WO2017006961A1 - Blood test kit and blood analysis method - Google Patents

Abstract

The present invention addresses the issue of providing: a blood test kit whereby components in a microscopic amount of blood can be freighted in a stable state, as a result of controlling the impact of external temperature during freighting; and a highly accurate blood analysis method using said blood test kit and having a reduced fluctuation coefficient for measurement values. As a result of the present invention, a blood test kit can be provided that includes: a diluent for diluting a blood sample; a separation means for recovering a blood plasma component from the diluted blood sample; a container for housing the blood plasma component recovered from the diluted blood sample; and a cool-insulation means for keeping the container cool.

Description

Blood test kit and blood analysis method

The present invention relates to a blood test kit and a blood analysis method for performing a blood test by mailing a component of a trace amount of blood collected by a subject in a stable state.

In general, for blood collection, a general qualified blood sample is collected by a doctor or other qualified person using a syringe to collect blood from the vein, and the subject is self-collected by inserting a blood collection needle into his / her finger or the like. There is blood sampling.

Blood collected by general blood collection is transported to a medical institution or inspection in a state of being sealed in a collection container, where it is inspected. In the case where blood is transported without being separated into blood cells and plasma, a test is performed after blood is separated into blood cells and plasma by a centrifuge at a medical institution or inspection institution. In the self-collection performed by the subject to be examined, the collected blood is separated into blood cells and plasma by a separation membrane and transported to the examination site in this separated state, where the examination is performed.

In Patent Document 1, the amount of a component to be analyzed in a sample is measured, and further, the amount of a standard component originally present in the sample other than the above is measured. A quantitative analysis method is described in which the amount of a sample is determined from the known concentrations of the standard components, and the concentration of the analysis target component in the sample is determined from the sample amount and the analysis target component amount.

Patent Document 2 describes a method for examining a blood sample collected by self blood collection. Specifically, 1) Prepare a sample for quantification consisting of an unknown volume of a biological sample containing a component to be quantified collected without quantifying the volume and a fixed amount of an aqueous solution containing a fixed amount of an indicator substance. ₂ ) The biological sample dilution rate (C ₁ ) in a fixed amount of aqueous solution containing a specified amount of indicator substance and the concentration (C ₂ ) of the indicator substance in the sample for quantification Step for obtaining a), 3) Step for obtaining the concentration (Y) of the component to be quantified in the sample for quantification, 4) Biological sample dilution ratio obtained in 2) above and for quantification obtained in 3) above A method for quantifying a component to be quantified in a biological sample is described which includes a step of determining a component to be quantified in a biological sample from the concentration (Y) of a substance to be quantified in the sample.

Further, Patent Document 3 describes that a small amount of blood is collected from a human or animal using a blood dilution quantification instrument and is supplied as it is or after dilution to supply a constant amount to another device, container or reagent. Further, Patent Document 4 describes a method of quantifying the concentration of a component to be quantified in a biological sample using the absorbance of an indicator substance in an aqueous solution for dilution.

On the other hand, when a test subject collects a blood sample, it is collected with a lancet equipped with a small knife and is used for quantifying the concentration of an arbitrary component in the blood. Usually, a 100 μL blood sample is collected. There is a need to do.

JP 2001-330603 A JP 2003-161729 A JP 2009-128202 A JP 2009-109196 A

The method of analyzing blood by diluting it with a buffer solution is a biological component stored in a buffer solution with physiological conditions of pH 7.4 and excellent in stability during transportation, but controls the influence of external temperature during mailing In particular, in the summer mailing, because some of the sample components are affected by this, a decrease or increase in the measured value occurs, and it is difficult to perform a highly accurate test. In particular, the self-collected trace blood is collected and delivered to a blood analysis center or the like by means of mail or the like, so that there has been a problem that a change in blood components during that time affects the accuracy and a correct test result cannot be obtained. If only the method for determining the dilution factor described in Patent Documents 1 and 2 is employed, it is difficult to perform a highly accurate inspection when transporting as described above.

The present invention is a blood test kit for diluting a trace amount of blood with a diluent, determining a dilution ratio, and analyzing a measurement target component in the blood, and controlling the influence of an external temperature during mailing to control the trace amount An object to be solved is to provide a blood test kit capable of mailing components in blood in a stable state. Furthermore, this invention makes it the subject which should be solved to provide the accurate blood analysis method which prevented the influence by the temperature of a measured value using said blood test kit.

As a result of intensive studies to solve the above problems, the present inventors have found that a diluted solution for diluting a blood sample, a separating means for recovering plasma components from the diluted blood sample, and a diluted blood sample It has been found that a blood test kit that can solve the above problems can be provided by including a cold insulation means for keeping the container cold in a blood test kit that includes a container for containing a plasma component recovered from the blood. Furthermore, the present inventors use the blood test kit described above to determine the dilution factor using a standard component that is constantly present in blood and / or a standard component (internal standard substance) in a diluent. As a result of analyzing the components to be measured by the blood analysis method including it, it was found that the blood analysis with high accuracy can be performed while preventing the influence of the measurement value on the temperature. The present invention has been completed based on the above findings. That is, according to the present invention, the following inventions are provided.

(1) a diluent for diluting the blood sample;
Separation means for recovering plasma components from the diluted blood sample;
A container for containing plasma components recovered from a diluted blood sample;
A blood test kit, comprising: a cold insulation means for keeping the container cold.
(2) The blood test kit according to (1), wherein the cold insulation means includes a cold insulation agent and a cold insulation bag.
(3) The blood test kit according to (1) or (2), wherein the cold insulation means further includes an accommodating member having a thickness of 35 mm or less that can accommodate a container for accommodating the collected plasma component.
(4) The blood test kit according to any one of (1) to (3), wherein the blood test kit includes a member that records a temperature history.
(5) The blood test kit according to any one of (1) to (4), wherein the blood test kit includes instructions describing how to use the cold insulation means.
(6) The blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, and the diluent does not contain a standard component. The blood test kit according to any one of (1) to (5).
(7) The blood test kit according to (6), wherein the standard component is sodium ion or chloride ion.
(8) The blood test kit according to (6) or (7), wherein the standard components are sodium ions or chloride ions and at least one standard component.
(9) The blood test kit according to (8), wherein the at least one standard component is a standard component selected from total protein or albumin.
(10) The blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood and verifying the analysis. The blood test kit according to (8) or (9), which does not contain a standard component.
(11) The diluent includes a standard component that does not exist in blood, and the blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using the standard component that is not present in blood. The blood test kit according to any one of (1) to (10).
(12) Using the blood test kit according to any one of (6) to (9), plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the diluted plasma is diluted with blood. A blood analysis method for determining the dilution ratio of plasma using a standard component that is constantly present, and analyzing the concentration of the target component in a blood sample.
(13) Using the blood test kit according to (10), plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the standard component that is present in the blood constantly in the diluted plasma Is used to determine the dilution ratio of plasma, analyze the concentration of the target component in the blood sample, and determine the dilution ratio of plasma using a standard component that is different from the above-mentioned standard component that is constantly present in blood. A blood analysis method that verifies the analysis of the concentration of the target component.
(14) Using the blood test kit according to (11), plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the diluted plasma is used with standard components that are not present in the blood. A blood analysis method for determining a dilution ratio of plasma and analyzing a concentration of a target component in a blood sample.
(15) Using the blood test kit according to (11), which includes a diluent that does not contain standard components that are constantly present in blood, plasma is collected from the blood sample, and the collected plasma is diluted with the diluent. In the diluted plasma, the standard component that is constantly present in the blood and the standard component that is not present in the blood are used to determine the dilution ratio of the plasma and analyze the concentration of the target component in the blood sample. Analysis method.
(16) The blood analysis method according to any one of (12) to (15), wherein the amount of diluted plasma is 100 μL or more and 1000 μL or less.

According to the blood test kit of the present invention, components in a minute amount of blood can be mailed in a stable state, and highly accurate blood analysis can be performed while preventing the influence of the measured value on temperature. According to the blood analysis method of the present invention, it is possible to perform highly accurate blood analysis in which the influence of the measurement value due to temperature is prevented.

FIG. 1 shows an example of the configuration of a container for containing a plasma component collected from a diluted blood sample. FIG. 2 is a schematic diagram of a moisture-proof case. FIG. 3 shows the linearity of the sodium enzymatic assay.

Hereinafter, the present invention will be described in detail.
A standard component that is constantly present in blood is called an external standard substance or external standard.
A standard component that does not exist in blood is called an internal standard substance or internal standard.

・ Patients and examiners usually go to a medical institution or examination place for treatment, diagnosis, or health management of a disease, and blood may be collected from a vein for examination. However, it is necessary to wait for a long time in order to know the test result, and collecting blood from a vein may be a burden for children. In addition, if the movement for inspection is included, it takes more than half a day, so there is a problem that the opportunity to receive the inspection is missed due to the circumstances of one's work or the like, and inconvenience occurs in one's own work. The above-described problems are solved by allowing patients and examiners to collect a small amount of blood at home or in a laboratory, and to store and transport the blood stably. For example, Patent Documents 1 and 2 describe a method for testing a blood sample separated by self blood collection.

Components that are constantly present in blood as standard components are sodium ions (Na +), chloride ions (Cl−), potassium ions (K +), magnesium ions (Mg2 +), calcium ions (Ca2 +), total protein (TP ) And the like. In order to detect the above-mentioned standard component with sufficient accuracy when a very small amount of blood component is diluted at a high dilution rate, it is preferable to measure a standard component present at a high concentration in a very small amount of blood. As such a standard component, sodium ion (Na +) or chloride ion (Cl−) is preferable, and sodium is the highest amount in the blood among the components that are constantly present in the blood. Ion (Na +) is most preferred. By the way, after absorbing blood in a filter paper or a porous material, it is dried and mailed, and the method of extracting blood components may denature the components during the drying process or mailing. According to Patent Document 1, it is necessary to use a buffer solution to which a biological component is extracted from a dried sample, to which NaOH, NaCl, or HCl is added for pH adjustment or biological component stabilization. Therefore, it cannot be used to correct the concentration of other diluted biological components using the concentration of sodium ions and chloride ions having the most homeostasis among the components of the sample and the small difference between individuals as an external standard.

Patent Document 2 discloses a method for accurately determining the dilution rate of blood by containing an internal standard substance in a buffer solution when diluted with a buffer solution.

On the other hand, in the method of diluting with a buffer solution, biological components are stored in a buffer solution having a physiological condition of pH 7.4, and are excellent in stability during transportation, for example, mail transportation. Since the influence could not be controlled, especially in the summer mailing, some specimen components were affected by the influence and decreased or increased, making it difficult to conduct highly accurate examinations. In addition, since the collected trace blood is collected and delivered to the blood analysis center or the like by mail or the like, there is a problem that a correct test result cannot be obtained due to changes in blood components during that time. In the above situation, the determination of the dilution rate with the external standard substance described in Patent Document 1 or the determination of the dilution ratio with the internal standard substance described in Patent Document 2 can be carried out for a long period of one week or longer. It was difficult.

In the present invention, a standard component (external standard substance) and / or a standard component (internal standard substance) that are constantly present in blood and / or using a blood test kit including a cold insulation means for keeping the container cold. It was found that the measurement value component can be analyzed by a blood analysis method that includes determining the dilution rate using () to reduce the variation in the measured value and enable highly accurate blood analysis. Conventionally, it has not been known at all to combine the cold preservation means with the determination of the dilution factor using the standard component constantly present in the blood and / or the standard component in the diluted solution. It is completely unexpected that blood analysis with high accuracy has become possible.

According to the blood test kit and the blood analysis method of the present invention, when a patient collects blood by himself / herself, for example, in summer or in a hot and humid area, the measurement target component is affected by the influence of the external temperature during mailing. Even in a situation where an increase is likely to occur, the influence of the outside temperature during mailing can be easily controlled, and fluctuations in the measurement target component can be suppressed, and a highly accurate inspection can be performed. The collection of micro blood using the blood test kit of the present invention is not limited in time and place, so it can be applied to cases that do not have time to go to medical institutions, disasters, telemedicine, health care, etc. Because it can be detected early, it can contribute to the reduction of medical costs. In addition, according to the present invention, a large amount of sample (for example, 65 μL) can be used for many tests such as 13 biochemical tests, tumor markers, and hepatitis tests. Can be measured. The test data measured using the blood test kit of the present invention can be used in a system for daily health management and early detection of diseases by transmitting it to a smartphone.

[1] Blood test kit The blood test kit of the present invention comprises a diluent for diluting a blood sample, a separating means for recovering plasma components from the diluted blood sample, and a blood sample collected from the diluted blood sample. A container for containing the plasma component, and a cold insulation means for keeping the container cold.

(1) Diluent for diluting a blood sample The blood test kit of the present invention is for diluting blood collected by a patient and transporting it to a medical institution or a test institution to analyze a component to be measured. There is a possibility of being left for a long time from blood collection to analysis. In the meantime, it is preferable to prevent decomposition and denaturation of the target component in the diluted blood. The pH of blood is usually kept constant at about pH 7.30 to 7.40 in healthy individuals. Accordingly, in order to prevent decomposition and denaturation of the target component, the diluent is preferably pH 6.5 to pH 8.0, more preferably pH 7.0 to pH 7.5, and still more preferably pH 7.3 to A buffer solution having a pH of 7.4 and containing a buffer component that suppresses fluctuations in pH is preferable.

The types of buffer include acetate buffer (Na), phosphate buffer (Na), citrate buffer (Na), borate buffer (Na), tartrate buffer (Na), Tris (Tris (hydroxy Methyl) aminoethane) buffer (Cl), HEPES ([2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid]) buffer, phosphate buffered saline (Na), etc. are known ing. Among these, phosphate buffers, Tris buffers, and HEPES buffers are representative examples of buffers around pH 7.0 to pH 8.0. However, the phosphate buffer contains a sodium salt of phosphate. Since the Tris buffer has a dissociated pKa (Ka is an acid dissociation constant) of 8.08, it is usually used in combination with hydrochloric acid in order to have a buffering ability in the vicinity of pH 7.0 to pH 8.0. The pKa of HEPES sulfonic acid dissociation is 7.55, and a mixture of sodium hydroxide, sodium chloride and HEPES is usually used to prepare a buffer solution with a constant ionic strength. Although these are useful as buffers having the action of keeping the pH constant, they contain sodium ions or chloride ions, which are preferably used as external standard substances in the present invention. It is not preferable. Therefore, the present inventors have intensively studied and found a new buffer solution that does not contain sodium ions or chloride ions.

The diluent containing no sodium ion and chloride ion that can be used in the present invention is preferably 2-amino-2-methyl-1-propanol (AMP), 2-ethylaminoethanol, N-methyl-D-glucamine. , Diethanolamine, and at least one amino alcohol compound selected from the group consisting of triethanolamine, and Good's buffer (Good buffer), which is also referred to as HEPES, which is a buffer having a pKa of around 7.4. 4- (2-hydroxyethyl-1-piperazinyl) ethanesulfonic acid) (pKa = 7.55), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pKa = 7.50), also referred to as TES, 3-morpholinopropanesulfonic acid (pKa = 7.20), also referred to as MOPS, and (N, N-bis (2-hydroxyethyl) -2-aminoethane, also referred to as BES A dilute solution containing at least one buffer selected from the group consisting of sulfonic acids (pKa = 7.15), among which 2-amino-2-methyl-1-propanol (AMP) and HEPES, A combination of TES, MOPS or BES is preferred, and a combination of 2-amino-2-methyl-1-propanol (AMP) and HEPES is most preferred.

In order to prepare the above buffer solution, the concentration ratio of amino alcohol and Good's buffer solution is 1: 2 to 2: 1, preferably 1: 1.5 to 1.5: 1, more preferably 1: 1. Can be mixed. The concentration of the buffer is not limited, but the concentration of amino alcohol or Good's buffer is 0.1 to 1000 mmol / L, preferably 1 to 500 mmol / L, more preferably 10 to 100 mmol / L.

In the buffer solution, chelating agents, surfactants, antibacterial agents, preservatives, coenzymes, saccharides and the like may be contained for the purpose of keeping the components to be analyzed stable. Examples of chelating agents include ethylenediaminetetraacetate (EDTA), citrate, and oxalate. Examples of the surfactant include a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant. Examples of the preservative include sodium azide and antibiotics. Examples of the coenzyme include pyridoxal phosphate, magnesium, zinc and the like. Examples of the saccharide of the erythrocyte stabilizer include mannitol, dextrose, oligosaccharide and the like. In particular, by adding antibiotics, it is possible to suppress the growth of bacteria partially mixed from the finger surface at the time of hand blood collection, to suppress the degradation of the biological components by bacteria, and to stabilize the biological components.

Components diluted with these buffers do not interfere with the measurement even with various measurement methods using biochemical / immuno-automated analyzers, and blood cells do not undergo hemolysis, and biological components can be stored as stably as possible even at 37 ° C. preferable.

When whole blood is used as a biological sample, it is necessary to filter the blood cell components in the diluted blood, so that the osmotic pressure of the buffer solution is equivalent to that of blood (285 mOsm / kg (mOsm / kg: 1 kg of solution water has It is possible to prevent hemolysis of blood cells by setting the osmotic pressure to the number of millimoles of ions)) or more. The osmotic pressure can be adjusted to be isotonic with salts, sugars, buffers, etc. that do not affect the measurement of the target component and the measurement of the standard component that is constantly present in the blood.

A first example of a diluent for diluting a blood sample is a diluent that does not contain a substance that is constantly present in blood (hereinafter also referred to as a homeostatic substance) that is used when determining the dilution factor. In this specification, “does not contain” means “does not contain substantially”. Here, “substantially free” means that the homeostatic substance used when determining the dilution factor is not included at all, or even if it is included, the homeostatic substance in the diluted solution after the blood sample is diluted It means the case where it is contained at a very small concentration that does not affect the measurement of the above. When sodium ions or chloride ions are used as the homeostatic substance, a diluent that does not substantially contain sodium ions or chloride ions is used as the diluent.

When the blood test kit of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, the diluent is the standard component. It is a diluted solution that does not contain.

A second example of a diluent for diluting a blood sample is a diluent containing an internal standard substance. The internal standard substance can be added to a diluent used for diluting a biological sample so as to have a predetermined concentration. As the internal standard substance, a substance which is not contained in the blood sample at all or is contained in a trace amount even if it is contained can be used. Internal standards include substances that do not interfere with the measurement of target components in blood samples, substances that do not degrade due to the action of biological enzymes in blood samples, substances that are stable in buffer solutions, and do not permeate the blood cell membrane. It is preferable to use a substance that is not contained in blood cells, a substance that is not adsorbed in a buffer storage container, or a substance that can use a detection system that can measure with high accuracy.

As the internal standard substance, a substance that is stable even if stored for a long time in a state of being added to a diluent that is a buffer solution is preferable. Examples of internal standard materials include glycerol triphosphate, Li, Rb, Cs, or Fr as alkali metals, and Sr, Ba, or Ra as alkaline earth metals, among which glycerol triphosphate Or Li is preferable. These internal standard samples are colored by adding a second reagent at the time of concentration measurement after blood dilution, and the concentration in diluted blood can be determined from the color density. For example, the measurement of the internal standard substance of lithium added to the buffer solution uses a chelate colorimetric method (halogenated porphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin). Using a biochemical automatic analyzer, a lot of samples can be easily measured.

When the blood test kit of the present invention is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is not present in blood, the diluent is present in the blood. This is a diluted solution containing standard components that do not.

A third example of a diluent for diluting a blood sample is a diluent that does not contain a standard component that is constantly present in the blood used when determining the dilution factor, and that contains an internal standard substance. .

(2) Separation means for recovering plasma components from a diluted blood sample and a container for storing plasma components recovered from the diluted blood sample Separation for recovering plasma components from a diluted blood sample As a means, an embodiment which is a separation membrane is preferable, and a filter having pores capable of separating blood cell components is more preferable.
The shape and size of the container for containing the plasma component collected from the diluted blood sample are not particularly limited.
The material of the container is preferably a synthetic resin from the viewpoint of resistance to breakage, hygiene, and price. For example, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polyurethane, polyethylene terephthalate, polylactic acid, acrylonitrile butadiene styrene resin (ABS resin), acrylonitrile styrene resin (AS resin), acrylic resin (PMMA) , Polycarbonate, silicone resin, silicone rubber and the like.

As an example of the blood test kit of the present invention, a diluting solution for diluting a blood sample, a first containing device containing the diluting solution, and a separation for separating and collecting plasma from the blood sample diluted with the diluting solution A device, a holding device for holding the separating device, a second containing device for containing the collected plasma, a sealing device for maintaining the contained plasma in the second containing device, and damaging the skin It is equipped with a needle that bleeds blood outside the skin, a lancet, an adhesive bandage or disinfecting member (for example, a non-woven fabric impregnated with isopropanol (70% by mass isopropanol) or ethanol), an instruction manual, etc. be able to.

The first storage device and the second storage device may be used as a first storage device and a second storage device, or may be provided with separate devices. In order to make it possible for the patient or the measurer who measures the dilution factor and the analysis of the analysis target component to confirm the diluted solution in which the blood in the storage device is diluted, the first storage device and the second storage device The instrument is preferably made of a transparent material. The term “transparent” as used in the present invention is not limited as long as the observer can confirm the amount of liquid inside, and is a concept including translucency.

The embodiment in which the holding device for holding the separation device is a gasket is preferable. In addition, as the sealing device, when the storage device is a tube-shaped device, a cap that can cover the opening, a cover having a spiral groove, or a rubber plug is used. can do.

A first storage device containing a diluent, a separation device for separating and collecting plasma from a blood sample diluted with the diluent, a holding device for holding the separation device, and a first device for containing the collected plasma Specific examples of the configuration of the second storage device and the sealing device for maintaining the stored plasma in the second storage device are described, for example, in FIGS. 1 to 13 of Japanese Patent No. 3597827. Can be used. FIG. 1 of Japanese Patent No. 3597827 is incorporated as FIG. 1 of the present application.

The blood separation instrument 1 includes a blood collection container 2 (first accommodation instrument in which a diluent is accommodated), a cylinder 3 (second accommodation instrument for accommodating the collected plasma) that can be inserted into the blood collection container 2, and The cap piston 4 that can be attached to the cylindrical body 3 and a sealing lid 5 (sealing device) provided at the lower end of the cap piston 4, and before use, as shown in FIG. The upper end opening is sealed with a cap 6 via a packing 7. The container for storing the diluted blood sample in the present invention corresponds to the combination of the blood collection container 2 and the cylinder 3 in the configuration of FIG. That is, the container for storing the diluted blood sample may be one or a combination of two or more.

The blood collection container 2 is made of a transparent material and has a cylindrical shape. A screw portion 8 is formed on the outer surface of the blood collection container 2 and an engaging portion 9 is projected on the inner surface. In addition, an inverted conical bottom portion 10 is formed at the lower end portion of the blood collection container 2, and a cylindrical leg portion 11 is formed around the bottom portion 10. The legs 11 have the same outer diameter as the sample cup used at the time of blood analysis and testing, and preferably, slit grooves 12 are formed in the vertical direction at positions opposite to the lower ends thereof. Further, as shown in FIG. 1, a required amount, for example, a diluted solution 13 of 500 mm ³ may be placed in the blood collection container 2 in advance.

The cylindrical body 3 is made of a transparent material and has a cylindrical shape, and an enlarged diameter portion 14 is formed at an upper end portion thereof. The enlarged diameter portion 14 is connected to the main body portion 16 through a thin portion 15. A reduced diameter portion 18 is formed at the lower end of the cylindrical body 3, and a locking projection 19 is formed on the inner surface of the reduced diameter portion 18. Furthermore, the outer flange portion 20 (holding device) is formed at the lower end portion of the reduced diameter portion 18, the lower end opening portion of the outer flange portion 20 is covered with a filtration membrane 21 (separation device), and the filtration membrane 21 is in the blood. It allows passage of plasma and prevents passage of blood cells.

A silicon rubber cover 22 is attached to the outer periphery of the reduced diameter portion 18 (FIG. 1).

The cap piston 4 includes a substantially cylindrical knob 26 and a mandrel 27 that is concentric with the knob 26 and extends downward. A cylindrical space 28 into which the enlarged diameter portion 14 of the cylindrical body 3 can be fitted is formed at the inner upper end portion of the knob portion 26, and the lower portion thereof is screwed and can be screwed into the screw. The lower end portion 29 of the mandrel portion 27 is formed in a pin shape, and the sealing lid 5 is detachably provided on the lower end portion 29 (see FIG. 1). The sealing lid 5 is made of silicon rubber.

The details of the blood separation method using the above-mentioned instrument are described in Japanese Patent No. 3597827, paragraph numbers 0023 to 0026, and FIGS. 12 and 13, the contents of which are cited in this specification.

(3) Cooling means for keeping the container cold The preferred temperature at the time of mailing the blood sample is −10 ° C. or more and 20 ° C. or less, and the particularly preferred temperature is 0 ° C. or more and 10 ° C. or less. In the present invention, the above-described cold insulation can be achieved by including a cold insulation means in the blood test kit.

As the cooling means, a cooling agent can be used. The surface of the bag containing the cooling agent or the instructions may describe the cooling agent in advance in a freezer or the like, and the cooling time until the cooling agent can be cooled. Moreover, the usage method etc. of the cold insulating member which accommodates the collect | recovered plasma component and a cold insulating agent may be described. Thus, the blood sampler can collect blood in consideration of the cooling time, and can store the blood sample in a predetermined cold insulation member.
The cryogen is not particularly limited. For example, examples of the aqueous solution of the inorganic salt include sodium chloride, ammonium chloride, and magnesium chloride. Examples of the polyhydric alcohol include ethylene glycol and propylene glycol. Examples of the gelling agent include carboxy. There are hydrophilic polymers such as methylcellulose, polyvinyl alcohol, sodium polyacrylate, polyacrylamide, etc., with the addition of silver iodide, copper sulfide, xanthan gum, α-phenazine, sodium pyrophosphate as nucleating agents for freezing the cryogen , Etc. can be used. In particular, the cryogens that are generally used in bags and used and marketed (for example, trade name: Ice Pack, registered trademark) are about 99% water and highly water-absorbent resin (sodium polyacrylate), preservatives, and shape-stable. Since it contains an agent and has no cooling effect when it is thawed at room temperature, it should be first frozen in a freezer before use. The cryogen can be used over and over again after it has been warmed up or by freezing it.

Dilution with a buffer solution and subsequent separation and collection of plasma are important in reducing hemolysis of blood cells and elution effects of substances from blood cells. In this case, cooling keeps the influence of substances in the buffer solution on blood components to a minimum, so that stabilization of the state after plasma separation is achieved in terms of maintaining the stability of the blood components of interest. The purpose is to produce a synergistic effect on stabilization by designing an appropriate buffer (diluent).

Containers for storing specimens in the form of a solution obtained by diluting a minute amount of blood used for blood tests with a diluent, or containers for specimens in which plasma and blood cells are separated immediately after blood is collected and diluted can be cooled as described above. It is preferable to cover or surround with a state-of-the-art cryogen. Furthermore, it is preferable that the above container is placed in a cold insulation bag having a metal foil such as an aluminum film having a heat shielding property in order to extend the cold insulation time of the cold insulation agent, and the temperature caused by light absorption due to shielding of external light. It is preferable at the point which prevents a raise. That is, preferably, the cold insulation means includes a cold insulation agent and a cold insulation bag. In addition, it is particularly preferable to cover with a heat insulating sheet after being covered with a cold insulating agent or after being enclosed and put into a heat insulating cold bag having a metal foil such as an aluminum film from the viewpoint of preventing cold air leakage of the cold insulating agent. Moreover, it is preferable that the structure has not only a cold insulation but also an impact mitigating effect by transportation.

For blood collectors, even if they are covered with a cold storage agent and put in a cold storage bag in this way, if they go to the post office, etc. In the case of being far away from the nearest post office or the elderly, the micro blood sampling kit of the present invention may be one factor that may be avoided.

For this purpose, it is preferable that the size is such that it can be inserted into the post entry port. For example, if it is a post packet (size 340 mm × 250 mm × thickness 35 mm, weight 1 kg or less), it is not necessary to go to the post office, and it can be posted directly on the post. Moreover, it can be transported at a low cost, and there is a mail tracking service, which is preferable in terms of price and security.

As a device for keeping the thickness within 35 mm, for example, a housing member that accommodates a container having a specimen having a thickness of 35 mm or less is included in the blood test kit as one of the cold insulation means, and the cold preservation agent is cooled in advance. By sandwiching this container with a cryogen, it is possible to secure a space for accommodating the container having the specimen in the accommodating member even when the cryogen is solidified and hardened so that it can be cryogenized. Therefore, even if the container having the specimen is sandwiched with a hardened cold-retaining agent, a total thickness of 35 mm or less can be achieved.

In another preferred embodiment of the present invention, a container for storing plasma components after separation of blood cells and plasma using a cryogen having a two-layer structure of a part where the cryogen is solidified by cooling and a part where the cryogen does not solidify is provided. By covering, a thickness of 35 mm or less can be realized, and mailing can be performed easily and inexpensively.

(4) Other elements A preferred embodiment of the blood test kit of the present invention is an embodiment having a member for recording a temperature history after use of the cold insulation means. As a member for recording the temperature history, it is preferable to use a thermo label or an RFID (radio frequency identifier) tag. By attaching a thermolabel or the like capable of detecting an appropriate temperature to the container having the sample, the maximum temperature before and after arrival at the analysis center can be checked, so that the status of the cold-retention effect during mailing can be grasped later. Alternatively, by placing an RFID tag equipped with a temperature sensor in the vicinity of a container that contains a blood sample, temperature changes during mailing can be managed in detail at the analysis center.

Also, in order to reuse the cooling agent, it is preferable that the bag containing the cooling agent can be washed and the surface is provided with an antibacterial coating or the like.

A preferred embodiment of the blood test kit of the present invention is an embodiment including an instruction describing how to use the cold insulation means. For example, a kit that includes a determination as to whether or not to use a cryogen during mailing is also a preferred embodiment. For example, when a thermolabel is included in the kit and the temperature reached by the reversible thermolabel before mailing is 25 ° C. or higher (summer, high temperature and high humidity), the kit is described in the instruction to use a cold insulator. Is preferred. In addition, the kit includes an irreversible thermolabel that can be used by removing the release paper, and by packaging the diluted blood and the irreversible thermolabel that has been used at the time of mailing, It becomes possible to grasp the maximum temperature at the time of mailing after blood collection. Therefore, if you purchase the kit in the summer and collect and mail it at low temperatures in the winter, or if you store the kit in a place where it is exposed to direct sunlight, the highest temperature that can be achieved after mail collection Is preferable because it is possible to appropriately grasp the above.

(5) Providing form of blood test kit The number of each element included in the blood test kit of the present invention is not particularly limited, and may be one each or two or more.
The blood test kit of the present invention contains a diluent for diluting a blood sample, separation means for recovering plasma components from the diluted blood sample, and a plasma component recovered from the diluted blood sample Therefore, it is possible to provide a container for storing the cooling container and a cooling means for cooling the container in a storage container that stores them.

[2] Blood Analysis Method A blood sample can be collected using the blood test kit of the present invention, and the analysis target component in the blood sample can be measured. The blood analysis method of the present invention may be carried out by self-collection in which the subject himself collects blood, or may be carried out in general blood collection in which a qualified person such as a doctor collects blood using a syringe. Good.

In the present invention, the biological sample to be analyzed is blood, and blood is a concept including serum or plasma. Preferably, plasma or serum obtained by collecting a small amount of blood from a subject, diluting with a buffer solution, and then separating blood cells by a filter or centrifugation can be used.

The origin of the blood sample is not limited to humans, but may be animals other than humans (mammals, birds, fishes, etc.). Examples of animals other than humans include horses, cows, pigs, sheep, goats, dogs, cats, mice, bears, pandas, and the like. Preferably, the blood sample originates from a human.

As a preferred embodiment, the patient himself / herself collects the blood that has come out of the skin by damaging a fingertip or the like using an instrument with a knife such as a lancet. In order to reduce the burden on the patient, it is preferable to collect blood with low invasiveness, and it is desirable that blood can be collected with no pain or very little pain when collecting blood, in which case the depth and size of the wound Is desirable to be small. Therefore, the amount of blood collected from the patient used in the analysis method of the present invention is preferably 100 μL or less in consideration of the load on the patient. Preferably, the amount of diluted plasma after dilution with a diluent is preferably 100 μL or more and 1000 μL or less. If it is 100 μL or more, there is no restriction such that the frequency of hemolysis at the time of plasma separation is reduced and a highly airtight container is required even when mailing takes time. If it is 1000 μL or less, a dilution rate effective for measurement can be realized, and measurement with high accuracy becomes possible.

As a first aspect of the blood analysis method of the present invention, a component that is constantly contained in a blood sample is used as a standard component. Specific examples include sodium ion (Na +), chloride ion (Cl−), potassium ion (K +), magnesium ion (Mg2 +), calcium ion (Ca2 +), total protein (“TP”), albumin and the like. . The concentration of these standard components contained in the blood sample is such that the Na concentration is 134 to 146 mmol / liter (average value: 142 mmol / liter), the Cl concentration is 97 to 107 mmol / liter (average value: 102 mmol / liter), K concentration is 3.2 to 4.8 mmol / liter (average value: 4.0 mmol / liter), Mg concentration is 0.75 to 1.0 mmol / liter (average value: 0.9 mmol / liter), Ca concentration Is 4.2 to 5.1 mmol / liter (average value: 4.65 mmol / liter), the total protein concentration is 6.7 to 8.3 g / 100 mL (average value: 7.5 g / 100 mL), and the albumin concentration is 4.1 to 5.1 g / 100 mL (average value: 4.6 g / 100 mL). Among these, in order to be able to detect these homeostatic standard components with sufficient accuracy when a small amount of blood components are diluted at a high dilution ratio, it is necessary to measure standard substances present in blood at high concentrations. In addition, a standard substance present in a high concentration in blood is highly resistant to the effects of contamination even if unintentional components other than blood are mixed in the diluted solution, and the test accuracy is high. It is considered that the decrease can be suppressed. As such a standard component, sodium ion or chloride ion is preferable, and among the standard components that are constantly present in blood, sodium ion having the highest amount present in blood is most preferable. Na ion accounts for 90% or more of the total cation in plasma at a standard value (normal value) of 142 mmol / liter.

In the blood analysis method according to the first aspect, plasma is collected from a blood sample using a blood test kit containing a diluent that does not contain standard components that are constantly present in blood, and the collected plasma is diluted. It is possible to analyze the concentration of the target component in the blood sample by diluting with a liquid, determining the dilution ratio of plasma using the standard component in the diluted plasma that is constantly present in the blood.

Sodium ion concentration and chloride ion concentration can be measured, for example, by flame photometry, atomic absorption, glass electrode, titration, ion selective electrode, enzyme activity, and the like.

In the present invention, a sample obtained by collecting a small amount of blood from a finger and diluting with a buffer solution is only about 150 μL, and more than 10 items of biochemical components and immunological test items are measured. It is preferable that it can be measured by. In addition, since it is necessary to analyze a large number of samples, it is preferable to be able to adapt to a commercially available biochemical / immunological automatic analyzer.

In the present invention, the preferred standard substance is sodium ion, the measured value (concentration X) of sodium ion in the diluted solution after blood dilution, and the known concentration value (concentration Y; concentration of sodium ion as the standard component in the blood sample). 142 mmol / liter), the dilution rate (Y / X) of the blood sample is calculated. By multiplying this dilution factor by the measured value (concentration Z) of the analysis target component in the diluted blood sample, the concentration [Z × (Y / X)] of the analysis target component actually contained in the blood sample is obtained. Is possible.

In addition, in order to verify whether blood dilution and plasma separation / recovery are normally performed, the dilution factor is independently determined for each of the two or more blood specimens containing different constitutively contained components as standard components. It is preferable to confirm that the values match. The coincidence means that in two measured values (a, b), the ratio of their difference to their average value, that is, | a−b | / {(a + b) / 2} × 100 is 20% or less. Preferably, it is 10% or less, more preferably 5% or less. As a preferred embodiment, the dilution rate is determined from the measured value of the sodium ion concentration in the blood plasma dilution of the blood sample and the known concentration value (142 mmol / liter) of sodium ion constantly contained in the plasma, In the analysis of the concentration of the target component in the blood sample using this dilution factor, the dilution factor obtained from the standard component constantly contained in the plasma other than sodium ions is the dilution factor obtained from the sodium ion concentration. By comparing and confirming that they match, it is possible to verify that the analysis of the components contained in the plasma of the blood sample is performed normally. Here, as an example of a standard component that is constantly present in plasma other than sodium ion or chloride ion, it is preferably selected from total protein or albumin, and more preferably selected from total protein. Methods for measuring total protein include known methods such as the Burette method, the ultraviolet absorption method, the Breadford method, the Raleigh method, the bicinchoninic acid (BCA) method, and the fluorescence method. A method to be used as appropriate can be selected according to the amount and the like.

As a second aspect of the blood analysis method of the present invention, the dilution concentration is determined using standard components that are not present in the blood. In this case, using a blood test kit containing a diluent containing a standard component that is not present in blood, plasma is collected from the blood sample, the collected plasma is diluted with the diluent, and the diluted plasma is diluted with blood. The dilution factor of plasma is determined using the standard component that is not present, and the concentration of the target component in the blood sample can be analyzed.

It is known that sodium in blood has very high homeostasis and little variation between individuals. Moreover, since the median concentration is 142 mmol / L, which is a high biological concentration, the concentration can be measured with high accuracy even if diluted with a buffer solution. Moreover, since the internal standard in the buffer for dilution can be set to a high concentration, the concentration can be measured with high accuracy.

As a third aspect of the blood analysis method of the present invention, plasma is collected from a blood sample using a blood test kit containing a diluent containing standard components not present in blood, and the collected plasma is diluted with the diluent. In the diluted plasma, the above-mentioned standard component that is constantly present in the blood and the standard component that is not present in the blood are used to determine the dilution ratio of the plasma and analyze the concentration of the target component in the blood sample. be able to. By combining the measured absorbance of the internal standard solution with the measured absorbance of the external standard that is a highly constitutive component of the sample as described above, the above-mentioned two quantitative methods can be complemented as a reliable quantitative method with high measurement accuracy. A high dilution component determination method can be obtained.

The dilution rate of the blood sample component is calculated by any one of the following formulas 1 to 4, and the concentration of the target component in the blood sample component is calculated by multiplying the concentration of the analysis target component in the diluted solution by the dilution rate. Is preferably analyzed.

In the above formula, A, B, C, D, B ′ and X are defined as follows.
A: Measured absorbance of a diluted solution containing an internal standard substance B: Absorbance obtained by subtracting the absorbance of a diluted solution obtained by diluting plasma components from A C: Measured absorbance D having a sodium ion concentration of 142 mmol / L as a homeostatic substance : Absorbance of sodium ions in a diluted solution obtained by diluting plasma components B ': Correction value of absorbance of standard components not present in blood in diluted plasma by dilution factor calculated from absorbance of plasma sodium X: Multiplex of plasma dilution

As another calculation method for obtaining the dilution rate, the blood sample component is calculated by calculating with Equation 5 using the root-mean-square method, and multiplying the concentration of the analysis target component in the diluted solution by the dilution rate calculated with Equation 5. An embodiment in which the concentration of the target component is analyzed is also preferable.

The present invention relates to a blood test kit for collecting blood by a patient, transporting the collected blood to a medical institution or testing institution, and performing a test, and a blood analysis method using the blood test kit. Therefore, there is a possibility of being left in a diluted state for a long time after blood collection until the test. If, for example, hemolysis of erythrocytes occurs during that time, substances or enzymes with high concentrations in the blood cells will become plasma or serum. If the sample is eluted and affects the test result, or the analysis target component is measured by color tone, hemoglobin may affect the test. Therefore, it is necessary to prevent hemolysis, blood coagulation, and the like during transportation. In the present invention, it is preferable to perform a step of separating blood cells from blood after diluting the blood collected by the patient with a diluent.

There is no particular limitation on the method of collecting blood cells by separating blood cells from blood. However, plasma separation after blood collection is preferably performed immediately after the blood is diluted with a buffer solution. After collecting blood with an anticoagulant blood collection tube, the blood is separated into blood cells and plasma components by centrifugation, and transported in a separated state, or the blood components are pressurized and passed through a separation membrane such as a filtration membrane. For example, a method of separating a blood cell component from blood by capturing the blood cell component on a separation membrane is used. In this case, it is preferable to use an anticoagulant. Further, in order to ensure the accuracy of measurement, it is preferable to physically separate it from the blood solution part excluding blood cell components. In this case, specifically, backflow prevention described in JP-A-2003-270239 is preferred. It is preferable to use a biological sample separation instrument having means.

In the present invention, to analyze the concentration of a target component in a blood sample is to determine the concentration of the target component (that is, to quantify the target component), or whether the concentration of the target component is equal to or higher than a predetermined reference value. The form of analysis is not particularly limited, including determining whether it is below a predetermined reference value, performing qualitative detection that includes a certain level of concentration, and the like.

The analysis target component is not limited, and any substance contained in a biological sample is targeted. Examples include biochemical test items in blood used for clinical diagnosis, markers for various diseases such as tumor markers and hepatitis markers, and include proteins, sugars, lipids, low molecular weight compounds, and the like. Further, the measurement includes not only the substance concentration but also the activity of substances having an activity such as an enzyme. Each target component can be measured by a known method.

An example of the blood analysis method of the present invention will be described below.
Add 65μL of micro blood sample to 280μL of internal standard addition buffer, mix, filter blood cells with filter, and measure each concentration of internal standard, external standard and biological components with biochemical automatic analyzer using diluted plasma as sample To do.

According to one embodiment of the present invention, a method for efficiently analyzing a large number of samples with a commercially available biochemical / immunological autoanalyzer for quantitative determination of enzyme components of diluted plasma biological sample of unknown concentration in collected blood and enzyme activity It is. Prepare an external standard that uses sodium plasma or the like that maintains a constant concentration in a biological sample, or an internal standard that does not pass through the blood cell membrane and is a component that is not or hardly contained in plasma. This is added to the buffer. This internal standard is preferably stable in a buffer solution for a long time and can be easily quantified. Specific examples include lithium and glycerol triphosphate. Also, sodium, which is the external standard of the measurement sample, is stable because it is an element.

Sodium measurement is based on the fact that the enzyme activity of the enzyme galactosidase is activated by sodium ions, and it is possible to measure a sample of very low concentration sodium (24 mmol / L or less) diluted with a buffer solution in several μL. Simple enzymatic assays can be used. This measurement method has a proportional relationship between the sodium concentration of the sample diluted with the buffer and the galactosidase activity, and can be applied to biochemical / immunological automatic analyzers, and does not require a separate measuring instrument for sodium measurement. It is a particularly preferable measurement method because it is highly efficient and economical.

The following examples explain the present invention, but the present invention is not limited to the examples.

Example 1
About 10 mL of blood collected with a syringe from a vein after informed consent from a volunteer patient was obtained in a blood collection tube. Prepare a sponge capable of absorbing about 65 μL of blood, and analyze GPT (glutamate pyruvate transaminase) and HbA1c (hemoglobin A1c) using DEMECAL (registered trademark) blood test kit (Regier Inc.) containing 300 μL of diluent. It went as follows. First, about 65 μL of blood is aspirated into the sponge using a blood collection device to which the sponge prepared as described above is attached using the above kit, and after elution into a diluted solution, plasma and blood cells are separated into a container. Accommodated. After that, we prepared samples that were stored for 3 days in an environment of 25 ° C to 40 ° C without cooling, assuming storage and transportation of bottle samples in summer. A thermolabel capable of detecting an environment of 40 ° C. was attached to the surface of the container.

Separately, a horizontally long housing member having a thickness of 30 mm, which has a space for a container containing a plasma / diluted plasma solution after separation of blood plasma and blood cells, and a space capable of storing a cryogen in an adjacent state on both sides of the container The case was prepared, and a container containing a plasma / diluted plasma diluted solution was stored, and stored in a form sandwiched by a refrigeration agent (ice pack: registered trademark) cooled from both sides. A thermolabel was affixed to the surface of the container containing the plasma dilution. The case was sealed in a heat-insulating cold bag having an aluminum film that can be shielded from light, and the cold bag was packaged so that it could be mailed and stored in the same environmental conditions for 3 days.

The thermo label of the container that does not use the cold storage means displayed a 40 ° C. history. On the other hand, the thermolabel of the container using the cold insulation means did not display a 40 ° C. history, and the actual temperature of the sample when using the cold insulation means was measured and found to be 3 ° C. to 9 ° C.

Using the two types of samples prepared as described above, the maximum and minimum values of the blood analysis results and the coefficient of variation when the dilution ratios of (Method 1) to (Method 3) below were corrected were determined. For each correction method, the same experiment was repeated 8 times, and statistical values were obtained.

(Method 1) Measure the sodium ion concentration in the diluted mixture, and calculate the dilution factor from this value and the concentration value for the sodium ion concentration of 142 mmol / L, which is normally evaluated as a component that is constantly present in the blood. It was.
(Method 2) Using the glycerol monophosphate added in the diluent, the dilution ratio of plasma according to the method described in “Clinical Pathology Vol. 56, No. 7 (July 2008) Supplement 577-583” Asked.
(Method 3) Using the value of the dilution factor obtained by the above method 1 and method 2, the dilution factor was determined according to the following method.

(Method 3: Measurement of dilution ratio by combined use of external standard method and internal standard method)
Examples of the external standard method using sodium ions are described below. Table 1 shows measurement reagents for detecting sodium ions that do not contain sodium ions.

When 65 μL of whole blood is added to 280 μL of diluent, plasma (about 30 μL) in whole blood is diluted about 10 times. The diluted plasma was diluted 5-fold with purified water, and 3 μL was added with 52 μL of the first reagent for sodium ion measurement reagent, kept at 37 ° C. for 5 minutes, and then added with 26 μL of the second reagent for sodium ion measurement reagent. The change in absorbance was measured at a main wavelength of 410 nm and a sub-wavelength of 658 nm with a JCA-BM-6050 type biochemical automatic analyzer, for a minute. As a result of preparing a calibration curve showing the sodium ion concentration and the amount of change in absorbance, as shown in FIG. 3, linearity passing through the origin up to 24 mmol / L was obtained, and the quantitative property of sodium was recognized.

Table 2 shows the configuration of the internal standard addition diluent that does not contain sodium ions. For the osmotic pressure, a value measured using OSMOATAT OM-6040 (manufactured by ARKRAY, Inc.) was displayed.

Using the concentration of glycerol triphosphate, which is an internal standard substance, and the sodium ion concentration, which is a homeostatic substance, the dilution factor (X) was determined according to equation (1). For the measurement, plasma and diluted plasma dilution ratio were obtained using JEOL Ltd. JCA-BM6050 type automatic analyzer.

A: Measured absorbance of a dilute solution containing glycerol triphosphate B: Absorbance obtained by subtracting the absorbance of the mixture obtained by mixing blood plasma components and dilute solution from the absorbance obtained in A: C concentration of sodium ion as a homeostatic substance Absorbance D of a solution of 142 mmol / L: Absorbance of sodium ion concentration of a diluted solution obtained by diluting plasma components X: Plasma dilution rate

The concentration of the biochemical component in the diluted plasma is obtained by multiplying the measured value of the concentration of the reaction product using a known enzyme activity by the dilution factor determined by the above formula (1) to obtain the biochemical component in the original plasma. Was quantified.

Table 3 shows the maximum value, minimum value, and coefficient of variation CV (coefficient-of-variation:%) of the quantitative values of GPT and HbA1C obtained using the dilution ratios obtained in Method 1 to Method 3 above.

As shown in Table 3, according to the configuration of the present invention, the difference between the maximum value and the minimum value of the measurement and the reduction of the coefficient of variation (CV%) were confirmed, and the effect that the accuracy of each measurement was high could be confirmed. .

(Example 2)
After blood collection, when diluted with a buffer solution, plasma / blood cell separation was performed immediately after dilution using the plasma / blood cell separation device of DEMECAL blood test kit (Regier Co., Ltd.). Next, a case with a thickness of 30 mm that can be kept cold is prepared in the same way as in Example 1, and after being made cold so that it can be kept in the same way as in Example 1, it is packaged so that it can be mailed. Vibration was applied, and blood analysis was performed after 3 days at 25 ° C. The dilution factor was determined using Method 1 or Method 3 described in Example 1. As test items, GOT (glutamate oxaloacetate transaminase), HbA1C and CRE (creatinine) were measured.

As a comparison, plasma and blood cells are not separated immediately after blood dilution, but are stored in a similarly coolable case, and the same packaging is applied to provide transport vibration equivalent to 2500Km land transportation in Japan. Plasma and blood cells were separated by a separation method, and blood analysis was performed.

Table 4 shows the average value and coefficient of variation% of the quantitative results.

As a result of cooling, the difference in the variability with and without plasma / blood cell separation is small, but by separating the blood sample immediately after dilution of the blood sample, the effect of transport vibration is slightly reduced, It was found that analysis results with little variation can be obtained.

(Example 3)
An example of a method for determining the dilution rate of blood plasma will be described. In the composition shown in Table 2, an internal standard addition dilution solution containing no sodium ion was prepared by changing glycerol triphosphate to 1 mmol / L of lithium chloride. The internal standard substance of lithium added to the buffer was measured using chelate colorimetry (halogenated porphyrin chelate method: perfluoro-5,10,15,20-tetraphenyl-21H, 23H-porphyrin). It can be obtained by measuring.

As described above, the dilution rate of plasma with a buffer solution can be obtained by the following formula (2).
X = (A + C) / (B + D) (2)
A: Measured absorbance B of a diluted solution containing lithium B: Absorbance obtained by subtracting the absorbance of a diluted solution obtained by diluting plasma components from A C: Measured absorbance D of a solution having a sodium ion concentration of 142 mmol / L as a homeostatic substance : Absorbance of sodium ion concentration of diluted solution diluted with plasma component X: Dilution ratio of plasma

Using blood collected from a plurality of patients whose blood coagulation has been prevented by adding EDTA as a sample, in the same manner as in Example 1, a container containing a plasma / blood cell separated plasma-diluted solution is sandwiched with a cryogen. Prepare a container containing plasma diluted solution with a thermolabel attached to the surface, put it in a heat-insulating cold-insulated bag with an aluminum film that can be shielded from light, and make it possible to mail this cool-down bag The product was packaged and stored in an environment of 25 ° C. to 40 ° C. under the same environmental conditions for 3 days. Thereafter, the biochemical component in the diluted plasma was measured, the dilution factor of plasma was calculated using equation (2), and the value (A) of the biochemical component contained in the plasma was calculated. Separately, a biochemical component value (B) measured using the same blood sample collected by adding EDTA without diluting plasma obtained by separating blood cells by centrifugation was obtained. The correlation coefficient of each acquired value was calculated using the following formula (3).

Correlation coefficient = (covariance of value (A) and value (B)) / {(standard deviation of value (A)) × (standard deviation of value (B))}
.... (3)

The closer the correlation coefficient is to 1.000, the more consistent the two data are. In addition, a scatter diagram was created from the two data, and a regression equation (y = ax ± b) was obtained as a statistical formula of the distribution using the least square method. a indicates that the proportionality of the two data is good when the slope of the regression equation is in the range of 0.95 to 1.05. In addition, b indicates that the error is small when it is a numerical value close to 0 in the intercept of the regression equation. The results are shown in Table 5. From the results in Table 5, it was found that the correlation between diluted plasma and plasma was a good result.

Example 4
In Example 1, the concentration of total protein was measured by the following method in addition to the measurement of sodium ion concentration, with respect to the plasma diluted solution stored in the container using the cold insulation means.

(Measurement of total protein concentration in plasma dilution)
Measurements were performed using the burette method as the measurement principle. Biuret reagent: 3.0 mmol / L copper sulfate 400 μL, potassium sodium tartrate 21.3 mmol / L, NaOH 0.75 mol / L were prepared and mixed with the plasma diluent. After mixing, leave at 37 ° C. for 10 minutes, wait until a complex of 540 to 560 nm blue-purple is formed by protein and copper ions in the plasma dilution under alkalinity, measure absorbance at 545 nm, The total protein concentration in the plasma dilution was quantified using a calibration curve obtained from the absorbance of.

Diluted solution after blood dilution with respect to the dilution factor of the blood sample obtained from the measured value of sodium ion concentration in the plasma diluted solution and the average value 142 mmol / L of the concentration constantly contained in the blood sample The dilution factor obtained from the measured value of total protein in the blood sample and the average value of 7.5 g / 100 mL of the constant concentration of total protein, which is the standard component in the blood sample, is the same value. Results were obtained. Thereby, it turned out that the measurement using the dilution rate calculated | required from the sodium ion density | concentration is performed normally, and it turned out that the verification of a measurement is possible.

(Example 5)
As a return container for the DEMECAL blood test kit (Rieger Co., Ltd.), from the viewpoint of light shielding properties, heat shielding properties, and prevention of leakage of diluted plasma, an outer shape of 21.0 cm × 17.0 cm × 3. A 4 cm moisture-proof case (outside made of printable and printable paper) was prepared. A schematic diagram of the moisture-proof case is shown in the lower part of FIG. In the moisture-proof case 101, a package 102 of a DEMECAL kit and a case 103 with a return cooling agent are accommodated and returned.

The case 103 with the cooling agent for return (the upper part of FIG. 1) will be described below. Inside the aluminum film 104 is a rectangular parallelepiped of outer dimensions 10.0 cm × 16.5 cm × 2.8 cm (the arrow in the figure indicates about 2.8 cm). A non-solid cold-retaining agent 105 made of a water-soluble resin (sodium polyacrylate), an antiseptic, and a shape stabilizer is placed in a resin bag 106 having a moisture-proof cushioning property. A space 107 for storing a 7 mm columnar bottle after plasma separation is provided. The bottle was stored in the space 107 so as to be sandwiched by a cold insulation agent, and a cold insulation pack that could be folded in two and stored in a moisture-proof case for return was produced. When the temperature inside the bottle during cold storage was measured by attaching an RF (radio frequency) tag equipped with a temperature sensor to the bottle surface, it was 6 ° C.

In this packaging form, a moisture-proof case containing a blood sample and a DEMECAL blood test kit after use was placed in a post box and mailed.

DESCRIPTION OF SYMBOLS 1 Blood separation instrument 2 Blood collection container 3 Cylindrical body 4 Cap piston 5 Sealing lid 6 Cap 7 Packing 8 Screw part 9 Locking part 10 Bottom part 11 Leg part 12 Slit groove 13 Diluted solution 14 Wide diameter part 15 Thin part 16 Main body part 18 Shrinkage Diameter portion 19 Locking projection portion 20 Outer flange portion 21 Filtration membrane 22 Cover 26 Picking portion 27 Mandrel portion 28 Space 29 Lower end portion 31 Stepped portion 33 Upper end portion 34 Top portion 101 Moisture proof case 102 Package 103 Returned cooler containing case 104 Aluminum film 105 Coolant 106 Resin bag 107 Space

Claims (16)

1. A diluent for diluting the blood sample;
   Separation means for recovering plasma components from the diluted blood sample;
   A container for containing plasma components recovered from a diluted blood sample;
   A blood test kit comprising a cold insulation means for keeping the container cold.
2. The blood test kit according to claim 1, wherein the cold insulation means includes a cold insulation agent and a cold insulation bag.
3. The blood test kit according to claim 1 or 2, wherein the cold-retaining means further includes an accommodating member having a thickness of 35 mm or less that can accommodate a container for accommodating the collected plasma component.
4. The blood test kit according to any one of claims 1 to 3, wherein the blood test kit includes a member that records a temperature history.
5. The blood test kit according to any one of claims 1 to 4, wherein the blood test kit includes an instruction describing how to use the cold insulation means.
6. The blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, and the diluent does not contain the standard component. The blood test kit according to any one of claims 1 to 5.
7. The blood test kit according to claim 6, wherein the standard component is sodium ion or chloride ion.
8. The blood test kit according to claim 6 or 7, wherein the standard components are sodium ions or chloride ions and at least one standard component.
9. The blood test kit according to claim 8, wherein the at least one standard component is a standard component selected from total protein or albumin.
10. The blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using a standard component that is constantly present in blood, and verifying the analysis, The blood test kit according to claim 8 or 9, which does not contain the standard component.
11. The dilution liquid contains a standard component not present in blood, and the blood test kit is a blood test kit for analyzing the concentration of a target component in a blood sample using the standard component not present in blood. The blood test kit according to any one of claims 1 to 10, wherein:
12. Using the blood test kit according to any one of claims 6 to 9, plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the diluted plasma is constantly in the blood. A blood analysis method for determining the dilution ratio of plasma using the existing standard component and analyzing the concentration of the target component in the blood sample.
13. Using the blood test kit according to claim 10, plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the standard component in the diluted plasma that is constantly present in the blood is used. Determining the dilution ratio of the plasma, analyzing the concentration of the target component in the blood sample, and determining the dilution ratio of the plasma using a standard component different from the standard component constantly present in the blood, A blood analysis method that verifies the analysis of the concentration of the target component.
14. Using the blood test kit according to claim 11, plasma is collected from a blood sample, the collected plasma is diluted with a diluent, and the plasma is diluted with the standard component that is not present in the diluted plasma. A blood analysis method for determining a dilution factor and analyzing a concentration of a target component in a blood sample.
15. Using the blood test kit according to claim 11, which contains a diluent that does not contain a standard component that is constantly present in blood, plasma is collected from the blood sample, and the collected plasma is diluted with a diluent. A blood analysis that determines the dilution ratio of plasma and analyzes the concentration of a target component in a blood sample using the standard component that is constantly present in blood and the standard component that is not present in blood Method.
16. The blood analysis method according to any one of claims 12 to 15, wherein the amount of diluted plasma is 100 µL or more and 1000 µL or less.

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