Blood analyzer and blood analysis method

Abstract

A blood analyzer (1) according to the present invention comprises: an introduction unit (11) into which a blood sample is introduced; a first reagent holder (12) for storing a first reagent for measuring a first measurement item of the blood sample; a first pump (18) for controlling the delivery of the first reagent; a second reagent holder (13) for storing a second reagent for measuring a second measurement item of the blood sample; a transport control means (14) that is connected to the introduction unit (11), the first pump (18), and the second reagent holder (13), and that controls the transport of the blood sample and the second reagent; a first pipe (15) which is connected to the transport control mechanism (14) and into which at least a portion of the blood sample is introduced; a liquid chromatography column (16) that is connected to the first pipe (15); and a flow cell-type optical detector (17) that is connected to the liquid chromatography column (16) and measures the first measurement item and the second measurement item.

Description

Technical Field

[0001] The present invention relates to a blood analysis device and a blood analysis method.

[0002] This application claims priority to Japanese Patent Application No. 2023-046792 filed in Japan on March 23, 2023, and Japanese Patent Application No. 2023-143702 filed in Japan on September 5, 2023, the contents of which are incorporated herein by reference. Background Art

[0003] Hemoglobin non-enzymatically binds to sugars or their metabolites present in the blood to form glycated hemoglobin. Hemoglobin A1 is a glycated form of hemoglobin A, which accounts for the majority of total hemoglobin. Hemoglobin A1 is further classified into A1a, A1b, and A1c. Hemoglobin A1c (hereinafter sometimes simply referred to as HbA1c) persists in the blood for approximately 120 days, the lifespan of red blood cells. Because the glycation reaction of hemoglobin is slow, the ratio of glycated hemoglobin to the total hemoglobin (hereinafter sometimes simply referred to as glycated hemoglobin % or HbA1c %) is less susceptible to temporary increases in blood sugar levels caused by, for example, eating or drinking. Therefore, measuring HbA1c % can be used to estimate blood sugar levels over the past one to two months. Thus, HbA1c % can be used to monitor changes in blood sugar levels over a longer period of time, making it clinically meaningful.

[0004] The Japanese Diabetes Association, in its Diabetes Diagnosis and Treatment Guidelines 2019, lists a method for confirming the type of diabetes as a method for diagnosing diabetes. The type of diabetes is confirmed by two methods: (1) blood glucose level and (2) HbA1c. Specifically, (1) a fasting blood glucose level of 126 mg / dL or higher, a blood glucose level of 200 mg / dL or higher after 2 hours in a glucose tolerance test, or a blood glucose level of 200 mg / dL or higher at any time, and (2) an HbA1c level of 6.5% or higher is considered to be a type of diabetes. The type of diabetes is confirmed twice, one of which must be confirmed by blood glucose level.

[0005] Patent Documents 1 and 2 disclose blood analyzers capable of measuring both HbA1c% and blood sugar levels.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2012 / 043444

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2014-95715 Summary of the Invention

[0010] Technical problem solved by the invention

[0011] The ability to simultaneously measure HbA1c% and blood sugar levels with a single device contributes to faster diabetes diagnosis. Furthermore, the ability to simultaneously measure not only blood sugar levels but also diabetes-related markers such as the ratio of glycated albumin to total albumin and 1,5-anhydro-D-glucitol (also known as 1,5-AG) along with HbA1c% within a single device also contributes to understanding various medical conditions, including diabetes.

[0012] The blood analyzer disclosed in Patent Document 1 uses a test strip loaded with an enzymatic reagent to measure both HbA1c% and blood glucose levels. This method optically measures the extent of the enzymatic reaction at a single site, but requires the preparation of two separate test strips for measuring HbA1c% and blood glucose levels, which is complex. Furthermore, since the blood sample is applied to the test strip and then manually mounted on the measuring device, controlling the reaction temperature and time is difficult, despite the enzymatic reaction, leading to issues with measurement accuracy.

[0013] The blood analyzer disclosed in Patent Document 2 is configured to perform both HbA1c% and blood glucose level measurements using a single blood sample. Furthermore, HbA1c% measurement is based on liquid chromatography and offers higher accuracy than measurements using test strips. However, HbA1c% is measured using an absorbance meter after liquid chromatography, while glucose is measured using an enzyme electrode method. Therefore, separate enzyme electrodes for glucose measurement and detectors for glycated hemoglobin measurement are required. Enzyme electrodes are consumables and require more frequent replacement than the light source of an absorbance meter, making maintenance complex. Furthermore, enzyme electrodes require the use of rare metals such as platinum or silver, requiring complex separation procedures after disposal. Furthermore, when whole blood is used as the blood sample and glucose is measured using the enzyme electrode method, a more complex process is required to separate the plasma components by passing the blood sample through a blood cell separation membrane in order to remove blood cell components.

[0014] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a blood analysis apparatus and a blood analysis method that can analyze at least two measurement items and have a simplified detection mechanism.

[0015] Technical means to solve the problem

[0016] The present invention has the following aspects.

[0017] [1] A blood analysis device comprising:

[0018] an introduction portion for introducing a blood sample;

[0019] a first reagent holder storing a first reagent for measuring a first measurement item of the blood sample;

[0020] a first pump for controlling the delivery of the first reagent;

[0021] a second reagent holder storing a second reagent for measuring a second measurement item of the blood sample;

[0022] a transport control mechanism connected to the introduction portion, the first pump, and the second reagent holder, and controlling the transport of the blood sample and the second reagent;

[0023] a first pipe connected to the transport control mechanism and configured to introduce at least a portion of the blood sample;

[0024] a liquid chromatography column connected to the first pipe; and

[0025] A flow cell type optical detector is connected to the liquid chromatography column and measures the first measurement item and the second measurement item.

[0026] [2] The blood analysis device according to [1], further comprising:

[0027] The second pipe connects the transport control mechanism and the flow cell type optical detector.

[0028] [3] The blood analysis device according to [2], wherein:

[0029] The delivery control mechanism includes a flow path switching portion between the introduction portion and the second pipe.

[0030] The second pipe connects the flow path switching unit and the flow cell type optical detector.

[0031] The flow path switching section switches so as to disconnect the introduction section from the liquid chromatography column and connect the introduction section to the second pipe.

[0032] [4] The blood analysis device according to [3], wherein:

[0033] The delivery control mechanism further comprises:

[0034] a second pump connected to the flow path switching unit; and

[0035] An injection port is located between the flow path switching portion and the introduction portion.

[0036] [5] The blood analyzer according to any one of [1] to [4], wherein:

[0037] The flow cell type optical detector detects absorbance.

[0038] [6] The blood analyzer according to any one of [1] to [5], further comprising:

[0039] a third reagent holder storing a third reagent for measuring the first measurement item;

[0040] a third pump that controls the delivery of the third reagent; and

[0041] a mixer connected to the first pump and the third pump to mix the first reagent and the third reagent;

[0042] The mixer is connected to the conveying control mechanism,

[0043] The mixed solution of the first reagent and the third reagent is transported to the liquid chromatography column via the transport control mechanism.

[0044] [7] The blood analyzer according to any one of [1] to [6], further comprising:

[0045] a diluent holder storing a reagent for diluting the blood sample,

[0046] The diluent holder is connected to the introduction portion via the transport control mechanism.

[0047] [8] The blood analysis device according to any one of [1] to [7], further comprising:

[0048] The first temperature adjustment mechanism adjusts at least the temperature of the flow cell type optical detector.

[0049] [9] The blood analysis device according to [8], further comprising:

[0050] The second temperature adjustment mechanism adjusts the temperature of at least one of the first reagent holder and the second reagent holder.

[0051]

[10] The blood analysis device according to any one of [1] to [9], further comprising:

[0052] a fourth reagent holder storing a fourth reagent for measuring the second measurement item;

[0053] The fourth reagent holder is connected to the transport control mechanism,

[0054] The fourth reagent is introduced from the fourth reagent holder into the introduction portion via the transport control mechanism.

[0055]

[11] The blood analyzer according to any one of [1] to

[10] , wherein:

[0056] The first measurement item is HbA1c%.

[0057]

[12] The blood analyzer according to any one of [1] to

[11] , wherein:

[0058] The second measurement item is glucose concentration.

[0059]

[13] A blood analysis method comprising:

[0060] a step of introducing a portion of the blood sample and a first reagent into a liquid chromatography column;

[0061] a step of separating the first component of the blood sample in the liquid chromatography column;

[0062] a step of measuring the concentration of the first component using a flow cell optical detector; and

[0063] A step of measuring the concentration of the second component of the blood sample using the remaining portion of the blood sample and at least a second reagent and using the flow cell optical detector.

[0064]

[14] The blood analysis method according to

[13] , wherein:

[0065] The step of measuring the concentration of the second component using the flow cell optical detector includes:

[0066] a step of adding the second reagent to the remaining portion of the blood sample to prepare a mixed solution.

[0067]

[15] The blood analysis method according to

[13] or

[14] , wherein:

[0068] The step of preparing the mixed solution is performed during or after the step of separating the first component.

[0069]

[16] The blood analysis method according to any one of

[13] to

[15] , wherein:

[0070] The step of measuring the concentration of the second component using the flow cell type optical detector is performed after the step of measuring the first component.

[0071]

[17] The blood analysis method according to any one of

[13] to

[16] , wherein:

[0072] The first component is HbA1c.

[0073]

[18] The blood analysis method according to any one of

[13] to

[17] , wherein:

[0074] The second component is glucose.

[0075]

[19] The blood analysis method according to

[18] , further comprising:

[0076] A step of calculating the total hemoglobin amount based on the chromatogram of the blood sample separated by the liquid chromatography column using the flow cell optical detector; and

[0077] a step of correcting the glucose concentration based on the total hemoglobin amount.

[0078]

[20] The blood analysis method according to any one of

[13] to

[19] , wherein:

[0079] The blood sample is whole blood.

[0080]

[21] The blood analysis method according to any one of

[13] to

[20] , wherein:

[0081] The step of measuring the concentration of the second component uses an enzymatic method as the measurement principle.

[0082]

[22] The blood analysis method according to

[21] , wherein:

[0083] The step of measuring the concentration of the second component includes the step of measuring the amount of hydrogen peroxide generated by the reaction of the second component and the enzyme.

[0084]

[23] The blood analysis method according to

[22] , wherein:

[0085] In the step of measuring the concentration of the second component, Trinder's reagent is used.

[0086]

[24] The blood analysis method according to

[23] , wherein:

[0087] The Trinder's reagent is a reagent that reacts with hydrogen peroxide and has a color peak at 580 nm to 900 nm.

[0088]

[25] The blood analysis method according to

[23] or

[24] , wherein:

[0089] The Trinder's reagent contains MAOS, which has the CAS number 82692-97-5.

[0090]

[26] The blood analysis method according to any one of

[23] to

[25] , wherein:

[0091] The Trinder's reagent is contained in the second reagent.

[0092]

[27] The blood analysis method according to any one of

[13] to

[26] , wherein:

[0093] The step of introducing a portion of the blood sample and the first reagent into the liquid chromatography column includes the step of diluting the blood sample with a diluent.

[0094]

[28] The blood analysis method according to

[27] , wherein:

[0095] The step of introducing a portion of the blood sample and the first reagent into the liquid chromatography column includes the step of diluting the blood sample with a diluent,

[0096] The diluent contains one or more enzymes selected from the group consisting of a color developing agent and a coupling agent used in the enzymatic reaction.

[0097]

[29] The blood analysis method according to

[28] , wherein:

[0098] The diluent contains a coupling agent or a color developing agent.

[0099]

[30] The blood analysis method according to any one of

[13] to

[29] , wherein:

[0100] In the step of measuring the concentration of the second component in the blood sample using the flow cell optical detector, a fourth reagent is further used.

[0101]

[31] The blood analysis method according to

[30] , wherein:

[0102] The step of measuring the concentration of the second component uses an enzymatic method as the measurement principle.

[0103] The fourth reagent includes one or more enzymes, a color developing agent, and a coupling agent used in the enzymatic reaction.

[0104] Effects of the Invention

[0105] According to the above aspect, a blood analysis device and a blood analysis method having a simplified detection mechanism can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] [ Figure 1 ] is a schematic diagram of one embodiment of a blood analysis device.

[0107] [ Figure 2 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0108] [ Figure 3 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0109] [ Figure 4] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0110] [ Figure 5 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0111] [ Figure 6 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0112] [ Figure 7 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0113] [ Figure 8 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0114] [ Figure 9 ] is a schematic diagram of one form of a blood analysis device in another embodiment.

[0115] [ Figure 10 ] is a graph plotting the OD value at 505 nm relative to the measurement time when measuring the glucose aqueous solution B.

[0116] [ Figure 11 ] is a graph plotting the values ​​obtained by integrating the OD value at 505 nm when measuring the glucose aqueous solution B with respect to the glucose concentration.

[0117] [ Figure 12 ] is a graph in which the OD value at 505 nm is plotted against the measurement time when a glucose-added whole blood sample is measured.

[0118] [ Figure 13 ] is a graph plotting the values ​​obtained by integrating the OD value at 505 nm when measuring a glucose-added whole blood sample with respect to the glucose concentration.

[0119] [ Figure 14 ] is the absorption spectrum of whole blood diluted 101 times with hemolysis cleaning solution.

[0120] [ Figure 15 ] is a graph in which the OD value at 630 nm in a whole blood sample is plotted against the measurement time.

[0121] [ Figure 16 ] is a graph plotting the values ​​obtained by integrating the OD values ​​at 630 nm of the whole blood sample with respect to the glucose concentration.

[0122] [ Figure 17 ] is a graph in which the OD value at 660 nm in a whole blood sample is plotted against the measurement time.

[0123] [ Figure 18 ] is a graph plotting the values ​​obtained by integrating the OD values ​​at 660 nm of the whole blood sample with respect to the glucose concentration.

[0124] [ Figure 19 ] are the results of comparison of (a) the elution peak of HbA1c and (b) the elution peak of HbA0 when a whole blood sample was diluted with a conventional RC20 hemolysis and washing solution or a diluent containing 4-aminoantipyrine. DETAILED DESCRIPTION

[0125] Hereinafter, one embodiment of the present invention will be described with reference to the drawings.

[0126] In this specification, glycated hemoglobin refers to any of hemoglobin A1, hemoglobin A1a, hemoglobin A1b, and hemoglobin A1c. From the perspective of facilitating understanding of the state of blood sugar control, stable hemoglobin A1c (hereinafter referred to as HbA1c) is preferred.

[0127] In this specification, "HbA1c%" refers to the ratio of HbA1c to the total amount of hemoglobin contained in a blood sample.

[0128] In this specification, the glucose concentration refers to the mass of glucose relative to the volume of plasma contained in a blood sample (unit: mg / dL).

[0129] <Blood Analyzer>

[0130] A blood analysis device in one embodiment of the present invention includes an introduction portion, a first reagent holder, a first pump, a second reagent holder, a transport control mechanism, a first piping, a liquid chromatography column, and a flow cell type optical detector. The introduction portion introduces a blood sample. The first reagent holder stores a first reagent for measuring a first measurement item of the blood sample. The pump controls the transport of the first reagent. The second reagent holder stores a second reagent for measuring a second measurement item of the blood sample. The transport control mechanism is connected to the introduction portion, the first pump, and the second reagent holder to control the transport of the blood sample and the second reagent. The first piping is connected to the transport control mechanism to introduce at least a portion of the blood sample. The liquid chromatography column is connected to the first piping. The flow cell type optical detector is connected to the liquid chromatography column to measure the first measurement item and the second measurement item.

[0131] In this specification, "A is connected to B" encompasses both direct and indirect connections between A and B. Indirect connection between A and B means that A is connected to B via C. For example, "a flow cell optical detector is connected to a liquid chromatography column" encompasses connections between the flow cell optical detector and the liquid chromatography column via tubing, etc. On the other hand, in this specification, "A is connected to B" refers to a state in which a liquid, such as a sample, can flow between A and B.

[0132] Figure 1 This is a schematic diagram of one form of the blood analyzer according to this embodiment. Figure 1 The blood analyzer 1 shown includes an introduction section 11, a first reagent holder 12, a second reagent holder 13, a transport control mechanism 14, a first piping 15, a liquid chromatography column 16, a flow cell optical detector 17, a pump 18, a waste liquid tank 19, and a control unit 20. The transport control mechanism 14 includes a first flow path switching unit 141, a pump 142, and an injection port 143. The introduction section 11 is connected to the injection port 143 of the transport control mechanism 14. The first reagent holder 12 is connected to the injection port 143 via the pump 18. The second reagent holder 13 is connected to the first flow path switching unit 141. The first piping 15 connects the injection port 143 and the liquid chromatography column 16. The liquid chromatography column 16 is connected to the flow cell optical detector 17. The waste liquid tank 19 is connected to the flow cell optical detector 17. The control unit 20 controls the transport control mechanism 14, the flow cell optical detector 17, and the pump 18.

[0133] In this embodiment, an example in which the first measurement item is HbA1c% and the second measurement item is glucose concentration will be described.

[0134] The introduction section 11 is a portion for introducing a sample containing blood-derived components (hereinafter, sometimes simply referred to as a blood sample). The blood sample can be blood or a sample diluted with blood. In the case where the blood sample is blood, a sample cup can be provided in the introduction section 11, a blood collection tube can be provided, or a blood sample collected using a lancet can be provided. In addition, the introduction section 11 can be a needle such as a sample probe or an injection method using a sample loop. In the case of continuously processing multiple samples, the introduction section 11 can also be an automatic sampler including an injection port 143 described below.

[0135] The first reagent holder 12 stores a first reagent for measuring a first measurement item. In this embodiment, the first measurement item is HbA1c%, and a liquid chromatography column eluent is stored as the first reagent. The first reagent holder 12 is provided with a pump 18 for aspirating the first reagent from the first reagent holder. A degassing device (also referred to as a degasser) may be provided between the first reagent holder 12 and the pump 18, or between the pump 18 and the transport control mechanism 14.

[0136] The eluent can be selected to have a composition suitable for eluting hemoglobin. Examples of the eluent include buffer solutions or organic solvents containing known salt compounds. For example, examples of eluents include organic acids such as citric acid, succinic acid, tartaric acid, and malic acid, and their salts; amino acids such as glycine, taurine, and arginine; inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, and acetic acid, and their salts; and Good's buffer solutions. Other commonly added substances, such as surfactants, various polymers, and hydrophilic low molecular weight compounds, may also be appropriately added to the buffer solution.

[0137] In addition, as the eluent, different eluents such as pH and salt concentration can be used. The number of eluents can be 2 or 3. In this case, Figure 2 As shown, the blood analyzer 1' may further include: a third reagent holder 21 for storing a third reagent having a different pH and salt concentration from the first reagent; a mixer 22 for performing gradient elution; and a pump 23 for sucking the third reagent from the third reagent holder 21. A degassing device may also be provided between the third reagent holder 21 and the pump 23, or between the pump 23 and the mixer 22. Figure 2 In the blood analyzer 1 ′ shown, the control unit 20 controls the operations of the transport control mechanism 14 , the flow cell optical detector 17 , the pump 18 , the mixer 22 , and the pump 23 .

[0138] The second reagent holder 13 stores a second reagent for measuring a second measurement item. In this embodiment, the second measurement item is glucose concentration. Details of the second reagent are described in detail in <Blood Analysis Method>.

[0139] The delivery control mechanism 14 is connected to the introduction part 11, the pump 18 and the second reagent holder 13. The control unit 20 controls the delivery of the blood sample and the second reagent by operating the delivery control mechanism 14. The delivery control mechanism 14 preferably has a first flow path switching unit 141 and a pump 142 for controlling the delivery of the blood sample and the second reagent. In addition, the delivery control mechanism 14 preferably has an injection port 143 in order to transport the blood sample, the first reagent and the second reagent to the first pipe 15. As the first flow path switching unit 141, an automatic valve and a solenoid valve can be cited. As the pump 142, a syringe pump can be cited. The injection port 143 can use a known injection port. In addition, the delivery control mechanism 14 can also include a degassing device. In addition, the delivery control mechanism 14 can also include a mechanism for controlling the pressure of the entire liquid chromatography column 16 below.

[0140] The control unit 20 operates the first flow path switching unit 141 to connect the introduction unit 11 and the pump 142, thereby aspirating a portion of the blood sample via the pump 142. The aspirated blood sample may be retained within the pump 142, or a separate portion for retaining a solution may be provided between the introduction unit 11 and the pump 142 and retained therein. The portion for retaining the solution may be a sample loop or the same component as the first tubing.

[0141] The first reagent is aspirated from the first reagent holder 12 by the pump 18 and continuously introduced into the first tube 15 and the liquid chromatography column 16 via the injection port 143. While the first reagent is being introduced into the first tube 15 and the liquid chromatography column 16, the aspirated blood sample is also introduced into the first tube 15 and the liquid chromatography column 16 via the injection port 143.

[0142] Next, the control unit 20 switches the first flow path switching unit 141 to connect the second reagent holder 13 and the pump 142, and the pump 142 aspirates the second reagent from the second reagent holder 13. In this case, the second reagent may be retained in a separate portion for retaining a solution provided between the first flow path switching unit 141 and the pump 142. The portion for retaining the solution may be a sample loop or the same component as the first tubing.

[0143] Next, the control unit 20 connects the first flow path switching unit 141 to the introduction unit 11, and injects the second reagent drawn by the pump 142 into the introduction unit 11. A needle such as a sample probe may be used to inject the second reagent into the introduction unit 11. Alternatively, after the second reagent is injected into the introduction unit 11, the blood sample and the second reagent may be mixed by repeatedly aspirating and ejecting the second reagent from the needle. It should be noted that mixing the blood sample and the second reagent may also be achieved by vibrating the second reagent using an ultrasonic generator or the like.

[0144] The mixed liquid of the blood sample and the second reagent is introduced into the first tube 15 via the injection port 143. In this embodiment, as described above, a portion of the blood sample and the first reagent are introduced into the liquid chromatography column 16 via the first tube 15. Thereafter, the remaining mixture of the blood sample and the second reagent is introduced into the liquid chromatography column 16 via the first tube 15. The liquid chromatography column 16 separates HbA1c and other blood components in the blood sample, specifically, separates hemoglobin other than HbA1c.

[0145] The liquid chromatography column 16 is a column filled with a stationary phase having cation exchange capacity. A column filled with a resin having cation exchange groups bonded thereto can be used as the liquid chromatography column 16. Examples of cation exchange groups include carboxyl groups, phosphoric acid groups, and sulfonic acid groups, with sulfonic acid groups being preferred.

[0146] The filler particles of the stationary phase having cation exchange capacity are not particularly limited as long as they are used as filler particles for ion exchange liquid chromatography, and examples include inorganic particles and organic particles. Examples of inorganic particles include particles composed of silica or zirconium oxide. Examples of organic particles include natural polymer particles such as cellulose, polyamino acids, and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate. Preferred examples of stationary phases having cation exchange groups include the filler disclosed in Japanese Patent Application Laid-Open No. 2011-047858, which comprises cross-linked polymer particles obtained by polymerizing a mixture of a non-cross-linked hydrophilic acrylic monomer and a polyglycidyl ether, and a layer of acrylic monomer having cation exchange groups polymerized on the surface of the cross-linked polymer particles.

[0147] The liquid chromatography column 16 may also be stored in a temperature control mechanism such as a column oven.

[0148] The flow cell optical detector 17 is connected to the liquid chromatography column 16. The blood sample that has passed through the liquid chromatography column 16 is measured for the first and second measurement parameters in the flow cell optical detector 17. In this embodiment, the flow cell optical detector 17 detects glycated hemoglobin and other blood components separated by the liquid chromatography column 16 to generate chromatogram data. The resulting chromatogram data can be used to measure HbA1c%. Furthermore, the flow cell optical detector 17 detects the glucose concentration contained in the mixture of the blood sample and the second reagent.

[0149] The flow cell optical detector 17 can use an absorbance meter that is included as a detector in a high performance liquid chromatography (HPLC) apparatus. In other words, the flow cell optical detector 17 can also detect absorbance. It should be noted that the flow cell optical detector 17 can also measure scattered light, fluorescence, or chemiluminescence.

[0150] The first reagent, the second reagent, and the blood sample that have passed through the flow cell optical detector 17 are discharged to the waste liquid tank 19 .

[0151] The control unit 20 controls the operation of the transport control mechanism 14, the flow chamber optical detector 17, and the pump 18. The control unit 20 is constructed using an information processing device. That is, the control unit 20 includes a CPU (Central Processor Unit), a memory, and an auxiliary storage device connected via a bus. The control unit 20 operates by executing a program. The control unit 20 can also be implemented by a computer. In this case, the program for implementing the control function can also be recorded in a computer-readable recording medium, and the computer system can read and execute the program recorded in the recording medium.

[0152] It should be noted that the "computer system" referred to here refers to the computer system built into the blood analyzer, including hardware such as the operating system and peripheral devices. Furthermore, a "computer-readable recording medium" refers to removable media such as floppy disks, magneto-optical disks, ROMs, and CD-ROMs, as well as storage devices such as hard disks built into the computer system. Furthermore, a "computer-readable recording medium" may also include media that dynamically store programs for a short period of time, such as communication lines used when transmitting programs via networks such as the Internet or communication lines such as telephone lines, or media that store programs for a certain period of time, such as volatile memory within a computer system that serves as a server or client in this case. Furthermore, the program may be a program for implementing a portion of the functions described, or a program that can implement the functions by combining with programs already stored in the computer system. Furthermore, a portion of the control unit of the blood analyzer in the above embodiment may be implemented as an integrated circuit such as an LSI (Large Scale Integration). The control unit of the blood analyzer may be processor-based, or part or all of it may be integrated to form a processor. Furthermore, the integrated circuit method is not limited to LSI; implementation may also be achieved using a dedicated circuit or a general-purpose processor. Furthermore, if an integrated circuit technology replacing LSI emerges due to advancements in semiconductor technology, an integrated circuit based on this technology may also be used.

[0153] The blood analyzer 1 described above can measure both HbA1c% and glucose concentration using the flow cell optical detector 17, thereby simplifying the detection mechanism and miniaturizing the entire device. Furthermore, since only one detection mechanism is required, maintenance is facilitated.

[0154] <Modification 1 of Blood Analyzer>

[0155] Figure 3 2 is a schematic diagram of a blood analyzer according to Modification 1 of the present invention. The blood analyzer 2 further includes a second pipe 25. The remaining configuration of the blood analyzer 2 is the same as that of the blood analyzer 1, and therefore, description thereof will be omitted.

[0156] The second pipe 25 is provided between the transport control mechanism 14 and the flow cell optical detector 17. In the present embodiment, the second pipe 25 is connected to the first flow path switching unit 141.

[0157] The blood analyzer 2' differs from the blood analyzer 1 in that the blood sample and the second reagent are transported as follows. The control unit 20 operates the first flow path switching unit 141 to connect the introduction unit 11 and the pump 142, allowing the pump 142 to aspirate a portion of the blood sample. The first reagent is aspirated from the first reagent holder 12 by the pump 18 and continuously introduced into the first tubing 15 and the liquid chromatography column 16 via the injection port 143. While the first reagent is being introduced into the first tubing and the liquid chromatography column 16, the aspirated blood sample is also introduced into the first tubing 15 and the liquid chromatography column 16 via the injection port 143.

[0158] The first flow path switching section 141 is switched so as to connect the second reagent holder 13 and the pump 142, and the second reagent is aspirated from the second reagent holder 13 by the pump 142. Next, the first flow path switching section 141 is switched so as to connect the pump 142 and the introduction section 11, and the aspirated second reagent is injected into the introduction section 11, thereby mixing the blood sample and the second reagent.

[0159] The mixed liquid of the blood sample and the second reagent is sucked by the pump 142, and the mixed liquid is maintained between the first flow path switching section 141 and the pump 142. Thereafter, the first flow path switching section 141 is operated in a manner that connects the pump 142 and the second piping 25. The mixed liquid of the blood sample and the second reagent sucked by the pump 142 is transported to the second piping 25 and introduced into the flow cell type optical detector 17. After the mixed liquid is introduced into the flow cell type optical detector 17, the pump 142 can be stopped for a predetermined time in order to ensure time for the blood sample to react with the component used to measure the glucose concentration. After the predetermined reaction time has passed, the pump 142 can be operated again to discharge the mixed liquid introduced into the flow cell type optical detector 17.

[0160] Figure 4 This is a schematic diagram of a blood analyzer according to a modified example 1' of the present invention. The blood analyzer 2' further includes a second flow path switching unit 144, a third flow path switching unit 145, and a second pipe 25. The remaining configuration of the blood analyzer 2' is the same as that of the blood analyzer 1, and therefore description thereof is omitted.

[0161] The second flow path switching section 144 and the third flow path switching section 145 are provided in the first pipe 15. The second flow path switching section 144 is connected to the third flow path switching section 145 via the second pipe 25. The third flow path switching section 145 is located between the liquid chromatography column 16 and the flow cell optical detector 17. The second flow path switching section 144 and the third flow path switching section 145 can be the same components as the first flow path switching section 141. A check valve can also be used for the third flow path switching section 145.

[0162] The blood analyzer 2' differs from the blood analyzer 1 in that the blood sample and the second reagent are transported as follows. The first flow path switching unit 141 is operated to connect the introduction unit 11 and the pump 142, and a portion of the blood sample is aspirated by the pump 142. Subsequently, the second flow path switching unit 144 is operated to connect the injection port 143 and the liquid chromatography column 16, and the third flow path switching unit 145 is operated to connect the liquid chromatography column 16 and the flow cell optical detector 17. The first reagent is aspirated from the first reagent holder 12 by the pump 18 and continuously introduced into the first tubing 15 and the liquid chromatography column 16 via the injection port 143. While the first reagent is being introduced into the first tubing 15 and the liquid chromatography column 16, the aspirated blood sample is introduced into the first tubing 15 and the liquid chromatography column 16 via the injection port 143.

[0163] The first flow path switching section 141 is switched so as to connect the second reagent holder 13 and the pump 142, and the second reagent is aspirated from the second reagent holder 13 by the pump 142. Next, the first flow path switching section 141 is switched so as to connect the pump 142 and the introduction section 11, and the aspirated second reagent is injected into the introduction section 11, thereby mixing the blood sample and the second reagent.

[0164] The second flow path switching section 144 separates the first pipe 15 from the liquid chromatography column 16 and connects the first pipe 15 to the second pipe 25. The third flow path switching section 145 separates the liquid chromatography column 16 from the flow cell optical detector 17 and connects the second pipe 25 to the flow cell optical detector 17. The mixed blood sample and second reagent are introduced into the flow cell optical detector 17 by the pump 142 via the injection port 143, the second flow path switching section 144, the second pipe 25, and the third flow path switching section 145. It should be noted that in the blood analyzer 2', the second pipe 25 is connected to the flow cell optical detector 17 via the third flow path switching section 145, but the present invention is not limited to this. For example, the second pipe 25 may be directly connected to the flow cell optical detector 17.

[0165] In Modifications 1 and 1', the mixture of the blood sample and the second reagent is not introduced into the liquid chromatography column 16 but is introduced into the flow cell optical detector 17 via the second pipe 25. This prevents contamination of the liquid chromatography column 16 by the mixture of the blood sample and the second reagent and shortens the measurement time.

[0166] <Variation 2 of Blood Analyzer>

[0167] Figure 5 This is a modified example 2 of the blood analyzer of the present invention. Figure 2The blood analyzer 1' shown further includes a diluent holder 26. The other configurations of the blood analyzer 3 are the same as those of the blood analyzer 1', and therefore description thereof will be omitted.

[0168] The diluent holder 26 stores a diluent for diluting blood. As the diluent, a hypotonic solution is used, specifically, a buffer solution that may contain a preservative or a surfactant. When the blood sample introduced into the introduction portion 11 is blood, the first flow path switching unit 141 is operated in a manner such that the diluent holder 26 is connected to the introduction portion 11, and the diluent is injected from the diluent holder 26 into the introduction portion 11 by the pump 142. When the diluent is injected into the introduction portion 11, a needle such as a sample probe may also be used. After the diluent is injected into the introduction portion 11, the blood sample and the diluent may be mixed by repeatedly sucking and ejecting the diluent from the needle. It should be noted that the mixing of the blood sample and the diluent may also be carried out by vibrating the diluent using an ultrasonic generator or the like.

[0169] Figure 6 The blood analyzer 3 ′ shown is a modified example of the blood analyzer 3 , and includes a pump 24 between the second reagent holder 13 and the first flow path switching unit 141 . In addition, a pump 27 is provided between the diluent holder 26 and the first flow path switching unit 141 .

[0170] According to this modification, the blood sample can be diluted with a diluent and introduced into the blood analyzer.

[0171] <Variation 3 of Blood Analyzer>

[0172] Figure 7 This is a modified example 3 of the blood analyzer of the present invention. Figure 5 The blood analyzer 3 shown also includes a fourth flow path switching unit 146. As the fourth flow path switching unit 146, the same component as the first flow path switching unit 141 can be used. The other components of the blood analyzer 4 are the same as those of the blood analyzer 3, and therefore the description thereof is omitted. The fourth flow path switching unit 146 connects the first flow path switching unit 141 to the second reagent holder 13 or the diluent holder 26. The fourth flow path switching unit 146 and the first flow path switching unit 141 are operated so that the diluent holder 26 is connected to the introduction section 11, and the diluent is injected from the diluent holder 26 into the introduction section 11. In addition, when mixing the second reagent and the blood sample, the fourth flow path switching unit 146 and the first flow path switching unit 141 are operated so that the second reagent holder 13 is connected to the introduction section 11, and the second reagent is injected from the second reagent holder 13 into the introduction section 11.

[0173] According to this modification, the number of ports of the first flow path switching unit 141 can be suppressed.

[0174] <Variation 4 of Blood Analyzer>

[0175] Figure 8 This is a modified example 4 of the blood analyzer of the present invention. Figure 3 The blood analyzer 2 shown further includes a first temperature regulating mechanism 28 and a second temperature regulating mechanism 29. The other configurations of the blood analyzer 5 are the same as those of the blood analyzer 2, and thus description thereof will be omitted.

[0176] The first temperature regulating mechanism 28 regulates the temperature of the flow chamber type optical detector 17 to at least 20 to 50°C. In this way, the output of the flow chamber type optical detector 17 can be stabilized. In addition, when the second reagent reacts with the blood sample, it can be maintained at an optimal temperature to improve the reactivity. In addition, the reaction rate of the second reagent with the blood sample is not easily affected by the environment such as the temperature around the device. In addition to the flow chamber type optical detector 17, the first temperature regulating mechanism 28 can also regulate the temperature of the liquid chromatography column 16. In addition, the first temperature regulating mechanism 28 can also regulate the temperature of one or more of the second piping 25, the introduction part 11, the first piping 15 and the injection port 143. The first temperature regulating mechanism 28 can also be a column temperature oven.

[0177] The second temperature regulating mechanism 29 regulates the temperature of at least one of the first reagent holder 12 and the second reagent holder 13 to 0 to 30°C. This can suppress the degradation of the components contained in each reagent, maintain the separation capability of the liquid chromatography, or maintain the reactivity of each component with the blood sample. The second temperature regulating mechanism 29 is particularly preferably used to regulate the temperature of the second reagent holder 13, which is a reagent containing an enzyme. The second temperature regulating mechanism 29 can also regulate the temperature of the diluent holder 26 at the same time. Figure 8 , an example is shown in which the second temperature adjustment mechanism 29 adjusts the temperature of the second reagent holder 13 .

[0178] It should be noted that, while the above description has been given of a blood analyzer 5 including both the first temperature control mechanism 28 and the second temperature control mechanism 29, the present invention is not limited thereto. The blood analyzer of the present invention may also include at least one of the first temperature control mechanism 28 and the second temperature control mechanism 29.

[0179] <Variation 5 of the Blood Analyzer>

[0180] Figure 9 This is a modified example 5 of the blood analyzer of the present invention. Figure 5 The blood analyzer 3 shown in the figure further includes a second pipe 25 and a fourth reagent holder 30. The second pipe 25 and the fourth reagent holder 30 are connected. Figure 3Since the blood analyzer 6 is the same as the blood analyzer 2 shown, its description is omitted. Since the other configurations of the blood analyzer 6 are the same as those of the blood analyzer 3, its description is omitted.

[0181] The fourth reagent holder 30 stores a fourth reagent for measuring the second measurement item of the blood sample. That is, in this variation, the reagent for measuring the second measurement item is stored separately in the second reagent holder 13 and the fourth reagent holder 30. Like the second reagent holder 13, the fourth reagent holder 30 is connected to the first flow path switching unit 141 of the transport control mechanism 14.

[0182] The following describes the operation of the blood analyzer 6. The operation from the injection of the second reagent from the second reagent holder 13 into the introduction portion 11 to the mixing of the blood sample and the second reagent is the same as that described in <Blood Analyzer>.

[0183] Next, the fourth reagent is introduced from the fourth reagent holder into the introduction section 11 via the transport control mechanism 14. More specifically, the first flow path switching section 141 of the transport control mechanism 14 is switched so as to connect the fourth reagent holder 30 and the pump 142, and the fourth reagent is aspirated from the fourth reagent holder 30 via the pump 142. Next, the first flow path switching section 141 is switched so as to connect the pump 142 and the introduction section 11, and the aspirated fourth reagent is injected into the introduction section 11, further mixing the fourth reagent with the mixture of the blood sample and the second reagent.

[0184] The mixed solution of the blood sample, the second reagent, and the fourth reagent is aspirated by pump 142 and held between first flow path switching section 141 and pump 142. First flow path switching section 141 is then operated to connect pump 142 to second piping 25. The mixed solution of the blood sample, the second reagent, and the fourth reagent is introduced into flow cell optical detector 17 through second piping 25.

[0185] It should be noted that, in the above description, blood analyzer 6 is described as a blood analyzer including the fourth reagent holder, but the present invention is not limited thereto. The blood analyzer may be configured such that any of blood analyzers 1, 2, 4, and 5 further includes the fourth reagent holder.

[0186] Furthermore, the blood analyzer may combine at least two configurations of the blood analyzers 1 to 6. For example, modifications 1 and 2 may be combined, modifications 1 to 2 and 4 may be combined, or modifications 2, 4, and 5 may be combined.

[0187] <Other components>

[0188] The blood analyzer of the present invention may also include any configuration necessary for the operation of the device. For example, it may include a cleaning liquid tank that stores cleaning liquid for cleaning the introduction portion 11 and the injection port 143. The location of the cleaning liquid tank is not particularly limited, as long as it can clean the introduction portion 11 and the injection port 143. The cleaning liquid stored in the cleaning liquid tank can be drawn out using the pump 142, or a dedicated pump for the cleaning liquid may be included. A waste liquid tank may also be included for discharging waste liquid after cleaning, and a flow path may be included that allows waste liquid to be recovered using the waste liquid tank 19.

[0189] With regard to the cleaning solution stored in the cleaning solution tank, a liquid comprising at least one cleaning component (e.g., surfactant) can be used. In addition, the cleaning solution can include preservatives such as 2-phenoxyethanol, sodium azide, or both.

[0190] Alternatively, a cleaning liquid tank for storing cleaning liquid may be provided for cleaning the flow cell optical detector 17. The cleaning liquid tank may be positioned so that the cleaning liquid can pass between the liquid chromatography column 16 and the flow cell optical detector 17. A pump may be provided to allow the cleaning liquid to pass through. A waste liquid tank for discharging waste liquid after cleaning may also be provided, and a flow path for recovering waste liquid using the waste liquid tank 19 may also be provided.

[0191] In addition, for example, a pump may be provided, or a flow path may be provided for purging (also called purging) the flow path. A suppression valve (also called a self-closing valve) may be provided in the flow path to prevent backflow. A pump may also be provided for draining the solution from the flow path.

[0192] The above configuration can be combined with any one of the blood analysis devices 1 , 1 ′, 2 , 2 ′, 3 , 3 ′, 4 , 5 , and 6 .

[0193] <Measurement Items>

[0194] In the above embodiment, an example is described in which the first measurement item is HbA1c% and the second measurement item is glucose concentration, but the present invention is not limited to this. For example, the first measurement item may be HbA1c% and the second measurement item may be glycated albumin%. In this case, the first reagent holder stores a reagent for measuring HbA1c% and the second reagent holder stores a reagent for measuring glycated albumin%, respectively. Glycated albumin% refers to the ratio of glycated albumin to total albumin.

[0195] The device configuration for measuring HbA1c% is the same as that in the example where the first measurement item is HbA1c% and the second measurement item is glucose concentration.

[0196] The second measurement item, glycated albumin percentage measurement, measures total albumin and glycated albumin separately, and calculates the ratio of glycated albumin to total albumin. Total albumin and glycated albumin can be measured by combining the measurement principles shown in Table 1. In Table 1, HPLC stands for high performance liquid chromatography.

[0197] [Table 1]

[0198]

[0199] In the case of measurement principle combination 1, albumin and glycated albumin can be separated using liquid chromatography column 16, or a second liquid chromatography column connected to a separately provided tubing can be used for separation. The second liquid chromatography column can be installed midway in the second tubing 25 shown in the blood analyzer 2' of Modification 1'. The second liquid chromatography column is connected to a flow cell optical detector 17, and the separated albumin and glycated albumin are detected by the flow cell optical detector 17. An eluent for glycated albumin measurement is stored as a reagent for glycated albumin measurement. Two or more eluents may be used.

[0200] In the case of measurement principle combination 2, for example, a total albumin measurement reagent and a glycated albumin measurement reagent, both liquid reagents, are stored in the second reagent holder 13. A pump for aspirating each reagent may be provided. Total albumin is measured using a biochemical reagent, while glycated albumin is measured using an enzymatic reagent.

[0201] use Figure 3 The blood analyzer 2 shown here illustrates an example of a measurement procedure for the case of combination 2 as the measurement principle. First, to measure HbA1c%, a portion of the blood sample placed in the introduction section 11 and an eluent serving as the first reagent are introduced into the liquid chromatography column 16 through the aforementioned operation. A total albumin measurement reagent is added from the second reagent holder 13 to the blood sample remaining in the introduction section 11. A portion of the mixture of the blood sample and the total albumin measurement reagent is aspirated by the pump 142 and reaches the flow cell optical detector 17 via the second piping 25 for total albumin detection. A glycated albumin measurement reagent is added from a separate reagent holder provided parallel to the second reagent holder 13 to the blood sample further remaining in the introduction section 11. A portion of the mixture is aspirated and reaches the flow cell optical detector 17 via the second piping 25 for glycated albumin detection. It should be noted that the order of total albumin and glycated albumin detection can also be reversed. After a portion of the blood sample is introduced into the liquid chromatography column 16 for HbA1c% measurement, a portion of the blood sample remaining in the introduction portion 11 may be transferred to a separate sample cup, and then a total albumin measurement reagent or a glycated albumin measurement reagent may be added.

[0202] Combinations 3 and 4 of the measurement principles can be performed by combining the HPLC, biochemical method, and enzymatic method described in the description of combinations 1 and 2. Combinations 3 and 4 of the measurement principles can also be used in which the blood analyzer including the second liquid chromatography column described in combination 1 of the measurement principle further includes a third pipe connecting the first flow path switching unit 141 and the flow cell optical detector 17.

[0203] Alternatively, for example, the first measurement item may be HbA1c% and the second measurement item may be 1,5-AG. In this case, the first reagent holder 12 and the second reagent holder 13 store the HbA1c% measurement reagent and the 1,5-AG measurement reagent, respectively. In this case, the configuration is the same as in the example where the second measurement item is glucose concentration, except that the reagent stored in the second reagent holder is the 1,5-AG measurement reagent.

[0204] <Blood Analysis Method>

[0205] A blood analysis method according to one embodiment of the present invention includes: introducing a portion of a blood sample and a first reagent into a liquid chromatography column; separating a first component of the blood sample in the liquid chromatography column; measuring the concentration of the first component using a flow cell optical detector; and measuring the concentration of a second component of the blood sample using the remaining portion of the blood sample and at least a second reagent using the flow cell optical detector. Furthermore, when measuring the concentration of the second component of the blood sample using the flow cell optical detector, the method may include adding at least the second reagent to the remaining portion of the blood sample to prepare a mixed solution.

[0206] A blood analysis method according to one embodiment of the present invention will be described with reference to the components of the blood analyzer 1. In this embodiment, an example in which the first measurement item is HbA1c% and the second measurement item is glucose concentration will be described.

[0207] A blood sample is introduced into the introduction portion 11. The blood sample can be blood or hemolyzed and diluted blood. Even if the blood sample is not hemolyzed and diluted, the blood sample can be hemolyzed and diluted using a blood analyzer equipped with the diluent tank of Modification 2. For example, if the blood sample is whole blood, it is diluted 50-200 times. A hypotonic solution, specifically a buffer solution that may contain a preservative or surfactant, is used for dilution.

[0208] The first flow path switching unit 141 is switched to connect the introduction unit 11 and the pump 142. A portion of the blood sample is sucked by the pump 142 and introduced into the liquid chromatography column 16 along with the first reagent via the first tube 15. In the liquid chromatography column 16, HbA1c and other blood components, specifically various hemoglobins other than HbA1c, in the blood sample are separated.

[0209] After a portion of the blood sample and the first reagent are introduced into the liquid chromatography column 16, the transport control mechanism 14 causes the pump 142 to add the second reagent from the second reagent holder 13 to the remaining portion of the blood sample in the introduction section 11, thereby preparing a mixed solution of the blood sample and the second reagent. To shorten the measurement time, the mixed solution is preferably prepared during or after the introduction of a portion of the blood sample into the liquid chromatography column 16 and the separation of HbA1c. For example, the amount of blood sample introduced into the liquid chromatography column 16 may be predetermined, and when a predetermined amount of blood sample is discharged from the introduction section 11, the discharge is stopped, and the pump 142 is controlled to add the second reagent from the second reagent holder 13 to the introduction section 11.

[0210] The second reagent may include a reagent based on a known assay principle. Examples include enzymatic methods, immunoturbidimetric methods, latex immunoturbidimetric methods (also known as LTIA), chemiluminescent immunoassays, and electrochemiluminescent immunoassays. When multiple components are used in these assay principles, the second reagent may include at least one of the multiple components.

[0211] The enzymatic method is a method in which a substance to be measured contained in a blood sample is reacted with a specific enzyme, and a reagent that develops color is used to measure the reaction product or a substance derived from the reaction product.

[0212] When the assay object substance is glucose, the assay principle based on the enzymatic method is roughly divided into two. The first is to make glucose react with specific enzymes and produce hydrogen peroxide, by the effect of the hydrogen peroxide produced and peroxidase (also referred to as POD), the developer added separately to the reagent is developed, and the method for measuring absorbance variation is measured. As such method, there are known glucose oxidase (GOD)-peroxidase method (also referred to as GOD-POD), mutarotase-GOD-POD method and pyranose oxidase-POD method etc. that further added mutarotase in the GOD-POD method. That is, in the first assay principle, at least enzyme, POD and developer are needed, which are included in the reagent, i.e. eluent (the first reagent or the third reagent) and diluent added to the blood sample when measuring the second assay item and at least one and the second reagent. In the case of comprising the component required for the first assay principle in the first reagent, by arranging the pipe 3 connecting pump 18 and the first stream switching unit 141, pump 142 can be used to attract the first reagent. When the third reagent contains components required for the first measurement principle, the third reagent can be aspirated using pump 142 by providing piping 4 connecting pump 23 and first flow path switching unit 141. The method for mixing the first and third reagents with the blood sample is the same as the method for mixing the second reagent with the blood sample.

[0213] In the second measurement principle, glucose is reacted with a specific enzyme in the presence of ATP (adenosine triphosphate). When the resulting substance further reacts with other enzymes, NADP (oxidized nicotinamide adenine dinucleotide phosphate) is converted to NADPH (reduced nicotinamide adenine dinucleotide phosphate), and the increase in NADPH is measured by absorbance changes. Glucose-6-phosphate dehydrogenase (G6-PDH) is commonly used as an enzyme that reacts with glucose. Known methods for this include the hexokinase-G6-PDH method and the glucokinase-G6-PDH method. That is, in the second measurement principle, at least ATP, two enzymes, and NADP are required. These are contained in the reagents added to the blood sample when measuring the second measurement item, namely, at least one of the eluent (first reagent or third reagent) and the diluent, as well as the second reagent.

[0214] NADPH has a maximum absorption at 340nm. When whole blood is used as a blood sample, the absorption spectrum of NADPH overlaps with the absorption spectrum of various hemoglobins including HbA1c, making it difficult to detect NADPH. In addition, colorimetric or fluorescence detection of the generated NADPH can also be further performed using known methods, but the reagent composition becomes complicated. Therefore, regarding the determination principle of glucose, the reagent composition is simple and the storage stability of the reagent is expected to be high, so the first determination principle is preferred. In the first determination principle, the GOD-POD method or the mutarotase-GOD-POD method is preferred.

[0215] When hydrogen peroxide and POD are used to develop the color of the developer, phenols or anilines are preferably used as the developer. Phenols or anilines function as hydrogen donors. Phenolic hydrogen donors are generally referred to as Trinder's reagents. Aniline hydrogen donors are generally referred to as modified Trinder's reagents (Tamaoku et al., "New Water-soluble Hydrogen Donors for the Enzymatic Photometric Determination of Hydrogen Peroxide. II. N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline Derivatives", Chem. Pharm. Bull. 30 (7) 2492-2497, 1982). The Trinder's reagent used in the present invention may be modified Trinder's reagent. In the color development step, modified Trinder's reagent may be used instead of Trinder's reagent. Trinder's reagent undergoes a condensation reaction with a coupling agent such as 4-aminoantipyrine (also known as 4-AA) or 3-methyl-2-benzothiazolinehydrazone to convert it into a pigment with a specific maximum absorption wavelength.

[0216] Examples of the color developing agent include phenol, phenol derivatives, Trinder's reagent, and new Trinder's reagent. Examples of the new Trinder's reagent include ADOS (CAS No. [82692-96-4], the maximum absorption wavelength of the oxidative condensation dye with 4-AA is 542 nm), ADPS (CAS No. [82611-88-9], the maximum absorption wavelength of the oxidative condensation dye with 4-AA is 540 nm), ALPS (CAS No. [82611-85-6], the maximum absorption wavelength of the oxidative condensation dye with 4-AA is 561 nm), DAOS (CAS No. [83777-30-4], the maximum absorption wavelength of the oxidative condensation dye with 4-AA is 593 nm), and HDAOS (CAS No. [82692-88-4], the maximum absorption wavelength of the oxidative condensation dye with 4-AA is 540 nm). The maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 583nm), MAOS (CAS number [82692-97-5], the maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 630nm), TOOS (CAS number [82692-93-1], the maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 555nm), TOPS (CAS number [40567-80-4], the maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 550nm), MADB (CAS number [209518-16-1], the maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 630nm) and TODB (CAS number [1044537-70-3], the maximum absorption wavelength of the oxidative condensation pigment with 4-AA is 550nm), etc.

[0217] Because it is less susceptible to the absorption spectra of plasma components and hemoglobin contained in blood samples, Trinder's reagent preferably has a maximum absorption wavelength of 550 nm or greater, and more preferably 580 to 800 nm, when reacting with a coupling agent to form a compound. Examples of reagents with a color development peak at 580 to 800 nm include DAOS, HDAOS, MAOS, and MADB.

[0218] The second reagent may further contain components for buffering or controlling pH, ionic strength, or osmotic pressure, etc. Examples of such components include acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, Good's buffer, and sodium, potassium, and calcium salts thereof.

[0219] The pH of the second reagent can be appropriately selected based on the optimal pH of the enzyme and the pH at which the pigment develops color appropriately.

[0220] The second reagent may also preferably contain a salt. As the type of salt, an inorganic salt is preferred. Examples of the inorganic salt include sodium chloride and potassium chloride. The concentration of the buffer or salt can be appropriately selected according to the type of enzyme as long as the concentration allows the enzyme to react appropriately.

[0221] The second reagent may further contain an enzyme or pigment stabilizing component. Examples of enzyme or pigment stabilizing components include proteins such as casein and bovine serum albumin; polymers such as polyethylene glycol, polyvinyl pyrrolidone, and phospholipid polymers; sugars; surfactants; oxidizing agents; reducing agents; and chaotropic agents. The enzyme or pigment stabilizing component may be a single component or a combination of multiple components.

[0222] In order to remove the components that affect the determination, the second reagent may further include specific compounds, enzymes, surfactants, etc. As such compounds, ferrocyanide ion (Japanese Patent Application Laid-Open No. 55-138656), EDTA-iron complex (Japanese Patent Application Laid-Open No. 57-71398), etc. can be enumerated. As such enzymes, ascorbic acid oxidase, bilirubin oxidase and catalase, etc. can be enumerated. As such surfactants, amphoteric surfactants (Japanese Patent Application Laid-Open No. 7-155196), nonionic surfactants (International Publication No. 2013 / 147309), etc. can be enumerated.

[0223] When the analyte is glucose, the components required for the measurement may react with each other in a solution state. To ensure that the component contents remain constant over time, the components may be prepared in multiple solutions. For example, when the second reagent comprises Trinder's reagent, the coupling agent and Trinder's reagent are preferably prepared as separate reagents to prevent a condensation reaction between the coupling agent and the Trinder's reagent during storage in the second reagent holder 13.

[0224] For example, a mode comprising a coupling agent, comprising Trinder's reagent in a kind of eluent (the first reagent or the third reagent) and a diluent, and a mode comprising Trinder's reagent in a kind of eluent (the first reagent or the third reagent) and a diluent, and a mode comprising a coupling agent can be prepared. In addition, a mode comprising a coupling agent, comprising Trinder's reagent in a kind of eluent (the first reagent or the third reagent) and a diluent, and a mode comprising a coupling agent can be prepared. As an example, in the reagent based on the mutarotase-GOD-POD method, they can be prepared according to a mode comprising Trinder's reagent, mutarotase and peroxidase in a kind of diluent as a coupling agent. When a blood sample is mixed with a diluent, the blood sample is mixed with 4-AA. In addition, by adding the second reagent thereto, Trinder's reagent, mutarotase, peroxidase are added, and an enzyme reaction is carried out to produce a color developing method that is dependent on glucose concentration.

[0225] The amount of glucose concentration-dependent color development is measured by the flow cell optical detector 17. The measurement method may be any method. For example, after mixing the blood sample and the second reagent, the increase in absorbance per unit time may be measured using a rate method after a certain period of time. Alternatively, the difference between the absorbance immediately after mixing the blood sample and the second reagent and the absorbance after a certain period of time may be measured using an endpoint method. When measuring using the endpoint method, before the mixed solution of the blood sample and the second reagent is passed through the flow cell optical detector 17, the eluent, diluent, and the required solutions in the second reagent may be separately passed through the flow cell optical detector 17 to measure the blank absorbance of each solution.

[0226] The combination of at least one of the eluent and the diluent and the components contained in the second reagent can be exemplified by (1) to (8) in Table 2. In Table 2, "at least one of the eluent and the diluent" is simply recorded as "eluent or diluent". In addition, in Table 2, "enzyme" means one or more selected from glucose oxidase (GOD), mutarotase and pyranose oxidase. Those skilled in the art can appropriately select the combination in consideration of the stability of the enzyme used, the interaction between the components, etc. Among them, in order to prevent contamination and deterioration of the liquid chromatography column, the component added to at least one of the eluent and the diluent is preferably added to the diluent, and combination 4 or 8 is further preferred. The reagent containing the enzyme and Trinder's reagent is preferably kept at a temperature by a second temperature regulating mechanism.

[0227] [Table 2]

[0228]

[0229] The components required for glucose determination can be divided into two reagents, namely, an eluent (the first reagent or the third reagent) and a diluent, and a second reagent for addition. In the case where the components required for glucose determination are divided into two reagents for addition, the two reagents are preferably a diluent and a second reagent. In addition, the components required for glucose determination can also be divided into three reagents for addition. In the case where the components required for glucose determination are divided into three reagents for addition, the three reagents can be any two selected from the first reagent, the third reagent, and the diluent, and the second reagent. In the case where the components required for glucose determination are divided into three reagents for addition, the three reagents are preferably a third reagent, a diluent, and the second reagent.

[0230] The above description describes an example in which, when the components required for measuring the second measurement item are added separately from the second reagent, the components required for measuring the second measurement item are added to reagents used for other purposes such as diluent, first reagent, and third reagent.

[0231] Alternatively, in order to prevent the components required for measurement from reacting with each other in a solution state, the components required for the second measurement item can be prepared separately as a second reagent and a fourth reagent. For example, the components required for glucose measurement can be prepared separately in three solutions: the second reagent, the fourth reagent, and the diluent.

[0232] The fourth reagent mainly contains the second measurement item, and in the above example, contains a reagent for measuring glucose. The components contained in the fourth reagent are the same as those in the second reagent, and therefore description thereof will be omitted.

[0233] As an example, in the GOD-POD method, use 4-AA, peroxidase, glucose oxidase and Trinder's reagent as coupling agent as reagent.Considering the stability of these 4 kinds of compositions, the interaction of composition each other etc., can separately prepare each composition in these 3 kinds of solutions of the 2nd reagent, the 4th reagent and diluent.For example, can prepare them in the mode of comprising peroxidase, glucose oxidase and Trinder's reagent in the 4-AA, the 2nd reagent as coupling agent, the 4th reagent in the diluent.When blood sample is mixed with diluent, blood sample mixes with 4-AA.In addition, by wherein adding the 2nd reagent and adding peroxidase, and then by adding the 4th reagent and adding glucose oxidase and Trinder's reagent, carry out enzyme reaction, produce the color developing of glucose concentration dependency.

[0234] In the case of the GOD-POD method, the combination of the components contained in the second reagent, the fourth reagent and the diluent can be appropriately selected by considering the stability of the enzyme used, the interaction between the components containing the ferrocyanide ion, EDTA-iron complex, ascorbic acid oxidase, bilirubin oxidase, etc. Among them, in order to prevent contamination and deterioration of the liquid chromatography column, it is preferred to include an enzyme or Trinder's reagent in the second reagent or the fourth reagent. In addition, it is known that ferrocyanide ions interact with various components. In the case of adding ferrocyanide ions, it is particularly preferred to add Trinder's reagent to a reagent different from the ferrocyanide ion. Specific combinations of the components contained in the second reagent, the fourth reagent and the diluent can be cited in Tables 3 (1) to (6). Among them, combination (4) is preferred. The reagent containing the enzyme and Trinder's reagent is preferably kept at a temperature by a second temperature regulating mechanism.

[0235] [Table 3]

[0236]

[0237] When separating HbA1c using the liquid chromatography column 16, whole blood is typically diluted approximately 101-fold. For glucose concentration measurement, the blood sample is typically diluted approximately 80-fold before measurement. Since the dilution ratios of the blood in the blood sample are similar for HbA1c% and glucose measurements, both measurements can be performed simultaneously.

[0238] When the second measurement target item is glycated albumin percentage, total albumin and glycated albumin are measured separately, and the ratio of glycated albumin to total albumin is calculated. When glycated albumin percentage is measured by high performance liquid chromatography, albumin and glycated albumin can be separated using the liquid chromatography column 16, or albumin and glycated albumin can be separated using a second liquid chromatography column connected to the second pipe 25.

[0239] When total albumin is measured by a biochemical method, known reagents such as the BCG (bromocresol green) method and the modified BCP (bromocresol purple) method can be used for measurement.

[0240] When measuring glycated albumin by an enzymatic method, the measurement can be performed, for example, by the following steps. (1) Glycated albumin in a blood sample is digested with a protease to produce fructosyl amino acids. (2) Ketoamine oxidase or fructosamine oxidase is allowed to act on the fructosyl amino acids in the presence of oxygen to produce hydrogen peroxide. (3) The hydrogen peroxide reacts with POD in the presence of a color developer to develop color, and the glycated albumin is quantified by absorbance measurement.

[0241] When the second measurement item is 1,5-AG, the reagent for measuring 1,5-AG may be an enzymatic reagent, and the measurement may be performed, for example, by the following steps. (1) ADP (adenosine diphosphate)-dependent hexokinase and adenosine-5'-diphosphate are brought into contact with 1,5-AG in the sample to generate 1,5-AG 6-phosphate. (2) 1,5-AG dehydrogenase is allowed to act on the 1,5-AG 6-phosphate and NADP to generate NADPH. (3) The NADPH is detected to quantify the amount of 1,5-AG.

[0242] When preparing the mixture, in order to promote the color reaction, such as Figure 8 As shown, it is preferably maintained at 20 to 50° C. by the first temperature adjustment mechanism 28 .

[0243] The HbA1c% separated in the liquid chromatography column 16 is measured using a flow cell optical detector 17. Specifically, HbA1c% is calculated based on the optical density (also known as OD value) at a wavelength of 300 to 600 nm, where hemoglobin exhibits absorption. Hemoglobin exhibits characteristic absorption at wavelengths of 350 to 450 nm and 520 to 590 nm. Therefore, the measurement wavelengths are preferably 350 to 450 nm and 520 to 590 nm, more preferably 350 to 450 nm, and most preferably 380 to 425 nm.

[0244] After the HbA1c concentration is measured, the first flow path switching section 141 is switched so that the introduction section 11 is connected to the pump 142. The mixed solution of the blood sample and the second reagent is aspirated from the introduction section 11 using the pump 142. The mixed solution is then introduced into the first piping 15 via the injection port 143, and the glucose concentration of the mixed solution is measured using the flow cell optical detector 17. In the case of Modification 1, after the mixed solution of the blood sample and the second reagent is aspirated using the pump 142, the first flow path switching section 141 connects the pump 142 to the second piping 25, and the mixed solution is introduced into the second piping 25. The glucose concentration of the mixed solution is then measured using the flow cell optical detector 17. The measurement wavelength in the flow cell optical detector 17 is set based on the color peak of the mixed solution based on the second reagent used.

[0245] The OD value corresponding to the glucose concentration measured by the above method may be affected by the absorbance of hemoglobin and other components contained in the mixed solution, resulting in the measured value being higher than the actual glucose concentration. Furthermore, the glucose concentration is calculated as the mass of glucose relative to the volume of plasma. Since the quantitative ratio of blood cell components to plasma components in whole blood varies from person to person, the amount of plasma components contained in the sample placed in the introduction section 11 may vary from sample to sample. To avoid these effects, it is preferable to correct the OD value corresponding to the concentration of hemoglobin and other components.

[0246] One correction method involves performing a correction based on the total hemoglobin area of ​​the chromatogram of each hemoglobin separated by the liquid chromatography column 16. The absorption coefficient of hemoglobin at the measurement wavelength of each hemoglobin is well known. Using this absorption coefficient and the total hemoglobin area, the absorbance value derived from hemoglobin at the wavelength for measuring glucose concentration is calculated. The OD value can be corrected by subtracting the absorbance value derived from hemoglobin from the absorbance during glucose measurement.

[0247] In addition, when the glucose concentration is low or high, the actual glucose concentration and the degree of deviation of the OD value are sometimes different. In this case, the OD value under the wavelength at which the OD value varies depending on the total hemoglobin content in the blood sample and the wavelength at which the glucose concentration is measured can be used to calculate the regression equation and perform correction. Using the regression equation as a correction equation, the OD value corresponding to the glucose concentration of the blood sample can be corrected. The regression equation can be a linear equation, a quadratic equation, or a cubic or higher equation. In addition, the regression equation can also use multiple equations according to the hemoglobin content.

[0248] As described above, the blood analysis method according to one embodiment of the present invention can measure the first and second components of a blood sample using one blood sample and one flow cell optical detector. This allows for simple measurement of two different components.

[0249] Example

[0250] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following description.

[0251] [Example 1]

[0252] The glucose concentration of the sample was measured using an HPLC apparatus equipped with an absorbance meter as a flow cell type optical detector.

[0253] 1. Sample Preparation

[0254] Glucose aqueous solutions A were prepared with glucose concentrations of 0, 300, 600, 1200, 1800, 2400, and 3000 mg / dL. Whole blood samples collected from volunteers (i.e., whole blood samples refrigerated for more than one week after blood collection) were mixed with the glucose aqueous solution A at a ratio of 5:1 to prepare whole blood samples with glucose concentrations of 0, 50, 100, 200, 300, 400, and 500 mg / dL (referred to as glucose-spiked whole blood samples). Separately, glucose aqueous solutions A and purified water were mixed at a ratio of 5:1 to prepare glucose aqueous solutions B with glucose concentrations of 0, 50, 100, 200, 300, 400, and 500 mg / dL.

[0255] 2. Preparation of Glucose Assay Reagents

[0256] As a glucose assay reagent, Glucose CII Test Wako (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used, which utilizes the mutarotase GOD-POD method. (1) 17.5 mL of a buffer solution containing phosphate buffer (pH 7.1) and phenol as a color developer was used to dissolve (2) a color developer (glucose assay reagent A) containing mutarotase, GOD, POD, 4-aminoantipyrine, and ascorbate oxidase. The concentration was 20 times that specified in the reagent package insert.

[0257] 3. Glucose Determination Reaction

[0258] 3 μL of each glucose-supplemented whole blood sample or glucose aqueous solution B was added to 300 μL of a solution containing phenol added to a final concentration of 0.1% by weight in RC20 Hemolysis and Washing Solution (manufactured by Sekisui Medical Co., Ltd.), diluted 101-fold to prepare the measurement sample. 15 μL of glucose assay reagent A was added to the measurement sample and the mixture was allowed to stand at 37°C for 5 minutes to obtain a reaction solution. The RC20 Hemolysis and Washing Solution is a solution that lyses red blood cells in whole blood.

[0259] 4.HPLC determination

[0260] The mixed solution was measured using an HPLC apparatus (Shimadzu Corporation, Model: LC20A) without a liquid chromatography column. Eluent A for the RC20, manufactured by Sekisui Medical Co., Ltd., a glycosylated hemoglobin analyzer, was used as the eluent. The reaction solution was measured under the following conditions: an injection volume of 5 μL, a flow rate of 1.1 mL / min, and a measurement wavelength of 505 nm.

[0261] 5. Results and Investigation

[0262] Figure 10 This is a graph plotting the OD value at 505 nm versus the measurement time when glucose aqueous solution B is used as a sample. Figure 11 This is a graph plotting the integrated OD value at 505 nm relative to the glucose concentration when the glucose aqueous solution B was used as a sample. Figure 12 This is a graph plotting the OD value at 505 nm versus measurement time when a glucose-supplemented whole blood sample is used as a sample. Figure 13 This is a graph plotting the integrated OD value at 505 nm relative to the glucose concentration when a glucose-supplemented whole blood sample is used as a sample.

[0263] Figure 11The graph of has good linearity, and therefore it can be seen that the glucose concentration can be measured by measuring the mixed solution of the glucose aqueous solution B and the glucose measuring reagent A using a flow cell optical detector, which is a detector of the HPLC apparatus.

[0264] also, Figure 13 In the graph, a rise in the blank derived from hemoglobin in the whole blood sample was observed, indicating good linearity. This indicates that the glucose concentration in the whole blood sample can be measured using a flow cell optical detector, which is a detector in an HPLC device.

[0265] [Example 2]

[0266] In order to verify a glucose measurement method that is less susceptible to the influence of components other than glucose contained in whole blood, the color developing agent in the glucose measurement reagent was changed to MAOS.

[0267] Figure 14 The absorption spectrum of whole blood diluted to 101 times with RC20 hemolysis cleaning solution manufactured by SEKISUI MEDICAL Co., Ltd. is shown (measured using an HPLC apparatus, manufactured by Shimadzu Corporation, model: LC20A). It can be said that the absorption spectrum of whole blood has low absorbance at wavelengths above 600nm. Therefore, MAOS, whose color development peak in a mixed solution with glucose is above 600nm, was used as a chromogenic substrate.

[0268] 1. Sample Preparation

[0269] Whole blood samples having glucose concentrations of 0, 50, 100, 200, 300, 400, 500, and 1000 mg / dL (referred to as glucose-supplemented whole blood samples) were prepared in the same manner as in Example 1.

[0270] 2. Preparation of Glucose Assay Reagents

[0271] A glucose assay reagent B having the following composition was prepared. MAOS was purchased from Dojin Chemical Laboratories Co., Ltd. In addition, commercially available enzymes were purchased and used.

[0272] 20 mM phosphate buffer (pH 7.2)

[0273] 4-Aminoantipyrine: 85 mg / mL

[0274] MAOS: 40 mg / mL

[0275] Glucose oxidase: 340 units / mL

[0276] Peroxidase: 350 units / mL

[0277] Ascorbate oxidase: 60 units / mL

[0278] Mutarotase: 100 units / mL

[0279] 3. Glucose Determination Reaction

[0280] 3 μL of glucose-spiked whole blood sample was diluted 101-fold with RC20 hemolysis and washing solution manufactured by Sekisui Medical Co., Ltd. to prepare a measurement sample. 15 μL of glucose assay reagent B was added to the measurement sample and the mixture was allowed to stand at 37°C for 5 minutes to obtain a reaction solution.

[0281] 4-1.HPLC determination-1

[0282] The measurement was performed in the same manner as in Example 1, except that the measurement wavelength was set to 630 nm. At this wavelength, a substance in which MAOS and 4-aminoantipyrine as a coupling agent are bound can be measured.

[0283] 4-2.HPLC determination-2

[0284] The mixed solution was measured using an HPLC apparatus (manufactured by SEKISUI MEDICAL Co., Ltd., model: RC20) without installing a liquid chromatography column. Eluent A for the RC20 was used as the eluent. Glucose-spiked whole blood samples were measured at an injection volume of 4 μL, a flow rate of 0.6 mL / min, and a measurement wavelength of 660 nm. At this wavelength, substances formed by the combination of MAOS and 4-aminoantipyrine as a coupling agent can also be measured.

[0285] 5. Results and Investigation

[0286] Figure 15 This is a graph in which the OD value at 630 nm is plotted against the measurement time when a glucose-supplemented whole blood sample is used as a sample. Figure 16 This is a graph in which the OD value at 630 nm of the whole blood sample is plotted against the integrated value of the added glucose concentration.

[0287] exist Figure 13 When converted to glucose concentration, a blank (intercept) equivalent to about 148 mg / dL was generated. Figure 16 In the figure, when converted to glucose concentration, the blank is reduced to about 14 mg / dL. It is believed that this intercept originates from components such as hemoglobin in the whole blood sample. This shows that the use of a chromogenic substrate such as MAOS, which has a color peak of 600 nm or more when mixed with glucose, is effective in suppressing the increase in the blank. Figure 16 The good linearity indicates that even when MAOS is used as a color developing agent, the glucose concentration contained in a whole blood sample can be measured using a flow cell optical detector as a detector of an HPLC apparatus.

[0288] Figure 17 This is a graph in which the OD value at 660 nm of a glucose-added whole blood sample is plotted against the measurement time. Figure 18 This is a graph in which the OD value at 660 nm of a glucose-supplemented whole blood sample is plotted against the integrated value of the glucose concentration.

[0289] exist Figure 18 In the figure, the rise in the blank derived from whole blood is equivalent to approximately 19 mg / dL when converted into a glucose concentration. Furthermore, it can be seen that due to the excellent linearity, the glucose concentration contained in the whole blood sample can be measured using an absorbance meter as a detector in an HPLC apparatus. As can be seen from the above, the glucose concentration contained in the whole blood sample can be measured using an absorbance meter as a detector in an HPLC apparatus, regardless of the specific HPLC apparatus.

[0290] [Example 3]

[0291] A reagent configuration was proposed in which the diluent contained 4-aminoantipyrine and the second reagent contained mutarotase, GOD, POD, or Trinder's reagent. The effect of 4-aminoantipyrine on HbA1c% measurement was evaluated by HPLC analysis of whole blood samples diluted with the diluent containing 4-aminoantipyrine.

[0292] 1. Preparation of Reagents and Samples

[0293] 4-Aminoantipyrine (hereinafter referred to as 4AA added diluent) was added to the RC20 hemolysis and washing solution in such a way that the final concentration became 1 mmol / L. 300 μL of the 4AA added diluent was added to 3 μL of each whole blood sample from three volunteers to prepare blood samples. As a control, a blood sample was prepared by diluting the whole blood sample at the same ratio with the RC20 hemolysis and washing solution. As for whole blood, three different donors were studied. It should be noted that 4-aminoantipyrine was not added to the RC20 hemolysis and washing solution.

[0294] 2.HPLC determination

[0295] A RC20 column was installed in an HPLC apparatus (manufactured by SEKISUI MEDICAL Co., Ltd., Model: RC20). A diluted whole blood sample (i.e., blood sample) was measured using RC20 eluent A as the first reagent and RC20 eluent B as the third reagent. Measurements were performed under the following conditions: an injection volume of 4 μL of diluted sample, a flow rate of 1.1 mL / min, and a gradient conforming to the RC20 program.

[0296] 3. Results and Investigation

[0297] Figure 19 This is a chromatogram plotting the results of the RC20 measurement. The vertical axis represents the absorbance (OD) obtained by subtracting the absorbance at 660 nm from the absorbance at 415 nm. Figure 19 (a) shows a peak with a retention time of about 39 seconds, and the peak of HbA0 is as follows Figure 19 (b) shows a peak with a retention time of approximately 84 seconds. The chromatograms of various hemoglobins including HbA1c were comparable when using the RC20 hemolysis and washing solution without 4AA and when using the diluent with 4AA.

[0298] Table 4 shows the results of HbA1c% measurement values ​​calculated from the chromatograms. For samples from three volunteers, the HbA1c% values ​​measured using the standard RC hemolysis and washing solution and the HbA1c% values ​​measured using the 4AA-supplemented diluent were highly consistent.

[0299] [Table 4]

[0300]

[0301] Depend on Figure 19 As shown in Table 4, the addition of 4-aminoantipyrine to the diluent has no effect on the HbA1c% measurement, and HbA1c% can be measured with high accuracy even when the diluent contains a component for glucose measurement.

[0302] The above demonstrates that a blood sample can be diluted with a diluent containing a component for glucose measurement and then introduced into a liquid chromatography column to separate hemoglobins, and that HbA1c% can be measured with high precision using a flow cell optical detector. Furthermore, by further adding a component for glucose measurement to the diluted sample, glucose can be measured with high precision using the flow cell optical detector.

[0303] Industrial Applicability

[0304] According to the above aspect, a blood analysis device and a blood analysis method having a simplified detection mechanism can be provided.

[0305] Explanation of symbols

[0306] 1, 1', 2, 2', 3, 3', 4, and 5...blood analyzer, 11...introduction unit, 12...first reagent holder, 13...second reagent holder, 14...transfer control mechanism, 15...first piping, 16...liquid chromatography column, 17...flow cell optical detector, 18, 23, 24, 27, 142...pump, 19...waste liquid tank, 20...control unit, 21...third reagent holder, 22...mixer, 25...second piping, 26...diluent holder, 28...first temperature control mechanism, 29...second temperature control mechanism, 30...fourth reagent holder, 141...first flow path switching unit, 143...injection port, 144...second flow path switching unit, 145...third flow path switching unit, 146...fourth flow path switching unit.

Claims

1. A blood analysis device comprising: an introduction portion for introducing a blood sample; a first reagent holder storing a first reagent for measuring a first measurement item of the blood sample; a first pump for controlling the delivery of the first reagent; a second reagent holder storing a second reagent for measuring a second measurement item of the blood sample; a transport control mechanism connected to the introduction portion, the first pump, and the second reagent holder, and controlling the transport of the blood sample and the second reagent; a first pipe connected to the transport control mechanism and configured to introduce at least a portion of the blood sample; a liquid chromatography column connected to the first pipe; and A flow cell type optical detector is connected to the liquid chromatography column and measures the first measurement item and the second measurement item.

2. The blood analysis device according to claim 1, further comprising: The second pipe connects the transport control mechanism and the flow cell type optical detector.

3. The blood analyzer according to claim 2, wherein: The delivery control mechanism includes a flow path switching portion between the introduction portion and the second pipe. The second pipe connects the flow path switching unit and the flow cell type optical detector. The flow path switching section switches so as to disconnect the introduction section from the liquid chromatography column and connect the introduction section to the second pipe.

4. The blood analyzer according to claim 3, wherein: The delivery control mechanism further comprises: a second pump connected to the flow path switching unit; and An injection port is located between the flow path switching portion and the introduction portion.

5. The blood analyzer according to claim 1 or 2, wherein: The flow cell type optical detector detects absorbance.

6. The blood analysis device according to claim 1 or 2, further comprising: a third reagent holder storing a third reagent for measuring the first measurement item; a third pump that controls the delivery of the third reagent; and a mixer connected to the first pump and the third pump to mix the first reagent and the third reagent; The mixer is connected to the conveying control mechanism, The mixed solution of the first reagent and the third reagent is transported to the liquid chromatography column via the transport control mechanism.

7. The blood analysis device according to claim 1 or 2, further comprising: a diluent holder storing a reagent for diluting the blood sample, The diluent holder is connected to the introduction portion via the transport control mechanism.

8. The blood analysis device according to claim 1 or 2, further comprising: The first temperature adjustment mechanism adjusts at least the temperature of the flow cell type optical detector.

9. The blood analysis device according to claim 8, further comprising: The second temperature adjustment mechanism adjusts the temperature of at least one of the first reagent holder and the second reagent holder.

10. The blood analysis device according to claim 1 or 2, further comprising: a fourth reagent holder storing a fourth reagent for measuring the second measurement item; The fourth reagent holder is connected to the transport control mechanism, The fourth reagent is introduced from the fourth reagent holder into the introduction portion via the transport control mechanism.

11. The blood analyzer according to claim 1 or 2, wherein: The first measurement item is HbA1c%.

12. The blood analyzer according to claim 1 or 2, wherein: The second measurement item is glucose concentration.

13. A blood analysis method comprising: a step of introducing a portion of the blood sample and a first reagent into a liquid chromatography column; a step of separating the first component of the blood sample in the liquid chromatography column; a step of measuring the concentration of the first component using a flow cell optical detector; and A step of measuring the concentration of the second component of the blood sample using the remaining portion of the blood sample and at least a second reagent and using the flow cell optical detector.

14. The blood analysis method according to claim 13, wherein: The step of measuring the concentration of the second component using the flow cell optical detector includes: a step of adding the second reagent to the remaining portion of the blood sample to prepare a mixed solution.

15. The blood analysis method according to claim 14, wherein: The step of preparing the mixed solution is performed during or after the step of separating the first component.

16. The blood analysis method according to claim 13 or 14, wherein: The step of measuring the concentration of the second component using the flow cell type optical detector is performed after the step of measuring the first component.

17. The blood analysis method according to claim 13 or 14, wherein: The first component is HbA1c.

18. The blood analysis method according to claim 13 or 14, wherein: The second component is glucose.

19. The blood analysis method according to claim 18, further comprising: A step of calculating the total hemoglobin amount based on the chromatogram of the blood sample separated by the liquid chromatography column using the flow cell optical detector; and a step of correcting the glucose concentration based on the total hemoglobin amount.

20. The blood analysis method according to claim 13 or 14, wherein The blood sample is whole blood.

21. The blood analysis method according to claim 13 or 14, wherein: The step of measuring the concentration of the second component uses an enzymatic method as the measurement principle.

22. The blood analysis method according to claim 13 or 14, wherein: The step of measuring the concentration of the second component includes the step of measuring the amount of hydrogen peroxide generated by the reaction of the second component and the enzyme.

23. The blood analysis method according to claim 22, wherein: In the step of measuring the concentration of the second component, Trinder's reagent is used.

24. The blood analysis method according to claim 23, wherein: The Trinder's reagent is a reagent that reacts with hydrogen peroxide and has a color peak at 580 nm to 900 nm.

25. The blood analysis method according to claim 24, wherein The Trinder's reagent contains MAOS, which has the CAS number 82692-97-5.

26. The blood analysis method according to claim 23, wherein: The Trinder's reagent is contained in the second reagent.

27. The blood analysis method according to claim 13 or 14, wherein: The step of introducing a portion of the blood sample and the first reagent into the liquid chromatography column includes the step of diluting the blood sample with a diluent.

28. The blood analysis method according to claim 21, wherein The step of introducing a portion of the blood sample and the first reagent into the liquid chromatography column includes the step of diluting the blood sample with a diluent, The diluent contains one or more enzymes selected from the group consisting of a color developing agent and a coupling agent used in the enzymatic reaction.

29. The blood analysis method according to claim 28, wherein The diluent contains a coupling agent or a color developing agent.

30. The blood analysis method according to claim 13 or 14, wherein In the step of measuring the concentration of the second component in the blood sample using the flow cell optical detector, a fourth reagent is further used.

31. The blood analysis method according to claim 30, wherein: The step of measuring the concentration of the second component uses an enzymatic method as the measurement principle. The fourth reagent includes one or more enzymes, a color developing agent, and a coupling agent used in the enzymatic reaction.

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