Method for preparing serum samples and serum sample preparation kit

Abstract

The present invention provides a method for preparing serum samples and a serum sample testing kit that enable the easy preparation of high-quality serum samples without the need for centrifugation. [Solution] A method for preparing a serum sample, comprising: (a) preparing a blood mixture by adding a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator to blood, and coagulating the blood cell components; and (b) filtering the blood mixture after step (a) to remove coagulated material. Also, a serum sample preparation kit comprising a filtration filter, a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator.

Description

Technical Field

[0006] , ,

[0001] The present invention relates to a method for preparing a serum sample and a serum sample preparation kit.

Background Art

[0002] In blood chemistry tests, plasma or serum is prepared from blood and various components are analyzed. In blood collection performed at medical institutions such as hospitals, several mL to several tens of mL of blood is collected by venipuncture. Therefore, the preparation of plasma or serum from blood does not require any particular difficulty.

[0003] On the other hand, in self-blood collection performed using a painless needle or the like, the amount of blood that can be obtained is about several tens of μL. It is difficult to prepare plasma or serum from such a small amount of blood.

[0004] Patent Document 1 describes a method for separating serum or plasma from collected whole blood containing microparticles. In the method described in Patent Document 1, collected whole blood containing microparticles is mixed with a polymer flocculant, and the mixture is subjected to centrifugation to precipitate blood cells and microparticles aggregated by the polymer flocculant. The method described in Patent Document 1 is applicable to whole blood containing a microparticle preparation administered into the blood vessel such as an artificial enzyme carrier, and cannot be applied to blood not containing the microparticle preparation. Further, in the method described in Patent Document 1, since centrifugation is used, it is difficult to carry out at home.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] If high-quality plasma or serum samples can be prepared at home from small amounts of blood obtained through self-collection, patients will not need to go to a medical institution for blood tests. Therefore, patients can undergo blood tests more easily.

[0007] In a home setting, it is difficult to use specialized equipment such as centrifuges. Therefore, there is a need for a technology that allows for the simple preparation of high-quality plasma or serum samples from small amounts of blood without the need for specialized equipment.

[0008] Therefore, the present invention aims to provide a method for preparing serum samples and a serum sample testing kit that can easily prepare high-quality serum samples without performing centrifugation. [Means for solving the problem]

[0009] The present invention includes the following embodiments. [1] A method for preparing a serum sample, comprising: (a) preparing a blood coagulation treatment composition by adding a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure modifier to blood, and coagulating the blood cell components; and (b) filtering the blood coagulation treatment composition after step (a) to remove the coagulated material. [2] The method for preparing a serum sample according to [1], wherein the blood coagulation promoter is thrombin. [3] The method for preparing a serum sample according to [1] or [2], wherein the polyalkylene glycol is polyethylene glycol having a number average molecular weight of 10,000 or more and 500,000 or less. [4] A method for preparing a serum sample according to any one of [1] to [3], wherein the osmotic pressure adjusting agent is added to the blood such that the osmotic pressure of the blood coagulation treatment composition is higher than that of the plasma. [5] The method for preparing a serum sample according to any one of [1] to [4], wherein the osmotic pressure adjusting agent is an alkali metal salt. [6] A serum sample preparation kit comprising a filtration filter, a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator. [7] The serum sample preparation kit according to [6], wherein the blood coagulation promoter is thrombin. [8] The serum sample preparation kit according to [6] or [7], wherein the polyalkylene glycol is polyethylene glycol having a number average molecular weight of 10,000 or more and 100,000 or less. [9] A serum sample preparation kit according to any one of [6] to [8], wherein the osmotic pressure adjusting agent is an alkali metal salt. [Effects of the Invention]

[0010] According to the present invention, a method for preparing serum samples and a serum sample testing kit are provided that enable the easy preparation of high-quality serum samples without the need for centrifugation. [Brief explanation of the drawing]

[0011] [Figure 1] An example of the steps for preparing a serum sample according to one embodiment is shown. [Figure 2] This shows the correlation between the concentration of C-reactive protein (CRP) in serum samples prepared by Experimental Example 1 and the CRP concentration in serum samples prepared by centrifugation. [Modes for carrying out the invention]

[0012] [Method for preparing serum samples] A first aspect of this disclosure is a method for preparing a serum sample. The method for preparing a serum sample includes (a) preparing a blood coagulation treatment composition by adding a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator to blood, and coagulating the blood cell components; and (b) filtering the blood coagulation treatment composition after step (a) to remove the coagulated material.

[0013] The first embodiment of the method for preparing a serum sample is a method for preparing a serum sample from blood collected from a subject. The method of this embodiment allows for the preparation of a serum sample from a small amount of blood without the use of special equipment such as a centrifuge. A serum sample is a sample from which blood cell components and most coagulation factors have been removed.

[0014] <Process (a)> In step (a), a blood coagulation treatment composition is prepared by adding a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator to blood, thereby causing the blood cell components to coagulate.

[0015] (blood) Whole blood is used. Whole blood is blood collected from a subject and contains plasma and blood cells. The amount of blood used is not particularly limited. In the method of this embodiment, for example, serum samples can be prepared from small amounts of blood such as 500 μL or less, 400 μL or less, 300 μL or less, 200 μL or less, 100 μL or less, 90 μL or less, 80 μL or less, 70 μL or less, or 60 μL or less. The amount of blood used can be, for example, 10 μL or more, 20 μL or more, 30 μL or more, or 40 μL or more. The above upper and lower limits can be combined arbitrarily. Examples of blood quantities used include 10-500 μL, 10-400 μL, 10-300 μL, 10-200 μL, 10-100 μL, 10-90 μL, 10-80 μL, 10-70 μL, and 10-60 μL.

[0016] (Blood coagulation accelerator) Blood coagulation promoters are drugs that promote blood coagulation. Blood coagulation promoters promote the formation of fibrin and promote the coagulation of blood cell components through the action of fibrin. Examples of blood coagulation promoters include thrombin, thrombin activators (activated factor X (Xa), activated factor IX (IXa), etc.), and silica. Thrombin is preferred as a blood coagulation promoter. The animals from which thrombin and thrombin activators are derived are not particularly limited. Examples of animals from which they are derived include mammals (humans, mice, cattle, etc.).

[0017] The blood coagulation accelerator may be used alone or in combination of two or more kinds.

[0018] The amount of the blood coagulation accelerator used may be any concentration that can promote blood coagulation. When the blood coagulation accelerator is thrombin or a thrombin activating factor, examples of the concentration in the blood coagulation treatment composition include 0.1 to 500 units / mL, 1 to 100 units / mL, 5 to 50 units / mL, or 5 to 30 units / mL. When the blood coagulation accelerator is silica, examples of the concentration in the blood coagulation treatment composition include 0.01 to 1.5 mg / mL.

[0019] (Polyalkylene glycol) A polyalkylene glycol having a number average molecular weight of 10,000 or more (hereinafter sometimes simply referred to as "polyalkylene glycol") is used as a flocculant for coagulated blood cell components. By using a polyalkylene glycol having a number average molecular weight of 10,000 or more, fine aggregates can be formed in the coagulated blood cell components. When fine aggregates are formed, clogging of the filter can be avoided during the filter filtration in the subsequent step (b), and the coagulum can be efficiently removed.

[0020] Examples of the polyalkylene glycol include polyethylene glycol and polypropylene glycol. The polyalkylene glycol may have two or more monomer units. For example, the polyalkylene glycol may have an ethylene oxide unit (-C2H4O-) and a propylene oxide unit (-C3H6O-). Polyethylene glycol is preferred as the polyalkylene glycol.

[0021] Examples of the number-average molecular weight of polyalkylene glycol include 10,000 to 500,000. The number-average molecular weight of polyalkylene glycol is 10,000 or more, and may be 15,000 or more, or 16,000 or more. Examples of the upper limit of the number-average molecular weight of polyalkylene glycol include 500,000 or less, and may be 300,000 or less, 200,000 or less, 100,000 or less, 80,000 or less, 70,000 or less, 60,000 or less, 50,000 or less, 40,000 or less, or 350,000 or less. The above upper and lower limits can be combined in any way. Examples of the number-average molecular weight ranges for polyalkylene glycols include 10,000-500,000, 10,000-300,000, 10,000-200,000, 10,000-100,000, 10,000-80,000, 10,000-70,000, 10,000-60,000, 10,000-50,000, and 10,000-40,000. The number-average molecular weight can be calculated using the polystyrene-based number-average molecular weight obtained by GPC (gel permeation chromatography).

[0022] Polyalkylene glycols may be used individually or in combination of two or more types.

[0023] The amount of polyalkylene glycol used should be such that it can aggregate the coagulated material formed in the blood coagulation treatment composition to form fine aggregates. Examples of the concentration of polyalkylene glycol in the blood coagulation treatment composition include 0.1 to 20% (w / v), and may also be 0.5 to 15% (w / v), 1 to 10% (w / v), or 3 to 8% (w / v). "% (w / v)" represents the concentration expressed as a percentage of the mass (g) of solute contained in 1 mL of solution. 1% (w / v) is equivalent to 0.01 g / mL.

[0024] (Osmotic pressure regulator) Osmotic regulators are used to adjust the osmotic pressure of blood coagulation treatment compositions. Preferably, the osmotic regulator is added to the blood so that the osmotic pressure of the blood coagulation treatment composition is higher than that of the plasma. The osmotic pressure of plasma is 285 mOsm / L. Therefore, it is preferable that the osmotic regulator is added to the blood so that the osmotic pressure of the blood coagulation treatment composition is higher than 285 mOsm / L. By adjusting the osmotic pressure of the blood coagulation treatment composition to be higher than that of the plasma, high-quality serum samples with suppressed hemolysis can be prepared.

[0025] When serum samples are subjected to biochemical analysis, it is preferable to use osmotic regulators that do not interfere with the biochemical analysis. Examples of osmotic regulators include inorganic salts, organic salts, and sugars. Examples of inorganic salts include salts of alkali metals (sodium, potassium, etc.) and inorganic acids, salts of alkaline earth metals (calcium, magnesium, etc.) and inorganic acids, and ammonium salts of inorganic acids. Examples of inorganic acids include hydrochloric acid, phosphoric acid, sulfuric acid, carbonic acid, nitric acid, and boric acid. Specific examples of inorganic salts include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, potassium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate. Examples of organic salts include salts of alkali metals (sodium, potassium, etc.) and organic acids, salts of alkaline earth metals (calcium, magnesium, etc.) and organic acids, and ammonium salts of organic acids. Examples of organic acids include acetic acid, citric acid, and tartaric acid. Specific examples of organic salts include, but are not limited to, sodium acetate, potassium acetate, sodium citrate, and sodium tartrate. Sugars can be monosaccharides or disaccharides. Specific examples of sugars include glucose, sucrose, mannitol, and sorbitol. The osmotic pressure regulator is preferably an alkali metal salt. The alkali metal salt is preferably at least one selected from the group consisting of sodium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0026] Osmotic pressure regulators may be used individually or in combination of two or more types.

[0027] It is preferable to use an amount of osmotic pressure regulator such that, when an aqueous solution of the osmotic pressure regulator is prepared in the same volume as the blood coagulation treatment composition, the osmotic pressure of the aqueous solution is between 450 and 1300 mOsm / L. In other words, assuming an aqueous solution of the osmotic pressure regulator is prepared in the same volume as the blood coagulation treatment composition, it is preferable to add an amount of osmotic pressure regulator to the blood such that the osmotic pressure of the aqueous solution is within the range of 450 to 1300 mOsm / L.

[0028] The osmotic pressure of the aqueous solution can be calculated using the following formula (1). Osmotic pressure (mOsm / L) = (M × I) / V (1) M: Number of moles (mmol) of osmotic regulator I: Number of ion particles generated from one molecule of osmotic pressure regulator (1 if the substance does not ionize) V: Volume of aqueous solution

[0029] In formula (1) above, V can be replaced with the volume of the blood coagulation treatment composition. It is preferable to use an amount of osmotic pressure adjusting agent such that the osmotic pressure calculated by formula (1) above is 450 to 1300 mOsm / L. When two or more osmotic pressure adjusting agents are used, the osmotic pressure can be calculated by summing the values ​​calculated by (M × I) for each osmotic pressure adjusting agent and dividing by V.

[0030] (Addition method) The method of adding a blood coagulation accelerator, polyalkylene glycol, and osmotic pressure regulator to blood is not particularly limited. For example, a solution containing a blood coagulation accelerator, polyalkylene glycol, and osmotic pressure regulator (hereinafter also referred to as a "blood coagulation accelerator composition") may be prepared and added to the blood. Alternatively, the blood coagulation accelerator, polyalkylene glycol, and osmotic pressure regulator may each be dissolved in water to prepare aqueous solutions, and these aqueous solutions may be added to the blood.

[0031] A blood coagulation accelerator composition may be prepared, for example, by dissolving a blood coagulation accelerator and a polyalkylene glycol in an aqueous solution containing an osmotic pressure regulator. The aqueous solution containing the osmotic pressure regulator may also be a buffer solution containing the osmotic pressure regulator. Specific examples of aqueous solutions containing the osmotic pressure regulator include potassium phosphate buffer (a buffer solution obtained by dissolving potassium dihydrogen phosphate and dipotassium hydrogen phosphate in water) and an aqueous sodium chloride solution. A specific example of potassium phosphate buffer is 0.2 M potassium phosphate buffer. The pH of the potassium phosphate buffer can be, for example, pH 7 to 8, with pH 7.4 being a specific example. A specific example of an aqueous sodium chloride solution is a 1.5 to 4.0% (w / v) aqueous sodium chloride solution.

[0032] A blood coagulation accelerator composition may be prepared by dissolving a blood coagulation accelerator, polyalkylene glycol, and an osmotic pressure regulator in water.

[0033] Examples of the mixed volume ratio of blood to the blood coagulation accelerator composition include blood:blood coagulation accelerator composition = 1:10 to 1:50, but may also be 1:10 to 1:40, 1:10 to 1:30, 1:10 to 1:20, 1:15 to 1:40, 1:15 to 1:30, or 1:15 to 1:20.

[0034] A blood coagulation accelerator, polyalkylene glycol, and osmotic pressure regulator are added to blood, and then the mixture is stirred and mixed to prepare a blood coagulation treatment composition. The blood coagulation treatment composition is a composition in which a blood coagulation accelerator, polyalkylene glycol, and osmotic pressure regulator are added to blood. Blood coagulation treatment is performed using the blood coagulation treatment composition.

[0035] The blood coagulation treatment is carried out by preparing a blood coagulation treatment composition and allowing the blood coagulation treatment composition to stand. The standing time for the blood coagulation treatment composition is preferably 1 minute or more, and more preferably 2 minutes or more. The upper limit of the standing time is not particularly limited, but for example, it is 30 minutes or less. The standing time for the blood coagulation treatment composition can be 1 to 30 minutes, and may also be 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, or 1 to 5 minutes.

[0036] By allowing the blood coagulation treatment composition to stand, fibrin formation progresses, and the interaction between fibrin and blood cells leads to the coagulation of blood cells.

[0037] Step (a) can be carried out entirely at room temperature. Room temperature is approximately 10 to 35°C, preferably 15 to 30°C, and more preferably 20 to 30°C.

[0038] <Process (b)> In step (b), the blood coagulation treatment composition after step (a) is filtered to remove the coagulated material.

[0039] The filter used for filtration should be capable of removing the coagulated material formed in step (a). Since the size of red blood cells is approximately 7 μm, the pore size of the filter should preferably be less than 7 μm. For example, the pore size of the filter may be 5 μm or less. For example, the lower limit of the pore size of the filter may be 0.5 μm or more, or 1 μm or more. If the pore size of the filter is greater than or equal to the lower limit, clogging of the filter and hemolysis can be suppressed. For example, the range of pore size of the filter may be 0.5 to 5 μm and 1 to 5 μm. As the filter, a syringe filter that can be attached to a syringe can be used.

[0040] The blood coagulation treatment composition after step (a) can be filtered by placing it in a syringe fitted with a filter at its tip and pressing down the syringe piston. Alternatively, the blood coagulation composition can be filtered by connecting a syringe to a dropper-shaped container and pressing down on the dropper container. The filtrate obtained by filter filtration can be collected in a suitable container. For example, the filtrate can be collected in a microtube or the like. This filtrate can be used as a serum sample.

[0041] Figure 1 shows an example of the flow of the method according to this embodiment. In the example in Figure 1, a blood coagulation accelerator composition is prepared. Next, the blood coagulation accelerator composition and blood are mixed and stirred to prepare a blood coagulation treatment composition. Next, the blood coagulation treatment composition is allowed to stand. After standing, the blood coagulation treatment composition is filtered and the filtrate is obtained as a serum sample.

[0042] In this embodiment, serum samples can be prepared from a small amount of blood without using special equipment such as centrifugation. This embodiment also allows for the production of high-quality serum samples with minimal hemolysis.

[0043] Serum samples prepared by the method of this embodiment can be used for serum biochemical analysis, such as serum protein analysis.

[0044] [Serum sample preparation kit] A second aspect of this disclosure is a serum sample preparation kit. The serum sample preparation kit comprises a filtration filter, a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator.

[0045] The filtration filter, blood coagulation accelerator, polyalkylene glycol with a number-average molecular weight of 10,000 or more, and osmotic pressure regulator can be the same as those described in the [Method for Preparing Serum Samples] above.

[0046] A specific example of a filtration filter is a syringe filter with a pore size of 0.5 to 5 μm, preferably 1 to 5 μm. A specific example of a blood coagulation accelerator is thrombin. Polyalkylene glycols with a number average molecular weight of 10,000 or more include polyethylene glycols with a number average molecular weight of 10,000 to 500,000.

[0047] Specific examples of osmotic regulators include sodium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. The osmotic regulator may be provided in the form of an aqueous solution. For example, when the osmotic regulator is potassium dihydrogen phosphate and dipotassium hydrogen phosphate, it may be provided as a potassium phosphate buffer. The concentration of potassium phosphate in the potassium phosphate buffer is preferably 0.16 M or higher, and more preferably 0.2 M or higher. The pH of the potassium phosphate buffer is typically 7 to 8. For example, when the osmotic regulator is sodium chloride, it may be provided as an aqueous sodium chloride solution. The concentration of sodium chloride in the aqueous sodium chloride solution is typically 1.5 to 4.0% (w / v).

[0048] <Other configurations> The kit of this embodiment may include other components in addition to the above configuration. Examples of other components include a blood collection device, a container for preparing a blood coagulation treatment composition, a container for collecting serum samples, a diluent, and so on.

[0049] Publicly known blood collection devices can be used. Preferably, the blood collection device is one that allows for self-collection of blood. Examples of blood collection devices include blood collection lancets equipped with painless needles.

[0050] A container for preparing a blood coagulation treatment composition is used to prepare a blood coagulation treatment composition by mixing blood with a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure regulator. The blood coagulation accelerator, the polyalkylene glycol with a number average molecular weight of 10,000 or more, and the osmotic pressure regulator may be pre-filled in the container for preparing the blood coagulation treatment composition. The container for preparing the blood coagulation treatment composition may be in the shape of a dropper to which a filter can be connected, making it easy to supply the blood coagulation treatment composition to the filter. The container for preparing the blood coagulation treatment composition may also be a syringe to which a filter can be connected.

[0051] A serum sample collection container is used to collect the filtrate after filtration. The serum sample collection container may be, for example, a microcentrifuge tube.

[0052] Diluents are used to dilute blood coagulation accelerators, polyalkylene glycols, osmotic pressure regulators, and / or blood. Examples of diluents include water (deionized water, distilled water, etc.) and buffer solutions (phosphate-buffered saline, etc.).

[0053] The kit of this embodiment can be used to prepare a serum sample from a small amount of blood. The kit of this embodiment can be used to carry out the serum sample preparation method according to the first embodiment described above. [Examples]

[0054] The present invention will be explained below with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0055] [Reagents, etc.] Filtration filter (ABLUO syringe filter, 25mm diameter, 5μm pore size) Polyethylene dropper (Used by: Mano Chemical Container Co., Ltd., Urine Collection Dropper, 10 mL) Thrombin (derived from bovine, manufactured by Fuji Pharmaceutical Co., Ltd., Thrombin Oral / Topical Preparation 10,000 "F") 0.2M potassium phosphate buffer solution, pH 7.4 (Muto Chemical Co., Ltd.)

[0056] [Experimental Example 1] <Preparation of serum samples> (Preparation of blood coagulation promoter compositions) 945 μL of 0.2 M potassium phosphate buffer containing 5% (w / v) polyethylene glycol 35000 (number average molecular weight 35,000, Sigma-Aldrich) was mixed with 5 μL of thrombin (2000 units / mL) dissolved in physiological saline. This prepared a blood coagulation accelerator composition.

[0057] (Blood coagulation process) 950 μL of blood coagulation accelerator composition was placed in a polyethylene dropper. Next, 50 μL of whole blood sample was placed in the same polyethylene dropper. The blood coagulation accelerator composition and whole blood sample in the polyethylene dropper were mixed by inversion. The polyethylene dropper was then left to stand for 5 minutes. This obtained a blood coagulation treatment composition.

[0058] (Removal of solidified material) A polyethylene dropper, which had been left to stand for 5 minutes, was connected to a filter, and the blood coagulation treatment composition was filtered manually. This removed the coagulated material from the blood coagulation treatment composition, and a serum sample was obtained.

[0059] [Experimental Example 2] In preparing the blood coagulation accelerator composition, serum samples were prepared using the same method as in Experimental Example 1, except that phosphate-buffered saline (PBS) was used instead of 0.2 M potassium phosphate buffer and thrombin was not added.

[0060] [Experimental Example 3] In preparing the blood coagulation accelerator composition, serum samples were prepared using the same method as in Experimental Example 1, except that thrombin was not added.

[0061] [Experimental Examples 4-6] In preparing the blood coagulation accelerator composition, serum samples were prepared in the same manner as in Experimental Example 1, except that the concentration of potassium phosphate buffer was changed to 0.02 M, 0.05 M, or 0.1 M.

[0062] [Experimental Examples 7-13] Serum samples were prepared in the same manner as in Experimental Example 1, except that 0.9% (w / v), 1.5% (w / v), 2.0% (w / v), 3.0% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v) aqueous sodium chloride solutions were used instead of 0.2M potassium phosphate buffer in the preparation of the blood coagulation accelerator composition.

[0063] [Experimental Examples 14-16] In preparing the blood coagulation accelerator composition, serum samples were prepared in the same manner as in Experimental Example 1, except that dextran with a number-average molecular weight of 40,000, 60,000, or 200,000 was used instead of polyethylene glycol 35,000. The concentration of dextran in the blood coagulation accelerator composition was 5% (w / v), as in Experimental Example 1.

[0064] [Experimental Examples 17-19] Serum samples were prepared using the same method as in Experimental Example 1, except that the standing time in the blood coagulation process was changed to 30 seconds, 1 minute, or 2 minutes.

[0065] <Evaluation of hemolysis> The serum samples from each of the above experimental examples were visually inspected for hemolysis. The results are shown in Tables 1-4. The evaluation criteria are as follows.

[0066] Evaluation criteria: -: No hemolysis (serum sample is clear). +: Slight hemolysis (serum sample is pale red). ++: Significant hemolysis present (serum sample is red).

[0067] [Table 1]

[0068] [Table 2]

[0069] [Table 3]

[0070] [Table 4]

[0071] In Tables 1-4, "Composition" refers to the components used in the preparation of the blood coagulation accelerator composition. Each notation in Tables 1-4 refers to the following components: 0.2M KPi: 0.2M potassium phosphate buffer (pH 7.5) 0.02M KPi: 0.02M potassium phosphate buffer (pH 7.4) 0.05M KPi: 0.05M potassium phosphate buffer (pH 7.4) 0.1M KPi: 0.1M potassium phosphate buffer (pH 7.4) 0.9% NaCl: 0.9% (w / v) sodium chloride aqueous solution 1.5% NaCl: 1.5% (w / v) sodium chloride aqueous solution 2.0% NaCl: 2.0% (w / v) sodium chloride aqueous solution 3.0% NaCl: 3.0% (w / v) sodium chloride aqueous solution 4.0% NaCl: 4.0% (w / v) sodium chloride aqueous solution 4.5% NaCl: 4.5% (w / v) sodium chloride aqueous solution PEG35000: Polyethylene glycol (number average molecular weight 35,000) DEX35000: Dextran with a number-average molecular weight of 35,000 DEX40000: Dextran with a number-average molecular weight of 40,000 DEX60000: Dextran with a number-average molecular weight of 60,000 DEX200000: Dextran with a number-average molecular weight of 200,000

[0072] Table 5 shows the osmotic pressures of each solvent used in the preparation of the blood coagulation accelerator composition.

[0073] [Table 5]

[0074] The results in Tables 1-4 confirm that using a blood coagulation accelerator is effective in preparing serum samples with minimal hemolysis. It was also confirmed that polyethylene glycol is preferable to dextran as the polymer compound in the preparation of the blood coagulation accelerator composition.

[0075] In the preparation of blood coagulation accelerator compositions, it has been confirmed that it is preferable to use a solvent with an osmotic pressure of approximately 500 to 1400 mOsm / L. The osmotic pressure of physiological saline (0.9% (w / v) sodium chloride aqueous solution) is 308 mOsm / L. The osmotic pressure of plasma is 285 mOsm / L. Therefore, it is considered preferable to use an osmotic pressure regulator so that the osmotic pressure of the blood coagulation treatment composition prepared by adding the blood coagulation accelerator composition to blood is higher than the osmotic pressure of plasma. It is considered preferable to use an amount of osmotic pressure regulator such that, when an aqueous solution of the same volume as the blood coagulation treatment composition is prepared, the osmotic pressure of the aqueous solution becomes 450 to 1300 mOsm / L.

[0076] [Experimental Example 20] (Preparation of blood coagulation promoter compositions) The blood coagulation accelerator composition was prepared and the blood coagulation treatment was performed using the same method as in Experimental Example 1.

[0077] [Experimental Examples 21-25] In the preparation of the blood coagulation accelerator composition, polyethylene glycol with number average molecular weights of 200, 2,000, approximately 3,100 (PE-74), approximately 3,500 (PE-75), or approximately 16,000 (PE-108) was used instead of polyethylene glycol 35,000, except that the blood coagulation accelerator composition was prepared and the blood coagulation treatment was performed in the same manner as in Experimental Example 1. The concentration of polyethylene glycol in the blood coagulation accelerator composition was 5% (w / v), as in Experimental Example 1.

[0078] [Experimental Example 26] In the preparation of the blood coagulation accelerator composition, PBS was used instead of 0.2M potassium phosphate buffer, and polyethylene glycol 35000 was not added. The blood coagulation accelerator composition was prepared and the blood coagulation treatment was performed in the same manner as in Experimental Example 1.

[0079] <Evaluation of aggregate formation> The blood coagulation treatment composition was visually observed after the blood coagulation treatment, and the formation of agglutination clumps was evaluated. The results are shown in Tables 6 and 7. The evaluation criteria are as follows:

[0080] Evaluation criteria: A: Formation of fine aggregates. B: A huge blood clot formed.

[0081] [Table 6]

[0082] [Table 7]

[0083] In Table 6, "Composition" refers to the components used in the preparation of the blood coagulation accelerator composition. The notations in Table 6 are the same as those in Tables 1-4. Notations not listed in Tables 1-4 refer to the following components. PEG200: Polyethylene glycol (number average molecular weight 200) PEG2000: Polyethylene glycol (number average molecular weight 2,000) PEG3100: Polyethylene glycol (PE-74, number average molecular weight approximately 3,100) PEG3500: Polyethylene glycol (PE-75, number average molecular weight approximately 3,500) PEG16000: Polyethylene glycol (PE-108, number average molecular weight approximately 16,000)

[0084] The results shown in Table 6 confirm that using polyethylene with a number-average molecular weight of 10,000 or more results in the formation of fine aggregates. It is believed that the formation of these fine aggregates helps to prevent filter clogging during filtration.

[0085] <Evaluation of inflammatory marker (CRP) concentration in serum samples> The concentration of C-reactive protein (CRP) in serum samples prepared using Experimental Example 1 was compared with the CRP concentration in serum samples prepared using the conventional centrifugation method. The results are shown in Figure 1.

[0086] As shown in Figure 1, the CRP concentration in the serum sample prepared by Experimental Example 1 was found to be linearly correlated with the CRP concentration in the serum sample prepared by centrifugation. This result confirms that the serum sample prepared by Experimental Example 1 can be used as a serum sample for serum component analysis. [Industrial applicability]

[0087] According to the present invention, a method for preparing serum samples and a serum sample testing kit are provided that enable the easy preparation of high-quality serum samples without the need for centrifugation.

Claims

1. (a) A step of preparing a blood coagulation treatment composition by adding a blood coagulation accelerator, a polyalkylene glycol with a number average molecular weight of 10,000 or more, and an osmotic pressure modifier to blood, and coagulating the blood cell components, (b) A step of filtering the blood coagulation treatment composition after step (a) to remove coagulated material, A method for preparing serum samples, including [the specified component].

2. The method for preparing a serum sample according to claim 1, wherein the blood coagulation promoter is thrombin.

3. The method for preparing a serum sample according to claim 1 or 2, wherein the polyalkylene glycol is polyethylene glycol having a number average molecular weight of 10,000 or more and 500,000 or less.

4. The method for preparing a serum sample according to claim 1 or 2, wherein the osmotic pressure adjusting agent is added to the blood such that the osmotic pressure of the blood coagulation treatment composition is higher than the osmotic pressure of the plasma.

5. The method for preparing a serum sample according to claim 1 or 2, wherein the osmotic pressure adjusting agent is an alkali metal salt.

6. Filtration filter and, Blood coagulation accelerators, Polyalkylene glycols with a number average molecular weight of 10,000 or more, Osmotic pressure regulators, A serum sample preparation kit, including the following:

7. The serum sample preparation kit according to claim 6, wherein the blood coagulation promoter is thrombin.

8. The serum sample preparation kit according to claim 6 or 7, wherein the polyalkylene glycol is polyethylene glycol having a number average molecular weight of 10,000 or more and 100,000 or less.

9. The serum sample preparation kit according to claim 6 or 7, wherein the osmotic pressure adjusting agent is an alkali metal salt.

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