Blood coagulation time shortening agent for blood sample lacking blood coagulation factor XII

Abstract

Provided is an agent for shortening the blood coagulation time of a blood sample deficient in blood coagulation factor XII in activated partial thromboplastin time measurement, the agent containing, as an active ingredient, a polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit.

Description

Technical Field

[0001] The present invention relates to an agent for shortening the coagulation time of a blood sample lacking coagulation factor XII in an activated partial thromboplastin time (APTT) measurement. Background Art

[0002] Coagulation test is a test used to diagnose the patient's coagulation ability by adding a specified reagent to the patient's blood sample and measuring the coagulation time. Typical examples of coagulation time include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, etc. Abnormal coagulation ability causes the extension of coagulation time. The causes of the extension of coagulation time include coagulation inhibitors (heparin, etc.), a decrease in components involved in coagulation, congenital coagulation factor deficiency (hemophilia, etc.), autoantibodies that inhibit coagulation reactions (lupus anticoagulant: LA, anticoagulation factor antibodies, etc.), etc.

[0003] In a coagulation test, a reagent is added to a blood sample, the subsequent coagulation reaction is measured, and the coagulation time is determined by the coagulation reaction. When a prolonged coagulation time is confirmed, it is determined to be an abnormal coagulation ability. Coagulation test reagents contain components for causing coagulation under certain conditions. For example, APTT assay reagents contain coagulation factor activators such as ellagic acid, phospholipids, buffers, etc. On the other hand, there are various types of commercially available APTT assay reagents, and the sensitivity to various coagulation abnormalities is different among these reagents.

[0004] Patent document 1 discloses an APTT assay reagent containing manganese chloride as an active enhancer and glycine as a suspension enhancement means. Patent document 2 records that aluminum chloride, ferric chloride, or a mixture of manganese chloride and ellagic acid activates the coagulation of the sample, but ferric chloride generates a precipitate of ellagic acid. Patent document 3 records the use of glycylglycine or diglycylglycine as a coagulation factor stabilizer in the APTT assay, but if they are added at a high concentration, the coagulation time is prolonged. Patent document 4 records that Good's buffers such as HEPES, PIPES, POPSO, ACES, TAPSO, EPPS, Tricine, and CHES can be used as specimen diluents for coagulation ability assays. Patent document 5 records that an APTT assay reagent containing specified concentrations of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine has improved sensitivity to LA and has appropriate sensitivity to heparin. Patent Document 6 describes that the quality of an APTT assay reagent is stabilized by using a polymer containing a (meth)acrylic acid monomer unit having a phosphoserine residue.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 59-91899

[0008] Patent Document 2: Japanese Patent Application Publication No. 2002-156379

[0009] Patent Document 3: Japanese Patent Publication No. 6-50999

[0010] Patent Document 4: Japanese Patent Application Publication No. 2013-145238

[0011] Patent Document 5: Japanese Patent Application Publication No. 2020-56578

[0012] Patent Document 6: Japanese Patent Application Publication No. 2021-181929 Summary of the invention

[0013] In coagulation tests, it is desirable to accurately detect samples with coagulation abnormalities (coagulation factor deficiency, LA positivity, coagulation inhibitor positivity, etc.). For example, it is desirable to adjust the coagulation time of these blood samples so that blood samples with coagulation abnormalities can be accurately detected based on the coagulation time.

[0014] The present invention provides a clotting time shortening agent for a sample deficient in blood coagulation factor XII (FXII) in APTT assay, an APTT assay method using the agent, and a method for shortening the clotting time of a sample deficient in FXII in APTT assay.

[0015] The following aspects are provided as embodiments of the present invention.

[0016] [1] A coagulation time shortening agent for a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time measurement, the agent comprising a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit as an active ingredient.

[0017] [2] The shortening agent according to [1], wherein the polymer is a copolymer of 2-methacryloyloxyethylphosphorylcholine and another monomer having a hydrophobic group, an anionic group or a cationic group.

[0018] [3] The shortening agent according to [2], wherein the surface tension of a 0.1 wt % aqueous solution of the copolymer at 25° C. is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the above copolymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

[0019] [4] The shortening agent according to any one of [1] to [3], further comprising at least one selected from a metal salt compound and an amino acid as an active ingredient.

[0020] [5] A shortening agent according to [4], wherein the metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and hydrates thereof.

[0021] [6] The shortening agent according to [4] or [5], wherein the amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

[0022] [7] A reagent for measuring activated partial thromboplastin time, comprising the coagulation time shortening agent according to any one of [1] to [6].

[0023] [8] A method for measuring an activated partial thromboplastin time, comprising measuring the coagulation time of a sample solution containing a blood sample and the coagulation time shortening agent according to any one of [1] to [6].

[0024] [9] A method for shortening the coagulation time of a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time measurement, comprising measuring the coagulation time of a sample solution containing the blood sample and the coagulation time shortening agent according to any one of [1] to [6].

[0025]

[10] Use of a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit for shortening the coagulation time of a blood sample lacking coagulation factor XII in an activated partial thromboplastin time assay.

[0026]

[11] The use according to

[10] , wherein the polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers having a hydrophobic group, anionic group or cationic group.

[0027]

[12] The use according to

[11] , wherein the surface tension of a 0.1 wt % aqueous solution of the copolymer at 25° C. is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the above copolymer at 25°C is 70 to 75×10 -3N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

[0028]

[13] The use according to any one of

[10] to

[12] , wherein at least one selected from a metal salt compound and an amino acid is used together with the polymer.

[0029]

[14] The use according to

[13] , wherein the metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and their hydrates.

[0030]

[15] The use according to

[13] or

[14] , wherein the amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

[0031]

[16] A polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit, for use in shortening the coagulation time of a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time measurement.

[0032]

[17] The polymer according to

[16] , which is a copolymer of 2-methacryloyloxyethylphosphorylcholine and another monomer having a hydrophobic group, an anionic group or a cationic group.

[0033]

[18] The polymer according to

[17] , wherein the surface tension of a 0.1 wt % aqueous solution of the copolymer at 25° C. is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the above copolymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

[0034]

[19] The polymer according to any one of

[16] to

[18] , which is used in combination with at least one selected from a metal salt compound and an amino acid.

[0035]

[20] The polymer according to

[19] , wherein the metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and hydrates thereof.

[0036]

[21] The polymer according to

[19] or

[20] , wherein the amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

[0037]

[22] Use of a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit for producing a coagulation time shortening agent for a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time assay.

[0038]

[23] The use according to

[22] , wherein the polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers having a hydrophobic group, anionic group or cationic group.

[0039]

[24] The use according to

[23] , wherein the surface tension of a 0.1 wt % aqueous solution of the copolymer at 25° C. is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the above copolymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

[0040]

[25] The use according to any one of

[22] to

[24] , wherein at least one selected from a metal salt compound and an amino acid is used together with the polymer.

[0041]

[26] The application according to

[25] , wherein the metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and their hydrates.

[0042]

[27] The use according to

[25] or

[26] , wherein the amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

[0043] According to the present invention, the coagulation time of a sample lacking FXII in the APTT assay can be shortened, thereby enabling accurate detection of such blood samples based on the coagulation time. DETAILED DESCRIPTION

[0044] The present invention provides a clotting time shortening agent for a specimen lacking FXII in an APTT assay (hereinafter, also referred to as "the clotting time shortening agent of the present invention"). In addition, the present invention provides an APTT assay reagent containing the clotting time shortening agent. Furthermore, the present invention provides an APTT assay method using the clotting time shortening agent and a method for shortening the clotting time of a specimen lacking FXII in an APTT assay.

[0045] The "FXII-deficient sample" in the present specification refers to a blood sample deficient in coagulation factor XII (FXII), and examples thereof include blood samples collected from FXII-deficient patients and commercially available FXII-deficient plasma.

[0046] In the following description, "blood specimen" and "coagulation time" are sometimes referred to as "specimen" and "coagulation time", respectively. Therefore, in the following description, "coagulation time shortening" and "coagulation time shortening agent" are also referred to as "coagulation time shortening" and "coagulation time shortening agent", respectively.

[0047] In the APTT assay, a specimen and an APTT assay reagent are mixed to prepare a sample solution, and the coagulation reaction of the sample solution is measured. As the specimen, the plasma of the subject is preferably used. An anticoagulant commonly used in coagulation tests may be added to the specimen. For example, blood is collected using a blood collection tube filled with sodium citrate, and plasma is obtained by centrifugation. The APTT assay reagent is generally composed of a first reagent containing an activator and a second reagent containing a coagulation initiator. The specimen can be diluted with a diluent before being mixed with the APTT assay reagent as needed. The first reagent is added to the specimen, and after incubation for a specified time, the second reagent is added to initiate the coagulation reaction. The coagulation time of the sample solution after the second reagent is added is measured.

[0048] The present invention provides a coagulation time shortening agent for a FXII-deficient specimen in an APTT assay. In one embodiment, the coagulation time shortening agent of the present invention contains a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit as an active ingredient for shortening the coagulation time of a FXII-deficient specimen.

[0049] The above-mentioned polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit (hereinafter sometimes referred to as a polymer) is not particularly limited as long as the constituent monomer has 2-methacryloyloxyethyl phosphorylcholine. As a polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit, it can be a polymer of 2-methacryloyloxyethyl phosphorylcholine alone, or it can be a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers. The other monomer may have a hydrophobic group, an anionic group or a cationic group. Or the other monomer may have both a hydrophobic group and a hydrophilic group. Preferably, the other monomer is a monomer having a hydrophobic group, an anionic group or a cationic group bonded to the 2-methacryloyloxy group, and the copolymer has a methacryloyl group in the main chain and phosphorylcholine and a hydrophobic group, anionic group or cationic group in the side chain.

[0050] The polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit can be, for example, a polymer produced according to a known synthesis method such as that shown in Japanese Patent Publication No. 2004-189678. 2-methacryloyloxyethyl phosphorylcholine monomer is sold by NOF Corporation, for example, and a person skilled in the art can polymerize it with any monomer to produce a polymer having the desired properties. In addition, the polymer can use a commercially available product, such as the LIPIDURE (registered trademark) series sold by NOF Corporation. As the polymer, the LIPIDURE (registered trademark)-BL series sold as a product in the field of diagnostic drugs is preferred, but is not limited thereto. The LIPIDURE-BL series is a copolymer formed by copolymerizing various monomers with the 2-methacryloyloxyethyl phosphorylcholine monomer. For example, LIPIDURE (registered trademark)-BL200 series copolymerized with a monomer having a hydrophobic group, LIPIDURE (registered trademark)-BL400 series copolymerized with a monomer having an anionic group, LIPIDURE (registered trademark)-BL500 series copolymerized with a monomer having a cationic group, LIPIDURE (registered trademark)-BL800 series copolymerized with a monomer having a hydrophobic group, LIPIDURE (registered trademark)-BL1000 series, LIPIDURE (registered trademark)-BL1100 series, LIPIDURE (registered trademark)-BL1200 series, and LIPIDURE (registered trademark)-BL1300 series. More specific examples include LIPIDURE (registered trademark)-BL403, BL405, BL503, BL504, BL802, BL1002, BL1003, BL1103, BL1201, BL1301, and BL205.

[0051] The weight average molecular weight of the polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit is not particularly limited as long as the desired performance can be obtained. The lower limit of the weight average molecular weight of the polymer is, for example, 1000, preferably 2000, more preferably 5000, and further preferably 10000. The upper limit of the weight average molecular weight is, for example, 2000000, preferably 1000000, more preferably 700000, further preferably 100000, and further preferably 70000. The weight average molecular weight can be arbitrarily set within the range of any lower limit to any upper limit mentioned above. The weight average molecular weight of the polymer can be determined by conventional methods such as gel permeation chromatography. In addition, when the polymer is a copolymer, the constituent ratio of 2-methacryloyloxyethyl phosphorylcholine to other monomers in the copolymer varies depending on the structure of the monomers used, and generally, the content of 2-methacryloyloxyethyl phosphorylcholine in the copolymer is preferably 5 mol% or more. As for the weight average molecular weight and monomer constituent ratio of the polymer, a person skilled in the art can refer to the above-mentioned Lipidure (registered trademark)-BL series and select according to the purpose.

[0052] The polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit preferably has a surface tension of 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the above polymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 As for the surface tension and kinematic viscosity of the polymer, those skilled in the art can refer to the above-mentioned Lipidure (registered trademark)-BL series and select according to the purpose.

[0053] The above-mentioned polymers having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit may be used alone or in combination of any two or more.

[0054] The above polymer is used to shorten the coagulation time of a specimen lacking FXII in the APTT assay. The total amount of the polymer used in the APTT assay is not particularly limited as long as it is an effective amount for shortening the coagulation time of a specimen lacking FXII in the APTT assay. For example, the total amount of the polymer used in the APTT assay is preferably 0.0005 to 0.2% by mass, more preferably 0.001 to 0.1% by mass, more preferably 0.0075 to 0.075% by mass, and more preferably 0.01 to 0.05% by mass, based on the concentration of the polymer in the sample solution for the APTT assay.

[0055] In one embodiment, the clotting time shortening agent of the present invention may contain at least one selected from the following metal salt compounds and amino acids as an active ingredient for shortening the clotting time of a specimen lacking FXII. These metal salt compounds and amino acids may be used alone as an active ingredient of the clotting time shortening agent, or may be used in combination with the above-mentioned polymer as an active ingredient of the clotting time shortening agent.

[0056] As examples of the above-mentioned metal salt compounds, yttrium chloride, iron chloride (III), aluminum chloride, indium chloride (III), cupric chloride, potassium aluminum sulfate, ammonium iron sulfate (III), copper sulfate, zinc chloride, manganese chloride and their hydrates can be cited. As the hydrate, yttrium chloride hexahydrate, iron chloride hexahydrate (III), indium chloride tetrahydrate (III), copper chloride (II) dihydrate, potassium aluminum sulfate dodecahydrate, ammonium iron sulfate dodecahydrate (III), copper sulfate pentahydrate, etc. can be cited. The above-mentioned metal salt compounds can be used in any one or in combination of any two or more.

[0057] The above-mentioned metal salt compound is used to shorten the coagulation time of the specimen lacking FXII in the APTT assay. The total amount of the metal salt compound used in the APTT assay is not particularly limited as long as it is an effective amount for shortening the coagulation time of the specimen lacking FXII in the APTT assay. For example, the total amount of the metal salt compound used in the APTT assay is preferably 0.0001 to 0.15 mg / mL, more preferably 0.001 to 0.05 mg / mL, further preferably 0.005 to 0.04 mg / mL, and further preferably 0.0075 to 0.0225 mg / mL, based on the concentration of the metal salt compound in the sample solution for the APTT assay.

[0058] Examples of the amino acids include α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine, and tricin. These amino acids may be used alone or in combination of two or more.

[0059] The above amino acid is used to shorten the clotting time of a specimen lacking FXII in the APTT assay. The total amount of the amino acid used in the APTT assay is not particularly limited as long as it is an effective amount for shortening the clotting time of a specimen lacking FXII in the APTT assay. For example, the total amount of the amino acid used in the APTT assay is preferably 10 to 500 mM, more preferably 20 to 300 mM, further preferably 25 to 140 mM, and further preferably 30 to 135 mM, based on the concentration of the amino acid in the sample solution for the APTT assay.

[0060] The coagulation time shortening agent of the present invention may further contain a buffer, a polysaccharide, or an inclusion compound as an active ingredient for adjusting the coagulation time of a specimen lacking FXII, in addition to the above-mentioned polymer, metal salt compound, or amino acid. By using the buffer, polysaccharide, or inclusion compound in the coagulation time shortening agent of the present invention, the coagulation time of the specimen can be appropriately adjusted, for example, prolonged or shortened.

[0061] The buffering agent may be at least one selected from PIPES, ACES, HEPES, TAPSO, POPSO, EPPS and CHES. The total amount of the buffering agent used in the APTT assay is preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, further preferably 1 to 200 mM, and further preferably 10 to 100 mM, based on the concentration in the sample solution.

[0062] As the above-mentioned polysaccharides, at least one polysaccharide selected from dextran, cyclodextrin, sulfated polysaccharides and salts thereof can be cited. As sulfated polysaccharides, dextran sulfate and cyclodextrin sulfate can be cited. As salts of sulfated polysaccharides, dextran sulfate sodium and cyclodextrin sulfate sodium can be cited. As the above-mentioned inclusion compounds, cyclodextrin and its modified forms, and calixarene derivatives can be cited. As cyclodextrin and its modified forms, α-cyclodextrin sulfate, β-cyclodextrin sulfate, γ-cyclodextrin sulfate, and salts thereof can be cited. As salts, sodium salts can be cited. As calixarene derivatives, calixarene (6) hemisulfuric acid and calixarene (8) hemisulfuric acid can be cited. Any one of the above-mentioned polysaccharides or inclusion compounds can be used or any two or more of them can be used in combination. The total amount of the polysaccharide or inclusion compound used in the APTT measurement is preferably 0.0001 to 0.15 mg / mL, more preferably 0.001 to 0.05 mg / mL, and even more preferably 0.001 to 0.01 mg / mL in terms of concentration in the sample solution.

[0063] The above-mentioned buffers and polysaccharides or inclusion compounds may be used alone or in combination of any two or more.

[0064] The clotting time shortening agent of the present invention is used to shorten the clotting time of a specimen lacking FXII in APTT assay. The clotting time shortening agent of the present invention can be added alone to the above-mentioned specimen, diluent or sample solution for APTT assay, or the APTT assay reagent containing the clotting time shortening agent of the present invention can be added to the above-mentioned specimen, diluent or sample solution.

[0065] Therefore, the present invention provides an APTT assay reagent containing the clotting time shortening agent of the present invention. In one embodiment, the APTT assay reagent of the present invention contains at least one selected from the above-mentioned polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit, a metal salt compound, and an amino acid. The APTT assay reagent of the present invention preferably contains the above-mentioned polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit. In one embodiment, the APTT assay reagent of the present invention contains the above-mentioned polymer and at least one selected from the above-mentioned metal salt compound and an amino acid. In one embodiment, the APTT assay reagent of the present invention contains the above-mentioned polymer and the above-mentioned metal salt compound. In one embodiment, the APTT assay reagent of the present invention contains at least one selected from the above-mentioned buffer and a polysaccharide or inclusion compound in addition to at least one selected from the above-mentioned polymer, metal salt compound, and amino acid. The APTT assay reagent of the present invention can shorten the clotting time of a specimen lacking FXII.

[0066] The APTT assay reagent of the present invention is preferably a kit comprising a first reagent containing an activator and a second reagent containing a coagulation initiator. In the APTT assay reagent, the coagulation time shortening agent of the present invention is preferably contained in the first reagent, but may be contained in the kit separately from the first reagent and the second reagent.

[0067] The content of the clotting time shortening agent of the present invention in the APTT assay reagent of the present invention can be adjusted so that the concentration of the active ingredient of the clotting time shortening agent of the present invention contained in the sample solution obtained by mixing the APTT assay reagent and the specimen (for example, the total amount of the above-mentioned polymer, metal salt compound and amino acid) is within the above-mentioned effective amount range for shortening the clotting time of the specimen lacking FXII in the APTT assay. The same applies to the content of the above-mentioned buffer, polysaccharide or inclusion compound in the APTT assay reagent.

[0068] The APTT assay reagent provided by the present invention may contain, in addition to the coagulation time shortening agent of the present invention, various components required for activation or coagulation of a blood sample, etc. For example, in the APTT assay reagent, the first reagent may contain a buffer, an activator, a phospholipid, and the like.

[0069] The buffer can be appropriately selected and used in the range of pH 4 to 9, preferably pH 6 to 8. Examples of the buffer include the above-mentioned buffer, or amino acids, citric acid, phosphoric acid, acetic acid, imidazole, barbital, GTA, etc. The amino acid as the active ingredient of the above-mentioned coagulation time shortening agent of the present invention can also be used as a buffer. Any one of these buffers can be used or any two or more can be used in combination. The amount of the buffer used is not particularly limited as long as it is an amount that exerts the buffering capacity. As the concentration in the reagent, it is preferably 0.1 to 1000 mM, more preferably 0.5 to 500 mM, further preferably 1 to 200 mM, and further preferably 10 to 100 mM.

[0070] Examples of the activator include ellagic acid, kaolin, diatomaceous earth, colloidal silica, polyphenol compounds, anhydrous silicic acid, and metal ions. Examples of the metal ions include Zn 2+ , Mn 2+ , Cu 2+ , Fe 2+ 、Al 3+ Etc. The metal ion may be contained in the above reagent in the form of a salt. The metal salt compound which is the active ingredient of the above-mentioned coagulation time shortening agent of the present invention may also be used as an activator. Any one of the above-mentioned activators may be used or any two or more of them may be used in combination. The amount of the activator used is not particularly limited, and is preferably 0.001 to 2 mg / mL, more preferably 0.001 to 0.5 mg / mL, further preferably 0.001 to 0.1 mg / mL, and further preferably 0.005 to 0.05 mg / mL, based on the concentration in the reagent.

[0071] As examples of the above-mentioned phospholipids, natural or synthetic phospholipids can be cited. As the natural phospholipids, phospholipids from natural substances, such as rabbit brain, bovine brain, human placenta, soybean, egg yolk, etc. can be cited. As the synthetic phospholipids, for example, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), sphingomyelin (SM), cardiolipin, etc. can be cited. Any one of these phospholipids can be used or any two or more can be used in combination. For example, a variety of phospholipids can be mixed and used as liposomes. The amount of the phospholipid used is not particularly limited, and is preferably 0.005 to 2 mM, more preferably 0.02 to 0.5 mM, in terms of the concentration in the reagent. In the APTT assay reagent, the phospholipids as platelet substitutes are added as components required for the coagulation reaction. In the intrinsic coagulation reaction, phospholipids form a complex with activated factors IX and VIII, which forms fibrin through a common factor system (factors V, II and I) below activated factor X.

[0072] The second reagent of the APTT assay reagent may contain a component that triggers blood coagulation, such as calcium ions. As calcium ions, water-soluble calcium compounds such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, and calcium tartrate can be used. These calcium compounds can be used alone or in combination of any two or more. The amount of the calcium compound used is not particularly limited, and is preferably 5 to 100 mM, more preferably 10 to 50 mM, based on the concentration in the reagent.

[0073] The first reagent and the second reagent may further contain additives for improving the preservation or stability of the reagent. Examples of such additives include preservatives, antioxidants, dispersants, stabilizers, etc. Examples of the preservative include ciprofloxacin, propionic acid, sodium benzoate, sodium azide, a mixture of 2-methyl-1,2-thiazol-3(2H)-one and 5-chloro-2-methyl-1,2-thiazol-3(2H)-one (e.g., Proclin 300), etc. Examples of the antioxidant include citric acid, butylated hydroxyanisole, etc. Examples of the dispersant include phenol, collagen peptide, etc. Examples of the stabilizer include polyethylene glycol, polyvinyl pyrrolidone, high molecular weight polysaccharides such as dextran or polysucrose (e.g., Ficoll), salts such as sodium chloride, amino acids, sugars, etc.

[0074] The coagulation time shortening agent and the APTT assay reagent of the present invention may be in the form of a liquid or frozen product thereof, or may be in a dry form. The dry form reagent may be dissolved in water, a buffer solution, etc. to prepare a liquid reagent when used. The concentrations of the above-mentioned various components represent the concentrations in the liquid reagent.

[0075] The present invention provides a method for measuring a coagulation reaction (APTT assay) using the coagulation time shortening agent or APTT assay reagent of the present invention. The APTT assay method of the present invention can shorten the coagulation time of a specimen lacking FXII by using the coagulation time shortening agent or APTT assay reagent of the present invention.

[0076] The APTT assay method of the present invention can be implemented according to conventional methods except that the clotting time shortening agent or APTT assay reagent of the present invention is used. In a preferred embodiment, the first reagent containing the clotting time shortening agent of the present invention is mixed in the specimen diluted as needed, and the obtained mixed solution is heated. The heating conditions are, for example, 30°C to 40°C, preferably 35°C to 39°C. Thereafter, the second reagent is added to the mixed solution to induce a coagulation reaction. The coagulation reaction of the mixed solution (sample solution) after the second reagent is added is measured. The measurement can be performed using an optical method for measuring the amount of scattered light, transmittance or absorbance of the sample solution, or a mechanical method for measuring the viscosity of the sample solution. The temperature of the sample solution in the measurement is, for example, 30°C to 40°C, preferably 35°C to 39°C. The APTT assay reagent is preferably added to the specimen at a concentration such that the clotting time (APTT) of normal plasma falls within the range of 15 to 80 seconds, preferably 15 to 60 seconds, and more preferably 20 to 50 seconds.

[0077] The reaction start time of the coagulation reaction can be typically defined as the time when the second reagent is mixed in the specimen to induce the coagulation reaction. Alternatively, other times can be used as the reaction start time. The time for continuously measuring the coagulation reaction can be, for example, from the time when the specimen and the second reagent are mixed for several tens of seconds to about 7 minutes. The measurement time can be an arbitrarily determined fixed value, or it can be until the time when the coagulation reaction of each specimen is detected to be finished. The measurement of the progress of the coagulation reaction (for example, the measurement of the amount of scattered light) can be repeatedly performed at specified intervals within the measurement time. For example, it is sufficient to measure at intervals of 0.1 seconds. The clotting time (APTT) can be determined according to a known method based on the measured coagulation reaction.

[0078] The series of operations in the APTT assay method of the present invention can be performed using an automatic analyzer. Alternatively, a portion of the operations can be performed manually. For example, the specimen can be prepared by a person, and the subsequent operations can be performed by an automatic analyzer.

[0079] Example

[0080] The present invention will be described in further detail below with reference to Examples, but the present invention is not limited to these Examples.

[0081] Test 1

[0082] 1) Specimen

[0083] The following specimens were used.

[0084] FXII-deficient specimens (specimens from FXII-deficient patients, N=6)

[0085] 2) APTT assay reagent

[0086] 2-1) Basic formula

[0087] Reagent 1 (R1)

[0088]

[0089]

[0090] Reagent 2 (R2)

[0091]

[0092] *1) Prepared according to the method described in International Publication No. 2003 / 015753.

[0093] 2-2) Test reagents

[0094] · Test reagents 1-1 to 1-22: A polymer having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit (LIPIDURE (registered trademark)-BL series; BL403, BL405, BL503, BL504, BL802, BL1002, BL1003, BL1103, BL1201 or BL1301) was added to the basic formula R1 under the conditions described in Table 1.

[0095] Test reagents 2-1 to 2-10: In the basic formula R1, yttrium chloride hexahydrate, manganese (II) chloride tetrahydrate, iron (III) chloride hexahydrate, aluminum chloride (anhydrous), indium (III) chloride tetrahydrate, copper (II) chloride dihydrate, or ammonium iron (III) sulfate dodecahydrate were added under the conditions described in Table 2 instead of 0.015 mg / mL copper sulfate pentahydrate, or copper sulfate pentahydrate was used at the concentration described in Table 2. As a control, R1 containing zinc chloride instead of copper sulfate pentahydrate was prepared.

[0096] Test reagents 3-1 to 3-14: In the basic formulation R1, amino acids were added in place of glycine under the conditions described in Table 3. As a control, R1 containing lysine in place of glycine was prepared.

[0097] 2-3) Preparation of R1 and R2

[0098] The above components were mixed to give predetermined concentrations to prepare each test reagent and control R1 and R2. The mixing of R1 was performed under ice cooling.

[0099] 3) Determination of coagulation time

[0100] After heating 50 μL of the specimen at 37°C in a cuvette for 45 seconds, 50 μL of the first reagent (R1) is added and further heated for 171 seconds. 50 μL of the second reagent (R2) is added to the heated mixture to initiate a coagulation reaction. The reaction is carried out at 37°C. The specimen (sample solution) to which R2 is added is irradiated with light of a wavelength of 660 nm, and the optical change (change in scattered light intensity) caused by the coagulation reaction is measured. The coagulation time is calculated from the measured coagulation reaction. The coagulation time determination is carried out using a coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). The change rate (%) of the coagulation time (APTT) using the test reagent relative to the coagulation time (APTT) using the control (basic formula) is calculated [change rate (%) = (APTT using the test reagent) / APTT using the control × 100]. A change rate of less than 100% indicates a shortened coagulation time.

[0101] The results (average value of the change rate: N=6) are shown in Tables 1 to 3. The clotting time of the FXII-deficient specimen was shortened by the test reagent compared with the control.

[0102] [Table 1]

[0103]

[0104] [Table 2]

[0105]

[0106] [Table 3]

[0107] Test reagents Concentration (mass %) Amino Acids Change rate (%) Comparison 3 133 Lysine 100 3-1 133 α-Alanine 47 3-2 133 Arginine 47 3-3 30 Asparagine 48 3-4 133 Aspartic acid 51 3-5 133 Glutamate 52 3-6 133 Glycine 44 3-7 133 Glycylglycine 47 3-8 40 Methionine 54 3-9 36 Phenylalanine 49 3-10 133 Proline 52 3-11 133 Serine 49 3-12 105 Threonine 50 3-13 81 Valine 48 3-14 133 Triticum 51

[0108] Test 2

[0109] 1) Specimen

[0110] The following specimens were used.

[0111] FXII-deficient specimens (specimens from patients with FXII deficiency, N=3)

[0112] 2) APTT assay reagent

[0113] 2-1) Basic formula

[0114] The same formulation as in Experiment 1, wherein the copper sulfate pentahydrate in the first reagent (R1) was replaced with zinc chloride.

[0115] 2-2) Test reagents

[0116] Test reagent 4-1: LIPIDURE (registered trademark)-BL205 was added to the basic formulation R1 under the conditions described in Table 4. The case where LIPIDURE (registered trademark) was not added was used as a control.

[0117] Test reagents 5-1 to 5-3: The zinc chloride in the basic formula R1 was changed to the metal salt compounds listed in Table 5, and LIPIDURE (registered trademark)-BL205 was further added. Test reagent 4-1 was used as a control.

[0118] 2-3) Reagent preparation and clotting time determination

[0119] R1 and R2 were prepared by the same procedure as in Test 1, and the clotting time (APTT) was measured to determine the change rate (%) of APTT using the test reagent relative to the control.

[0120] The results (average value of the variation rate: N=3) are shown in Tables 4 to 5. By using a polymer (LIPIDURE (registered trademark)) and zinc chloride in combination, the coagulation time of the FXII-deficient specimen was shortened compared to the control compared to zinc chloride alone. In addition, when manganese (II) chloride tetrahydrate, yttrium chloride (hexahydrate) or indium (III) chloride tetrahydrate was used in combination with the polymer instead of zinc chloride, the coagulation time was further shortened.

[0121] [Table 4]

[0122]

[0123] [Table 5]

[0124]

Claims

1. A coagulation time shortening agent for a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time measurement, comprising a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit as an active ingredient.

2. The shortening agent according to claim 1, wherein The polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers having a hydrophobic group, an anionic group or a cationic group.

3. The shortening agent according to claim 2, wherein The surface tension of a 0.1 wt% aqueous solution of the copolymer at 25°C is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the copolymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

4. The shortening agent according to claim 1, wherein Furthermore, at least one selected from the group consisting of metal salt compounds and amino acids is used as an active ingredient.

5. The shortening agent according to claim 4, wherein The metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and hydrates thereof.

6. The shortening agent according to claim 4, wherein The amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

7. A reagent for measuring activated partial thromboplastin time, comprising the blood coagulation time shortening agent according to any one of claims 1 to 6.

8. A method for measuring an activated partial thromboplastin time, comprising measuring the coagulation time of a sample solution containing a blood sample and the coagulation time shortening agent according to any one of claims 1 to 6.

9. A method for shortening the coagulation time of a blood sample deficient in coagulation factor XII in an activated partial thromboplastin time measurement, comprising measuring the coagulation time of a sample solution containing the blood sample and the coagulation time shortening agent according to any one of claims 1 to 6.

10. Use of a polymer having 2-methacryloyloxyethylphosphocholine as a constituent unit for shortening the coagulation time of a blood sample lacking coagulation factor XII in an activated partial thromboplastin time measurement.

11. The use according to claim 10, wherein: The polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine and other monomers having a hydrophobic group, an anionic group or a cationic group.

12. The use according to claim 11, wherein: The surface tension of a 0.1 wt% aqueous solution of the copolymer at 25°C is 44 to 66×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 0.5 to 6 m 2 / s; or the surface tension of a 0.1 wt% aqueous solution of the copolymer at 25°C is 70 to 75×10 -3 N / m, and the kinematic viscosity of a 5 wt% aqueous solution at 25°C is 2 to 7.3 m 2 / s.

13. The use according to claim 10, wherein: At least one selected from the group consisting of a metal salt compound and an amino acid is used together with the polymer.

14. The use according to claim 13, wherein: The metal salt compound is at least one selected from yttrium chloride, iron (III) chloride, aluminum chloride, indium (III) chloride, copper chloride, potassium aluminum sulfate, ammonium iron (III) sulfate, copper sulfate, zinc chloride, manganese chloride and hydrates thereof.

15. The use according to claim 13, wherein: The amino acid is at least one selected from α-alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, glycylglycine, methionine, phenylalanine, proline, serine, threonine, valine and tricin.

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