyl trimellitate, and tri-iso-decyl trimellitate.

[0072] Examples of the pyromellitate include tetra-iso-octyl pyromellitate.

[0073] Examples of commercially available products of the trimellitate include "MONOSIZER W700" and "MONOSIZER W-750" manufactured by DIC Corporation, and "SANSOSIZER TOTM" and "SANSOSIZER TITM" manufactured by Shin Nippon Rika Co., Ltd.

[0074] Examples of commercially available products of the pyromellitate include "MONOSIZER W-7010" manufactured by DIC Corporation.

[0075] The alkyl benzenepolycarboxylate derivative is preferably a phthalate, a trimellitate, or a pyromellitate, and more preferably a trimellitate.

[0076] In addition, as the organic component, a liquid mixture of poly-α-pinene polymer and chlorinated hydrocarbon, a liquid mixture of chlorinated polybutene and epoxidized animal and vegetable oil, a liquid mixture of vinylidene chloride trifluoride or an alkyl ester derivative of benzene polycarboxylic acid and polyoxyalkylene glycol, a mixture of a liquid / liquid or solid / liquid combination containing a petroleum resin or a dicyclopentadiene resin and an alkyl ester derivative of benzene polycarboxylic acid, etc. can be cited.

[0077] The specific gravity of the organic component at 25°C is preferably 1.020 or more, more preferably 1.030 or more, preferably 1.080 or less, more preferably 1.060 or less, and further preferably 1.050 or less. When the specific gravity is below the upper limit, the specific gravity of the composition for mononuclear cell plasma separation can be prevented from being too high, and the partition wall can be formed well. When the specific gravity is above the lower limit, the specific gravity of the composition for mononuclear cell plasma separation can be adjusted well without adding a large amount of inorganic fine powder. If a large amount of inorganic fine powder is added, the fluidity of the composition for mononuclear cell plasma separation decreases, and sometimes the partition wall cannot be formed well.

[0078] The specific gravity of the organic component at 25°C is measured by sequentially dropping one drop of the organic component into the 25°C saline solution whose specific gravity has been adjusted in a gradient of 0.002 and observing whether it floats or sinks in the saline solution.

[0079] <Inorganic fine powder>

[0080] The composition for mononuclear cell plasma separation of the present invention contains two or more kinds of inorganic fine powders. As the inorganic fine powders, only two kinds can be used, or three or more kinds can be used.

[0081] Examples of the inorganic fine powder include silica fine powder, titanium oxide powder, zinc oxide powder, aluminum oxide powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titanium dioxide powder, zirconium powder, etc.

[0082] From the viewpoint of further effectively exerting the effects of the present invention, the inorganic fine powder preferably contains silica fine powder. That is, it is preferred that one of the two or more kinds of inorganic fine powders is silica fine powder. From the viewpoint of more effectively exerting the effects of the present invention, the inorganic fine powder more preferably contains silica fine powder and an inorganic fine powder different from the silica fine powder. The inorganic fine powder different from the silica fine powder is preferably an inorganic fine powder having a specific gravity of 3 or more. When only silica fine powder is used as the inorganic fine powder and the specific gravity of the composition for separating mononuclear cell-containing plasma is adjusted to be 1.060 or more and 1.080 or less, a large amount of silica fine powder needs to be mixed, so that the viscosity and thixotropic index of the composition for separating mononuclear cell-containing plasma may be excessively increased. By using a combination of silica fine powder and an inorganic fine powder different from silica fine powder and having a specific gravity of 3 or more as the inorganic fine powder, it is possible to easily control the specific gravity of the composition for separating mononuclear cell-containing plasma while appropriately maintaining the viscosity and thixotropic index of the composition for separating mononuclear cell-containing plasma.

[0083] Examples of the silica fine powder include natural silica and synthetic silica. Examples of the synthetic silica include hydrophilic silica and hydrophobic silica. From the viewpoint of stable quality, the silica fine powder is preferably synthetic silica, and more preferably synthetic silica prepared by the vapor phase method. In the present invention, the silica fine powder is counted as one kind of inorganic fine powder.

[0084] Hydrophilic silica has the function of imparting thixotropy to the composition for separating mononuclear cell-containing plasma by forming hydrogen bonds between the hydroxyl groups on the particle surface and at the same time adjusting the specific gravity. On the other hand, hydrophobic silica has a smaller effect of imparting thixotropy compared to hydrophilic silica.

[0085] From the viewpoint of keeping both the specific gravity and thixotropy of the composition for separating mononuclear cell-containing plasma within an appropriate range, the silica fine powder preferably contains hydrophilic silica, and more preferably contains hydrophilic silica and hydrophobic silica. The silica fine powder preferably contains at least hydrophilic silica.

[0086] In 100% by weight of the composition for mononuclear cell-containing plasma separation, the content of hydrophilic silica is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, further preferably 0.3% by weight or more, preferably 2.50% by weight or less, and more preferably 2.00% by weight or less. When the content of the hydrophilic silica is above the lower limit and below the upper limit, the specific gravity and thixotropy of the composition for mononuclear cell-containing plasma separation can be further maintained within an appropriate range. When the content of the hydrophilic silica exceeds 2.50% by weight, compared with the case of 2.50% by weight or less, the viscosity and thixotropy index of the composition for mononuclear cell-containing plasma separation may increase excessively, and the partition wall may not be formed well during centrifugation.

[0087] The average particle size of the silica fine powder is not particularly limited. The average particle size of the silica fine powder can be 1 nm or more, can be 10 nm or more, can be 500 nm or less, and can be 100 nm or less.

[0088] The average particle size of the silica fine powder and the inorganic fine powder different from the silica fine powder described below is the average particle size measured on a volume basis, which is the value of the median particle size (D50) of 50%. The volume average particle size (D50) can be measured by methods such as laser diffraction / scattering method, image analysis method, COULTER method, and centrifugal sedimentation method. The volume average particle size (D50) is preferably measured by the laser diffraction / scattering method or the image analysis method.

[0089] Examples of commercially available products of the hydrophilic silica include AEROSIL series (manufactured by Nippon Aerosil Co., Ltd.) such as AEROSIL (registered trademark) 90G, 130, 200, 300, 200CF, 300CF, etc., REOLOSIL series (manufactured by Tokuyama Corporation) such as REOLOSIL (registered trademark) QS-10, QS-20, QS-30, etc., and WACKER HDK series (manufactured by Wacker Asahikasei Silicone Co., Ltd.) such as WACKER HDK S13, N20, T30, etc. The commercially available products of the hydrophilic silica are hydrophilic silica prepared by the gas phase method and are easy to use.

[0090] As commercially available products of the hydrophobic silica, for example, AEROSIL series such as AEROSIL R972, R974, R805, R812, etc. (manufactured by Nippon Aerosil Co., Ltd.), REOLOSIL series such as REOLOSIL MT-10, DM-30S, HM-30S, KS-20S, PM-20, etc. (manufactured by Tokuyama Corporation), WACKER HDK series such as WACKER HDK H15, H18, H30, etc. (manufactured by Wacker Asahikawa Silicone Co., Ltd.) can be cited. The commercially available products of the hydrophobic silica are hydrophobic silica prepared by the vapor phase method and are easy to use.

[0091] As the inorganic fine powder different from the silica fine powder and having a specific gravity of 3 or more, titanium oxide powder, zinc oxide powder, aluminum oxide powder, etc. can be cited.

[0092] From the viewpoint of maintaining thixotropy and specific gravity within an appropriate range, the inorganic fine powder having a specific gravity of 3 or more is preferably titanium oxide powder or zinc oxide powder.

[0093] The specific gravity of the inorganic fine powder having a specific gravity of 3 or more is preferably 3.5 or more, more preferably 4 or more. The larger the specific gravity of the inorganic fine powder having a specific gravity of 3 or more, the better. When the specific gravity is at least the lower limit, the specific gravity of the composition for mononuclear cell-containing plasma separation can be effectively increased.

[0094] The average particle diameter of the inorganic fine powder having a specific gravity of 3 or more is not particularly limited. The average particle diameter of the inorganic fine powder having a specific gravity of 3 or more can be 10 nm or more, can be 100 nm or more, can be 10 μm or less, and can be 1 μm or less.

[0095] In 100% by weight of the composition for mononuclear cell-containing plasma separation, the content of the inorganic fine powder having a specific gravity of 3 or more is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, further preferably 1% by weight or more, preferably 10% by weight or less, and more preferably 5% by weight or less. When the content of the inorganic fine powder having a specific gravity of 3 or more is at least the lower limit and at most the upper limit, the effects of the present invention can be further effectively exerted. In addition, when the content of the inorganic fine powder having a specific gravity of 3 or more is at least the lower limit and at most the upper limit, the specific gravity of the composition for mononuclear cell-containing plasma separation can be effectively increased.

[0096] <Other components>

[0097] The composition for mononuclear cell plasma separation of the present invention may contain other components other than the above components within the scope not impairing the effects of the present invention. The composition for mononuclear cell plasma separation of the present invention may contain, for example, an organic gelling agent, a thermoplastic elastomer, a polyalkylene glycol, a silicone oil, a co-solvent, an antioxidant, a colorant, water, etc. as the other components. Each of the other components may be used alone or in combination of two or more.

[0098] Examples of the organic gelling agent include dibenzylidene sorbitol, dibenzylidene sorbitol derivatives, fatty acid amides, etc. By containing the organic gelling agent, thixotropy can be further improved.

[0099] Examples of commercially available products of the dibenzylidene sorbitol and dibenzylidene sorbitol derivatives include "GELOL MD" and "GELOL D" manufactured by Shin Nippon Rika Co., Ltd.

[0100] Examples of the thermoplastic elastomer include styrene-butadiene-styrene copolymer (SBS), styrene-isoprene-styrene copolymer (SIS), styrene-ethylene-butene-styrene copolymer (SEBS), styrene-butadiene-butene-styrene copolymer (SBBS), styrene-ethylene-propylene-styrene copolymer (SEPS), styrene-butadiene copolymer (SB), styrene-isoprene copolymer (SI), styrene-ethylene-butene copolymer (SEB), styrene-butadiene-butene copolymer (SBB), styrene-ethylene-propylene copolymer (SEP), and modified products thereof.

[0101] Examples of the polyalkylene glycol include polybutylene glycol, polypropylene glycol, polyoxypropylene glycerol ether, polyoxypropylene sorbitol, polyglycerol, polyoxypropylene diglycerol ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene glycerol ether, polyoxypropylene butyl ether, polyoxypropylene monoether, polyoxypropylene alkyl ether, and modified products thereof.

[0102] Examples of the silicone oil include dimethyl silicone oil, methylphenyl silicone oil, methylhydrogen silicone oil, alkyl-modified silicone oil, aralkyl-modified silicone oil, fluorine-modified silicone oil, polyether-modified silicone oil, amino-modified silicone oil, epoxy-modified silicone oil, phenol-modified silicone oil, carboxyl-modified silicone oil, methacrylate-modified silicone oil, and alkoxy-modified silicone oil.

[0103] Examples of the co-solvent include toluene, N,N-dimethylformamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, etc. By using the co-solvent, for example, it is possible to easily mix an organic gelling agent and a thermoplastic elastomer, etc.

[0104] The composition for mononuclear cell-containing plasma separation may or may not contain water. The water is preferably purified water. The less the content of water, the better.

[0105] <Method for manufacturing a composition for mononuclear cell-containing plasma separation>

[0106] The method for manufacturing the composition for mononuclear cell-containing plasma separation is not particularly limited. The composition for mononuclear cell-containing plasma separation can be manufactured, for example, by mixing the organic component having fluidity at 25°C, the inorganic fine powder, and other components mixed as needed. There is no particular limitation on the mixing method, and it can be mixed by a known kneader such as a planetary mixer, a ball mill, a disperser, etc.

[0107] <Blood collection container>

[0108] The blood collection container of the present invention includes a blood collection container main body and the composition for mononuclear cell-containing plasma separation, and the composition for mononuclear cell-containing plasma separation is accommodated in the blood collection container main body.

[0109] The composition for mononuclear cell-containing plasma separation is preferably accommodated at the bottom of the blood collection container main body. In the present invention, even when the composition for mononuclear cell-containing plasma separation is accommodated at the bottom of the blood collection main body, a partition can be formed well during centrifugation.

[0110] There is no particular limitation on the raw material of the blood collection container main body. For example, thermoplastic resins such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), polymethyl methacrylate, polyacrylonitrile, polyamide, acrylonitrile-styrene copolymer, ethylene-vinyl alcohol copolymer, etc.; thermosetting resins such as unsaturated polyester resin, epoxy resin, epoxy-acrylate resin, etc.; modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, ethyl chitin, etc.; silicates such as soda-lime glass, phosphosilicate glass, borosilicate glass, quartz glass, etc., glass, combinations thereof, or substances mainly composed of these, etc. are known raw materials.

[0111] The blood collection container main body can be sealed by a sealing member such as a stopper or an aluminum seal.

[0112] The internal pressure of the blood collection container is not particularly limited. The blood collection container can be used as a vacuum blood collection tube that is evacuated inside and sealed by the sealing member. In the case of a vacuum blood collection tube, quantitative blood collection can be easily performed regardless of the technical difference of the blood collector.

[0113] From the viewpoint of preventing bacterial infection, the interior of the blood collection container is preferably sterilized with reference to the standards of ISO and JIS.

[0114] On the inner wall of the blood collection container main body, a known reagent such as an anti-blood adhesion agent may be adhered for the purpose of preventing blood adhesion and the like.

[0115] Preferably, an anticoagulant is accommodated in the blood collection container main body. In this case, the anticoagulant may be adhered to the inner wall of the blood collection container main body. It should be noted that the anticoagulant may be added to the collected blood. Examples of the anticoagulant include heparin, ethylenediaminetetraacetic acid (EDTA), and citric acid.

[0116] <Method for Separating Mononuclear Cell-Containing Plasma>

[0117] The mononuclear cell-containing plasma separation composition of the present invention can be used to separate mononuclear cell-containing plasma from blood. The method for separating mononuclear cell-containing plasma preferably includes: a step of collecting blood into the blood collection container main body (blood collection step); and a step of centrifuging the blood collection container containing the blood (centrifugation step).

[0118] In the blood collection step, it is preferable that the anticoagulant is accommodated in the blood collection container main body, or the blood added with the anticoagulant is collected into the blood collection container main body.

[0119] The centrifugation conditions in the centrifugation step are not particularly limited as long as a partition can be formed by the mononuclear cell-containing plasma separation composition and the mononuclear cells can be separated from blood cell components other than mononuclear cells. Examples of the centrifugation conditions include centrifugation conditions of 400 G or more and 4000 G or less for 10 minutes or more and 120 minutes or less. In the centrifugation step, red blood cells and granulocytes are located below the partition formed by the mononuclear cell-containing plasma separation composition, and mononuclear cell-containing plasma is located above.

[0120] Hereinafter, examples will be given to explain the present invention in more detail. The present invention is not limited to the following examples.

[0121] As materials for the mononuclear cell-containing plasma separation composition, the following materials are prepared.

[0122] (Materials of organic components having fluidity at 25°C)

[0123] Resin:

[0124] Petroleum resin ("RIGARAITO S5090" manufactured by EASTMAN CHEMICALS)

[0125] Dicyclopentadiene resin 1 (“SUKORETTSU SU500” manufactured by COLON Co., Ltd.)

[0126] Dicyclopentadiene resin 2 (“SUKORETTSU SU90” manufactured by COLON Co., Ltd.)

[0127] Organic compound:

[0128] Trimellitate (alkyl benzenepolycarboxylate derivative, “MONOSIZER W700” manufactured by DIC Corporation)

[0129] (Inorganic fine powder)

[0130] Hydrophilic silica (silica fine powder, “200CF” manufactured by Nippon Aerosil Co., Ltd., specific gravity 2.2)

[0131] Hydrophobic silica (silica fine powder, “R974” manufactured by Nippon Aerosil Co., Ltd., specific gravity 2.2)

[0132] Titanium oxide powder (“A - 100” manufactured by Ishihara Sangyo Co., Ltd., specific gravity 4)

[0133] Zinc oxide powder (“Zncox Super F - 2” manufactured by HAKUSUI TECH Co., Ltd., specific gravity 5.6)

[0134] (Other components)

[0135] Organic gelling agent (“GELOL D” manufactured by Shin Nippon Rika Co., Ltd.)

[0136] 1 - Methyl - 2 - pyrrolidone (auxiliary solvent)

[0137] Thermoplastic elastomer (“3421C” manufactured by Zeon Corporation, Japan)

[0138] (Example 1)

[0139] Preparation of an organic component having fluidity at 25°C:

[0140] Mix the materials of the organic component having fluidity at 25°C as described in Table 1, heat and dissolve at 130°C, and mix to prepare an organic component having fluidity at 25°C.

[0141] Preparation of a composition for mononuclear cell - rich plasma separation:

[0142] Mix the organic component having fluidity at 25°C, inorganic fine powder, and other components in the mixing ratio described in Table 1 to prepare a composition for mononuclear cell - rich plasma separation.

[0143] (Examples 2 to 5 and Comparative Examples 1 to 3)

[0144] The types and amounts of the mixed components were changed as shown in Table 1. Except for this, the composition for mononuclear cell plasma separation was prepared in the same manner as in Example 1.

[0145] (Evaluation)

[0146] (1) Specific gravity

[0147] One drop of the obtained organic component having fluidity at 25°C or the obtained composition for mononuclear cell plasma separation was successively dropped into the 25°C saline solution whose specific gravity was adjusted in a gradient at intervals of 0.002, and the measurement was carried out according to the floating and sinking in the saline solution.

[0148] (2) Viscosity

[0149] Regarding the viscosity at 25°C of the obtained composition for mononuclear cell plasma separation, it was measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., "TVE-35") under the conditions of 25°C and a shear rate of 1.0 second -1 conditions.

[0150] (3) Thixotropic index

[0151] Using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., "TVE-35"), for the obtained composition for mononuclear cell plasma separation, it was measured under the conditions of 25°C and a shear rate of 1.0 second -1 conditions to obtain the first viscosity value. Similarly, using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., "TVE-35"), for the obtained composition for mononuclear cell plasma separation, it was measured under the conditions of 25°C and a shear rate of 2.0 seconds -1 conditions to obtain the second viscosity value. Calculate the ratio of the first viscosity value to the second viscosity value (first viscosity value / second viscosity value), and set it as the thixotropic index at 25°C of the composition for mononuclear cell plasma separation.

[0152] (4) Flow

[0153] 1.0 g of the obtained composition for mononuclear cell plasma separation was placed at the bottom of a transparent PET bottomed tube (blood collection container main body) with an inner diameter of 14 mm and a length of 100 mm, and the blood collection container was prepared by plugging it with a butyl rubber stopper. Then, the blood collection container was tilted so that the open end side of the blood collection container main body was tilted downward at 45° and the bottom side was tilted upward at 45°, and in this state, it was left standing in an oven maintained at 55°C for 1 day. The distance that the composition for mononuclear cell plasma separation flowed downward along the inner wall of the blood collection container before and after standing was measured using a vernier caliper.

[0154] (5) Formability of partition wall

[0155] Preparation of blood collection container:

[0156] Prepare a PET bottomed tube (blood collection container main body) with a length of 100 mm and an inner diameter of 14 mm at the opening. Accommodate 1.0 g of the mononuclear cell-containing plasma separation composition obtained in the bottom of the blood collection container main body. In addition, attach ethylenediaminetetraacetic acid (EDTA) to the inner wall of the blood collection container main body, reduce the pressure inside the blood collection container to 50 kPa, and seal it with a butyl rubber stopper. Thus, a blood collection container containing the mononuclear cell-containing plasma separation composition in the blood collection container main body was prepared.

[0157] Prepare 3 portions of blood and perform the following steps in sequence.

[0158] Blood collection step:

[0159] Collect 4 mL of blood into the blood collection container main body of the obtained blood collection container.

[0160] Centrifugation step:

[0161] Centrifuge the blood collection container at 1500 G for 30 minutes.

[0162] Visual observation:

[0163] Confirm by naked eye observation whether the mononuclear cell-containing plasma separation composition accommodated in the bottom of the blood collection container main body before centrifugation moves between the red blood cell layer and the plasma layer and forms a partition wall well after centrifugation. Specifically, evaluate the formation of the partition wall by the following method.

[0164] Test (1): Observe the blood collection container after centrifugation with the naked eye. The case where the mononuclear cell-containing plasma separation composition is located between the red blood cell layer and the plasma layer is judged as good. On the other hand, the case where all of the mononuclear cell-containing plasma separation composition is located below the red blood cell layer is judged as bad.

[0165] Test (2): Place the blood collection container after centrifugation stationary in such a way that the bottom of the blood collection container faces upward at 90°. At this time, confirm by naked eye observation whether the blood cell components located more on the bottom side of the blood collection container than the mononuclear cell-containing plasma separation composition move to the side of the opening end of the blood collection container more than the mononuclear cell-containing plasma separation composition, and whether this blood cell component is mixed with the plasma. In the case where this blood cell component is not mixed with the plasma, it is judged as good. On the other hand, in the case where this blood cell component is mixed with the plasma, it is judged as bad.

[0166] [Judgment criteria for formation of partition wall]

[0167] ○: The judgment results of both Test (1) and Test (2) are good.

[0168] ×: The judgment result of Test (1) or Test (2) is bad.

[0169] (6) Recovery of mononuclear cell-containing plasma

[0170] After the centrifugation step in “(5) Formation of partition wall”, the blood cell components located on the partition wall formed by the composition for separating mononuclear cell-containing plasma are suspended in the plasma located above the blood cell components, and the plasma containing the blood cell components is recovered. It should be noted that in Comparative Example 3, no partition wall was formed, so this evaluation was not performed.

[0171] Using a multi-item automatic blood cell analyzer (SYSMEX Corporation's “XE5000”), the recovered liquid was analyzed to measure the cell counts of blood cell components (red blood cells, granulocytes (neutrophils, eosinophils, and basophils), mononuclear cells (lymphocytes and monocytes)) in the separated plasma. In addition, for the prepared blood (whole blood sample), similarly, the cell counts of blood cell components (red blood cells, granulocytes (neutrophils, eosinophils, and basophils), mononuclear cells (lymphocytes and monocytes)) in the whole blood sample were measured. It should be noted that the concentrations of the said blood cell components are the average values of the results obtained by evaluating the blood of 3 individuals prepared separately.

[0172] The removal rates of red blood cells, granulocytes, and the recovery rate of mononuclear cells were calculated respectively by the following formulas.

[0173] Removal rate of red blood cells (%) = 100 - [(number of red blood cells (pieces) contained in the separated plasma) / (number of red blood cells (pieces) contained in the whole blood sample) × 100]

[0174] Removal rate of granulocytes (%) = 100 - [(number of granulocytes (pieces) contained in the separated plasma) / (number of granulocytes (pieces) contained in the whole blood sample) × 100]

[0175] Recovery rate of mononuclear cells (%) = (number of mononuclear cells (pieces) contained in the separated plasma) / (number of mononuclear cells (pieces) contained in the whole blood sample) × 100

[0176] The composition and results are shown in Table 1 below.

[0177]

[0178] In the case of the composition for mononuclear cell plasma separation having a specific gravity of 1.060 or more and 1.080 or less obtained in Examples 1 to 5, it can be considered that the recovery rate of mononuclear cells is relatively high, and the removal rates of red blood cells and granulocytes are relatively high. In contrast, in the case of the composition for mononuclear cell plasma separation having a specific gravity of less than 1.060 obtained in Comparative Example 1, although the removal rates of red blood cells and granulocytes can be increased, the recovery rate of mononuclear cells is relatively low. In addition, in the case of the composition for mononuclear cell plasma separation having a specific gravity of more than 1.080 obtained in Comparative Example 2, although the recovery rate of mononuclear cells can be increased, the removal rates of red blood cells and granulocytes are relatively low.

[0179] In particular, in the case of the composition for mononuclear cell plasma separation having a specific gravity of 1.060 or more and 1.070 or less obtained in Examples 1 to 3, it can be considered that the removal rate of granulocytes can be particularly increased.

[0180] In addition, in the case of the composition for mononuclear cell plasma separation containing two or more kinds of inorganic fine powders (silica fine powder and inorganic fine powder having a specific gravity of 3 or more) obtained in Examples 1 to 5, the viscosity and thixotropic index of the composition for mononuclear cell plasma separation can be improved, the flow of the composition for mononuclear cell plasma separation during storage can be suppressed, and a partition wall can be formed well during centrifugation. In contrast, in the case of the composition for mononuclear cell plasma separation obtained in Comparative Example 3 using only silica fine powder (one kind of inorganic fine powder) as the inorganic fine powder, although the flow of the composition for mononuclear cell plasma separation during storage can be effectively suppressed, it cannot flow sufficiently during centrifugation and a partition wall cannot be formed.

Claims

1. A composition for mononuclear cell - rich plasma separation, comprising: an organic component that is fluid at 25°C, and two or more kinds of inorganic fine powders, wherein the specific gravity of the composition at 25°C is 1.060 or more and 1.080 or less.

2. The composition for mononuclear cell - rich plasma separation according to claim 1, wherein the organic component that is fluid at 25°C contains a resin.

3. The composition for mononuclear cell - rich plasma separation according to claim 2, wherein the organic component that is fluid at 25°C is a mixture of the resin and an alkyl benzenepolycarboxylate derivative.

4. The composition for mononuclear cell - rich plasma separation according to claim 2 or 3, wherein the resin contains a petroleum resin, a cyclopentadiene resin, or a polyester resin.

5. The composition for mononuclear cell - rich plasma separation according to any one of claims 2 - 4, wherein the resin contains a petroleum resin or a cyclopentadiene resin.

6. The composition for mononuclear cell - rich plasma separation according to any one of claims 1 - 5, wherein the inorganic fine powder contains silica fine powder.

7. The composition for mononuclear cell - rich plasma separation according to any one of claims 1 - 6, wherein the inorganic fine powder contains silica fine powder and an inorganic fine powder with a specific gravity of 3 or more.

8. The composition for mononuclear cell - rich plasma separation according to claim 6 or 7, wherein the silica fine powder contains hydrophilic silica.

9. The composition for mononuclear cell - rich plasma separation according to any one of claims 6 - 8, wherein the silica fine powder contains hydrophilic silica and hydrophobic silica.

10. The composition for mononuclear cell - rich plasma separation according to any one of claims 1 - 9, which contains an organic gelling agent.

11. A blood collection container, comprising: a blood collection container main body, and the composition for mononuclear cell - rich plasma separation according to any one of claims 1 - 10, wherein the composition for mononuclear cell - rich plasma separation is accommodated in the blood collection container main body.

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