Blood separation method, blood separation apparatus, and blood separation kit

Abstract

The present invention provides a blood separation method that can efficiently separate a sufficient amount of serum from a small amount of blood without causing hemolysis. [Solution] The blood separation method includes a containment step of containing a mixed sample containing a blood sample and a diluent in a container, a coagulation step of coagulating the mixed sample in the container, and a filtration step of separating serum from the blood clot formed by the coagulation of the mixed sample by bringing the contents of the container into contact with a filter after the coagulation step.

Description

Technical Field

[0001] The present invention relates to a blood separation method for separating serum from blood, a blood separation device, and a blood separation kit.

Background Art

[0002] For diagnosing the health condition of a subject, blood tests for analyzing the presence or concentration of specific substances contained in blood are widely performed. For example, by analyzing the content of various components contained in the liquid component in blood, the organ function etc. of the subject can be diagnosed.

[0003] In a general blood test, the blood sample is separated into a liquid component such as plasma or serum and a solid component such as cells using a centrifuge, a suction / pressure pump, etc. Usually, for centrifugation, a certain amount (for example, 5 mL or more) of blood is required. Therefore, a general blood test is performed by a doctor, a nurse, or a clinical laboratory technician after collecting blood from a subject, in a medical institution or a specialized inspection institution equipped with a centrifuge and other analysis instruments and a power supply facility for operating them.

[0004] On the other hand, development of a technique for separating plasma components without using a centrifuge, a suction / pressure pump, etc. has also been carried out (see, for example, Patent Documents 1 and 2).

[0005] In recent years, the need for so-called POCT (Point of Care Testing), which can be quickly tested at the point of medical treatment and care (Point of Care), has also been increasing. POCT can be used not only for rapid testing at the site of home medical care and emergency, but also for health management of people in areas with underdeveloped medical infrastructure such as disaster areas and developing countries, and for self-health management from the viewpoints of preventive medicine and healthy life expectancy. Therefore, a technique for separating liquid components from blood is required even in places without equipped facilities.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-205253 [Patent Document 2] Japanese Patent Publication No. 2004-344874 [Overview of the project] [Problems that the invention aims to solve]

[0007] In the plasma separation apparatus disclosed in Patent Document 1, there is a gap (flow channel) that generates capillary force through a combination of multiple hydrophilic members, and plasma is aspirated and extracted from this gap by capillary force.

[0008] However, when the inventors of this application examined the plasma separation device disclosed in Patent Document 1, they found that the yield of the amount of plasma collected was insufficient, and it was difficult to recover more than 30% of the total blood volume. In other words, depending on the amount of blood collected from the subject (for example, a small amount of blood of about several tens of μL collected by fingertip puncture), even a simple blood test device for POCT may not have enough sample volume to meet the requirements.

[0009] On the other hand, Patent Document 2 describes a filter device configured by connecting a plasma or serum separation membrane, which is a membrane for separating plasma or serum from blood and has a porosity of 30% or less, in series after a first filter that moves plasma faster than blood cells. Furthermore, Patent Document 2 discloses that the blood to be separated "may be a diluted blood sample" (see paragraph 0036 of Patent Document 2), that "if the blood flow direction and the filtration direction are the same, clogging may occur in the through-holes. However, clogging is less likely to occur when separating blood cells from diluted blood" (see paragraph 0038 of the same document), and that "during the separation process, filtration is completed when red blood cells clog the holes" (see paragraph 0039 of the same document).

[0010] For example, as disclosed in Patent Document 2, the amount of liquid component usable for testing can be increased by diluting a blood sample. However, when a blood sample is diluted, its viscosity decreases, which improves its water permeability, and it is possible that red blood cells may pass through the filter. Furthermore, if the gaps in the filter are adjusted to prevent red blood cells from passing through, the diluted blood sample may soak into the filter all at once, causing the gaps in the filter to become blocked by red blood cells and the filter to clog prematurely. In such a state, applying pressure to the blood sample by a piston or suction may cause hemolysis.

[0011] The present invention has been made in view of the above, and aims to provide a blood separation method, a blood separation apparatus, and a blood separation kit that can efficiently separate a sufficient amount of serum for testing from a small amount of blood without causing hemolysis. [Means for solving the problem]

[0012] A blood separation apparatus according to one aspect of the present invention includes a containment step of containing a mixed sample containing a blood sample and a diluent in a container, a coagulation step of coagulating the mixed sample in the container, and a filtration step of separating serum from the blood clot formed by the coagulation of the mixed sample by contacting the contents of the container with a filter after the coagulation step.

[0013] In the blood separation method described above, the dilution ratio of the blood sample in the mixed sample may be 10 times or more. In the blood separation method described above, the coagulation step may include letting the mixed sample stand for one minute or more.

[0014] In the blood separation method described above, the coagulation step may include adding a coagulation accelerator to the mixed sample. In the blood separation method described above, a coagulation accelerator may be added to the diluent beforehand.

[0015] In the above blood separation method, the coagulation accelerator may contain any one of silica, thrombin, thrombin-like enzyme, celite, and calcium. In the above blood separation device, the blood sample may be fingertip puncture blood.

[0016] Another aspect of the blood separation device of the present invention is a container capable of accommodating a mixed sample containing a blood sample and a diluent, the container having an opening provided at one end side, and a lid capable of closing the opening, the lid being provided with a discharge port capable of discharging liquid from the inside to the outside of the container, and a filter for filtering the contents of the container, the filter being configured not to contact the contents in a state where the closed container is upright, and being configured to contact the contents by reversing the top and bottom of the closed container.

[0017] In the above blood separation device, the contents include a blood clot and serum formed by coagulation of the mixed sample in the container, and the serum may be separated from the blood clot by contacting the contents with the filter by reversing the top and bottom of the closed container.

[0018] In the above blood separation device, the discharge port may be a nozzle provided so as to protrude outside the container. In the above blood separation device, the filter may be attached inside the lid.

[0019] In the above blood separation device, a space capable of accommodating the liquid that has passed through the filter may be provided between the filter and the discharge port.

[0020] The above blood separation device may further include a diluent accommodated in the container. In the above blood separation device, a coagulation accelerator may be added to the diluent.

[0021] Another aspect of the present invention is a blood separation kit, which includes the above blood separation device and a blood collection tool capable of transporting a blood sample with a dilution ratio of 10 times or more with respect to the diluent contained in the container.

Advantages of the Invention

[0022] According to the present invention, it becomes possible to efficiently separate a sufficient amount of serum from a small amount of blood without causing hemolysis.

Brief Description of the Drawings

[0023] [Figure 1] It is a cross-sectional view showing a schematic configuration of a blood separation device according to an embodiment of the present invention. [Figure 2] It is a cross-sectional view showing the configuration of the container shown in FIG. 1. [Figure 3] It is a cross-sectional view showing the configuration of the lid shown in FIG. 1. [Figure 4] It is a flowchart showing a blood separation method according to an embodiment of the present invention. [Figure 5] It is a schematic diagram for explaining a blood separation method according to an embodiment of the present invention. [Figure 6] It is a schematic diagram for explaining a blood separation method according to an embodiment of the present invention. [Figure 7] It is a schematic diagram for explaining a blood separation method according to an embodiment of the present invention. [Figure 8] It is a table showing the results of Experiment 1. [Figure 9] It is a graph showing the results of Experiment 2. [Figure 10] It is a table showing the results of Experiment 3. [Figure 11] It is a graph showing the results of Experiment 3.

Modes for Carrying Out the Invention

[0024] Hereinafter, a blood separation method, blood separation apparatus, and blood separation kit according to embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to these embodiments. Furthermore, in each drawing, the same parts are denoted by the same reference numerals.

[0025] The drawings referenced in the following description merely provide a schematic representation of the shape, size, and positional relationships to the extent necessary to understand the content of the present invention. That is, the present invention is not limited to the shapes, sizes, and positional relationships exemplified in each drawing. Furthermore, there may be differences in the dimensional relationships and proportions between drawings.

[0026] In this specification, "trace amount" refers to a very small amount, such as the amount of blood that can be collected by fingertip puncture, and specifically refers to an amount of about 10 μL to 50 μL. "Sufficient amount" refers to an amount required in a simple blood testing device for POCT, for example, an amount of about 1 μL to 1000 μL after dilution.

[0027] (Composition of blood separation device and blood separation kit) Figure 1 is a cross-sectional view showing a schematic configuration of a blood separation device according to an embodiment of the present invention. As shown in Figure 1, the blood separation device 1 according to this embodiment is a container 10 capable of containing a mixed sample including a blood sample and a diluent, and comprises a container 10 having an opening 10a on one end and a lid 20 capable of closing the opening 10a.

[0028] As will be described in detail later, the container 10 of the blood separation device 1 may contain a diluent beforehand, or a coagulation accelerator may be added to this diluent beforehand. Alternatively, a blood separation kit may be constructed by combining such a blood separation device 1 with a blood collection device used in combination with the blood separation device 1. By including a blood collection device capable of collecting a predetermined amount of blood in the kit, it becomes possible to keep the dilution ratio by the diluent contained in the container 10 roughly constant.

[0029] Figure 2 is a cross-sectional view showing the structure of the container 10. In this embodiment, the container 10 is a cylindrical shape with a bottom overall. However, the shape of the container 10 is not limited to this, and any shape that can accommodate a mixed sample and whose opening 10a can be closed by the lid 20 can be used.

[0030] The material used to form the container 10 is not particularly limited, but from the viewpoint of ease of handling, light weight, and manufacturing cost, the container 10 may be formed from resin materials such as polyethylene (high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE)), polyethylene terephthalate (PET), polyarylate (PAR), polycarbonate (PC), or polypropylene (PP). Furthermore, from the viewpoint of allowing the reaction inside the container 10 (for example, blood coagulation reaction) to be visually observed from the outside of the container 10, the container 10 may be formed from a transparent or translucent material.

[0031] In this embodiment, a threaded portion 11 is formed on the outer circumferential surface of the container 10 near the opening 10a, which can be screwed into a threaded portion 25 formed on the inner circumferential surface of the lid 20. By fastening the two together using a screw method when closing the container 10 with the lid 20, leakage of liquid from the fastening portion can be prevented even when the container 10 is closed and turned upside down. However, the two can be fastened together using a method other than a screw method, such as a press-fit method, as long as leakage of liquid from the fastening portion can be prevented. In addition, a sealing member such as an O-ring may be placed on the inner circumferential side of the lid 20 or on the outer circumferential side of the container 10.

[0032] The size of container 10 is not limited, but for example, if a mixed sample of 3000 μL is prepared by diluting 30 μL of blood 100 times for the purpose of testing a small amount of blood collected from a fingertip, and the inner diameter of container 10 is 9 mm, the liquid level will be approximately 47 mm. Therefore, if the height of container 10 is about 63 mm, the liquid level can be kept at about 3 / 4 of the height of container 10, and the liquid level can be prevented from touching the filter 23, which will be described later.

[0033] Figure 3 is a cross-sectional view showing the structure of the lid 20. The lid 20 has a lid body 21 and a nozzle 22 which serves as a discharge port capable of discharging liquid from the inside to the outside of the container 10. In addition, a threaded portion 25 is formed on the inner circumferential surface of the lid body 21, which can be screwed into the threaded portion 11 of the container 10.

[0034] The nozzle 22 is provided on the top surface 21a of the lid body 21 so as to protrude to the outside of the container 10. However, the discharge port capable of dispensing liquid does not necessarily have to have a nozzle shape; for example, a through hole communicating with the outside of the lid 20 may be formed on the top surface 21a of the lid body 21 or on the side surface near the top surface 21a to serve as a discharge port. Alternatively, the nozzle 22 may be provided on the side surface of the lid body 21.

[0035] In Figures 1 and 3, the nozzle 22 is always in an open state, but a cap that can be opened and closed may be attached to the tip of the nozzle 22.

[0036] The material used to form the lid body 21 and nozzle 22 is not particularly limited, but from the viewpoint of ease of handling, light weight, and manufacturing cost, the lid body 21 and nozzle 22 may be formed from resin materials such as polyethylene (high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE)), polyethylene terephthalate (PET), polyarylate (PAR), polycarbonate (PC), and polypropylene (PP). In addition, the lid body 21 may be formed from a transparent or translucent material so that the amount of liquid accumulated in the space 24 can be seen from the outside.

[0037] The filter 23 is for filtering the contents of the container 10, and in this embodiment, it is attached to the inside of the lid body 21. By attaching the filter 23 to the lid body 21, when the closed container 10 is upright, the contents of the container 10 do not come into contact with the filter 23, but by turning the closed container 10 upside down, the contents of the container 10 can come into contact with the filter 23.

[0038] Preferably, the filter 23 is mounted on the opening side 21b (opposite the nozzle 22) of the lid body 21, with a gap between it and the nozzle 22 (discharge port). This gap becomes a space 24 that can contain the liquid that has permeated through the filter 23. By providing such a space 24, the liquid that has permeated through the filter 23 can be temporarily stored in the space 24, and only the required amount can be discharged from the nozzle 22.

[0039] However, the filter for filtering the contents of container 10 does not necessarily have to be attached to the lid 20. For example, after placing a mixed sample containing a blood sample and a diluent in container 10, the filter may be attached to the opening 10a of container 10, and the lid 20 may be attached so as to cover the filter.

[0040] The filter 23 is made of a material that does not allow blood clots formed by coagulating a mixed sample containing a blood sample and a diluent to pass through, but allows serum separated from the blood clot to pass through. Specifically, the filter 23 can be formed using at least one of the following: nitrocellulose fibers, polysulfone fibers, glass fibers, urethane fibers, and sintered filters (also called sintered metal filters or metal fibers) formed by sintering metal particles. As an example, the filter 23 may be a sheet made of the various fibers described above. As another example, the filter 23 may be a material in which the various fibers described above are arranged on one or both sides of a sponge made of polyvinyl alcohol (PVA) or polyethylene.

[0041] The column volume of filter 23 can be appropriately determined according to the capacity of container 10. By determining the column volume based on the maximum capacity of container 10, it is possible to sufficiently filter the liquid contained in container 10 without clogging filter 23. Alternatively, if the container 10 contains a diluent beforehand, the column volume of filter 23 may be determined based on the amount of diluent contained in container 10.

[0042] As mentioned above, the container 10 may contain a diluent beforehand. Physiological saline, phosphate-buffered saline (PBS), etc., can be used as the diluent.

[0043] Furthermore, a coagulation accelerator may be added to the diluent beforehand. As the coagulation accelerator, any of the following can be used: silica, thrombin, thrombin-like enzyme, Celite, and calcium.

[0044] The configuration of the blood collection device used in combination with the blood separation device 1 in the blood separation kit is not particularly limited as long as it is capable of transporting a predetermined amount of blood. For example, a dropper, a platinum loop or aisle that holds liquid by surface tension, or a device that collects liquid into a cylinder using capillary action (see, for example, Japanese Patent Publication No. 2008-157783) can be used. Among these, a liquid collection device that has a cylindrical liquid holding part without a bottom or top and holds liquid inside the cylinder by surface tension (see, for example, Japanese Patent Publication No. 2023-162456) can be preferably used. With such a liquid collection device, the amount of blood collected can be kept relatively constant, and by immersing the entire cylindrical liquid holding part in the diluent, the amount remaining on the liquid holding part side can be almost eliminated.

[0045] (Blood separation method) Figure 4 is a flowchart illustrating a blood separation method according to an embodiment of the present invention. Figures 5 to 7 are schematic diagrams illustrating the same blood separation method.

[0046] Prior to blood separation, blood is first collected by fingertip puncture or the like. For example, as shown in Figure 5, when using a liquid collection device 30 equipped with a cylindrical liquid holding section 31 without a bottom or top, the lower end of the liquid holding section 31 is brought into contact with the blood (blood pool) on the fingertip, and the blood is drawn in by surface tension until it reaches the volume of the liquid holding section 31.

[0047] Next, the mixed sample containing the collected blood sample and diluent is placed in the container 10 (storage step S11). Physiological saline, phosphate-buffered saline (PBS), etc., can be used as the diluent. In this case, as shown in Figure 5, the mixed sample may be prepared by immersing the liquid-holding section 31 in the diluent already contained in the container 10, or the blood sample may be transferred to the container 10 and then the diluent may be added to the container 10. From the viewpoint of serum separation, a dilution ratio of 10 times or more is preferable, and a ratio of 100 times or more is also possible. In short, the dilution ratio should be appropriately determined within a range of 10 times or more, depending on the intended use of the separated serum (e.g., test items).

[0048] Next, as shown in Figure 6, the mixed sample containing the blood sample and diluent is coagulated with the lidded container 10 upright (i.e., so that the mixed sample does not come into contact with the filter 23) (coagulation step S12). Here, coagulation includes the phenomenon of a portion of the liquid mixed sample solidifying. In coagulation step S12, the mixed sample containing only diluted blood may be allowed to stand for a predetermined time to coagulate. In this case, the standing time will vary depending on the sample and environmental conditions, but it may be about 1 minute or more. Alternatively, the mixed sample containing diluted blood and a coagulation accelerator may be allowed to coagulate. As the coagulation accelerator, any of silica, thrombin, thrombin-like enzyme, Celite, and calcium can be used. The coagulation accelerator may be added to the diluent beforehand, or it may be added to the diluted blood later. When using a coagulation accelerator, the next step can be started without providing a specific standing time.

[0049] Next, as shown in Figure 7, the container 10 with the lid is inverted, and the contents of the container 10 are filtered by bringing them into contact with the filter 23 (filtration step S13). This separates the liquid component (diluted serum) 4 from the blood clot 5 formed by the coagulation of the mixed sample. The serum that has passed through the filter 23 is collected in the space 24 between the filter 23 and the nozzle 22 (step S14).

[0050] After the serum has accumulated in space 24, the serum is dispensed from nozzle 22 (step S15). If the serum is to be used for testing, it can be dispensed directly from nozzle 22 into the testing device.

[0051] As described above, according to the embodiments of the present invention, it is possible to efficiently separate a sufficient amount of serum for blood testing, such as POCT, from a small amount of blood without causing hemolysis. Therefore, it is possible to improve the convenience of simple tests in home healthcare, emergency situations, disaster areas, and developing countries. In particular, the blood separation device 1 (see Figure 1) according to this embodiment is small and serum can be directly dropped from the blood separation device 1 to a blood testing device, etc., making it convenient to carry and usable anywhere.

[0052] Furthermore, according to this embodiment, since the collected blood is diluted before separation, a sufficient amount of serum for clinical testing and the like can be recovered.

[0053] Furthermore, in this embodiment, the mixed sample after blood coagulation is brought into contact with the filter 23 to separate it into blood clot and serum. This prevents clogging caused by the mixed sample before coagulation seeping into the filter 23, and prevents hemolysis that may occur when red blood cells attempt to pass through the filter 23. Consequently, the liquid component can be efficiently passed through the filter 23 without clogging it.

[0054] Furthermore, according to this embodiment, it is not necessary to reduce the porosity of the filter 23 as in the conventional method in order to separate the blood clot from the liquid component. Therefore, from this point of view as well, it is possible to efficiently allow the liquid component to pass through while preventing clogging of the filter 23.

[0055] (Experiment 1) To evaluate the separation performance of the blood separation method according to this embodiment based on differences in filter material, the following experiment was conducted.

[0056] 1-1. Filter material used • Cellulose fiber ADVANTEC "Quantitative Filter Paper No. 7", 0.18mm thick sheet • Glass fiber Cytiva's "Fusion5" sheet, 0.37mm thick. • Polysulfone fiber Cytiva's "Vivid Plasma Separation GR," a 0.33mm thick sheet. • Fiberglass + sponge: The filter built into the "CDTopR-HN-FSGDF" filter tip manufactured by Shin Corporation (the sponge portion is made of polyvinyl alcohol (PVA) and polyethylene filter), 4mm thick.

[0057] 1-2. Experimental Conditions • Sample: Whole blood • Diluent: Physiological saline solution ·Container used Container (Reference: Container 10 in Figure 1): A cylindrical transparent container with an inner diameter of 9 mm and a height of 49 mm. Lid with nozzle (Reference: Lid body 21 and nozzle 22 in Figure 1): Inner diameter of lid body is 6 mm • Coagulation accelerator: None

[0058] 1-3. Experimental Method (1) Whole blood was diluted with physiological saline to prepare 100-fold diluted blood.

[0059] (2) 1000 μL each of 100-fold diluted blood was placed in five containers. Cellulose fiber, glass fiber, and polysulfone fiber filters were placed over the openings of three of the containers, and the filters were glued to the bodies of the containers. For the glass + sponge filters, a lid with a nozzle into which the filter was fitted (see Figure 3) was attached to the container.

[0060] (3) The container was left to stand for 3 minutes to allow the blood to coagulate. (4) For samples fitted with filters, the contents of each container (solid and liquid components) were filtered by inverting the container, and the liquid component was collected.

[0061] (5) A sample for measurement was prepared by mixing 50 μL of pure water with 50 μL of the collected liquid component. For samples that were not fitted with a filter, a sample for measurement was prepared by mixing 50 μL of pure water with 50 μL of the contents.

[0062] (6) For each sample to be measured, the absorbance of hemoglobin at 540 nm, which is one of the absorbance peaks of hemoglobin, is measured using a microplate reader (Abs 540nm The following measurements were taken. Note that since the optical path length of the cuvette used was 3 mm, the measurements were converted to a 10 mm cell length.

[0063] (7) For the five measurement samples, the absorbance at 540 nm was blank-corrected to obtain the corrected absorbance (ΔAbs). 540nm The following equation (1) was used to calculate the separation performance. In equation (1), ΔAbs no-filter This represents the corrected absorbance at 540 nm for the measurement sample without a filter. Separation performance (%)=(1-ΔAbs 540nm / ΔAbs no-filter ) × 100 …(1)

[0064] Here, a lower absorbance value indicates less hemoglobin eluted into the sample being measured. Separation performance indicates the degree to which hemoglobin is reduced in the sample filtered by various filters, compared to an unfiltered sample. In other words, a higher separation performance value indicates a higher ability to separate serum without causing hemolysis or allowing red blood cells to pass through.

[0065] 1-4. Experimental Results Figure 8 is a table showing the results of Experiment 1. As shown in Figure 8, the separation performance was 33% when a cellulose fiber filter was used. This indicates that hemolysis occurred or that red blood cells passed through the filter. In contrast, when glass fiber, polysulfone fiber, and glass fiber + sponge filters were used, the separation performance was 90% or higher. This indicates that blood clots and serum could be separated without causing hemolysis or allowing red blood cells to pass through the filter. However, in the case of the polysulfone fiber filter, it was visually confirmed that blood cells were present in the second drop of the liquid component that was dropped.

[0066] (Experiment 2) To investigate the necessary blood coagulation time in the blood separation method according to this embodiment, the following experiment was conducted.

[0067] 2-1. Experimental Conditions • Sample: Whole blood • Diluent: Physiological saline solution • Container used: Same as in Experiment 1 • Filter material: Glass fiber + sponge • Coagulation accelerator: Calcium chloride

[0068] 2-2. Experimental Method (1) Whole blood was diluted with physiological saline to prepare 100-fold diluted blood.

[0069] (2) 1000 μL each of 100-fold diluted blood was placed into eight containers. Calcium chloride was added to five of these containers to a concentration of 2.2 mM (millimolar). Furthermore, each container was fitted with a nozzle-equipped lid that had a glass + sponge filter pre-installed.

[0070] (3) Immediately after closing the lid (0 minutes), and at 1 minute, 2 minutes, 3 minutes, and 5 minutes later, the following procedure was performed. Specifically, the contents of each container were filtered by inverting the container, and the liquid components were collected by dripping them from a nozzle. The collected liquid components were then analyzed using a microplate reader to obtain the absorbance (Abs) at 540 nm.540nm The following measurements were taken. Note that the optical path length of the cuvette used was 3 mm, and conversion to a 10 mm cell length was not performed.

[0071] 2-3. Experimental Results Figure 9 is a graph showing the results of Experiment 2. As shown in Figure 9, when no coagulation accelerator was added, the absorbance was relatively high at 0 minutes, but after 1 minute the absorbance decreased sharply, and there were no significant fluctuations thereafter. From this, it can be said that when no coagulation accelerator is added, a standing time of 1 minute is sufficient for blood coagulation. On the other hand, when a coagulation accelerator was added, the absorbance had already decreased sufficiently at 0 minutes. From this, it was found that when a coagulation accelerator is added, there is no problem in proceeding to the filtration process immediately after addition.

[0072] (Experiment 3) To investigate the appropriate blood dilution ratio in the blood separation method according to this embodiment, the following experiment was conducted.

[0073] 3-1. Experimental Conditions • Sample: Whole blood • Diluent: Physiological saline solution • Container used: Same as in Experiment 1 • Filter material: Glass fiber + sponge • Coagulation accelerator: None

[0074] 3-2. Experimental Method (1) Diluted blood was prepared with dilution ratios of 1x (no dilution), 1.5x, 2x, 3x, 5x, 10x, and 100x.

[0075] (2) 300 μL of the diluted blood at the above ratio was placed in each of the seven containers, a lid with a nozzle fitted with a glass + sponge filter was attached, and the containers were left to stand for 3 minutes to allow the blood to coagulate.

[0076] (3) By inverting each container, the contents of the container were filtered through a filter, and the liquid components were collected by dripping them from a nozzle. Then, 50 μL of pure water was mixed with 50 μL of the collected liquid components to prepare a sample for measurement, and the absorbance at 540 nm (Abs) was measured using a microplate reader. 540nm The following measurements were taken. Note that since the optical path length of the cuvette used was 3 mm, the measurements were converted to a 10 mm cell length.

[0077] 3-3. Experimental Results Figure 10 is a table showing the results of Experiment 3. Figure 11 is a graph showing the results of Experiment 3. As shown in Figure 10, in the samples with dilution ratios of 1x, 1.5x, and 2x, the filter became clogged and the liquid component could not be recovered. In the samples with dilution ratios of 3x and 5x, the liquid component passed through the filter, but the recovered liquid was visibly red, and the absorbance at 540nm was high, above 4. Therefore, it is believed that red blood cells were present. In particular, in the 3x sample, partial blood coagulation was observed even in the liquid after it was dropped from the nozzle. In contrast, in the 10x and 100x samples, the recovered liquid was visibly clear, and the absorbance was low, below 0.1. From this, it can be said that if the dilution ratio is 10x or higher, blood clots and serum can be separated without causing hemolysis and without red blood cells passing through the filter. However, in the 3x sample, the first three drops of liquid dropped from the nozzle were clear, but thereafter a visibly red color was observed. On the other hand, in the 100x magnification sample, the liquid dropped from the nozzle remained transparent until the very end.

[0078] The present invention described above is not limited to the embodiments, and various inventions can be formed by appropriately combining the multiple components disclosed in the above embodiments. For example, the invention may be formed by excluding some components from all the components shown in the above embodiments, or by appropriately combining the components shown in the above embodiments. [Explanation of symbols]

[0079] 1...Blood separation device, 4...Liquid component, 5...Blood clot, 10...Container, 10a...Opening, 11...Screw part, 20...Lid, 21...Lid body, 21a...Top surface, 21b...Opening side, 22...Nozzle, 23...Filter, 24...Space, 25...Screw part, 30...Liquid collection device, 31...Liquid holding part

Claims

1. A storage step of placing a mixed sample containing a blood sample which is whole blood and a diluent into a container, A solidification step in which the mixed sample is solidified in the container, After the coagulation step, a filtration step is performed to separate serum from the blood clot formed by the coagulation of the mixed sample by bringing the contents of the container into contact with a filter and filtering it, Includes, The dilution ratio of the blood sample, which is whole blood, in the mixed sample is 10 times or more. Blood separation method.

2. The blood separation method according to claim 1, wherein the coagulation step includes letting the mixed sample stand for a period of 1 minute or more and 5 minutes or less.

3. The blood separation method according to claim 1, wherein the coagulation step includes adding a coagulation accelerator to the mixed sample.

4. The blood separation method according to claim 1, wherein a coagulation accelerator is added to the diluent beforehand.

5. The blood separation method according to claim 3 or 4, wherein the coagulation accelerator comprises any one of silica, thrombin, thrombin-like enzyme, Celite, and calcium.

6. The blood separation method according to any one of claims 1 to 4, wherein the blood sample is fingertip blood.

7. The blood separation method according to any one of claims 1 to 4, wherein the amount of the blood sample contained in the container is 0.01 mL or more and 0.05 mL or less.

8. A container capable of containing a mixed sample including a blood sample which is whole blood and a diluent, the container having an opening on one end, A lid capable of closing the aforementioned opening, the lid having a discharge port from which liquid can be discharged from the inside to the outside of the container, A filter for filtering the contents of the container, wherein the filter does not come into contact with the contents when the closed container is upright, but comes into contact with the contents when the closed container is turned upside down. Equipped with, The container is capable of containing the mixed sample in which the blood sample, which is whole blood, has a dilution ratio of 10 times or more. Blood separation device.

9. The contents include a blood clot and serum formed by the coagulation of the mixed sample within the container. The blood separation apparatus according to claim 8, wherein the serum is separated from the blood clot by inverting the closed container and bringing the contents into contact with the filter.

10. The blood separation apparatus according to claim 8 or 9, wherein the discharge port is a nozzle provided to protrude to the outside of the container.

11. The blood separation apparatus according to claim 8, wherein the filter is attached to the inside of the lid.

12. The blood separation apparatus according to claim 11, wherein a space capable of containing the liquid that has passed through the filter is provided between the filter and the discharge port.

13. The blood separation apparatus according to claim 8, further comprising a diluent contained in the container.

14. The blood separation apparatus according to claim 13, wherein a coagulation accelerator is added to the diluent.

15. The blood separation apparatus according to claim 8, wherein the amount of the blood sample is 0.01 mL or more and 0.05 mL or less.

16. A blood separation device according to claim 13 or 14, A blood collection device capable of transporting a blood sample, which is whole blood, to a dilution ratio of 10 times or more with respect to the diluent contained in the container, A blood separation kit equipped with the following features.

17. The blood separation kit according to claim 16, wherein the blood collection device is capable of transporting a blood sample in an amount of 0.01 mL or more and 0.05 mL or less.

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