JP2007047031A - Analytical method and analysis implement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method capable of being easily used and improving analysis accuracy. <P>SOLUTION: The analytical method is used for analyzing a sample solution containing both a specific component and a solvent component and comprises a separation process for separating at least part of the solvent component from the sample solution; a solvent component measurement process for measuring a known quantity from the solvent component separated in the separation process; a sample solution measurement process for measuring a known quantity from the sample solution; a dilution process for creating a diluted sample solution through the use of both a known quantity of the sample solution measured in the sample solution measurement process and a known quantity of the solvent component measured in the solvent component measurement process; and an analysis process for analyzing the specific component contained in the diluted sample solution. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analysis method and an analysis tool for analyzing a specific component in blood, for example.

  Analyzing specific components in the blood is effective in understanding the health condition of the human body or treating specific diseases. In order to analyze, for example, red blood cells and white blood cells as the specific component, an analysis method using a blood cell counter is performed.

  FIG. 23 shows a conventional blood cell counter used in an analysis method for analyzing red blood cells and white blood cells (see Patent Document 1). The blood cell counter X shown in the figure includes a blood cell measurement unit 94 and an immune measurement unit 95. The blood cell measuring unit 94 includes a white blood cell counting measurement cell 94a and a red blood cell counting measurement cell 94b, and is configured to count blood cells such as red blood cells and white blood cells contained in blood using an electrical resistance detection method. The immunoassay unit 95 is configured to measure C-reactive protein. In order to measure the blood cell count and the C reaction protein with the blood cell counter X, blood as a specimen is first injected into the specimen container 91. When counting white blood cells, a predetermined amount of blood is injected from the sample container 91 into the white blood cell counting measurement cell 94a by the nozzle 93a of the probe unit 93. In addition, a predetermined amount of diluent is injected from the diluent container 92a through the electromagnetic valve 92c and the nozzle 93a into the white blood cell count measurement cell 94a. Thereby, the blood is diluted in the white blood cell counting measurement cell 94a. Using this diluted blood, white blood cells are counted by the electric resistance detection method. Similarly, for red blood cell counting, the blood is diluted and counted in the red blood cell counting measurement cell 94b. Further, according to the blood cell counter X, it is possible to measure the C-reactive protein using the immunoassay unit 95 together with the blood cell count. These measurement results are processed by the MPU in the figure and then output to the display device or printer in the figure. The diluted blood after the measurement is sent to the waste liquid container 92b.

  FIG. 24 shows an example of a conventional analytical test element used in a method for analyzing a total hemoglobin value and a glycated hemoglobin value (see Patent Document 2). The analysis test element Y shown in the figure includes an introduction part 101 for introducing a blood sample 100, a separation chamber 102, a mixing chamber 103, a total hemoglobin measurement part 108, and a glycated hemoglobin measurement part 109. A part of the blood sample 100 introduced into the introduction unit 101 is made into plasma by removing red blood cells by the separation chamber 102. This plasma and a part of the blood sample 100 that has passed through the branch channel 103 are mixed in the mixing chamber 104. Thereby, a diluted blood sample is generated in the mixing chamber 104. A part of this diluted blood sample is sent to the total hemoglobin measuring unit 108 through the oxidation chamber 105, and the total hemoglobin value is measured. On the other hand, a part of the diluted blood sample is sent to the glycated hemoglobin measuring unit 109 via the first and second reaction chambers 106 and 107, and the glycated hemoglobin value is measured. The diluted blood sample that has been measured is collected into the collection chamber 110. As described above, according to the analysis test element Y, the total hemoglobin value and the glycated hemoglobin value can be measured in a state in which the blood sample is diluted.

  However, the blood cell counter X and the analysis test element Y have the following problems.

  First, the blood cell counter X needs to contain a diluent container 92a, dilution means such as the probe unit 93 and blood cell measuring unit 94, and a waste container 92b. If these are built in, the size of the blood cell counter X becomes large, which is not suitable for carrying. In addition, the nozzle 93a repeats blood suction and discharge a plurality of times. For this reason, it is necessary to clean the nozzle 93a with the nozzle cleaner 93b every time it is used. Since this cleaning solution contains blood, care must be taken to dispose of it properly.

  For counting white blood cells, for example, sample blood obtained by diluting blood about 100 times is used, and for measuring red blood cells, sample blood obtained by diluting blood about 10,000 times is used. For example, physiological saline is often used to generate these specimen blood. For this reason, not only leukocytes and erythrocytes but also the concentration of, for example, C-reactive protein is similarly reduced. Analyzing C-reactive protein and leukocytes at the same time provides more precise clinical data. However, if the concentration of the C-reactive protein is low, the analysis accuracy decreases. As a countermeasure, in the blood cell counter X, C-reactive protein measurement is performed by the immunoassay unit 95 using sample blood obtained by mixing a predetermined reagent with whole blood. This requires generation of a dedicated specimen blood separately from the diluted blood used for counting in the blood cell measuring unit 94, and further increases the size of the blood cell counter X.

  On the other hand, the test element Y for analysis only makes the blood sample 100 diluted from the state of whole blood, and cannot be diluted at an accurate magnification. Further, the amount of the diluted blood sample sent to the total hemoglobin measurement unit 108 and the glycated hemoglobin measurement unit 109 is not accurately measured. In order to count blood cells such as red blood cells and white blood cells, unlike the measurement of total hemoglobin value and glycohemoglobin value, if the dilution factor and the amount of diluted blood sample sent to the measurement unit are not precisely specified, an appropriate measurement is required. Is impossible to do. Therefore, it is necessary to prepare an analyzer capable of counting blood cells separately from the analysis test element Y.

Japanese Patent No. 3475056 JP 2004-239904 A

  The present invention has been conceived under the circumstances described above, and it is an object of the present invention to provide an analysis method and an analysis tool that are simple and can improve analysis accuracy.

  In order to solve the above problems, the present invention takes the following technical means.

  The analysis method provided by the first aspect of the present invention is an analysis method for analyzing a sample solution containing a specific component and a solvent component, and separating the at least part of the solvent component from the sample solution A solvent component metering step for metering a certain amount from the solvent component separated by the separation step, a sample solution metering step for metering a certain amount from the sample solution, and a certain amount metered by the sample solution metering step A dilution step for generating a diluted sample solution using the sample solution and a predetermined amount of the solvent component measured in the solvent component measurement step, and an analysis step for analyzing the specific component contained in the diluted sample solution; It is characterized by having.

  According to such a configuration, it is not necessary to prepare a dedicated diluent for generating the diluted sample solution. For this reason, it is advantageous for size reduction of the analyzer used for the said analysis method. The concentration of the solvent component in the diluted sample solution is almost the same as the concentration of the solvent component in the sample solution. For this reason, it is possible to analyze the solvent component with high accuracy using the diluted sample solution. Furthermore, the dilution ratio in the dilution step can be made accurate.

  In a preferred embodiment of the present invention, the dilution step includes a first dilution step of generating a first diluted sample solution using a part of the sample solution and the solvent component separated in the separation step; A second dilution step of generating a second diluted sample solution using the first diluted sample solution and the solvent component separated in the separation step. According to such a configuration, so-called two-stage dilution can be performed. If two-stage dilution is performed, the amount of the sample solution used can be made relatively small even in the case of performing high-magnification dilution. Therefore, it is suitable for downsizing of the analyzer used for the analysis method.

  In a preferred embodiment of the present invention, the sample solution is blood, and the solvent component is plasma. According to such a configuration, there is no possibility that hemolysis occurs in the dilution step.

  In a preferred embodiment of the present invention, a plasma separation filter is used in the separation step. According to such a configuration, the separation efficiency of the separation step can be increased.

  In a preferred embodiment of the present invention, the specific component is blood cells such as red blood cells, white blood cells, and platelets.

  In a preferred embodiment of the present invention, the method further comprises the step of analyzing hemoglobin or C-reactive protein.

  The analysis tool provided by the second aspect of the present invention includes a sample liquid introduction part, a first flow path having a sample liquid measurement means, and a solvent component from a part of the sample liquid introduced into the sample liquid introduction part. A second flow path having a separation means for separating the solvent component and a solvent component measurement means for weighing the solvent component separated by the separation means, connected to the first flow path and the second flow path, and Included in the dilution means for diluting the sample liquid by mixing the sample liquid measured in the flow path and the solvent component measured in the second flow path, and the diluted sample liquid diluted by the dilution means And an analysis unit for analyzing the specific component.

  According to such a configuration, the analysis method provided by the first aspect of the present invention can be appropriately implemented, and the effects of the analysis method can be sufficiently exhibited.

  In a preferred embodiment of the present invention, the dilution means includes a first dilution means for generating a first diluted sample liquid using a part of the sample liquid and the solvent component separated by the separation means, Second dilution means for generating a second diluted sample liquid using the first diluted sample liquid and the solvent component separated by the separation means. Such a configuration is suitable for so-called two-stage dilution.

  Other features and advantages of the present invention will become more apparent from the detailed description given below with reference to the accompanying drawings.

  Hereinafter, preferred embodiments of the present invention will be specifically described with reference to the drawings.

  FIG. 1 shows a flow of an example of an analysis method according to the first aspect of the present invention. The analysis method shown in the figure is an analysis method for analyzing red blood cells, white blood cells, hemoglobin (hereinafter referred to as Hb), and C-reactive protein (hereinafter referred to as CRP) contained in blood as a sample solution. This analysis method includes a blood introduction step, a plasma separation step, a blood measurement step, a plasma measurement step, a first dilution step, a first diluted sample liquid measurement step, a second dilution step, a red blood cell counting step, a white blood cell counting step, and a Hb photometric step. And CRP photometric process. Prior to the description of this analysis method, the configuration of the analysis apparatus used in this analysis method and the cartridge loaded therein will be described below.

  2 and 3 are examples of the analysis tool according to the second aspect of the present invention, and show an example of the analyzer cartridge loaded in the analyzer used in the analysis method shown in FIG. The cartridge A shown in the figure has a main body 1A and a printed wiring board 1B attached to each other, and includes a liquid introduction tank 2, a separation means 3, a dilution means 4, a plurality of analysis sections 5A, 5B, 5C, 5D, And two flow rate measuring units 6A and 6B.

  The main body 1A has a flat rectangular shape and is made of a transparent resin such as acrylic. On the lower surface of the main body 1A in FIG. 3, a plurality of recesses or grooves for forming channels and tanks to be described later are formed. The main body 1A has a size of about 70 mm square and a thickness of about 3 mm.

  In the printed wiring board 1B, a plurality of base materials made of epoxy resin or the like are laminated, and a wiring pattern made of copper foil or the like is formed between these base materials. A plurality of electrodes 51 and 62 described later are formed on the printed wiring board 1B. These electrodes 51 and 62 have a so-called through-hole structure. A connector 8 is formed on the extended portion of the printed wiring board 1B. The connector 8 is used to connect the cartridge A to an analyzer such as a blood cell counter (not shown). The main body 1A and the printed wiring board 1B are liquid-tightly bonded using, for example, an adhesive. Further, in each of the main body 1A and the printed wiring board 1B, at least the surface forming a flow path, which will be described later, is a hydrophobic surface with a water contact angle of 60 degrees or more.

  The liquid introduction tank 2 corresponds to an example of the sample liquid introduction section referred to in the present invention, and is a tank into which blood to be analyzed is introduced into the cartridge A. As shown in FIG. 3, the introduction port formed in the main body 1A. It leads to 2a. The liquid introduction tank 2 has a diameter of about 12 mm and a depth of about 2 mm, and its volume is about 200 μL.

  The separation means 3 is for separating plasma, which is a solvent component, from the blood introduced into the liquid introduction tank 2, and comprises a separation tank 31 and a plasma separation filter 32 as shown in FIG. The separation tank 31 has a rectangular parallelepiped shape with a plan view dimension of about 11 mm × 10 mm and a depth of about 2 mm. The plasma separation filter 32 is a filter that is built in the separation tank 31 and transmits the plasma contained in the blood. The plasma separation filter 32 has a size in plan view of about 10 mm square and a height of about 2 mm. The plasma separation filter 32 is a porous body made of a resin such as glass or polyethylene, and has a porosity of about 80 to 90%. Thereby, the plasma separation filter 32 allows plasma to pass through but does not pass blood cells such as red blood cells, white blood cells, and platelets. When pressure is applied before and after the plasma separation filter 32, plasma can be separated efficiently. As the plasma separation filter 32, a model number VF1, VF2, GMF150 manufactured by whatman, a model number AP25 manufactured by Millipore, a model number GA-200 manufactured by ADVANTEC, or the like may be used. Further, the separation filter 32 may be composed of a single filter having a uniform porosity, a filter having a different porosity depending on a part, or a plurality of filters having different porosity. The separation layer 31 is not limited to a rectangular parallelepiped shape, and may be a tapered shape whose cross-sectional shape changes from the entry side toward the exit side.

  The dilution means 4 is for diluting the blood introduced from the liquid introduction tank 2 to a concentration suitable for various analyses. The first and second dilution tanks 42A and 42B, the blood measurement means 43, and the plasma measurement means 44 are used. , And a first diluted sample solution measuring means 45. The diluting means 4 of the present embodiment is of a type capable of two-stage dilution using the first and second dilution tanks 42A and 42B as will be described later.

  The blood measurement means 43 is disposed between the liquid introduction tank 2 and the first dilution tank 42A, and includes an introduction flow path 43a, a measurement flow path 43c, and an overflow flow path 43d. The blood measurement means 43 corresponds to an example of the sample liquid measurement means in the present invention. The introduction flow path 43a is a flow path for introducing blood from the liquid introduction tank 2, has a width of about 250 μm, a depth of about 250 μm, and a width / depth of 1. A metering flow path 43c and an overflow flow path 43d extend from the introduction flow path 43a via a branching portion 43b. The measuring channel 43c is for temporarily retaining a predetermined amount of blood suitable for analysis. The measuring channel 43c has a length of about 8 mm and a volume of about 0.5 μL. An orifice 43e is provided between the measurement channel 43c and the first dilution tank 42A. The orifice 43e has a width of about 50 μm, and is intended to intentionally increase the pressure loss resistance from the measuring flow path 43c to the first dilution tank 42A. The overflow channel 43d is a meandering channel and is connected to the drain D1. An electrode 43f is provided in the overflow channel 43d.

  The plasma metering means 44 corresponds to an example of the solvent component metering means in the present invention, and is disposed on the downstream side of the separating means 3, and the first and second dilution tanks 42A and 42B are connected via valves V3 and V4. It is connected to each. The plasma metering means 44 includes an introduction channel 44a, a metering channel 44c, and an overflow channel 44d. The introduction channel 44 a is a channel for introducing plasma from the separation tank 31. A metering flow path 44c and an overflow flow path 44d extend from the introduction flow path 44a via a branching portion 44b. The measuring channel 44c is for temporarily retaining an accurate amount of plasma in order to dilute blood to a predetermined concentration. As shown in FIGS. 5 and 6, the metering flow path 44 c has a large cross section 44 ca and two tapered portions 44 cb. The large cross section 44ca has a width of about 2 mm and a depth of about 2 mm, and a volume of about 50 μL. The two taper portions 44cb are respectively connected to the front and rear ends of the large cross-section portion 44ca, and prevent the flow from being unduly disturbed when plasma flows into the large cross-section portion 44ca and out of the large cross-section portion 44ca. Is for. As shown in FIG. 2, the overflow channel 44d is connected to the drain D2.

  The first and second dilution tanks 42A and 42B are tanks in which blood is diluted. Each of the first and second dilution tanks 42A and 42B has a diameter of about 6 mm and a depth of about 2 mm, and has a volume of 50 μL or more. The first and second dilution tanks 42A and 42B correspond to examples of the first dilution means and the second dilution means, respectively, in the present invention. 42 A of 1st dilution tanks are connected with the blood measurement means 43 and the plasma measurement means 44, The blood measured by the blood measurement means 43 is diluted with the plasma measured by the plasma measurement means 44, and a 1st dilution sample is obtained. It is a tank in which a first sample blood as a liquid is generated. The second dilution tank 42B is connected to the first dilution tank 42A and the plasma measuring means 44, and the first specimen blood as the first diluted sample liquid diluted in the first dilution tank 42A is measured by the plasma measuring means 44. It is a tank in which the second specimen blood as the second diluted sample solution is generated by being diluted with the plasma. Between the first dilution tank 42A and the second dilution tank 42B, a first diluted sample liquid measuring means 45 including a measuring channel 45a is provided. In the present embodiment, since the dilution ratios in the first and second dilution tanks 42A and 42B are the same, the metering channel 45a is the same size as the metering channel 43c described above.

  In the cartridge A, the first flow path referred to in the present invention is configured by a portion including the introduction flow path 43a, the measurement flow path 43c, and the orifice 43e connected from the liquid introduction tank 2. Further, the second flow path referred to in the present invention is constituted by the part including the separation means 3, the introduction flow path 44a, and the measurement flow path 44c connected from the liquid introduction tank 2.

  The plurality of analysis units 5A, 5B, 5C, and 5D are parts where analysis of specific components in blood is performed. The first and second analysis units 5A and 5B are analysis units using an electrical resistance detection method, and the first analysis unit 5A is for white blood cells and the second analysis unit 5B is for red blood cells. On the other hand, the third and fourth analysis units 5C and 5D are analysis units using an optical technique, and the third analysis unit 5C is for Hb and the fourth analysis unit 5D is for CRP.

  The first analyzer 5A is connected to the first dilution tank 42A via the buffer tank 50, and is a part for counting white blood cells using the first sample blood generated in the first dilution tank 42A. As shown in FIGS. 7 and 8, the first analysis unit 5A has a pore 53 and a pair of electrodes 51 sandwiching the pore 53, so that counting using an electrical resistance detection method is possible. It is configured. The pore 53 has a narrow width of about 50 μm while the width of the flow path before and after the pore is about 250 μm. This width is determined so that the change in electrical resistance between the pair of electrodes 51 becomes significantly large when white blood cells pass. A pair of electrodes 51 are provided in the flow path portion expanded in a substantially circular shape around the pore 53. The pair of electrodes 51 is made of one or a plurality of types selected from, for example, gold, platinum, palladium, and carbon, and is formed by a printing technique. As shown in FIG. 8, each electrode 51 is electrically connected to the wiring pattern 22 through the through hole 52. The through hole 52 and the wiring pattern 22 are made of, for example, copper.

  The second analysis unit 5B is connected to the second dilution tank 42B, and is a part for counting red blood cells using the second sample blood generated in the second dilution tank 42B. The second analysis unit 5B has substantially the same structure as the first analysis unit 5A described with reference to FIGS.

  The third and fourth analysis units 5C and 5D are connected to the buffer tank 50 independently. As shown in FIG. 9 and FIG. 10, the third and fourth analysis units 5C and 5D have a reflection film 55 provided in the flow path portion enlarged in a substantially circular shape, and each of them is Hb by an optical method. And a site for measuring CRP. The reflection film 55 is made of one or more kinds selected from, for example, gold, platinum, and palladium, and is formed together with the electrode 51 by a printing method. As shown in FIG. 10, a reagent 56 is applied to the upper surface of the enlarged flow path portion in the figure. The reagent 56 is mixed with the first specimen blood and enables Hb or CRP to be measured by an optical method. In the present embodiment, the third and fourth analyzers 5C and 5D are irradiated with light through the transparent main body 1A, and the reflected light is detected to measure Hb and CRP. .

  The first and second flow measurement units 6A and 6B are connected to the first and second analysis units 5A and 5B, respectively. The first and second flow rate measuring units 6A and 6B are parts for measuring the flow rates of the first and second specimen blood that have passed through the first and second analyzing units 5A and 5B, respectively. And a plurality of electrodes 62. The meandering channel 62 has a sufficient volume while increasing the length in the flow direction. In the present embodiment, the meandering channel 62 is a storage means capable of storing at least 50 μL or more of the analyzed first and second specimen blood that has passed through the first analysis unit 5A or the second analysis unit 5B. The plurality of electrodes 62 are arranged at a constant pitch in the flow direction of the meandering channel 61. Each electrode 62 has the same structure as the electrode 51 described above.

  Next, an analysis method for blood analysis using the cartridge A will be described below.

  First, the blood introduction process shown in FIG. 1 is performed. The blood introduction step is performed by introducing blood as a sample liquid into the liquid introduction tank 2 from the introduction port 2a shown in FIG. The cartridge A into which blood has been introduced is loaded into an analyzer (not shown). In this loading, the connector 8 is connected to a connector (not shown) of the analyzer. At this time, the liquid introduction tank 2 and the drains D1 to D8 are connected to a plurality of air discharge nozzles or air suction nozzles connected to a pump provided in the analyzer. The analyzer is configured such that the connection state between the pump and the air discharge nozzle and air suction nozzle can be switched as appropriate.

  Next, the blood measurement process shown in FIG. 1 is performed. In this blood measurement step, blood measurement means 43 shown in FIG. 2 is used. This procedure will be described with reference to FIGS. First, as shown in FIG. 11, the valve V1 connected to the liquid introduction tank 2 is opened, and the valve V2 is closed. Further, the drain D3 is closed. Then, air is discharged from the liquid inlet 2a. Thereby, the blood S flows out from the liquid introduction tank 2 through the introduction flow path 43a to the measurement flow path 43c and the overflow flow path 43d. Since the measurement flow path 43c and the overflow flow path 43d have substantially the same cross-sectional area, the pressure loss resistance when the blood S flows is also substantially the same. Accordingly, the blood S flows through the measurement flow path 43c and the overflow flow path 43d so that the lengths in the flow direction of the blood S are substantially the same.

  If the discharge is continued, as shown in FIG. 12, the measuring flow path 43c is filled with the blood S. Since the orifice 43e is provided on the downstream side of the measuring channel 43c, the pressure loss resistance when the blood S flows is very large. On the other hand, since the overflow channel 43d has a uniform cross section in the flow direction, the pressure loss resistance is significantly smaller than that of the orifice 43e. Therefore, if the discharge is further continued, the blood S continues to flow in the overflow channel 43d while the blood S remains in the measurement channel 43c, and the tip of the blood S reaches the electrode 43f.

  Upon detecting that the tip of the blood S has reached the electrode 43f, as shown in FIG. 13, the valve V1 is closed, and the discharge of air from the liquid inlet 2a is stopped. Then, the drain D3 is opened, and air is discharged from the drain D3. Thereby, the blood S in the introduction flow path 43a is sent out to the downstream part of the overflow flow path 43d. The blood S in the measurement channel 43c remains stagnant.

  If the discharge is further continued, the state shown in FIG. 14 is obtained. In this figure, as a result of the continuation of the above discharge, air has entered the upstream portion of the introduction flow path 43a and the overflow flow path 43d instead of the blood S, and the blood Sa staying in the measurement flow path 43c is retained. Separated from blood S. When the rear end portion of the blood S in the overflow channel 43d has passed through the electrode 43f, the air discharge from the drain D3 is temporarily stopped.

  Then, as shown in FIG. 15, when the drain D1 is closed and air is discharged again from the drain D3, the blood Sa staying in the measuring channel 43c passes through the orifice 43e to the first dilution tank 42A. Spilled. With the above procedure, measurement of a predetermined amount of blood Sa is completed, and about 0.5 μL of blood Sa, which is a predetermined amount, stays in the first dilution tank 42A.

  Next, the plasma separation step and the plasma measurement step shown in FIG. 1 will be described with reference to FIGS. First, as shown in FIG. 16, the valve V1 is closed and the valve V2 is opened. Further, the drain D2 is closed and the drain D4 is opened. When air is discharged from the liquid introduction port 2a in this state, the blood S in the liquid introduction tank 2 flows to the separation tank 31. A differential pressure is applied to the plasma separation filter 32 by the pressure of the air discharge. Thereby, the plasma Bp contained in the blood S passes through the plasma separation filter 32 and is separated from the blood S.

  When the discharge is further continued, as shown in FIG. 17, the measurement flow path 44c is filled with the plasma Bp, and the plasma Bp goes to the drain D4. The fact that the tip portion of the plasma Bp has reached before the drain D4 is detected by, for example, an electric resistance means using an electrode (not shown) or an optical means using a reflective film (not shown). The discharge is stopped with this detection.

  Then, as shown in FIG. 18, the valve V2 is closed and the valve V3 is opened. Further, the drain D2 is opened and the drain D4 is closed. In this state, for example, air is discharged from the drain D2, or is sucked from any drain located downstream from the first dilution tank 42A. Thereby, the plasma Bp staying in the measurement flow path 44c is sent out to the first dilution tank 42A. The plasma measurement process is completed by the above procedure, and about 50 μL of plasma Bpa which is a predetermined amount stays in the first dilution tank 42A.

  Thereafter, the first dilution step shown in FIG. 1 is performed. In this step, about 0.5 μL of blood Sa and about 50 μL of plasma Bpa are mixed in the first dilution tank 42A to generate a first specimen blood as a first diluted sample liquid diluted 100 times. To do. This mixing may be performed, for example, by causing an iron ball built in the first dilution tank 42A to circularly move in the first dilution tank 42A using a magnetic force.

  After the first dilution step is completed, the white ball counting step, the Hb photometric step, and the CRP photometric step shown in FIG. 1 are performed. As shown in FIG. 2, a buffer tank 50 is connected to the first dilution tank 42A. The first specimen blood diluted 100 times is sent to the buffer tank 50. In order to count white blood cells, it is necessary to perform a hemolysis treatment that destroys red blood cells in the first specimen blood. In the present embodiment, a hemolytic agent is applied to the buffer tank 50, for example.

  The procedure of the white blood cell counting step will be described with reference to FIGS. For this counting, the first analysis unit 5A and the first flow rate measurement unit 6A provided on the downstream side thereof are used. FIG. 19 shows a state in which white blood cell counting is started, and the first sample blood DS diluted 100 times remains in the buffer tank 50. In this state, for example, air suction is started from the drain D5. Then, as shown in FIG. 20, the first sample blood DS flows out of the buffer tank 50 and flows through the first analyzer 5A. When the suction from the drain D5 is continued, the distal end portion of the first sample blood DS reaches the electrode 62a located on the most upstream side among the plurality of electrodes 62. For example, by monitoring the conduction between the electrode 62a and the electrode 51, it is possible to detect that the tip portion of the first sample blood DS has reached the electrode 62a. Using this detection as a guide, counting of white blood cells by the first analyzer 5A is started. As described above, since the pore 53 is narrow, when white blood cells pass through, the electrical resistance between the pair of electrodes 51 increases momentarily. As a result, when the electrical resistance between the pair of electrodes 51 is monitored in time series, a pulse signal is generated corresponding to the passage of white blood cells. The number of pulse signals is integrated.

  If the aspiration is continued while integrating the pulse signals, the distal end portion of the first sample blood DS reaches the second electrode 62b counted from the upstream side among the plurality of electrodes 62 as shown in FIG. To do. This arrival can be detected, for example, by monitoring conduction between the electrodes 62a and 62b. The flow rate of the first sample blood DS that has passed through the first analyzer 5A during the period from when the tip of the first sample blood DS reaches the electrode 62a to when it reaches the electrode 62b can stay between the electrodes 62a and 62b. The amount of the sample blood DS is the same. Since the flow direction distance between the electrodes 62a and 62b is known, the flow rate of the first sample blood DS that has passed through the first analysis unit 5A can be known. The number of white blood cells per unit volume of the first blood sample DS is obtained from this flow rate and the number of pulses integrated. Thereby, the number of white blood cells per unit volume of blood S can be counted.

  Thereafter, it is possible to further improve the accuracy of counting by continuing the above suction and repeating counting. Then, as shown in FIG. 22, for example, by detecting that the distal end portion of the first specimen blood DS has reached the electrode 62n located on the most downstream side among the plurality of electrodes 62, the first analyzer 5A What is necessary is just to complete the counting process. Further, as is clear from this figure, when the counting by the first analyzer 5A is completed, the analyzed sample blood DS is in a state of staying in the meandering flow path 61.

  On the other hand, the Hb photometric process and the CRP photometric process shown in FIG. 1 are performed using the third and fourth analyzers 5C and 5D shown in FIG. For example, after the above-described white blood cell counting step is completed, suction is performed from the drains D6 and D7, and the first sample blood DS is made to reach the reflective films 55 of the third and fourth analyzers 5C and 5D, respectively. At this time, the first sample blood DS reacts with the reagent 56 shown in FIG. 10 and becomes capable of analyzing each of Hb and CRP. In this state, light is emitted from the analyzer through the main body 1A to the respective reflection films 55, and the reflected light is received through the main body 1A by a light receiving element provided in the analyzer. By appropriately processing this light, Hb and CRP can be analyzed. Unlike the present embodiment, the printed wiring board 1B may be replaced with a substrate made of a transparent material. In this case, the reflective film 55 is unnecessary. The third and fourth analyzers 5C and 5D have a structure sandwiched between the transparent main body 1A and the substrate. Therefore, it is possible to analyze Hb and CRP by so-called transmission measurement.

  Next, the first diluted sample solution measurement step and the second dilution step shown in FIG. 1 will be described. In the first diluted sample liquid measuring step, about 0.5 μL from the first sample blood DS staying in the first dilution tank 42A shown in FIG. 2 is measured using the measuring channel 45a. The measurement of the first specimen blood DS using the measurement channel 45a is substantially the same as the measurement procedure described with reference to FIGS. Further, in the plasma measuring step described with reference to FIGS. 16 to 18, the valve V3 shown in FIG. 18 is closed, while the valve V4 is opened. Thereby, about 50 μL of plasma Bpa is delivered to the second dilution tank 42B. Next, a second dilution step is performed. In this step, about 0.5 μL of the first sample blood DS and about 50 μL of plasma Dpa are mixed in the second dilution tank 42B. Thereby, the 2nd sample blood as a 2nd dilution sample liquid equivalent to what diluted blood S 10,000 times is obtained.

  The red blood cell counting step shown in FIG. 1 is performed using the 10,000-fold diluted second sample blood obtained by the above procedure. In this step, the second analysis unit 5B is used. This step is almost the same as the leukocyte counting step by the first analyzer 5A. The point of measuring the flow rate using the second flow rate measurement unit 6B is the same as the flow rate measurement using the first flow rate measurement unit 6A.

  Next, the operation of the cartridge A and the analysis method using the same will be described.

  According to this embodiment, a diluent such as physiological saline is not used to generate the first sample blood DS and the second sample blood. For this reason, it is not necessary to incorporate a diluent in the cartridge A or the analysis device. Therefore, it is advantageous for miniaturization of the cartridge A and the analyzer. Thus, if the cartridge A and the analyzer loaded with the cartridge A are carried, it is possible to easily perform blood cell counting and the like on the go, etc.

  Further, since the first specimen blood DS and the second specimen blood that have been analyzed are stored in the cartridge A, the analyzer is not wet at all, and is completely dry during and before use. is there. This is suitable for keeping the analyzer clean. Further, since the analyzer is not required to be cleaned, troublesome operations such as disposal of the cleaning solution can be avoided.

  According to the present embodiment, two-stage dilution can be performed using the first and second dilution tanks 42A and 42B. Therefore, two types of dilution with relatively high magnification, for example, 100-fold dilution and 10,000-fold dilution, are possible. As a result, it is possible to collectively perform the analysis of significantly different dilution ratios suitable for each of the white blood cell count and the red blood cell count. Further, by providing the blood measuring means 43 and the plasma measuring means 44, it is possible to dilute at a sufficiently accurate dilution factor. Weighing using the large cross section 44ca is particularly effective for high magnification dilution.

  Furthermore, since the first sample blood DS and the second sample blood are diluted with plasma, components other than blood cells can have the same concentration as the blood S. For example, regarding CRP, the blood S and the first sample blood DS have almost the same concentration. Therefore, it is possible to analyze CRP much more accurately than in the case where blood is diluted with physiological saline. Further, there is no risk of hemolysis due to dilution, and blood cell counting can be performed reliably.

  The analysis method and analysis tool according to the present invention are not limited to the above-described embodiments. The specific configuration of the analysis method and the analysis tool according to the present invention can be changed in various ways.

  As a means for separating plasma, a configuration using a plasma separation filter is preferable for downsizing the cartridge A and improving separation efficiency, but is not limited thereto, and components such as plasma excluding blood cells are appropriately separated from blood. Any means may be used.

  The dilution factor in the diluting means can be further increased by appropriately setting the size of the flow path and the like. Moreover, it is not limited to two-stage dilution, For example, it is good also as a structure which performs the dilution of only 1 time, or the dilution 3 times or more.

  The analysis method and the analysis tool according to the present invention are not limited to blood cell count and the like, and can be used for analysis of various sample solutions.

It is a flowchart which shows an example of the analysis method which concerns on the 1st side surface of this invention. It is a whole top view which shows an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. It is a whole perspective view which shows an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. It is a principal part perspective view which shows the isolation | separation means of an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. It is a principal part top view which shows the measurement flow path of an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. FIG. 4 is a main part sectional view taken along line VI-VI in FIG. 3. It is a principal part top view which shows the electrical resistance type | mold analysis part of an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. It is principal part sectional drawing in alignment with the VIII-VIII line | wire of FIG. It is a principal part top view which shows the optical analysis part of an example of the cartridge for analyzers which concerns on the 2nd side surface of this invention. It is principal part sectional drawing which follows the XX line of FIG. It is a principal part top view which shows the blood introduction | transduction state in the blood measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the state with which the measurement flow path was filled in the blood measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the continuous inflow to an overflow flow path in the blood measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the state by which the blood was isolate | separated in the measurement flow path in the blood measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the state by which the blood was sent to the 1st dilution tank in the blood measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the plasma separation process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the state with which the measurement flow path was filled in the plasma measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. It is a principal part top view which shows the state by which the plasma was delivered to the 1st dilution tank in the plasma measurement process of an example of the analysis method which concerns on the 1st side surface of this invention. In the leukocyte counting process of an example of the analysis method concerning the 1st side of the present invention, it is a principal part top view showing the initial state. In the leukocyte count process of an example of the analysis method concerning the 1st side of the present invention, it is a principal part top view showing the count start state. In the leukocyte counting step of an example of the analysis method according to the first aspect of the present invention, FIG. FIG. 6 is a plan view of a principal part showing a state in which the tip portion of the first sample blood reaches the most downstream electrode in the white blood cell counting step of an example of the analysis method according to the first aspect of the present invention. It is a conceptual diagram which shows an example of the blood cell counter used for the conventional analysis method. It is a conceptual diagram which shows an example of the test | inspection element for analysis used for the conventional analysis method.

Explanation of symbols

A (Analyzer) cartridge Bp Plasma (solvent component)
Bpa Weighed plasma (solvent component)
D1 to D8 Drain DS First specimen blood (first diluted sample solution)
S blood (sample liquid)
Sa blood (weighed sample solution)
V1, V2, V3, V4 Valve 1A Body 1B Printed circuit board 2 Liquid introduction tank (sample liquid introduction part)
3 Separation means 4 Dilution means 5A First analysis section (electric resistance analysis section)
5B 2nd analysis part (electric resistance type analysis part)
5C 3rd analysis unit (optical analysis unit)
5D Fourth analysis unit (optical analysis unit)
6A 1st flow measurement part 6B 2nd flow measurement part 8 Connector 22 Wiring 31 Separation tank 32 Plasma separation filter 42A 1st dilution tank 42B 2nd dilution tank 43 Blood measurement means (sample liquid measurement means)
43a Introducing channel 43b Branching portion 43c Metering channel 43d Overflow channel 43e Orifice 44 Diluent metering means (solvent component diluting means)
44a Introducing channel 44b Branching portion 44c Metering channel 44ca Large cross section 44cb Taper portion 44d Overflow channel 45 First diluted sample liquid metering means 45a Metering channel 50 Buffer tank 51 Electrode 52 Through hole 53 Pore 55 Reflective membrane 56 Reagent 61 Meandering channel (storage means)
62 Electrode (Diluted sample solution detection means)

Claims (8)

An analysis method for analyzing a sample solution containing a specific component and a solvent component,
A separation step of separating at least a part of the solvent component from the sample solution;
A solvent component metering step for metering a certain amount from the solvent component separated by the separation step;
A sample solution measuring step for measuring a certain amount from the sample solution,
A dilution step for producing a diluted sample solution using a certain amount of the sample solution weighed by the sample solution weighing step and a certain amount of the solvent component weighed by the solvent component weighing step;
And an analysis step of analyzing the specific component contained in the diluted sample solution. The dilution step is
A first dilution step for producing a first diluted sample solution using a part of the sample solution and the solvent component separated in the separation step;
The analysis method according to claim 1, further comprising a second dilution step of generating a second diluted sample solution using the first diluted sample solution and the solvent component separated in the separation step.   The analysis method according to claim 1, wherein the sample solution is blood, and the solvent component is plasma.   The analysis method according to claim 3, wherein a plasma separation filter is used in the separation step.   The analysis method according to claim 3 or 4, wherein the specific component is blood cells such as red blood cells, white blood cells, and platelets.   The analysis method according to claim 3 or 4, further comprising a step of analyzing hemoglobin or C-reactive protein. A sample solution introduction part;
A first flow path having sample liquid measuring means;
A second flow path having a separation means for separating the solvent component from a part of the sample liquid introduced into the sample liquid introduction section, and a solvent component measurement means for measuring the solvent component separated by the separation means;
The sample liquid is connected to the first flow path and the second flow path, and the sample liquid is mixed with the sample liquid measured in the first flow path and the solvent component measured in the second flow path. Dilution means for dilution;
An analysis unit for analyzing a specific component contained in the diluted sample solution diluted by the dilution means;
An analytical tool comprising: The dilution means includes a first dilution means for generating a first diluted sample liquid using a part of the sample liquid and the solvent component separated by the separation means;
The analytical method instrument according to claim 7, further comprising: a second dilution unit that generates a second diluted sample solution using the first diluted sample solution and the solvent component separated by the separation unit.

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