Measurement method, measurement system and test reagent kit

Abstract

A measurement method for determining the concentration of reticulocytes or nucleated red blood cells contained in a blood specimen, the measurement method including a staining step, a removal step and a measurement step. In the staining step, reticulocytes or nucleated red blood cells, among red blood cells contained in the blood specimen, are stained. In the removal step, a surfactant is added to the blood specimen to take out hemoglobin from the red blood cells. In the measurement step, the concentration of the reticulocytes or nucleated red blood cells contained in the blood specimen is determined using the blood specimen after the staining and removal steps.

Description

Measurement method, measurement system, and test reagent kit

[0001] The present invention relates to a measurement method and measurement system for measuring the concentration of reticulocytes or nucleated red blood cells contained in a blood sample, and a test reagent kit used for measuring the concentration of reticulocytes or nucleated red blood cells.

[0002] Reticulocytes are immature red blood cells that have just entered the peripheral blood from the bone marrow and mature within a day. Erythroblasts in the bone marrow contain nuclei, but as they mature, they lose their nuclei. In immature reticulocytes, remnants of nuclear components, such as RNA (ribonucleic acid), are observed as reticular granular material. An increase in reticulocytes is an indicator of increased red blood cell production in the bone marrow and is observed, for example, during anemia treatment with iron. In contrast, in aplastic anemia, reticulocytes do not increase due to decreased hematopoietic capacity. Therefore, measuring the number (concentration) of reticulocytes is useful for diagnosing various types of anemia and monitoring the progress of treatment.

[0003] In measuring the concentration of reticulocytes, for example, in Patent Document 1, reticulocytes are stained with a staining reagent (K3EDTA, NMB (new methylene blue), NaCl), and then hemoglobin is eluted from the red blood cells using a red blood cell hemolysis reagent (KSCN, 1N H2SO4). The reduction in hemoglobin count improves the identification of reticulocytes and enables their counting by flow cytometry.

[0004] Special Publication No. 8-510330

[0005] In Patent Document 1, the osmotic pressure of the red blood cell hemolyzing reagent is set to 75 to 110 mOsm, preferably 82 to 105 mOsm. This is based on the following reason: if the osmotic pressure of the red blood cell hemolyzing reagent is low, red blood cells are damaged, resulting in a decrease in the reliability of the reticulocyte count. On the other hand, if the osmotic pressure is insufficient, red blood cells do not elute hemoglobin, which makes it difficult to distinguish reticulocytes from each other.

[0006] In addition, in Patent Document 1, the pH of the red blood cell hemolyzing reagent is adjusted to a range of about 1.0 to 3.0, preferably 1.0 to 2.0, because an acidic red blood cell hemolyzing reagent is thought to dissolve hemoglobin and facilitate the removal of hemoglobin from red blood cells.

[0007] In the method using a red blood cell hemolysis reagent to elute hemoglobin from red blood cells as in Patent Document 1, the osmotic pressure and pH of the red blood cell hemolysis reagent must be appropriately adjusted as described above in order to appropriately elute hemoglobin, resulting in complex pretreatment before measuring the reticulocyte concentration.

[0008] The problem of complicated pretreatment before concentration measurement can also occur when measuring the concentration of nucleated red blood cells. Because nucleated red blood cells contain nucleic acids such as RNA, the concentration of nucleated red blood cells can be measured using the same method as that for measuring the concentration of reticulocytes. Because nucleated red blood cells, like reticulocytes, are immature red blood cells, the detection of nucleated red blood cells in the peripheral blood of an adult is considered to indicate some kind of hyperhematopoiesis.

[0009] The present invention has been made to solve the above problems, and its object is to provide a measurement method, a measurement system, and a test reagent kit that can measure the concentration of reticulocytes or nucleated red blood cells with simple pretreatment.

[0010] A measurement method according to one aspect of the present invention is a method for measuring the concentration of reticulocytes or the concentration of nucleated red blood cells contained in a blood sample, and includes the steps of: a staining step of staining the reticulocytes or the nucleated red blood cells among the red blood cells contained in the blood sample using a staining reagent; a removal step of extracting hemoglobin from the red blood cells by adding a surfactant to the blood sample; and a measurement step of measuring the concentration of reticulocytes or the concentration of nucleated red blood cells contained in the blood sample using the blood sample that has undergone the staining step and the removal step.

[0011] A test reagent kit according to another aspect of the present invention includes a staining reagent for staining reticulocytes or nucleated red blood cells among red blood cells contained in a blood sample, and a surfactant for extracting hemoglobin from the red blood cells.

[0012] A measurement system according to yet another aspect of the present invention is a measurement system for measuring the concentration of reticulocytes or the concentration of nucleated red blood cells contained in a blood sample, and includes: a container for containing a staining reagent for staining the reticulocytes or nucleated red blood cells and a surfactant that is added to the blood sample to extract hemoglobin from the red blood cells contained in the blood sample; a flow cell into which the blood sample, the staining reagent, and the surfactant are introduced; a blood cell volume size measurement unit for measuring changes in the volume of blood cells in the blood sample introduced into the flow cell; an optical measurement unit for measuring the light transmittance of the blood cells in the blood sample introduced into the flow cell; and a concentration calculation unit for calculating the concentration of reticulocytes or the concentration of nucleated red blood cells contained in the blood sample based on measurement results from the blood cell volume size measurement unit and the optical measurement unit.

[0013] According to the present invention, the concentration of reticulocytes or nucleated red blood cells can be measured with simple pretreatment.

[0014] 1 is a perspective view showing the external configuration of a blood analyzer constituting a measurement system according to an embodiment of the present invention. FIG. 2 is an explanatory diagram schematically showing the internal configuration of a blood analyzer. FIG. 3 is a block diagram schematically showing another configuration of the measurement system. FIG. 4 is a flowchart showing the process flow when measuring the concentration of reticulocytes using a first measurement method. FIG. 5 is an explanatory diagram schematically showing models of red blood cells before and after ghosting using a surfactant. FIG. 6 is an explanatory diagram showing the general configuration of an LMNE chamber. FIG. 7 is a graph plotting results obtained by the electrical resistance measurement unit and the optical measurement unit for the same blood cells when no surfactant is added to the blood sample. FIG. 8 is a graph plotting results obtained by the electrical resistance measurement unit and the optical measurement unit for the same blood cells when a surfactant is added to the blood sample. FIG. 9 is a flowchart showing the process flow when measuring the concentration of reticulocytes using a second measurement method. FIG. 10 is a flowchart showing the process flow when measuring the concentration of reticulocytes using a third measurement method. FIG. 11 is a flowchart showing the process flow when measuring the concentration of reticulocytes using a fourth measurement method. FIG. 12 is a perspective view showing an example of a reticulocyte testing reagent kit. FIG. 13 is a perspective view showing another example of a reticulocyte testing reagent kit. FIG. 1 is an explanatory diagram showing a schematic configuration of an optical device for detecting reticulocytes using a fluorescent dye.

[0015] Hereinafter, exemplary embodiments of the present invention will be described with reference to the drawings.

[0016] 1. Overview of the Blood Analyzer Figure 1 is a perspective view showing the external configuration of a blood analyzer 1 that constitutes a measurement system 80 (see Figure 2) of this embodiment. The blood analyzer 1 has a display unit 3 located at the top front of a device main body 2. The display unit 3 is configured, for example, as a liquid crystal display device with a touch panel, and displays blood analysis results and the like, as well as accepts input of various information by an operator (e.g., a medical professional).

[0017] A specimen container loading section 4 is provided at the bottom of the device main body 2. The specimen container 10 can be loaded into the device main body 2 by opening the cover 4a of the specimen container loading section 4, setting a specimen container 10 containing a blood specimen, and closing the cover 4a.

[0018] A reagent container loading section 5 is provided on the side of the apparatus main body 2. By opening the door 5a of the reagent container loading section 5, containers containing reagents used in blood analysis (e.g., immunoassay reagents, reticulocyte staining reagents), surfactants, diluents, etc. can be loaded. Note that the above containers may, for example, contain the staining reagent, surfactant, and diluent described below all together, or may be prepared for each reagent used. Furthermore, the above reagents may be stored in a storage section 320 connected to the chamber 31 (see FIG. 2) via a conduit 321, as described below.

[0019] 2. Internal Configuration of Blood Analyzer Fig. 2 is an explanatory diagram that schematically shows the internal configuration of blood analyzer 1. Blood analyzer 1 includes needle 20, blood analysis unit 30, and control unit 40. Control unit 40 is configured by a central processing unit (computer), for example, called a CPU (Central Processing Unit), and controls the operation of each unit of blood analyzer 1.

[0020] The needle 20 aspirates and dispenses the blood sample contained in the sample container 10. The needle 20 pierces the lid of the sample container 10 as needed to aspirate and dispense the blood sample. The rear end of the needle 20 is connected to a metered dose syringe 22 via a pipe 21 shown by a dashed line and an electromagnetic valve device (not shown). The control unit 40 drives the metered dose syringe 22 and the electromagnetic valve device, causing the needle 20 to aspirate and dispense the blood sample.

[0021] The needle 20 is moved in the horizontal and vertical directions by the probe unit 23. The probe unit 23 includes a horizontal movement mechanism 24 and a vertical movement mechanism 25. The horizontal movement mechanism 24 and the vertical movement mechanism 25 include, for example, an endless belt, and a drive roller and a driven roller that move the endless belt. When the control unit 40 drives the drive rollers of the horizontal movement mechanism 24 and the vertical movement mechanism 25, the endless belt of the horizontal movement mechanism 24 moves in the horizontal direction, and the endless belt of the vertical movement mechanism 25 moves in the vertical direction. This allows the needle 20 supported by the vertical movement mechanism 25 to move in the horizontal and vertical directions.

[0022] The horizontal movement mechanism 24 and the vertical movement mechanism 25 may be configured as feed screw mechanisms. The feed screw mechanism rotates the feed screw with a stepping motor to move an engagement portion that engages with the feed screw in the horizontal or vertical direction. Therefore, by connecting the vertical movement mechanism 25 to the engagement portion of the horizontal movement mechanism 24 and holding the needle 20 in the engagement portion of the vertical movement mechanism 25, the needle 20 can be moved in the horizontal and vertical directions.

[0023] The blood analysis unit 30 performs blood analysis, such as counting blood cells and measuring immunity. The blood analysis unit 30 has a chamber 31 (container) that receives a blood sample aspirated and discharged from the specimen container 10 by the needle 20. A counting device 310 is provided in the chamber 31 for blood cell counting. The counting device 310 can perform measurement methods such as impedance analysis, flow cytometry, and focusing flow impedance analysis, depending on the blood cells to be counted. The control unit 40 can create a frequency distribution or the like by processing the measurement data acquired by the counting device 310. The counting device 310 may be provided outside the chamber 31 and connected to the chamber 31.

[0024] The chambers 31 are provided according to the type of blood cells to be counted. For example, a BASO chamber, an LMNE chamber, an RBC chamber, and a WBC chamber are separately provided as the chambers 31. The BASO chamber is provided for counting basophils, which are white blood cells. The LMNE chamber is provided for counting lymphocytes, monocytes, neutrophils, and eosinophils, which are white blood cells. The "LMNE" in the LMNE chamber is an abbreviation of the initials of lymphocyte, monocyte, neutrophil, and eosinophil. The RBC chamber is provided for counting red blood cells. The WBC chamber is provided for counting white blood cells and analyzing hemoglobin (HGB).

[0025] A CRP measurement chamber may be further provided as chamber 31. The CRP measurement chamber is configured to enable optical measurement of CRP values ​​according to the latex agglutination method. CRP is an abbreviation for C-reactive protein. The CRP measurement chamber is provided with a light irradiator and a light detector for CRP measurement on the lower wall surface of the chamber, and is configured to allow appropriate stirring of the liquid contained therein.

[0026] A plurality of reagent containers (not shown) are provided near the CRP measurement chamber. Each reagent container contains a hemolysis reagent, a diluent, an anti-human CRP-sensitized latex immunoreagent, etc. When CRP measurement is performed, the reagent is discharged from each reagent container at an appropriate time through a conduit (not shown) into the CRP measurement chamber.

[0027] The blood analysis unit 30 has a washing container 32. The washing container 32 is provided for washing the needle 20. Washing the needle 20 in the washing container 32 makes it possible to remove unwanted substances such as blood or reagents adhering to the needle 20 and to discard excess blood sample in the needle 20.

[0028] A discharge pipe 33 shown by a dashed line is connected to the lower end of the chamber 31 and the washing container 32. The waste liquid in the chamber 31 and the washing container 32 is sent to the waste liquid container 50 through an electromagnetic valve device (not shown) by driving a discharge part (not shown). The discharge part is composed of a discharge syringe or a discharge pump.

[0029] When a specimen container 10 containing a blood specimen is loaded into the specimen container loading section 4 (see FIG. 1 ) of the apparatus main body 2, the control section 40 drives the probe unit 23 to move the needle 20 horizontally and vertically. This allows the needle 20 to advance into and retreat from the specimen container 10 and chamber 31. In addition, the control section 40 drives the metering syringe 22 to aspirate and eject the blood specimen through the needle 20. Through these operations, blood analysis (blood cell counting, immunoassay, etc.) is performed in the blood analysis section 30.

[0030] The blood analysis unit 30 further includes a storage unit 320. The storage unit 320 stores the staining reagent, surfactant, and diluent, which will be described later. In this embodiment, the storage unit 320 stores the staining reagent, surfactant, and diluent together in a mixed state, but multiple storage spaces may be provided to store these separately. The storage unit 320 is connected to the chamber 31 (e.g., the LMNE chamber) via a conduit 321. This allows the staining reagent, surfactant, and diluent stored in the storage unit 320 to be supplied to the chamber 31 (e.g., the LMNE chamber) via the conduit 321.

[0031] In this embodiment, two LMNE chambers are provided. This is to clearly distinguish between the chambers used to count lymphocytes, monocytes, neutrophils, and eosinophils and the chamber used to measure the concentration of reticulocytes, which will be described later. Note that one LMNE chamber may be configured to serve both as a chamber for counting lymphocytes and a chamber for measuring the concentration of reticulocytes.

[0032] The blood analyzer 1 further includes a temperature adjustment unit 41. The temperature adjustment unit 41 is configured, for example, by a heater, and heats the chamber 31 and the washing container 32 to a desired temperature (e.g., 37°C). This prevents blood samples that tend to aggregate in cold temperatures from aggregating. While FIG. 2 illustrates a configuration in which a temperature adjustment unit 41 is provided for each of the chamber 31 and the washing container 32 and heats them separately, the temperature adjustment unit 41 may be provided to surround the chamber 31 and the washing container 32 together and heat them simultaneously.

[0033] The temperature adjustment unit 41 may be configured as a tank (also referred to as a buffer tank) wrapped around a heater. The tank contains, for example, a diluent. The temperature adjustment unit 41 is connected, for example, to another pipe (not shown) branching off from the pipe 21 via a switching valve (not shown). The diluent heated by the heater merges with the pipe 21 via the other pipe and the switching valve and is then ejected into the chamber 31 via the needle 20. At this time, the blood sample previously aspirated by the needle 20 from the sample container 10 is also ejected into the chamber 31 simultaneously with the heated diluent. Therefore, even in this case, the blood sample is heated to a desired temperature by mixing with the heated diluent in the chamber 31, thereby preventing aggregation of the blood sample. It is desirable that the temperature adjustment unit 41 also heats reagents other than the diluent (e.g., surfactants) to the desired temperature (e.g., 37°C) in order to promote appropriate reactions.

[0034] The blood analyzer 1 also includes a concentration calculation unit 42. The concentration calculation unit 42 calculates the concentration of reticulocytes contained in the blood sample based on the counting results (e.g., the number of reticulocytes described below) from the counting device 310 of the blood analysis unit 30 and the amount of the blood sample used for the counting. The concentration calculation unit 42 may be configured with the same CPU as the control unit 40 or a separate CPU, or may be configured with a processing circuit dedicated to concentration calculation.

[0035] Alternatively, concentration calculation unit 42 may be provided outside blood analyzer 1 and communicably connected to blood analyzer 1. Fig. 3 is a block diagram schematically showing another configuration of measurement system 80. Measurement system 80 shown in the figure is composed of blood analyzer 1 and terminal device 70. Terminal device 70 is communicably connected to blood analyzer 1 via a communication line. The communication line may be wired or wireless.

[0036] Terminal device 70 is configured, for example, by a personal computer, and includes an input unit such as a keyboard, a display unit, and a concentration calculation unit 42. Concentration calculation unit 42 is configured, for example, by a CPU provided in terminal device 70, and calculates the concentration of reticulocytes contained in the blood sample based on the measurement result (e.g., the number of reticulocytes) output from counting device 310 of blood analyzer 1 and the amount (volume) of the blood sample input on the terminal device 70 side.

[0037] 3. Reticulocyte Concentration Measurement Method (3-1. First Measurement Method) Next, a method for measuring reticulocyte concentration using measurement system 80 will be described. FIG. 4 is a flowchart showing the process flow for measuring reticulocyte concentration using the first measurement method. The first measurement method includes a staining step (S1), a removal step (S2), a dilution step (S3), a loading step (S4), and a measurement step (S5). Of these, steps S1 to S3 are also referred to as pre-steps because they are performed before the measurement step S5. Note that the first measurement method does not use reagents (such as staining reagents) contained in the storage unit 320, but uses a blood sample to which reagents have been added outside the device. This will be described in detail below.

[0038] (S1: Staining Step) In the staining step, reticulocytes among the red blood cells contained in the blood sample collected in the sample container 10 (see FIG. 1) are stained. NMB (New Methylene Blue: C 18 H 22 A staining reagent called ClNS is used. NMB is a non-fluorescent staining agent and a cationic (+) molecule. Reticulocytes contain RNA (ribonucleic acid)-ribosome complexes. When NMB is added to reticulocytes, the complexes in the reticulocytes aggregate into a net-like structure, engulfing other intracellular organelles. The RNA in the aggregated complexes then stains blue. In other words, the reticulocytes are stained.

[0039] The staining reagent used in the staining step is not limited to the above-mentioned NMB. For example, Azure B (:C 15 H 16ClN3S), chromophore, and fluorophore can also be used as staining reagents. In particular, NMB, Azure B, and chromophore are suitable as staining reagents.

[0040] (S2: Removal Step) In the removal step, hemoglobin is extracted from red blood cells by adding a surfactant to all or part of the blood sample stained in S1. This step of extracting hemoglobin from red blood cells is also called ghosting. From the viewpoint of achieving a good balance between the amount of blood sample and the amount of surfactant, it is desirable to add a surfactant to only part of the blood sample stained in S1.

[0041] Here, it is desirable to use a cationic (positively charged), zwitterionic, or nonionic surfactant as the surfactant. This is for the following reason: nucleic acids such as RNA are negatively charged, and NMB is positively charged. If the surfactant is anionic (negatively charged), there is a risk that the surfactant will bind to NMB before binding to the RNA, thereby inhibiting staining with NMB.

[0042] Examples of cationic surfactants that can be used include quarternary ammonium ion surfactants (quarternary ammonium salts; QAS), such as DTAB (dodecyltrimethylammonium bromide) and HTAC (hexadecyltrimethylammonium chloride).

[0043] A zwitterionic surfactant is a surfactant that has both positive and negative polarity functional groups. For example, an ammoniopropanesulfonate surfactant can be used as the zwitterionic surfactant. For example, an ammoniopropanesulfonate surfactant can be used, such as DDAPS (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate).

[0044] Examples of nonionic surfactants include Tergitol (a registered trademark of Union Carbide Corporation), Triton X-100 ("Triton" is a registered trademark of Union Carbide Corporation), and sugar fatty acid esters. Examples of sugar fatty acid esters include sucrose fatty acid esters (sugar esters) such as DK Ester SS (a registered trademark of Daiichi Kogyo Seiyaku Co., Ltd.).

[0045] Figure 5 shows a schematic diagram of a red blood cell model before and after ghosting using a surfactant. The surfactant creates holes in the red blood cell membrane M, allowing soluble proteins, including hemoglobin (Hgb), to escape from the red blood cell. In this state, when light is irradiated onto the red blood cell, the light absorption by hemoglobin is reduced, so red blood cells other than reticulocytes (ghost RBCs) appear transparent. However, the red blood cell membrane M remains intact. On the other hand, reticulocytes (ghost RETs) appear blue because the RNA in the aggregated complex is stained blue by NMB, as described above. The osmotic pressure and pH of the surfactant are not particularly limited and can be, for example, similar to those of blood.

[0046] (S3: Dilution Step) In the dilution step, the blood sample after hemoglobin extraction from red blood cells is diluted with a diluent, such as phosphate-buffered saline (PBS).

[0047] (S4: Loading Step) In the loading step, the specimen container 10 containing the blood specimen diluted in S3 is set in the specimen container loading unit 4 of the blood analyzer 1 shown in Figure 1. Instead of setting the specimen container 10 in the specimen container loading unit 4, the diluted blood specimen may be aspirated from the blood specimen 10 using a pipette, and the aspirated blood specimen may be directly injected into the chamber 31 (particularly the LMNE chamber) by opening a panel (not shown) of the blood analyzer 1. In this case, the loading step of setting the specimen container 10 in the specimen container loading unit 4 is unnecessary.

[0048] (S5: Measuring Step) In the measuring step, the concentration of reticulocytes contained in the blood sample is measured using a blood sample that has been stained, ghosted, and diluted. In this embodiment, the blood analyzer 1 is provided with an LMNE chamber as the chamber 31 of the blood analysis unit 30. The reticulocyte concentration can be measured using the LMNE chamber. In particular, when the blood analyzer 1 is provided with multiple LMNE chambers, as in this embodiment, the reticulocyte concentration can be measured by using any of the LMNE chambers. The blood sample used in the measuring step may be a blood sample contained in a sample container 10 set in the sample container loading unit 4 in S4, or a blood sample directly injected into the LMNE chamber with a pipette.

[0049] 6 is an explanatory diagram showing a schematic configuration of an LMNE chamber 31A serving as the chamber 31. The LMNE chamber 31A includes a flow cell 311, an electrical resistance measurement unit 312, an optical measurement unit 313, and the above-described counting device 310 (see FIG. 2).

[0050] A blood sample containing a staining reagent (NMB), a surfactant, and a diluent is introduced into the flow cell 311 (S5-1; introduction step). The blood sample is introduced into the flow cell 311 by suction from the sample container 10 using a needle 20 (see FIG. 2) and discharging into the flow cell 311. The blood sample introduced into the flow cell 311 is also referred to as a "sample SA" here. Note that in S5-1, the blood sample may also be directly injected into the flow cell 311 using a pipette.

[0051] The sample SA, surrounded by the sheath fluid S-1 and forming a thin stream, passes through an aperture 312a provided in the electrical resistance measurement unit 312. At this time, changes in electrical resistance are detected as pulses by platinum electrodes (not shown) installed on both ends of the aperture 312a. As a result, the electrical resistance measurement unit 312 measures the size of the cells (blood cells) that have passed through the aperture 312a (S5-2: electrical resistance measurement step).

[0052] Next, the sample SA is surrounded by sheath fluid S-2 and guided to the optical measurement unit 313. The optical measurement unit 313 includes a light source unit 313a (e.g., a halogen lamp), a detection unit 313b (a photocell), and an optical system 313c. The optical system 313c is located between the light source unit 313a and the detection unit 313b. The optical system 313c includes a lens and an optical filter. When light is irradiated from the light source unit 313a toward the sample SA (blood specimen) flowing through the flow cell 311, the light that passes through the blood cells (red blood cells and white blood cells) of the sample SA reaches the detection unit 313b. As a result, the detection unit 313b detects the light transmittance (light transmittance) of the blood cells (S5-3; optical measurement step). The counting device 310 counts the reticulocytes contained in the sample SA (blood specimen) based on the measurement results of the electrical resistance measurement unit 312 and the optical measurement unit 313. The concentration calculation unit 42 then calculates the reticulocyte concentration from the reticulocyte count result and the amount of the blood sample (S5-4: concentration calculation step). It is assumed that the amount (volume) of the blood sample is known in advance.

[0053] For example, Figure 7 plots the results obtained by the electrical resistance measurement unit 312 and the optical measurement unit 313 for the same blood cells when the concentration of Tergitol as a surfactant was 0 g / L, i.e., when Tergitol was not added to the blood sample in S2. Figure 8 plots the results obtained by the electrical resistance measurement unit 312 and the optical measurement unit 313 for the same blood cells when the concentration of Tergitol as a surfactant added in S2 was 0.6 g / L. In these figures, the horizontal axis represents the measurement results (blood cell particle diameter) by the electrical resistance measurement unit 312, with larger particle diameters toward the right. The vertical axis represents the measurement results (light transmittance of blood cells) by the optical measurement unit 313, with greater light absorption (lower light transmittance) toward the top.

[0054] Furthermore, by keeping the flow rate of the sample SA flowing through the flow cell 311 constant and keeping the time lag between the timing of measuring the electrical resistance and the timing of measuring the light transmittance constant, it is possible to obtain measurement results (particle diameter) from electrical resistance measurement and measurement results (amount of light absorption) from light transmittance measurement for the same blood cells.

[0055] In this embodiment, since the LMNE chamber 31A is used to measure the concentration of reticulocytes, the boundaries of the five types of white blood cells that can be detected by the LMNE chamber 31A are shown as they are in Figures 7 and 8, but these boundaries can be ignored here.

[0056] If a surfactant is not added to a blood sample, hemoglobin is not removed from red blood cells. Therefore, when a blood sample containing blood cells of various particle sizes is irradiated with light, the hemoglobin in the red blood cells absorbs a large amount of light. As a result, the optical measurement unit 313 optically detects not only reticulocytes but also mature red blood cells. Therefore, as shown in FIG. 7 , a distribution of detected light absorption amounts for blood cells of various particle sizes, including reticulocytes and mature red blood cells, is obtained. Therefore, it is difficult for the counting device 310 to accurately count only reticulocytes based on the measurement results of the electrical resistance measurement unit 312 and the optical measurement unit 313.

[0057] In contrast, when a surfactant is added to a blood sample, hemoglobin is removed from red blood cells, thereby reducing light absorption by the red blood cells. This allows the optical measurement unit 313 to optically detect only reticulocytes. Therefore, the counting device 310 can count only reticulocytes by counting each point plotted in FIG. 8 , i.e., each point corresponding to each measurement result by the electrical resistance measurement unit 312 and the optical measurement unit 313. This allows the concentration calculation unit 42 to accurately calculate the concentration of reticulocytes.

[0058] As described above, according to the first measurement method, hemoglobin is removed from red blood cells using a surfactant before measuring the concentration of reticulocytes contained in a blood sample. As described above, a surfactant with an osmotic pressure and pH similar to those of blood can be used. Therefore, hemoglobin can be easily removed from red blood cells simply by adding a surfactant to a stained blood sample. Therefore, pretreatment to appropriately adjust the osmotic pressure and pH of the red blood cell hemolysis reagent, as in conventional methods using a red blood cell hemolysis reagent, is not required. As a result, the pretreatment prior to measuring the reticulocyte concentration can be simplified. In other words, the reticulocyte concentration can be measured with simple pretreatment.

[0059] In particular, the surfactant may be cationic, zwitterionic, or nonionic. In this case, the surfactant can inhibit binding of NMB (positively charged) to the RNA (negatively charged) contained in reticulocytes. As a result, RNA can be reliably stained with NMB.

[0060] Furthermore, by carrying out the above-mentioned staining step (S1), removal step (S2), and dilution step (S3) in this order (three steps), the pretreatment can be carried out reliably one step at a time.

[0061] Furthermore, for example, if the surfactant concentration is high, after hemoglobin is extracted from the red blood cells, the membranes of the red blood cells may be ruptured during transfer to the flow cell 311, making it impossible to measure the reticulocyte concentration. By performing the dilution step (S3) as described above, the surfactant concentration is reduced, thereby reducing this possibility. Note that the dilution step is not essential for measuring the reticulocyte concentration. For example, if the surfactant concentration is low, it is possible to omit (skip) the dilution step and measure the reticulocyte concentration. This also applies to the following measurement methods.

[0062] The measurement step S5 includes an introduction step (S5-1), an electrical resistance measurement step (S5-2), an optical measurement step (S5-3), and a concentration calculation step (S5-4). This allows for semi-automation, in which steps S1 to S4 are performed manually and step S5 is performed by blood analyzer 1 (and terminal device 70).

[0063] The above describes a method of measuring the concentration of reticulocytes by introducing a blood sample into the LMNE chamber 31A as the chamber 31. However, if the blood analyzer 1 is equipped with a chamber dedicated to measuring the concentration of reticulocytes in addition to the LMNE chamber 31A, the blood sample may be introduced into the dedicated chamber to measure the concentration of reticulocytes.

[0064] In the above-described S5-2, an example was described in which the size of blood cells (i.e., the change in the volume of blood cells) was measured based on the change in electrical resistance as the blood cells passed through the aperture 312a (see FIG. 6). However, the size of blood cells may also be measured using optical techniques. For example, it is possible to measure the size of blood cells based on the amount of light received by the light-receiving unit as the blood cells pass between the light-emitting unit and the light-receiving unit. Therefore, the electrical resistance measurement step of S5-2 can be considered an example of a blood cell volume size measurement step that measures the change in the volume of blood cells in the blood sample flowing through the flow cell 311. Furthermore, the electrical resistance measurement unit 312 can also be considered an example of a blood cell volume size measurement unit 312V (see FIG. 6) that measures the change in the volume of blood cells in the blood sample flowing through the flow cell 311 by measuring the change in the electrical resistance of the blood cells.

[0065] (3-2. Second Measurement Method) In the first measurement method described above, the three steps S1 to S3 can be combined into one step. Below, a second measurement method, which is another example of a method for measuring the concentration of reticulocytes, will be described. Figure 9 is a flowchart showing the processing flow when measuring the concentration of reticulocytes using the second measurement method.

[0066] First, a mixture of a staining reagent (NMB), a surfactant, and a diluent is simultaneously added to a blood sample (S1-1: mixture addition step). This simultaneously stains reticulocytes contained in the blood sample, removes hemoglobin from red blood cells using the surfactant, and dilutes the blood sample. In other words, step S1-1 corresponds to the simultaneous execution of the staining step (S1), removal step (S2), and dilution step (S3) described above. After S1-1, step S5 is performed, as in FIG. 3 . In step S5-1 of S5, for example, the diluted blood sample is aspirated from the blood sample 10 using a pipette, and the aspirated blood sample is directly injected into the LMNE chamber of the blood analyzer 1. Alternatively, the sample container 10 may be loaded into the sample container loading unit 4, and the blood sample inside the sample container 10 may be aspirated using a needle 20 and dispensed into the LMNE chamber.

[0067] In S1-1, the staining step, removal step, and dilution step are performed simultaneously (in one step), so that the agent (reagent, etc.) only needs to be added to the blood sample once. Therefore, compared to the first measurement method, which involves three pretreatment steps, the pretreatment step prior to concentration measurement can be further simplified.

[0068] (3-3. Third Measurement Method) The reticulocyte concentration can also be measured based on the results of visual measurement. Hereinafter, a method of measuring the concentration by visually counting reticulocytes will be described as the third measurement method. Figure 10 is a flowchart showing the process flow when measuring the reticulocyte concentration using the third measurement method.

[0069] First, a mixture of a staining reagent (NMB), a surfactant, and a diluent is simultaneously added to a blood sample (S11: mixture addition step). Note that step S11 is exactly the same as step S1-1 in FIG. 9.

[0070] Next, a predetermined amount of the blood sample after adding the mixture in S1-1 is dropped onto a glass slide (S12: dropping step).The slide is then placed under a microscope, and the stained reticulocytes are visually counted. The number of reticulocytes counted per predetermined amount is calculated as the reticulocyte concentration (S13: concentration calculation step).

[0071] As in the first measurement method, the pretreatment may be performed in three steps (staining step, removal step, and dilution step) in order, followed by the steps from S12 onward in Fig. 10. That is, after performing the above three steps, a predetermined amount of the diluted blood sample may be dropped onto a glass slide (S12), the number of stained reticulocytes may be counted visually using a microscope, and the reticulocyte concentration may be calculated based on the count results.

[0072] Whether the pretreatment is performed in three steps or in one step, by subsequently performing steps S12 and S13, the reticulocytes can be visually counted and the reticulocyte concentration can be determined.

[0073] Alternatively, the field of view observed under the microscope may be photographed with a camera, the photographed image may be subjected to image processing, and reticulocytes may be identified and counted from the shape of the red blood cells obtained in the final image processing, thereby measuring the concentration of reticulocytes.

[0074] (3-4. Fourth Measurement Method) Reticulocyte concentration measurement can also be performed fully automatically using measurement system 80. A fully automated method for measuring reticulocyte concentration will be described below as the fourth measurement method. The fourth measurement method uses the reagents (a mixture of a staining reagent, a surfactant, and a diluent) contained in the container 320 shown in FIG. 2. This will be described in detail below.

[0075] 11 is a flowchart showing the process flow for measuring the concentration of reticulocytes by the fourth measurement method. The fourth measurement method includes a loading step (S21), a mixture introduction step (S22), and a measurement step (S23).

[0076] In the loading step of S21, the sample container 10 containing the blood sample is set in the sample container loading unit 4 (see FIG. 1) of the blood analyzer 1.

[0077] In the mixture introduction step S22, a mixture of a staining reagent, a surfactant, and a diluent is supplied from the storage unit 320 to the chamber 31 (particularly the LMNE chamber) via the conduit 321. The mixture introduction step S22 corresponds to the step of simultaneously performing the three steps S1 (staining step), S2 (removal step), and S3 (dilution step) in Fig. 4 in one step.

[0078] The measurement step of S23 includes an introduction step (S23-1), an electrical resistance measurement step (S23-2), an optical measurement step (S23-3), and a concentration calculation step (S23-4). The measurement step of S23 corresponds to the measurement step (S5) in FIG. 4. Therefore, the introduction step (S23-1), electrical resistance measurement step (S23-2), optical measurement step (S23-3), and concentration calculation step (S23-4) included in the measurement step of S23 correspond to the introduction step (S5-1), electrical resistance measurement step (S5-2), optical measurement step (S5-3), and concentration calculation step (S5-4) shown in FIG. 4, respectively. The measurement step of S23 measures the concentration of reticulocytes contained in the blood sample in the same manner as in the first measurement method.

[0079] In the introduction step of S23-1, a blood sample is aspirated by the needle 20 from the sample container 10 loaded in the sample container loading unit 4 and discharged into the chamber 31 (particularly the LMNE chamber). After a predetermined time (at least the time required for staining and hemoglobin removal) has passed since the blood sample reacted with a mixture of dyeing reagents and the like supplied to the chamber 31 via the conduit 321, the reactant in the chamber 31 moves to the flow cell 311.

[0080] As described above, in the fourth measurement method, the staining step, removal step, and dilution step are performed by the blood analyzer 1 equipped with the flow cell 311 (S22). This allows the measurement system 80 to perform the processes from staining reticulocytes to measuring their concentration in a fully automated manner.

[0081] In addition, when the storage unit 320 of the blood analyzer 1 is configured to store the staining reagent, surfactant, and diluent separately, the staining reagent, surfactant, and diluent can be supplied in sequence from the storage unit 320 to the chamber 31. In this case, a concentration measurement method can be realized in which the staining step, removal step, and dilution step are performed in three stages with staggered times, and in a fully automated manner.

[0082] 12 is a perspective view showing an example of a reticulocyte test reagent kit 100. The above-described staining reagent, surfactant, and diluent can be provided to a user (e.g., a medical professional) as a reticulocyte test reagent kit 100. The reticulocyte test reagent kit 100 includes a container 11 containing a mixture of the staining reagent, surfactant, and diluent packaged in a package 100a. That is, the reticulocyte test reagent kit 100 includes a staining reagent, a surfactant, and a diluent.

[0083] The user removes the storage container 11 from the packaging 100a of the reticulocyte test reagent kit 100. The user can then aspirate the mixture from the storage container 11 using a pipette or the like and inject it into the sample container 10 containing the blood sample outside the blood analyzer 1. This makes it possible to perform the second measurement method described above. The user can also drip the mixture aspirated using a pipette or the like onto a glass slide or directly inject it into the chamber 31 in the blood analyzer 1. In this case, it becomes possible to perform the third and fourth measurement methods described above.

[0084] 13 is a perspective view showing another example of a reticulocyte test reagent kit 100. The reticulocyte test reagent kit 100 may include, as the storage containers 10, a first container 11a containing a staining reagent, a second container 11b containing a surfactant, and a third container 11c containing a diluent.

[0085] As shown in the figure, the staining reagent, surfactant, and diluent are contained in individual containers (first container 11a, second container 11b, and third container 11c), so that the user can remove the individual containers from the package 100a of the reticulocyte test reagent kit 100 and then, outside the blood analyzer 1, sequentially inject the staining reagent, surfactant, and diluent into the sample container 10 containing the blood sample using a pipette or the like. This makes it possible to carry out the first measurement method described above.

[0086] The reticulocyte test reagent kit 100 may contain at least the staining reagent and the surfactant among the staining reagent, surfactant, and diluent. The diluent is commonly used to dilute blood samples, and therefore the user may already have it. Therefore, by including the staining reagent and the surfactant in the reticulocyte test reagent kit 100, the minimum materials necessary for measuring the reticulocyte concentration can be provided to the user, allowing the user to perform the reticulocyte concentration measurement of this embodiment. In particular, if the reticulocyte test reagent kit 100 further includes a diluent, all materials necessary for measuring the reticulocyte concentration can be provided to the user, allowing the user to reliably perform the reticulocyte concentration measurement of this embodiment.

[0087] 5. Detection of Reticulocytes Using Fluorescent Dye Reticulocytes can also be detected using fluorescent dye. FIG. 14 is an explanatory diagram showing the schematic configuration of an optical device 200 that detects reticulocytes using fluorescent dye. The optical device 200 is applicable to the measurement system 80 described above. The optical device 200 includes a laser light source 201, a detection optical system 202, and a detector 203. The laser light source 201 emits laser light of an arbitrary wavelength (e.g., 488 nm). The detection optical system 202 includes three condenser lenses 211, 212, and 213, optical filters 214a and 214b, and a spatial filter 215.

[0088] The optical filter 214a (first optical filter) is a filter that cuts light of the same wavelength as the laser light. The optical filter 214b (second optical filter) is a filter that cuts Raman scattered light (particularly the Raman wavelength of water). The optical filters 214a and 214b are arranged in this order from the laser light source 201 side between the two condenser lenses 211 and 212 that are closest to the laser light source 201. The spatial filter 215 is arranged between the two condenser lenses 212 and 213 and has an aperture (pinhole). The detector 203 is composed of, for example, a Si-PMT (photomultiplier tube).

[0089] After staining the RNA contained in reticulocytes with a fluorescent dye, the blood sample is introduced into the flow cell 311. When laser light from the laser light source 201 is applied to cells (fluorescently stained reticulocytes) flowing through the flow cell 311, light excited by the laser light (fluorescence) and elastically scattered light of the same wavelength as the laser light are generated from the cells. The elastically scattered light from the cells is filtered out by the optical filter 214a of the detection optical system 202, and only the fluorescence is guided to the detector 203. On the other hand, when the laser light is applied to the cells flowing through the flow cell 311, the laser light is also scattered by the liquid (e.g., water) surrounding the cells, generating Raman scattered light (inelastically scattered light) with a longer wavelength than the laser light. However, the generated Raman scattered light is filtered out by the optical filter 214b of the detection optical system 202. As a result, the detector 203 detects the fluorescence with the influence of the Raman scattered light reduced.

[0090] Thus, the detection accuracy of reticulocytes can be improved by using an inexpensive and simple optical system configuration that includes an optical filter 214b that cuts off Raman scattered light. In particular, the Si-PMT that constitutes the detector 203 has excellent sensitivity and is extremely advantageous in terms of improving the detection accuracy of reticulocytes. Furthermore, in a configuration using, for example, an avalanche photodiode (APD) as the detector 203, the APD's small size requires an expensive optical system or optical adjustment mechanism that focuses light minutely, as well as adjustment work. However, compared to an APD, the Si-PMT not only has superior sensitivity but can also have a larger light-receiving area, eliminating the need for the expensive optical system and adjustment work required for an APD, and allowing the use of an inexpensive, low-output laser light source 201.

[0091] [6. Regarding measurement of nucleated red blood cell concentration] The reticulocyte concentration measurement method and measurement system 80 of this embodiment and the reticulocyte test reagent kit 100 used for measuring reticulocyte concentration described above can also be used to measure the concentration of nucleated red blood cells. That is, as described above, nucleated red blood cells contain nucleic acids such as RNA, and therefore, the concentration of nucleated red blood cells can be measured by staining the nucleic acids, removing hemoglobin by adding a surfactant, and the like, in the same way as in the case of measuring the reticulocyte concentration.

[0092] Although the embodiments of the present invention have been described above, the scope of the present invention is not limited to these, and the invention can be expanded or modified without departing from the spirit of the invention.

[0093] The present invention can be used, for example, to measure the concentration of reticulocytes or nucleated red blood cells.

[0094] REFERENCE SIGNS LIST 1 Blood analyzer 20 Needle 42 Concentration calculation unit 80 Measurement system 100 Reticulocyte test reagent kit (test reagent kit) 201 Laser light source (light source) 202 Detection optical system 203 Detector 214a Optical filter (first optical filter) 214b Optical filter (second optical filter) 310 Counting device 311 Flow cell 312 Electrical resistance measurement unit 312V Blood cell volume size measurement unit 313 Optical measurement unit 320 Storage unit

Claims

1. A measuring method for measuring the concentration of reticulocytes or the concentration of nucleated red blood cells contained in a blood sample, comprising: a staining step of staining the reticulocytes or the nucleated red blood cells among the red blood cells contained in the blood sample using a staining reagent; a removal step of removing hemoglobin from the red blood cells by adding a surfactant to the blood sample; a measuring step of measuring the concentration of the reticulocytes or the concentration of the nucleated red blood cells contained in the blood sample using the blood sample after the staining step and the removal step.

2. The measuring method according to claim 1, further comprising a dilution step of diluting the blood sample.

3. The measuring method according to claim 2, wherein the staining step, the removal step, and the dilution step are performed in this order.

4. The measuring method according to claim 2, wherein the staining step, the removal step, and the dilution step are performed simultaneously.

5. The measuring step includes: an introduction step of introducing the blood sample into a flow cell; a blood cell volume size measuring step of measuring a change in the volume of blood cells in the blood sample flowing through the flow cell; an optical measuring step of measuring the light transmittance of the blood cells in the blood sample flowing through the flow cell; a concentration calculating step of calculating the concentration of the reticulocytes or the concentration of the nucleated red blood cells contained in the blood sample based on each measurement result in the blood cell volume size measuring step and the optical measuring step.

6. The measuring method according to claim 5, wherein the blood cell volume size measuring step includes an electrical resistance measuring step of measuring a change in the electrical resistance of the blood cells in the blood sample flowing through the flow cell to measure a change in the volume of the blood cells.

7. The measuring method according to claim 5, wherein the staining step and the removal step are performed using a blood analyzer equipped with the flow cell.

8. In the measuring step, a predetermined amount of the blood sample is dropped onto a slide glass, the number of the stained reticulocytes or the nucleated red blood cells is visually counted using a microscope, and the concentration of the reticulocytes or the concentration of the nucleated red blood cells is calculated based on the measurement result.

9. The measuring method according to any one of claims 1 to 4, wherein the surfactant is a cationic, zwitterionic, or nonionic surfactant.

10. Among the red blood cells contained in a blood specimen, a staining reagent for staining reticulocytes or nucleated red blood cells, and a surfactant for removing hemoglobin from the red blood cells, a reagent kit for inspection.

11. The reagent kit for inspection according to claim 10, further comprising a diluent for diluting the blood specimen.

12. A measurement system for measuring the concentration of reticulocytes or nucleated red blood cells contained in a blood specimen, comprising: a housing unit that houses a staining reagent for staining the reticulocytes or nucleated red blood cells and a surfactant that is introduced into the blood specimen to remove hemoglobin from the red blood cells contained in the blood specimen; a flow cell into which the blood specimen, the staining reagent, and the surfactant are introduced; a blood cell volume size measurement unit that measures a change in the volume of blood cells in the blood specimen introduced into the flow cell; an optical measurement unit that measures the light transmittance of the blood cells in the blood specimen introduced into the flow cell; a concentration calculation unit that calculates the concentration of the reticulocytes or the concentration of the nucleated red blood cells contained in the blood specimen based on each measurement result obtained by the blood cell volume size measurement unit and the optical measurement unit.

13. The measurement system according to claim 12, wherein the blood cell volume size measurement unit includes an electrical resistance measurement unit that measures a change in the volume of the blood cells by measuring a change in the electrical resistance of the blood cells in the blood specimen introduced into the flow cell.

14. The housing unit further houses a diluent for diluting the blood specimen, and the diluent is introduced into the flow cell in addition to the staining reagent and the surfactant. The measurement system according to claim 12 or 13.

15. a light source that emits light toward fluorescently stained cells flowing through the flow cell; a detection optical system; a detector that detects fluorescence excited by the irradiation of the cells with the light via the detection optical system, wherein the detection optical system has a first optical filter that cuts elastic scattered light having the same wavelength as the light from the cells generated by the irradiation of the light, and a second optical filter that cuts Raman scattered light having a longer wavelength than the light generated by scattering of the light by the liquid around the cells. The measurement system according to claim 12 or 13.