Analytical blood cell separator and blood cell separator

Abstract

To provide a blood cell separating agent and a blood cell separation method capable of separating blood cells in a short time from a sample containing blood cells collected for analysis. The problem is solved by a blood cell separating agent for analysis that contains a (co)polymer having at least one structural unit of a structure derived from a monoallylamine or a diallylamine having a secondary amino group or a secondary amide group or a structure that is an inorganic acid salt or an organic acid salt thereof.

Description

Technical Field

[0001] The present invention relates to a blood cell separation agent and a blood cell separation method, and more specifically, to a blood cell separation agent and a blood cell separation method capable of rapidly separating blood cells from a sample containing blood cells collected for analysis.

Background Art

[0002] Plasma and serum are widely used as analytical samples for various biochemical or immunological tests, and centrifugation is generally used for separating plasma or serum from blood cells. However, since centrifugation requires a relatively long time, simpler and faster blood cell separation methods that do not require centrifugation have been studied. For example, in order to separate red blood cells from a blood sample, it has been proposed to aggregate red blood cells with a solution containing a cholic acid-based surfactant and an acid (Patent Document 1).

[0003] In addition, a method has been proposed in which a specific quaternary ammonium sulfone polymer is added to collected blood to separate it into serum and a blood clot (Patent Document 2). However, this method requires a blood coagulation time of 30 minutes or more and is insufficient in terms of rapid blood cell separation. In addition, inventions of sanitary products to which a cationic polymer, which is a specific quaternary ammonium salt homopolymer or a quaternary ammonium salt copolymer, is applied have been proposed (Patent Documents 3 and 4). However, the inventions in these documents use the cationic polymer in sanitary products to improve the blood absorption rate in sanitary products, and do not relate to a technique for separating blood cells from a sample collected from a patient for analysis, diagnosis, etc.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

[0005] In view of the prior art described above, the object of the present invention is to provide a blood cell separation agent and a blood cell separation method that can separate blood cells from a sample containing blood cells collected for analysis in a short time. Furthermore, in a preferred embodiment of the present invention, the object is to provide a blood cell separation agent and a blood cell separation method that can separate blood cells from a sample containing blood cells collected for analysis in a short time and can provide a sample that has little impact on the analysis of biochemical or immunological tests. [Means for solving the problem]

[0006] As a result of diligent research, the inventors have discovered that by using a (co)polymer having at least one constituent unit of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic or organic salt thereof, blood cells can be separated in a short time that could not be achieved with prior art, and have completed the present invention. In other words, the present invention is [1] This invention relates to an analytical blood cell separator containing a (co)polymer having at least one constituent unit of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic or organic salt thereof.

[0007] Hereinafter, [2] to

[17] are all preferred embodiments or models of the present invention. [2] The (co)polymer is at least one diallylamine-based structural unit (a) having a structure represented by the following structural formula (Ia) or (Ib), or a structure that is an inorganic or organic acid salt thereof. [ka] [ka] The blood cell separator according to [1], which is a diallylamine (co)polymer having the following properties. [3] A blood cell separator according to [2], having a structure in which the diallylamine constituent unit (a) is a hydrochloride salt, carboxylate salt, sulfonate salt, or alkyl sulfate salt of the structure shown in the above structural formula (Ia) or (Ib). [4] The blood cell separator according to any one of [1] to [3], wherein the (co)polymer further comprises at least one constituent unit selected from the group consisting of an anionic constituent unit (a), a sulfur dioxide constituent unit (c), and a (di)allylamine constituent unit (d) that does not have a secondary amino group or a secondary amide group. [5] The blood cell separator according to any one of [1] to [4], wherein the weight-average molecular weight of the (co)polymer is 3,000 to 200,000. [6] The blood cell separator according to [4], wherein the (co)polymer has an anionic structural unit (a), and the anionic structural unit (a) is derived from a divalent carboxylic acid. [7] A blood cell separator according to any one of [1] to [6], wherein the (co)polymer is supported on an inorganic carrier. [8] The blood cell separator according to [7], wherein the (co)polymer is supported on the inorganic carrier in a form adsorbed by electrostatic interaction and / or covalently bonded. [9] The hematopoietic agent according to [8], wherein the covalent bond is achieved by silane coupling.

[10] A method for separating blood cells, comprising the step of contacting a sample containing blood cells with a blood cell separating agent described in any one of items [1] to [9].

[11] The method according to

[10] , comprising mixing a sample containing the blood cells with the blood cell separation agent according to any one of [1] to [9] to aggregate the blood cells and collecting the supernatant separated from the blood cells.

[12] The method according to

[11] , wherein the supernatant is collected 15 minutes after the mixing.

[13] The method according to any one of

[10] to

[12] , wherein the sample is a whole blood specimen.

[14] A method for producing a blood cell separation agent, comprising bonding a (co)polymer having at least one kind of constitutional unit of a structure derived from monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or an inorganic acid salt or an organic acid salt thereof, to an inorganic carrier.

[15] The method according to

[14] , wherein the (co)polymer is bonded to the inorganic carrier by electrostatic interaction.

[16] The method according to

[14] , wherein the (co)polymer is bonded to the inorganic carrier by covalent bond.

[17] The method according to

[16] , wherein the inorganic carrier, a silane coupling agent, and the (co)polymer are reacted to silane-couple the (co)polymer to the inorganic carrier. [Effect of the Invention]

[0008] According to the blood cell separation agent and the blood cell separation method of the present invention, blood cell separation can be performed in a shorter time, and since there is little influence on biochemical or immunological analysis, it is possible to more rapidly perform various analyses such as general-purpose automatic analysis, chromatographic analysis such as HPLC, and point-of-care testing at the clinical site, and a blood cell separation technology having high practical value can be provided. Further, in a preferred embodiment, not only red blood cells but also other blood cell components can be separated. Also, there is less influence in more items and it is applicable to more tests. [Brief Description of the Drawings]

[0009] [Figure 1]Figure 1 is a photograph showing the separation of blood cells and supernatant observed at 5, 10, 15, and 30 minutes after mixing, following the addition of 200 μL each of the polymer solutions from Examples 1 to 5 and Comparative Examples 1 to 5 to 1.0 mL of whole blood sample in a 2.0 mL microtube (made of PP), and subsequent inversion and mixing. [Modes for carrying out the invention]

[0010] The blood cell separating agent of the present invention contains a (co)polymer (hereinafter also referred to as "specific (co)polymer") having at least one constituent unit of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic salt or organic salt thereof. By containing a specific (co)polymer, the blood cell separation agent of the present invention can achieve excellent technical effects, such as superior blood cell separation performance. The blood cell separating agent of the present invention may contain only one specific (co)polymer, or it may contain a combination of two or more specific polymers.

[0011] Specific (co)polymer A specified (co)polymer is any (co)polymer having at least one constituent unit (hereinafter also referred to as "specified constituent unit") of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group in its molecule, or a structure that is an inorganic or organic salt thereof, and is not subject to any other restrictions. Furthermore, since structures derived from diallylamine do not contain a secondary amide group, the above phrase "structures derived from monoallylamine or diallylamine having a secondary amino group or a secondary amide group in its molecule" can also be expressed as "structures derived from monoallylamine having a secondary amino group or a secondary amide group in its molecule, or structures derived from diallylamine having a secondary amino group in its molecule." Here, "(co)polymer" is a concept that encompasses both homopolymers and copolymers. Therefore, a specific (co)polymer may be a homopolymer consisting only of specific constituent units, or it may be a copolymer having other constituent units.

[0012] A specific (co)polymer only needs to have one specific structural unit in its molecule; however, from the viewpoint of blood cell separation performance, it is preferable to have multiple specific structural units. More specifically, the proportion of the specific structural unit to the total structural units of the specific (co)polymer is preferably 1 mol% or more, more preferably 10 to 100 mol%, and particularly preferably 50 to 100 mol%. A specific (co)polymer may have only one of the specific structural units in its molecule, or it may have two or more specific structural units. If it has two or more specific structural units, the proportion of the above-mentioned specific structural units to the total structural units of the specific (co)polymer is calculated based on the total number of moles of the two or more specific structural units.

[0013] Diallylamine system constituent unit (A) The specific structural unit may be a structural unit derived from a monoallylamine having a secondary amino group or a secondary amide group, or a structural unit that is an inorganic or organic salt thereof, or a structural unit derived from a diallylamine having a secondary amino group, or a structural unit that is an inorganic or organic salt thereof. However, from the viewpoint of blood cell separation performance and the impact on biochemical or immunological analysis, it is preferable that the structural unit be a structural unit derived from a diallylamine having a secondary amino group, or a structural unit that is an inorganic or organic salt thereof (hereinafter also referred to as "diallylamine-based structural unit (a)").

[0014] More specifically, the diallylamine component (a) has a structure represented by the following structural formula (Ia) or (Ib), or a structure that is an addition salt thereof (inorganic salt or organic salt). [ka] [ka]

[0015] When the diallylamine system component (a) is an addition salt of the structure shown in the above structural formula (Ia) or (Ib), the structure is represented by the following structural formulas (I'a) or (I'b), respectively. In the formula, X is not particularly limited as long as it is a group that can form an inorganic or organic acid together with hydrogen. [ka] [ka]

[0016] From the viewpoint of ease of availability and controllability of the reaction, the inorganic or organic acid salt of the structure represented by the above structural formula (Ia) or (Ib) is preferably a hydrochloride salt, carboxylate salt, sulfonate salt, or alkyl sulfate salt, and is particularly preferably a hydrochloride salt. In other words, in the above structural formulas (I'a) and (I'b), X is preferably chlorine, a carboxylate group, a sulfonate group, or an alkyl sulfate group, and is particularly preferably chlorine.

[0017] Allylamine system constituent units Another option for the specific structural unit is a structure derived from a monoallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic or organic salt thereof (hereinafter also referred to as the "monoallylamine structural unit"). More specifically, the structural unit has the structure represented by the following formula (II), or the structure that is an addition salt thereof. In formula (II), R 1 R is an optional hydrocarbon group, where the hydrocarbon group may have heteroatoms such as oxygen and sulfur as a result of the substitution, and may be, for example, a substituted or unsubstituted acyl group. 1It is preferable that the group is an alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 5 to 6 carbon atoms, or an acyl group having 2 to 5 carbon atoms. R 1 The preferred C1-C12 alkyl group may be linear, branched, or aralkyl. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, octyl, decyl, dodecyl, and benzyl groups. 1 Other preferred options for cycloalkyl groups having 5 to 6 carbon atoms include, but are not limited to, cyclopentyl and cyclohexyl groups. 1 Other preferred options for acyl groups having 2 to 5 carbon atoms include unsubstituted acyl groups, alkoxy-substituted acyl groups, and amine-substituted acyl groups. More specifically, examples include, but are not limited to, methyl carbonyl groups, methoxy carbonyl groups, amino carbonyl groups, ethyl carbonyl groups, propyl carbonyl groups, butyl carbonyl groups, ethoxycarbonyl groups, propoxycarbonyl groups, and butoxycarbonyl groups. [ka]

[0018] When the monoallylamine system unit is an addition salt (inorganic or organic acid), its structure is represented by the following formula (II'). [ka] In formula (II'), HX represents an inorganic or organic acid, H is a hydrogen atom, and X is a group that can form an inorganic or organic acid together with hydrogen, but is not particularly limited. There are no particular restrictions on the type of addition salt, but from the viewpoint of availability and ease of reaction control, for example, hydrochloride, sulfate, phosphate, nitrate, sulfite, phosphate, nitrite, hydrobromide, acetate, amide sulfate, methanesulfonate, trifluoroacetate, p-toluenesulfonate, etc. can be used. Among these, hydrochloride salts, sulfate salts, phosphate salts, and amide sulfate salts are preferred, and hydrochloride salts, sulfate salts, phosphate salts, and amide sulfate salts with structures derived from monoallylamine are particularly preferred.

[0019] As described above, a specific (co)polymer may have only one specific structural unit in its molecule, or it may have two or more specific structural units. If it has two or more specific structural units, each of these two or more specific structural units may be a diallylamine structural unit (a), each may be a monoallylamine structural unit, or it may be a combination of a diallylamine structural unit (a) and a monoallylamine structural unit. When using a combination of diallylamine-based structural units (A) and monoallylamine-based structural units, there are no particular restrictions on the ratio of diallylamine-based structural units (A) to monoallylamine-based structural units, but a molar ratio of 100:1 to 1:100 is preferred, and a molar ratio of 10:1 to 1:10 is particularly preferred.

[0020] Other constituent units When a specific (co)polymer contains constituent units other than the specific constituent units, there are no particular restrictions on the constituent units other than the specific constituent units, and monomers copolymerizable with monoallylamine monomers or diallylamine monomers can be appropriately used to introduce constituent units other than the specific constituent units. Particularly preferred as such structural units are anionic structural units (a), sulfur dioxide structural units (c), and (di)allylamine structural units (d) that do not have a secondary amino group or a secondary amide group. At least one structural unit selected from these can be introduced into a specific (co)polymer. By introducing these structural units into the specific (co)polymer in addition to the specific structural unit, the blood cell separation ability of the blood cell separator can be improved and the impact on biochemical or immunological analysis can be reduced.

[0021] Anionic constituent unit (a) In this embodiment, by having an anionic structural unit (a), the specific (co)polymer becomes a so-called amphoteric polymer, which enables the realization of advantageous effects such as miscibility with blood, urine, etc., and hydrophilicity. It is also preferable from the viewpoint of improving blood cell separation ability and reducing the impact on biochemical or immunological analysis. In the embodiments of the present invention, the anionic constituent unit (a) may be any constituent unit capable of having a negative charge upon dissociation, and there are no other restrictions. However, from the viewpoint of further improving blood cell separation ability and further reducing the impact on biochemical or immunological analysis, it is preferable that it has a structure derived from a divalent carboxylic acid, and it is particularly preferable that it has a structure represented by the following structural formulas (III), (IV), or (V). [ka] [ka] [ka] However, in the above formula (III), R 2 is either hydrogen or a methyl group, and in (III), (IV), and (V), Y is independently hydrogen, Na, K, NH4, 1 / 2Ca, 1 / 2Mg, 1 / 2Fe, 1 / 3Al, or 1 / 3Fe for each carboxyl group to which it is bonded. The anionic structural unit (a) may have a structure that does not correspond to any of the above structural formulas (III), (IV), and (V), as long as it has an anionic group such as a carboxyl group.

[0022] If a specific (co)polymer has an anionic structural unit (a), one type of anionic structural unit (a) may be used alone, or multiple types of anionic structural units (a) with different structures may be used in combination. When using multiple types of mutually different anionic structural units (a), each anionic structural unit may have a structure different from the others within the range represented by the same general structural formula (III), (IV), or (V), or it may have a structure different from the others represented by different general structural formulas. In the former case, for example, multiple types of anionic structural units (a) may be used, each represented by general structural formula (III), but with different structures due to different elements of Y. In the latter case, for example, one anionic structural unit (a) having a structure represented by structural formula (III) and another anionic structural unit (a) having a structure represented by structural formula (IV) may be used.

[0023] From the viewpoint of further improving blood cell separation ability and further reducing the impact on biochemical or immunological analysis, it is preferable that at least a portion of the anionic constituent unit (a) is derived from maleic acid and fumaric acid, in which case the anionic constituent unit (a) will have the structure represented by the above formula (IV), where Y is hydrogen in all cases. When a specific (co)polymer has an anionic structural unit (i), there are no particular restrictions on the content of the anionic structural unit (i). However, when the anionic structural unit (i) is derived from an unsaturated dicarboxylic acid, the molar ratio of the specific structural unit to the anionic structural unit (i) is preferably specific structural unit / anionic structural unit (i) = 1 / 0.1 to 1 / 1, and particularly preferably 1 / 0.2 to 1 / 1. At least a portion of the anionic constituent unit (a) may be derived from an unsaturated monocarboxylic acid. When the anionic constituent unit (a) is derived from an unsaturated monocarboxylic acid, the ratio of specific constituent unit to anionic constituent unit (a) is preferably 1 / 100 to 100 / 1, and particularly preferably 1 / 10 to 10 / 1.

[0024] Sulfur dioxide constituent unit (c) In this embodiment, by having a sulfur dioxide constituent unit (c), the specific (co)polymer can be given advantageous properties such as improved hydrophobicity, improved separation performance of anionic components (e.g., blood cells), and reduced impact on biochemical or immunological analysis. In this embodiment, the sulfur dioxide constituent unit (c) in the (co)polymer is derived from sulfur dioxide having the structure represented by the following structural formula (VI). [ka]

[0025] It is preferable that the proportion of sulfur dioxide constituent unit (c) to the total constituent units of the (co)polymer of this embodiment is 1 mol% or more. In particular, by having a proportion of sulfur dioxide constituent unit (c) of 1 mol% or more, advantageous properties such as improved hydrophobicity, improved separation performance of anionic components (e.g., blood cells), and reduced impact on biochemical or immunological analysis can be more effectively imparted to the (co)polymer of this embodiment. The proportion of sulfur dioxide constituent units (c) is more preferably 1 to 50 mol%, and particularly preferably 10 to 50 mol%.

[0026] In this embodiment, there are no particular restrictions on the ratio of the specific structural unit to the sulfur dioxide structural unit (c), and any ratio is possible as long as copolymerization is possible. From the viewpoint of increasing the molecular weight of the copolymer, it is preferable that the ratios of the two structural units do not differ significantly. For example, the ratio of the specific structural unit (if there is a (di)allylamine structural unit (d) that does not have a secondary amino group or a secondary amide group, the sum of the specific structural unit and the (di)allylamine structural unit (d) that does not have a secondary amino group or a secondary amide group) to the sulfur dioxide structural unit (c) is preferably 99:1 to 50:50, and particularly preferably 90:10 to 50:50.

[0027] (E) Allylamine-based structural units that do not have a secondary amino group or a secondary amide group. In this embodiment, by having a (di)allylamine-based structural unit (e) that does not have a secondary amino group or a secondary amide group, it is possible to impart advantageous properties to a specific (co)polymer, such as improved blood cell separation ability.

[0028] (Di)allylamine constituent units (E) that do not have a secondary amino group or a secondary amide group are constituent units of structures derived from monoallylamine or diallylamine, or structures that are inorganic or organic salts thereof, and which do not contain a secondary amino group or a secondary amide group in their structure. (E) Allylamine-based structural units (E) that do not have a secondary amino group or a secondary amide group may be used individually or in combination of two or more types. When a specific (co)polymer has a (di)allylamine-based constituent unit (E) that does not have a secondary amino group or a secondary amide group, there are no particular restrictions on the content of the constituent unit (E). However, the molar ratio of the specific constituent unit to the constituent unit (E) is preferably specific constituent unit / constituent unit (E) = 1 / 100 to 100 / 1, and particularly preferably 1 / 10 to 10 / 1.

[0029] If a (di)allylamine constituent unit (E) that does not have a secondary amino group or a secondary amide group is a constituent unit of a structure derived from a monoallylamine, or a structure that is an inorganic or organic acid salt thereof, then the constituent unit (E) has a primary amino group and / or a tertiary amino group in its structure. Preferred examples of such constituent units (E) include constituent units derived from allylamine, allylamine hydrochloride, allylamineamide sulfate, allylamine acetate, dimethylallylamine, dimethylallylamine hydrochloride, dimethylallylamineamide sulfate, dimethylallylamine acetate, and the like.

[0030] If a (di)allylamine component (E) that does not have a secondary amino group or a secondary amide group is a component of a structure derived from diallylamine, or a structure that is an inorganic or organic acid salt thereof, then the component (E) has a tertiary amino group and / or a quaternary amino group in its structure. Preferred examples of such constituent units (E) include those derived from methyldiallylamine, methyldiallylamine hydrochloride, methyldiallylamine amide sulfate, methyldiallylamine acetate, diallyldimethylammonium chloride, diallylmethylethylammonium ethyl sulfate, and the like.

[0031] There are no particular restrictions on the molecular weight of the specific (co)polymer. Depending on the form of use of the blood cell separator and its relationship with other components, a specific (co)polymer with a suitable molecular weight can be obtained or polymerized as appropriate. However, from the viewpoint of blood cell separation ability and availability, a weight-average molecular weight (Mw) of 3,000 to 200,000 is usually used. From the viewpoint of achieving excellent blood cell separation ability, the weight-average molecular weight (Mw) of the specific (co)polymer is preferably 100,000 or less, more preferably 50,000 or less, particularly preferably 20,000 or less, even more preferably 15,000 or less, and even more preferably 5,000 or less. From the viewpoint of carrying out polymerization in a practically acceptable time and cost, the weight-average molecular weight (Mw) of the specific (co)polymer is preferably 1,000,000 or less, and more preferably 100,000 or less. The weight-average molecular weight (Mw) of a specific (co)polymer can be measured, for example, by gel permeation chromatography (GPC) using a liquid chromatograph. More specifically, it can be measured by the method described in the examples of this application. The molecular weight of a specific (co)polymer can be appropriately adjusted by adjusting the presence, type, and composition of comonomers, the temperature, time, and pressure in the polymerization process, and the type and amount of radical initiator used in the polymerization process.

[0032] There are no particular restrictions on the rotational viscosity [η] of the specific (co)polymer, and it can be set appropriately depending on the form of use of the blood cell separator and its relationship with other components, but it is preferably 10 to 700 mPa·s (25°C), and particularly preferably 10 to 60 mPa·s (25°C). The rotational viscosity [η] can be measured by methods commonly used in this industry, for example, by an AMETEK Brookfield DV-3T digital type B viscometer. Measurement is typically performed using a ULA adapter, with a liquid volume of 16 mL and a liquid temperature of 25°C. The rotational viscosity [η] can also be adjusted as appropriate by adjusting the dilution concentration, the presence, type and composition of comonomers, the temperature, time and pressure in the polymerization process, and the type and amount of radical initiator used in the polymerization process.

[0033] Method for producing specific (co)polymers There are no particular restrictions on the method for producing specific (co)polymers, and they can be produced by methods conventionally known in the art. For example, they can be produced by homopolymerizing monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or by inorganic or organic salt thereof, or optionally by copolymerizing with other monomers.

[0034] The solvent used when homopolymerizing or copolymerizing monoallylamines or diallylamines having a secondary amino group or a secondary amide group, or their inorganic or organic salts, with other monomers is not particularly limited. It may be an aqueous solvent or an organic solvent such as an alcohol, ether, sulfoxide, or amide, but an aqueous solvent is preferred. When monoallylamines or diallylamines having a secondary amino group or a secondary amide group, or their inorganic or organic acid salts, are homopolymerized or copolymerized with other monomers, the monomer concentration varies depending on the type of monomer and the type of solvent used for (co)polymerization, but is usually 10 to 75% by weight in the case of aqueous solvents. This (co)polymerization reaction is usually a radical polymerization reaction and is carried out in the presence of a radical polymerization catalyst. The type of radical polymerization catalyst is not particularly limited, but preferred examples include peroxides such as t-butyl hydroperoxide, persulfates such as ammonium persulfate, sodium persulfate, and potassium persulfate, and water-soluble azo compounds such as azobis and diazo compounds.

[0035] The amount of radical polymerization catalyst added is generally 0.1 to 20 mol%, preferably 1.0 to 10 mol%, relative to the total monomer. The polymerization temperature is generally 0 to 100°C, preferably 5 to 80°C, and the polymerization time is generally 1 to 150 hours, preferably 5 to 100 hours. Polymerization can be carried out in the atmosphere of air without causing significant problems, but it can also be carried out in an atmosphere of an inert gas such as nitrogen.

[0036] Blood cell separator The blood cell separation agent of the present invention contains a specific (co)polymer (a (co)polymer having at least one constituent unit of a structure derived from monoallylamine or diallylamine having a secondary amino group or secondary amide group, or a structure that is an inorganic or organic salt thereof). This specific (co)polymer may be used as a blood cell separation agent as is, but from the viewpoint of miscibility with the sample, it is preferable to use an aqueous solution in which it is dissolved in an aqueous solvent. As the aqueous solvent, pure water, physiological saline, etc., which do not affect the sample or analysis, are preferred. In addition to the specific (co)polymer, other blood cell separation components such as inorganic particles such as glass, silica, kaolin, zeolite, and bentonite, cholic acid, quaternary ammonium salt homopolymers, quaternary ammonium salt copolymers, and quaternary amine sulfone polymers can also be appropriately combined.

[0037] As described above, the blood cell separation agent of the present invention may consist of a specific (co)polymer alone, or the specific (co)polymer may be directly added to a sample containing blood cells such as blood. However, from the viewpoint of handling, reuse of the blood cell separation agent, or suppression of contamination of the blood cell separation agent into the sample separated from blood cells, a blood cell separation agent in which the specific (co)polymer is supported on an inorganic carrier may also be used. There are no particular restrictions on the shape of the inorganic carrier; for example, containers, the inner walls of tubes, inorganic particles, magnetic beads, etc., can be used as inorganic carriers. As for the material of the inorganic carrier, glass, silica, kaolin, zeolite, bentonite, etc., are examples of materials that possess blood cell separation ability themselves and can easily support specific (co)polymers. When using particulate inorganic carriers, there are no particular restrictions on the particle size of the inorganic carriers, but the primary particle size is preferably 0.05 to 500 μm, and particularly preferably 0.1 to 50 μm.

[0038] In this embodiment, there are no particular restrictions on the method of supporting the specific (co)polymer on the inorganic support, and any conventional method used for polymer support may be used as appropriate. However, it is particularly preferable to adsorb the specific (co)polymer onto the inorganic support by electrostatic interaction, to support the specific (co)polymer on the inorganic support by covalent bonding, or to use a combination of these methods. There are no particular restrictions on the method of supporting a specific (co)polymer on an inorganic support by covalent bonding, but it is preferable to use a silane coupling agent, and more specifically, it is particularly preferable to use epoxysilane, chlorosilane, etc.

[0039] Blood cell separation method When the aforementioned blood cell separating agent is brought into contact with a sample containing blood cells, the blood cells will agglutinate, allowing for the separation of blood cells from other components. Therefore, there are no particular restrictions on the blood cell separation method other than bringing the blood cell separating agent into contact with the blood cells; however, depending on the form of the blood cell separating agent, blood cells can be separated by various methods.

[0040] For example, when using the specific (co)polymer itself or a solution of the specific (co)polymer as a blood cell separator, these blood cell separators can be added to and mixed with a sample containing blood cells in a collection container, or a sample containing blood cells can be added to and mixed with a collection container that already contains the blood cell separator to agglutinate and precipitate the blood cells, separating them from other components. This method is one of the most versatile methods in clinical practice, and as will be demonstrated in the examples described later, the blood cell separator of the present invention exhibits excellent blood cell separation ability in this embodiment. In this embodiment, the solution of the specific (co)polymer is preferably a solution dissolved in pure water. Furthermore, it is preferable to add the specific (co)polymer itself or a solution of the specific (co)polymer in an amount of 1 μg to 500 μg per 1.0 mL of sample, more preferably 5 μg to 100 μg, and particularly preferably 5 μg to 20 μg.

[0041] Blood cells can also be separated by holding or impregnating a filter material such as filter paper with the specific (co)polymer itself or a solution of the specific (co)polymer, adding a sample containing blood cells to it, and collecting the sample that passes through the filter material. Alternatively, blood cells can be separated by mixing a blood cell separating agent with a sample containing blood cells, adding the mixture to a filter material such as filter paper, and collecting the sample that passes through the filter material. In these methods, it is preferable to pack the filter material such as filter paper as a support in a container such as a column, and then add the blood cell separating agent or a mixture of the blood cell separating agent and the sample to it. With such separation methods, the separation rate can be controlled to some extent by using a pump, etc., and the sample that passes through the above container can be used for analysis, making it highly compatible with HPLC, automated analyzers, and immediate clinical testing.

[0042] When using a blood cell separation agent in which a specific (co)polymer is supported on a particulate inorganic carrier, blood cells can be separated from other components in the same manner as when using the specific (co)polymer itself or a solution of the specific (co)polymer as the blood cell separation agent. In a blood cell separation agent in which a specific (co)polymer is supported on a particulate inorganic carrier, it is possible to impart blood cell separation ability to the inorganic carrier itself by devising the material of the inorganic carrier, thereby enhancing the blood cell separation ability. Furthermore, even with a blood cell separation agent in the form of a specific (co)polymer supported on a particulate inorganic carrier, by using a filtration material such as filter paper as a support, or even a container such as a column filled with this material, a separation method with excellent compatibility with HPLC, automated analyzers, and immediate clinical testing can be achieved.

[0043] When using a blood cell separation agent in which a specific (co)polymer is supported on the inner wall surface of a sample collection tube, it is convenient because blood cells can be separated simply by placing the sample in the collection tube and mixing it by inversion as needed. Another advantage is that the sample collection tube on which the blood cell separation agent is supported can be reused.

[0044] The blood cell separation agent according to the present invention exhibits a rapid rate of agglutination of blood cells, enabling the separation of blood cells within a timeframe unattainable with conventional blood cell separation agents. Specifically, after contacting or mixing the blood cell separation agent with a sample containing blood cells, the blood cells can be separated from other components in less than 30 minutes, preferably 20 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less. Therefore, the present invention provides a blood cell separation means that can respond to tests requiring rapid presentation of analytical results, such as immediate clinical testing. The method of the present invention is applicable to any sample containing blood cells, such as blood, urine, lymph, cerebral fluid, body cavity fluid, cell exudate, digestive fluid, and feces. A typical sample to which it can be applied is blood, which includes whole blood samples and blood samples containing anticoagulants such as heparin, with whole blood samples being the most typical sample.

[0045] The sample separated from blood cells by the method of the present invention can be used for analysis as a specimen for various tests. As demonstrated in the examples described later, the sample treated with the specific (co)polymer has little effect on the analytical results for a relatively large number of biochemical or immunological tests, and the specific (co)polymer of the preferred embodiment has little effect on the analytical results for most tests. Therefore, the method of the present invention can be used as a pretreatment for samples to be used for various tests. [Examples]

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0047] 1. Preparation of blood cell separator Cationic polymers having structures derived from monoallylamines having secondary amide groups (Examples 1 and 2), cationic polymers having structures derived from diallylamines having secondary amino groups (Examples 3 to 5), and cationic polymers having structures derived from diallylamines or monoallylamines having tertiary or quaternary amino groups (Comparative Examples 1 to 5) were dissolved in pure water to prepare polymer solutions with a polymer solids content of 10% by weight, and the pH was adjusted to 6.8 to 7.5 with 4 N NaOH. The pH was measured using a HORIBA benchtop pH meter. The same procedure was followed in the following examples.

[0048] [Table 1]

[0049] [Table 2]

[0050] [Table 3]

[0051] [Table 4]

[0052] (Method for measuring weight-average molecular weight) The weight-average molecular weight of each of the above cationic polymers was measured by the following method. The weight-average molecular weight (Mw) of cationic polymers containing anion constituent unit (a) (Comparative Examples 4 and 5) was measured by gel permeation chromatography (GPC) using a Hitachi L-2000 high-performance liquid chromatograph. A Hitachi L-2130 pump was used as the eluent flow channel pump, a Hitachi L-2490 RI detector as the detector, and two TOSOH TSKgel α-M columns (exclusion limit molecular weight 10,000,000) connected in series were used as the column. The sample was prepared to a concentration of 0.5 g / 100 mL in the eluent and 100 μL was used. The eluent used was a 0.15 mol / L sodium sulfate and 1 wt% acetic acid aqueous solution. The column temperature was 40°C and the flow rate was 1.0 mL / min. Calibration curves were obtained using pullulan with molecular weights of 5,900, 47,300, 212,000, and 788,000 as standard substances, and the weight-average molecular weight (Mw) of the cationic polymer was determined based on these calibration curves. The weight-average molecular weight (Mw) of the other cationic polymers was measured by gel permeation chromatography (GPC) using a Chromaster® 5450 high-performance liquid chromatograph manufactured by Hitachi High-Tech Science Corporation. The detector used was an RI differential refractive index detector, and the column consisted of two Shaudex Asahi Pack aqueous gel filtration type columns, GS-220HQ (exclusion limit molecular weight 3,000) and GS-620HQ (exclusion limit molecular weight 2,000,000), connected in series. The sample was prepared to a concentration of 0.5 g / 100 mL with the eluent, and 20 μL was used. A 0.4 mol / L sodium chloride aqueous solution was used as the eluent. The column temperature was 30°C, and the flow rate was 1.0 mL / min. Calibration curves were obtained using polyethylene glycol with molecular weights of 106, 194, 440, 600, 1,470, 4,100, 7,100, 10,300, 12,600, and 23,000 as standard substances, and the weight-average molecular weight (Mw) of the cationic polymer was determined based on these calibration curves.

[0053] 2. Removal of blood cells using polymer solution To 1.0 mL of whole blood sample in a 2.0 mL microtube (made of PP), 200 μL of each polymer solution was added and mixed by inversion to test whether blood cells could be separated by agglutination. Samples mixed by inversion with physiological saline instead of the polymer solution were used as a control. The blood cell separation ability of each polymer was evaluated by counting blood cells and visual observation. (1) Blood cell count At 15 and 30 minutes after inversion and mixing, the supernatant (allicote near the surface for non-agglutinated blood) was collected, and the number of white blood cells, red blood cells, and platelets was measured using a hematology analyzer (Sysmex XS-500i). The measured blood cell counts were compared with the control counts of each blood cell type, and the results were determined based on the following criteria. (Evaluation Criteria) ○: Compared to the control, the number of blood cell components in the supernatant decreased to less than 20% within 15 minutes after inversion and mixing. △: Compared to the control, the number of blood cell components in the supernatant decreased to less than 30% within 30 minutes after inversion and mixing. ×: Compared to the control, the number of blood cell components in the supernatant was 30% or more 30% at 30 minutes after inversion and mixing. (2) External observation The separation of blood cells and supernatant was observed at 5, 10, 15, and 30 minutes after inversion and mixing.

[0054] The test results are shown in Tables 5 and 6 and Figure 1 below. [Table 5]

[0055] [Table 6]

[0056] As shown in Figure 1, when PAA-AC5000 and PAA-N5000, cationic polymers having a structure derived from monoallylamines with a secondary amide group, and PAA-D11-HCl, PAS-92, and P(DAA / FMA), cationic polymers having a structure derived from diallylamines with a secondary amino group, were added to whole blood samples, separation of plasma and blood cells was visually confirmed at 10 minutes at the latest. Furthermore, as shown in Table 5 above, after 15 minutes of inversion mixing, the number of red blood cells in the supernatant decreased to less than 20% compared to the control in all cases. Furthermore, when PAA-AC5000, PAA-N5000, PAA-D11-HCl, PAS-92, and P(DAA / FMA) were added to whole blood samples, the number of red blood cells and platelets in the supernatant decreased to less than 20% compared to the control 15 minutes after inversion and mixing. Similarly, when PAA-AC5000, PAS-92, and P(DAA / FMA) were added to whole blood samples, the number of white blood cells, red blood cells, and platelets in the supernatant decreased to less than 20% compared to the control 15 minutes after inversion and mixing. On the other hand, as shown in Table 6 and Figure 1, when cationic polymers having structures derived from diallylamines or monoallylamines with tertiary or quaternary amino groups—PAS-2201CL (methyldiallylamine hydrochloride sulfur dioxide copolymer), PAS-A-5 (diallyldimethylammonium chloride sulfur dioxide copolymer), PAA-1112 (allylamine dimethylallylamine copolymer), PAS-2451 (diallylmethylethylammonium ethyl sulfate maleate copolymer), or PAS-2351 (diallyldimethylammonium chloride maleate copolymer)—were added to whole blood samples, none of the blood cells precipitated, and plasma could not be obtained.

[0057] 3. Effects of each polymer on biochemical and immunological tests of separated plasma. Biochemical and immunological tests were performed on a simulated high-level whole blood sample prepared by adding 100 μL of Aalto Control Level II α (Sinotest), dissolved in half the prescribed amount of pure water, and 15 μL of CRP antigen at a concentration of 100 mg / dL to 1.0 mL of whole blood sample in a 2.0 mL microtube. Alternatively, 200 μL of each polymer solution was added to 1.0 mL of the simulated high-level whole blood sample in a 2.0 mL microtube, and the supernatant was collected 15 minutes after inversion and mixing. Ten biochemical tests and three immunological tests were performed using the collected supernatant with a Hitachi 7180 automated analyzer (Hitachi, Ltd.). As a control, plasma obtained by centrifugation at 3,000 rpm for 10 minutes from blood collected in a heparinized plasma collection tube (Terumo) was used. To match the dilution ratio of the blood with the polymer solution, 200 μL of physiological saline was added to 1.0 mL of the obtained plasma. The reagents used for measuring 10 biochemical items and 3 immunological items are as follows: reagent AST:N-Assay L AST (manufactured by Nitto Boseki Medical Co., Ltd.) ALT:N-Assay L ALT (manufactured by Nitto Boseki Medical Co., Ltd.) LDH:N-Assay L LDH Nitto Boseki B-Type (manufactured by Nitto Boseki Medical) γ-GTP:N-Assay L γ-GTP-H Nitto Boseki B-Type (manufactured by Nitto Boseki Medical) ALB:N-Assay L ALB-S Nitto Boseki (manufactured by Nitto Boseki Medical) CRE:N-Assay L CRE-K (manufactured by Nitto Boseki Medical Co., Ltd.) LDL: Metabolead LDL-C (manufactured by Hitachi Chemical Diagnostics Systems) HDL: Metabolead HDL-C (manufactured by Hitachi Chemical Diagnostics Systems) TCHO:N-Assay L T-CHO-H (manufactured by Nitto Boseki Medical Co., Ltd.) TG: Determiner L TG II (manufactured by Hitachi Chemical Diagnostics Systems) CRP:N-Assay LA CRP-S Nitto Boseki D-Type (manufactured by Nitto Boseki Medical) IgG:N-Assay TIA IgG-SH (manufactured by Nitto Boseki Medical Co., Ltd.) C3:N-Assay TIA C3-SH Nitto Boseki (manufactured by Nitto Boseki Medical) Calibration was performed using Aalto Control Level II α (Sinotest) for 10 biochemical tests and calibrators specified by the reagent manufacturer for 3 immunological tests. The parameters for each test followed the instructions provided with the kit.

[0058] The test results for each test item of the samples obtained after treatment with each polymer are summarized below. Note that values ​​other than the control value are relative values ​​when the control value is set to 100. [Table 7] As described above, in samples treated with PAA-AC5000, a cationic polymer having a structure derived from monoallylamine with a secondary amide group, 5 out of 10 biochemical tests and 2 out of 3 immunological tests could be measured within an error range of ±20% or less. In contrast, plasma obtained by treating whole blood samples with PAS-92 and P(DAA / FMA), cationic polymers having a structure derived from diallylamine with a secondary amino group, showed that 9 or more out of 10 biochemical tests and all 3 immunological tests could be measured within an error range of ±20% or less. [Industrial applicability]

[0059] The blood cell separation agent and blood cell separation method using the present invention enable blood cell separation in a shorter time and have minimal impact on biochemical or immunological analysis. Therefore, they have high practical value, enabling faster execution of various analyses such as general-purpose automated analysis, chromatographic analysis such as HPLC, and immediate clinical testing, and thus have high applicability in various fields of the analytical instrument industry.

Claims

1. A hematopoietic cell separator for analysis, containing a (co)polymer having at least one constituent unit of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic or organic salt thereof.

2. The (co)polymer is at least one diallylamine-based structural unit (a) having a structure represented by the following structural formula (Ia) or (Ib), or a structure that is an inorganic or organic acid salt thereof. 【Chemistry 1】 【Chemistry 2】 The blood cell separating agent according to claim 1, which is a diallylamine (co)polymer having the following properties.

3. The blood cell separator according to claim 2, wherein the diallylamine constituent unit (a) is a hydrochloride salt, carboxylate salt, sulfonate salt, or alkyl sulfate salt of the structure shown in the above structural formula (Ia) or (Ib).

4. The blood cell separator according to any one of claims 1 to 3, wherein the (co)polymer further comprises at least one constituent unit selected from the group consisting of an anionic constituent unit (a), a sulfur dioxide constituent unit (c), and a (di)allylamine constituent unit (d) that does not have a secondary amino group or a secondary amide group.

5. The blood cell separating agent according to any one of claims 1 to 3, wherein the weight-average molecular weight of the (co)polymer is 3,000 to 200,000.

6. The blood cell separator according to claim 4, wherein the (co)polymer has an anionic structural unit (a), and the anionic structural unit (a) is derived from a divalent carboxylic acid.

7. The blood cell separator according to any one of claims 1 to 3, wherein the (co)polymer is supported on an inorganic carrier.

8. The blood cell separator according to claim 7, wherein the (co)polymer is supported on the inorganic carrier in a form adsorbed by electrostatic interaction and / or covalently bonded.

9. The blood cell separator according to claim 8, wherein the covalent bond is achieved by silane coupling.

10. A method for separating blood cells, comprising the step of contacting a sample containing blood cells with a blood cell separating agent described in any one of claims 1 to 3.

11. The method according to claim 10, comprising mixing a sample containing the blood cells with a blood cell separating agent according to any one of claims 1 to 3 to agglutinate the blood cells and collecting the supernatant separated from the blood cells.

12. The method according to claim 11, wherein the supernatant is collected 15 minutes after the mixing.

13. The method according to claim 10, wherein the sample is a whole blood sample.

14. A method for producing a blood cell separator, comprising attaching a (co)polymer having at least one constituent unit of a structure derived from a monoallylamine or diallylamine having a secondary amino group or a secondary amide group, or a structure that is an inorganic salt or organic salt thereof, to an inorganic carrier.

15. The method according to claim 14, wherein the (co)polymer is bonded to the inorganic support by electrostatic interaction.

16. The method according to claim 14, wherein the (co)polymer is covalently bonded to the inorganic support.

17. The method according to claim 16, wherein the inorganic carrier, the silane coupling agent, and the (co)polymer are reacted to silane coupling the (co)polymer to the inorganic carrier.

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