Bone marrow fluid analysis method, sample analysis device, and recording medium containing program

Abstract

The present invention relates to a bone marrow fluid analysis method, a sample analysis device, and a recording medium containing a program. The present invention provides a means for obtaining an index related to bone marrow nucleated cell density by measuring nucleated cells and lipid particles in bone marrow fluid. By counting the number of nucleated cells and lipid particles in bone marrow fluid and obtaining an index related to bone marrow nucleated cell density based on the nucleated cell and lipid particle counts, the aforementioned technical problems are solved.

Description

Technical Field

[0001] The present invention relates to a bone marrow fluid analysis method, a sample analysis device, and a recording medium containing the program. Background Art

[0002] Bone marrow examination is an important examination for diagnosing blood diseases such as hematopoietic dysfunction and hematopoietic organ tumors, as well as for determining the effectiveness of treatment. Among bone marrow examinations, bone marrow examination is a useful examination that can quickly obtain results and is cost-effective. In the bone marrow examination, the bone marrow fluid obtained by bone marrow puncture is smeared on a slide and stained, and the cells are classified, counted, and morphologically evaluated by microscopic observation. Preparation of smear specimens from the bone marrow and observation of cell morphology require professional knowledge and experience, so bone marrow examinations are performed by highly trained experts. For example, in Japan, bone marrow examinations are performed by qualified bone marrow certification technicians, while in foreign countries, bone marrow examinations are performed by hematopathology experts such as hematopathologists. Patent document 1 discloses a pretreatment method and device for preparing specimens for bone marrow examinations.

[0003] One piece of information obtained from a bone marrow examination is bone marrow nucleated cell density. Bone marrow cells are observed under a microscope, and the nucleated cell density is calculated by comparing the area ratio of nucleated cells to adipocytes in the smear (area of ​​adipocytes / area of ​​nucleated cells). In normal bone marrow fluid, the ratio of nucleated cells to adipocytes is roughly constant. However, the number of nucleated cells in the bone marrow fluid of patients with blood disorders can vary abnormally. For example, the number of nucleated cells in the bone marrow increases in diseases such as acute leukemia, while it decreases in diseases such as aplastic anemia. In clinical practice, the nucleated cell density calculated from this area ratio is used to categorize bone marrow status into hyperplasia, normal development, and hypoplasia, and is used to differentiate between blood disorders.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 4-20298. Summary of the Invention

[0007] Technical problem to be solved by the invention

[0008] Bone marrow fluid is collected from patients during a bone marrow aspiration for bone marrow examination. During a bone marrow aspiration, local anesthesia is administered, a needle is inserted into the bone marrow, and the bone marrow fluid is aspirated. Bone marrow aspiration is often quite painful and burdensome for the patient, making it difficult to perform bone marrow examinations frequently. Therefore, every bone marrow examination must provide highly accurate information. On the other hand, bone marrow aspiration involves microscopic observation and manual counting and classification of cells based on their morphology. Therefore, the accuracy of measurements such as bone marrow nucleated cell density depends on the examiner's ability and experience, and can also be influenced by subjective factors.

[0009] Therefore, an object of the present invention is to provide a bone marrow fluid analysis method, an analysis device, and a computer program that can provide an objective index related to bone marrow nucleated cell density and that does not depend on the ability and experience of the examiner.

[0010] Technical means to solve technical problems

[0011] The present inventors have discovered that indices based on the number of nucleated cells and the number of lipid particles surprisingly correlate well with the density of nucleated cells in bone marrow smears observed under a microscope, thereby completing the present invention.

[0012] A technical solution of the present invention provides a bone marrow fluid analysis method, which has the following steps: a step of counting the number of nucleated cells and lipid particles in the bone marrow fluid; a step of obtaining a bone marrow nucleated cell density-related index based on the number of nucleated cells and lipid particles.

[0013] Here, "bone marrow fluid" refers to bone marrow fluid collected from a subject by bone marrow puncture or bone marrow biopsy, and a sample containing the bone marrow fluid.

[0014] The term "nucleated cell count" refers to the total number of leukocytes and erythroblasts. Examples of leukocytes include myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, segmented leukocytes, eosinophils, basophils, lymphocytes, and monocytes. Examples of erythroblasts, also called nucleated red blood cells, include proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and normochromatic erythroblasts.

[0015] The "lipid particle number" refers to the total number of adipocytes and adipocyte-derived formed elements.

[0016] Generally, bone marrow nucleated cell density is defined as the area ratio calculated from the area of ​​nucleated cells and the area of ​​adipocytes obtained by microscopic observation of a bone marrow smear (adipocyte area / nucleated cell area). Here, "bone marrow nucleated cell density-related index" refers to new information related to bone marrow nucleated cell density discovered by the present invention, and refers to information obtained using the number of nucleated cells and the number of lipid particles.

[0017] In this technical solution, the bone marrow nucleated cell density-related index can be designed as a value related to the ratio of the number of nucleated cells to the number of lipid particles. In addition, in this technical solution, the bone marrow nucleated cell density-related index can be designed as a value of the ratio of the number of lipid particles to the number of nucleated cells.

[0018] In this technical solution, it can be designed to display indicators related to bone marrow nucleated cell density.

[0019] In this technical solution, it can be designed to further include a step of determining the state of the bone marrow based on the number of nucleated cells and the number of lipid particles.

[0020] In addition, the present invention can be designed to further include a step of determining the bone marrow status based on an index related to bone marrow nucleated cell density, and can be designed to determine at least one of aplasia, normal development, and hyperplasia as the bone marrow status.

[0021] In the above technical solution, the bone marrow status can be determined by comparing an indicator related to bone marrow nucleated cell density with a certain threshold. In the above technical solution, the bone marrow status can be determined as hypoplasia when the ratio of the number of lipid particles to the number of nucleated cells is higher than a first threshold; normal development when the ratio of the number of lipid particles to the number of nucleated cells is lower than the first threshold and higher than a second threshold; and hyperplasia when the ratio of the number of lipid particles to the number of nucleated cells is lower than the second threshold. The "first threshold" is the threshold that distinguishes hypoplasia from normal development and hyperplasia. The "second threshold" is the threshold that distinguishes hyperplasia from normal development and hypoplasia. In the above technical solution, the relevant information of the determined bone marrow status can be displayed.

[0022] In the present technical solution, it is possible to design the method such that, in the step of counting the number of nucleated cells and the number of lipid particles, the measurement sample is measured by flow cytometry.

[0023] This technical solution can be designed to further include a step of mixing bone marrow fluid with a reagent to prepare a measurement sample. This technical solution can be designed to prepare a measurement sample by mixing at least bone marrow fluid with a hemolytic agent. Furthermore, this technical solution can be designed to further include a fluorescent dye to prepare a measurement sample. Here, "hemolytic agent" refers to a substance that can lyse red blood cells. Furthermore, "fluorescent dye" refers to a fluorescent substance that can stain nucleic acids.

[0024] In this technical solution, the lipid particle number can be counted based on the fluorescence signal information, forward scattered light information, and side scattered light information obtained by flow cytometry. In this way, the lipid particle number can be obtained with high precision.

[0025] In the present technical solution, it can be designed as follows: the process of obtaining the number of nucleated cells and the number of lipid particles has the following steps: a process of measuring a first measurement sample containing bone marrow fluid and a first reagent, and counting the number of nucleated cells in the first measurement sample; a process of measuring a second measurement sample containing bone marrow fluid and a second reagent different from the first reagent, and counting the number of lipid particles in the second measurement sample.

[0026] In the present technical solution, it can be designed as follows: the first reagent includes a dissolving reagent with a pH of 2.0 or more and 4.5 or less, and the second reagent includes a dissolving reagent with a pH of 5.5 or more and 7.0 or less.

[0027] A technical solution of the present invention provides a sample analysis device, which includes: a sample preparation unit, which prepares a measurement sample from bone marrow fluid; a detection unit, which detects particles contained in the measurement sample; and a control unit, which obtains the number of nucleated cells and the number of lipid particles in the measurement sample based on information obtained by the detection unit; wherein the control unit obtains an index related to bone marrow nucleated cell density based on the number of nucleated cells and the number of lipid particles.

[0028] In this technical solution, it can be designed as follows: the detection part has: a flow chamber, which allows the measurement sample prepared by the sample preparation part to flow; a light source part, which irradiates the measurement sample flowing in the flow chamber with light; and an optical detection part, which obtains optical information obtained when the measurement sample is irradiated with light.

[0029] In the present technical solution, it can be designed as follows: the number of lipid particles is obtained based on the fluorescence signal information, forward scattered light information and side scattered light information obtained by the light receiving part.

[0030] In this technical solution, the sample analyzer may further include an output unit, and the control unit may output the bone marrow nucleated cell density-related index to the output unit. Here, the "output unit" includes a display device having an interface capable of displaying text, images, etc., a printer, an audio output device, and the like.

[0031] In the present technical solution, it can be designed that the bone marrow nucleated cell density-related index is a related value of the ratio of the number of nucleated cells to the number of lipid particles.

[0032] In this technical solution, the control unit can be designed to determine the bone marrow status based on the number of nucleated cells and the number of lipid particles. In this technical solution, the sample analyzer further includes an output unit, and the control unit outputs the determined bone marrow status related information to the output unit.

[0033] Another technical solution of the present invention provides a recording medium containing an executable program, which is a computer program for analyzing bone marrow fluid, and the computer program enables the computer to execute the following steps: a step of obtaining the number of nucleated cells and the number of lipid particles in the bone marrow fluid based on information obtained by a detection unit that detects particles contained in the bone marrow fluid; a step of obtaining an index related to the nucleated cell density of the bone marrow based on the number of nucleated cells and the number of lipid particles.

[0034] Effects of the Invention

[0035] The present invention can provide an objective index related to bone marrow nucleated cell density that is independent of the ability and experience of the examiner. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A Schematic diagram of a scatter plot obtained by flow cytometry (FCM) using reagents for counting nucleated red blood cells and white blood cells;

[0037] Figure 1B Schematic diagram of a scattergram obtained by FCM measurement using a leukocyte differential reagent;

[0038] Figure 1C Schematic diagram of the scatter plot obtained by FCM assay using universal reagents;

[0039] Figure 2A A schematic diagram of the structure of the sample analysis device of this embodiment;

[0040] Figure 2B A schematic diagram of the structure of the sample analysis device of this embodiment;

[0041] Figure 3 An oblique view of the structure of the flow chamber;

[0042] Figure 4 A block diagram of the structure of the parsing unit;

[0043] Figure 5 Flowchart of the operation flow of the sample analyzer of this embodiment;

[0044] Figure 6 Flowchart of the steps in the sample preparation process;

[0045] Figure 7 Flowchart of the steps of measurement data analysis processing;

[0046] Figure 8A Flowchart of the steps of the determination process based on bone marrow nucleated cell density;

[0047] Figure 8B Flowchart of the steps of the determination process based on bone marrow nucleated cell density;

[0048] Figure 8C Flowchart of the steps of the determination process based on bone marrow nucleated cell density;

[0049] Figure 9 A diagram showing an example display of analysis results;

[0050] Figure 10A Example of a scatter plot showing the distribution of particles in a measurement sample prepared using a reagent for counting nucleated red blood cells and white blood cells;

[0051] Figure 10B The first example of a scattergram showing the distribution of particles in a measurement sample prepared using a leukocyte differential reagent;

[0052] Figure 10C Second example of a scattergram showing the distribution of particles in a measurement sample prepared using a leukocyte differential reagent;

[0053] Figure 11 A graph showing the distribution of the ratio of the number of lipid particles to the number of nucleated cells for each subject categorized according to the density of nucleated cells in the bone marrow observed under a microscope;

[0054] Figure 12A ROC curve for determining whether bone marrow nucleated cell density is hypoplastic based on the ratio of lipid particle number to nucleated cell number;

[0055] Figure 12B ROC curve for determining whether bone marrow nucleated cell density is hyperplastic based on the ratio of the number of lipid particles to the number of nucleated cells. DETAILED DESCRIPTION

[0056] [1. Bone marrow fluid analysis method]

[0057] In the bone marrow fluid analysis method of this embodiment, the number of nucleated cells and the number of lipid particles in a measurement sample containing bone marrow fluid and reagents are first obtained. The method for obtaining the number of nucleated cells and the number of lipid particles in bone marrow fluid is described below.

[0058] (bone marrow fluid)

[0059] If the bone marrow fluid collected from the subject contains solid impurities such as bone fragments or blood cell aggregates that may interfere with cell analysis, the fluid can be filtered using a mesh or other means. If necessary, a chelating agent and / or anticoagulant can be added to the fluid. Examples of chelating agents include EDTA (ethylene diamine tetraacetic acid) salts. Examples of anticoagulants include heparin, citric acid, or citrate salts.

[0060] Bone marrow fluid typically contains nucleated cells and lipid particles. In a bone marrow examination, erythroblasts, leukocytes, plasma cells, reticular cells (macrophages), and megakaryocytes are generally counted as nucleated cells. Among the leukocytes, promyelocytes, myelocytes, and metamyelocytes are collectively referred to as immature granulocytes. Band-shaped leukocytes are mature neutrophils, and segmented leukocytes are neutrophils that have further matured from band-shaped leukocytes. Eosinophils include immature eosinophils and mature eosinophils. Basophils include immature basophils and mature basophils.

[0061] Leukocytes and erythroblasts include tumorous cells that appear and increase in hematopoietic organ tumors such as various leukemias and malignant lymphomas. For example, in acute lymphoblastic leukemia, lymphoblasts increase. Lymphocytes can also include atypical lymphocytes. Atypical lymphocytes are lymphocytes activated by antigenic stimulation and appear due to viral infection, for example.

[0062] In this embodiment, examples of lipid particles include adipocytes, all or part of damaged adipocytes, fat masses released from damaged adipocytes, etc. Although adipocytes have nuclei, they are also included in lipid particles.

[0063] It is known that in normal bone marrow, the combined number of leukocytes and erythroblasts accounts for 95% to 99% of all nucleated cells. In this embodiment, the nucleated cells measured are leukocytes and erythroblasts. That is, in the analysis method of this embodiment, the measurement sample prepared from bone marrow fluid is measured, and the sum of the number of leukocytes and erythroblasts is calculated as the nucleated cell count.

[0064] (Reagent)

[0065] In the analysis method of the present embodiment, the reagent used only needs to be a reagent that enables the measurement of nucleated cells and / or lipid particles, and there is no particular limitation. Preferably, the reagent contains at least one of a hemolytic agent and a fluorescent dye. In a preferred embodiment, the reagent contains a hemolytic agent and a fluorescent dye. At this time, the reagent may be a single reagent containing both a hemolytic agent and a fluorescent dye. In addition, it may also be a combination of a dissolving reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye, and the dissolving reagent containing a hemolytic agent and the staining reagent containing a fluorescent dye are prepared separately. Sometimes the storage stability of the fluorescent dye in an aqueous solution is not good, so it is preferred that the reagent consists of two reagents, a dissolving reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye.

[0066] The reagent used for the counting of nucleated cells and the counting of lipid particles can be one kind, or two or more kinds can be used in combination. In a preferred embodiment, it is a combination of a reagent capable of measuring nucleated cells and a reagent capable of measuring lipid particles, and the reagent capable of measuring nucleated cells and the reagent capable of measuring lipid particles are prepared separately. That is, the dissolving reagent and the staining reagent for measuring nucleated cells and the dissolving reagent and the staining reagent for measuring lipid particles are used separately. In the analysis method of the present embodiment, a reagent for counting nucleated red blood cells and white blood cells is used for the measurement of nucleated cells. In addition, a reagent for classifying white blood cells is used for the measurement of lipid particles. However, the above is only an example, and the reagents used for the measurement of nucleated cells and the measurement of lipid particles are not limited to this.

[0067] (Reagent for counting nucleated red blood cells and white blood cells) 1. Hemolytic agent used for the measurement of nucleated cells in bone marrow fluid

[0068] The reagent for counting nucleated red blood cells and white blood cells enables the counting of nucleated red blood cells and white blood cells (basophils and white blood cells other than basophils) in the sample. The reagent for counting nucleated red blood cells and white blood cells consists of two reagents, a dissolving reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye. As the first reagent above, it is preferred to use the reagent for counting nucleated red blood cells and white blood cells, but it is not limited to this.

[0069] The hemolytic agent of the reagent for counting nucleated red blood cells and white blood cells can be selected from, for example, the quaternary ammonium salt type cationic surfactant represented by the following formula (1) and the pyridine type cationic surfactant represented by the following formula (2). The cationic surfactant can be one kind, or two or more kinds can be used.

[0070]

Chemical 1

[0071]

[0072] In formula (1), R 1 , R 2 and R 3are the same or different and are hydrogen, an alkyl group having 1 to 8 carbon atoms, or an aralkyl group having 6 to 8 carbon atoms; R 4 is an alkyl group having 8 to 18 carbon atoms, an alkenyl group having 8 to 18 carbon atoms, or an aralkyl group having 6 to 18 carbon atoms; X - It is an anion.

[0073]

Chemistry 2

[0074]

[0075] In formula (2), R 5 is an alkyl group having 8 to 18 carbon atoms; X - It is an anion.

[0076] In the above formulas (1) and (2), examples of the alkyl group having 1 to 8 carbon atoms include methyl, ethyl, propyl, tert-butyl, n-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, heptyl, and octyl. Alkyl groups having 1 to 3 carbon atoms are preferred. Examples of the aralkyl group having 6 to 8 carbon atoms include benzyl and phenethyl.

[0077] Examples of alkyl groups having 8 to 18 carbon atoms include octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. Preferred are linear alkyl groups having 10 to 14 carbon atoms, such as decyl, dodecyl, and tetradecyl. Examples of alkenyl groups having 8 to 18 carbon atoms include octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, and octadecenyl. Examples of aralkyl groups having 6 to 18 carbon atoms include phenylpropene, phenylbutene, naphthylmethylene, naphthylethylene, naphthylpropene, biphenylmethylene, and biphenylethylene.

[0078] Anions include halogen ions (F - 、Cl - Br - or I - ), boron halide ions (BF4 - 、BCl4 - 、BBr4 - etc.), phosphorus compound ions, halogen oxygen acid ions, fluorosulfate ions, methyl sulfate ions, tetraphenylboron compound ions having a halogen or halogen-containing alkyl group as a substituent in the aromatic ring, etc. Among them, Br is preferred. - or BF4 - .

[0079] Examples of surfactants represented by the above formula (1) or (2) include octyltrimethylammonium bromide, octyltrimethylammonium chloride, decyltrimethylammonium bromide, decyltrimethylammonium chloride, dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, myristyltrimethylammonium bromide, myristyltrimethylammonium chloride, and dodecylpyridinium hydrochloride.

[0080] In reagents for counting nucleated red blood cells and white blood cells, it is preferred to use a nonionic surfactant as a hemolytic agent in conjunction with the cationic surfactant described above. By combining a cationic surfactant and a nonionic surfactant, excessive damage to nucleated red blood cells and white blood cells caused by the cationic surfactant can be suppressed. Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene sterols, and polyoxyethylene hydrogenated sterols. The nonionic surfactant may be used alone or in combination with two or more.

[0081] Examples of the nonionic surfactant include polyoxyethylene (16) oleyl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (20) polyoxypropylene (8) cetyl ether, polyoxyethylene (30) polyoxypropylene (6) decyltetradecyl ether, polyoxyethylene (20) castor oil, polyoxyethylene (20) hydrogenated castor oil, polyoxyethylene (50) hydrogenated castor oil, and polyoxyethylene (25) phytostanol. Among these, polyoxyethylene (16) oleyl ether and polyoxyethylene (20) polyoxypropylene (8) hexadecyl ether are particularly preferred.

[0082] In reagents for counting nucleated red blood cells and white blood cells, the concentration of the cationic surfactant in the lysis reagent is generally 300 to 9000 ppm, preferably 400 to 8000 ppm, and more preferably 500 to 7000 ppm. Furthermore, the concentration of the nonionic surfactant in the lysis reagent is generally 500 to 7000 ppm, preferably 800 to 6000 ppm, and more preferably 1000 to 5000 ppm.

[0083] The osmotic pressure of the lysis reagent containing the above-mentioned hemolytic agent is generally 150 mOsm / kg or less, preferably 120 mOsm / kg or less, and more preferably 100 mOsm / kg or less. The lower limit of the osmotic pressure is not particularly limited, but is, for example, 20 mOsm / kg or greater, preferably 30 mOsm / kg or greater, and more preferably 40 mOsm / kg or greater. The pH of the lysis reagent containing the above-mentioned hemolytic agent is preferably 2.0 or greater and 4.5 or less, and more preferably 2.5 or greater and 3.5 or less.

[0084] In order to make the osmotic pressure and pH of the dissolving reagent containing the above-mentioned hemolytic agent within the above-mentioned range, the dissolving reagent preferably contains electrolytes, sugars, buffers, aromatic organic acids, etc. In this specification, "aromatic organic acid" refers to an acid and its salt having at least one aromatic ring in the molecule. The electrolyte is preferably an inorganic salt, for example, sodium chloride, potassium chloride, etc. can be listed. The sugar can be one of a monosaccharide, a disaccharide, a polysaccharide and an oligosaccharide, for example, glucose, lactose, sucrose, etc. can be listed. The buffer can be any buffer having a pKa near the set pH ± 2.0, for example, malic acid, citric acid, maleic acid, tartaric acid, diglycolic acid, malonic acid, etc. can be listed. Aromatic organic acids include phthalic acid, salicylic acid, benzoic acid, hydroxybenzoic acid, aminobenzoic acid, hippuric acid, p-aminobenzenesulfonic acid, benzenesulfonic acid, and the above alkali metal salts (such as sodium salt and potassium salt). The above concentration is appropriately about 0.1 to 100 mM, for example, and preferably about 1 to 30 mM.

[0085] 2. Fluorescent dyes used for the determination of nucleated cells in bone marrow fluid

[0086] Examples of the fluorescent dye used in the reagent for counting nucleated red blood cells and white blood cells include compounds represented by one of the following formulas (3) and (4). The fluorescent dye may be one or two or more.

[0087]

Chemistry 3

[0088]

[0089] In formula (3), R 6 and R 7 the same or different, are alkyl;

[0090]

Chemistry 4

[0091] ;

[0092]

Chemistry 5

[0093] ; R 8 、R 9 、R 10 and R 11 are the same or different and are a hydrogen atom or an alkyl group; X - It is an anion.

[0094]

Chemistry 6

[0095]

[0096] In formula (4), R 12 and R 13 The same or different alkyl groups may have an acidic group;

[0097]

Chemistry 7

[0098] ;

[0099]

Chemistry 8

[0100] ; R 14 、R 15 、R 16 and R 17 The same or different, is a hydrogen atom or an acidic group, but R 12 ~R 17 There is an acidic group in one of the 12 ~R 17 The acidic groups in R 12 ~R 17 One of the acidic groups is a group that releases a proton.

[0101] The alkyl group in formula (3) or (4) may be linear or branched. The number of carbon atoms in the alkyl group is usually 1 to 20, preferably 1 to 10, and more preferably 1 to 6. Examples of the alkyl group include methyl, ethyl, propyl, tert-butyl, n-butyl, n-pentyl, and n-hexyl.

[0102] The anion in formula (3) can be F - 、Cl - Br - , I - Halogen ions and CF3SO3 - 、BF4 - 、ClO4 - The acidic groups that may be present in formula (4) include both proton-releasing groups and groups that are formed after the proton-releasing groups have released protons. Examples of proton-releasing groups include carboxyl groups, sulfonic acid groups, and phosphoric acid groups, with sulfonic acid groups being particularly preferred. The acidic groups may form salts. Examples of such salts include alkali metal salts such as sodium salts and potassium salts. Sodium salts are more preferred.

[0103] Examples of the fluorescent dye represented by the formula (3) or (4) include NK-529, NK-2670, NK-3750, NK-3383, NK-1840, NK-9001, NK-9003, NK-2929, NK-3375, NK-5056, NK-3266, and NK-3620. These fluorescent dyes can be obtained from Hayashibara Co., Ltd.

[0104] The concentration of the fluorescent dye in the reagent can be appropriately determined depending on the type of fluorescent dye, but is generally between 0.01 mg / L and 100 mg / L, preferably between 0.1 mg / L and 90 mg / L, and more preferably between 0.2 mg / L and 80 mg / L. When the reagent is a single reagent containing both the fluorescent dye and the hemolytic agent, the fluorescent dye can be dissolved in the lysis reagent containing the hemolytic agent. When the reagent is a combination of a lysis reagent containing the hemolytic agent and a staining reagent containing the fluorescent dye, the fluorescent dye can be dissolved in an appropriate organic solvent. The organic solvent is not particularly limited as long as it can dissolve the fluorescent dye. Examples include alcohols with 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, and dimethyl sulfoxide (DMSO).

[0105] (Reagents for Leukocyte Differentiation) 1. Hemolytic agent used for the measurement of lipid particles in bone marrow fluid

[0106] The leukocyte classification reagent is used to classify and count leukocytes in a sample into three types (granulocytes, lymphocytes, and monocytes), four types (neutrophils and basophils, eosinophils, lymphocytes, and monocytes), five types (neutrophils, basophils, eosinophils, lymphocytes, and monocytes), or six types (neutrophils, basophils, eosinophils, lymphocytes, and monocytes). It can also count lipid particles. The leukocyte classification reagent consists of two reagents: a lysis reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye. The second reagent is preferably a leukocyte classification reagent, but is not limited thereto.

[0107] Examples of hemolytic agents in the leukocyte classification reagent include combinations of cationic surfactants, nonionic surfactants, and aromatic organic acids. In a preferred embodiment, the hemolytic agent-containing lysis reagent includes a reagent containing a cationic surfactant, a nonionic surfactant, and an aromatic organic acid at a concentration of 20 mM to 50 mM. When the concentration of the aromatic organic acid is 20 mM to 30 mM, the pH of the reagent is 5.5 to 6.4; and when the concentration of the aromatic organic acid is 30 mM to 50 mM, the pH of the reagent is 5.5 to 7.0.

[0108] Examples of aromatic organic acids include phthalic acid, salicylic acid, benzoic acid, hydroxybenzoic acid, aminobenzoic acid, hippuric acid, p-aminobenzenesulfonic acid, benzenesulfonic acid, and their alkali metal salts (e.g., sodium and potassium salts). The aromatic organic acid may be a single acid or two or more. When the dissolving agent contains two or more aromatic organic acids, their combined concentrations may be between 20 mM and 50 mM.

[0109] When the concentration of the aromatic organic acid in the lysis reagent is 20 mM or more and less than 30 mM, the pH of the reagent is preferably 5.5 or more and 6.4 or less, more preferably 5.5 or more and 6.2 or less. Furthermore, when the concentration of the aromatic organic acid in the lysis reagent is 30 mM or more and 50 mM or less, preferably 40 mM or more and 50 mM or less, the pH of the reagent is 5.5 or more and 7.0 or less. Even more preferably, when the concentration of the aromatic organic acid in the reagent containing the hemolytic agent is 40 mM or more and 50 mM or less, the pH of the reagent is 5.5 or more and 6.2 or less.

[0110] The cationic surfactants in the leukocyte classification reagent are preferably quaternary ammonium salt-type cationic surfactants represented by the following formula (5) and pyridine-type cationic surfactants represented by the following formula (6). The cationic surfactants may be one or two or more.

[0111]

Chemistry 9

[0112]

[0113] In formula (5), R 18 is an alkyl or alkenyl group having 6 to 18 carbon atoms; R 19 and R 20 are the same or different and are alkyl or alkenyl groups with 1 to 4 carbon atoms; R 21 is an alkyl or alkenyl group or a benzyl group having 1 to 4 carbon atoms; X - It is a halide ion.

[0114] In formula (5), preferably R 18 It is an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and a linear alkyl group is particularly preferred. Specifically, octyl, decyl and dodecyl can be cited. 19 and R 20 is methyl, ethyl and propyl. 21 The halogen ions are methyl, ethyl and propyl. - 、Cl - Br - and I - .

[0115]

Chemistry 10

[0116]

[0117] In formula (6), R 22 is an alkyl group having 6 to 18 carbon atoms; X - is a halogen ion. 22It is an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and a linear alkyl group is particularly preferred. Specifically, octyl, decyl and dodecyl groups can be listed. Halogen ions include F - 、Cl - Br - and I - .

[0118] The nonionic surfactant of the reagent for leukocyte classification is preferably a polyoxyethylene nonionic surfactant represented by the following formula (7).

[0119] R 23 -R 24 -(CH2CH2O) n -H(7) In formula (7), R 23 is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms; R 24 is an oxygen atom, -COO- or

[0120]

Chemistry 11

[0121] ;

[0122] n is 10 to 50.

[0123] Examples of the nonionic surfactant include polyoxyethylene alkyl ethers, polyoxyethylene sterols, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkylamines, and polyoxyethylene polyoxypropylene alkyl ethers.

[0124] In the leukocyte classification reagent, the concentration of the nonionic surfactant in the lysis reagent is 10 to 100,000 ppm, preferably 100 to 10,000 ppm, and more preferably 1,000 to 5,000 ppm. The concentration of the cationic surfactant in the reagent is usually 10 to 10,000 ppm, and more preferably 100 to 1,000 ppm.

[0125] To maintain the pH within the above range, the lysis reagent in the leukocyte differential reagent may contain a buffer. Examples of such buffers include citrate, phosphate, and Good's buffers such as HEPES. Furthermore, the addition of a buffer is optional if an aromatic organic acid with a buffering effect is added to the lysis reagent. The osmotic pressure of the lysis reagent is not particularly limited; however, for efficient red blood cell hemolysis, it is preferably between 20 mOsm / kg and 150 mOsm / kg.

[0126] 2. Fluorescent dyes used for the determination of nucleated cells in bone marrow fluid

[0127] Fluorescent dyes for leukocyte differential reagents can be selected from the following groups, for example: propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium bisazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylenebis[3-[[4-[[(3-methylbenzothiazol-3-yl)-2-yl]methylene]-1,4-dihydroquinolin]-1-yl]propyl]dimethylammonium] tetraiodide, Bis[[3-[[4-[[(3-methylbenzothiazole-3-ium)-2-yl]methylene]-1,4-dihydroquinoline]-1-yl]propyl]dimethylaminium]tetraiodide)(TOTO-1)、4-[(3-methylbenzothiazole-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)propyl]quinolinium・diiodide(4-[(3-methylbenzothiazole-2(3H)-ylidene)methyl]-1-[3-(Trimethylaminio)propyl]Quinolinium・diiodide)(TO-PRO-1)、N,N,N',N'-tetramethyl- N,N'-bis[3-[4-[3-[(3-methylbenzothiazole-3-ium)-2-yl]-2-propenylidene]-1,4-dihydroquinoline-1-yl]propyl]-1,3-propanediaminium・tetraiodide (TOTO-3), 2-[3-[[1-[3-(trimethylammonium)propyl]-1,4-dihydroquinoline]-4- 2-[3-[[1-[3-(Trimethylaminio)propyl]-1,4-dihydroquinoline]-4-ylidene]-1-propenyl]-3-methylbenzothiazole-3-ium・diiodide (TO-PRO-3) and a fluorescent dye represented by the following formula (8).The fluorescent dye may be used alone or in combination of two or more.

[0128]

Chemistry 12

[0129]

[0130] In formula (8), R 25 and R 28 are the same or different and are a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, or a benzyl group which may have a substituent; R 26 and R 27 are the same or different and are hydrogen, hydroxy, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylsulfonyl or phenyl; Z is a sulfur atom, an oxygen atom, or has a methyl group; n is 0, 1, 2 or 3; X - It is an anion.

[0131] The alkyl group in formula (8) can be a straight chain or a branched chain. 25 and R 28 When one of R is an alkyl group having 6 to 18 carbon atoms, the other is preferably a hydrogen atom or an alkyl group having less than 6 carbon atoms. Among the alkyl groups having 6 to 18 carbon atoms, an alkyl group having 6, 8 or 10 carbon atoms is preferred. 25 and / or R 28 In the case of a benzyl group which may have a substituent, examples of the substituent include an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms. Among them, a methyl group or an ethyl group is preferred.

[0132] In formula (8), R 26 and R 27 Examples of the alkenyl group include alkenyl groups having 2 to 20 carbon atoms. 26 and R 27 Examples of the alkoxy group include alkoxy groups having 1 to 20 carbon atoms. Among them, methoxy and ethoxy groups are preferred. The anion in formula (8) includes F - 、Cl - Br - , I - Halogen ions and CF3SO3 - 、BF4 - 、ClO4 - wait.

[0133] The concentration and solvent of the fluorescent dye in the reagent for leukocyte differentiation are the same as those described for the reagent for counting nucleated red blood cells and leukocytes.

[0134] (Other reagents)

[0135] In this embodiment, different lysis reagents and staining reagents are used for the measurement of nucleated cells and lipid particles, respectively. However, this is not limiting. Nucleated cells and lipid particles can be measured using a common lysis reagent and staining reagent. For example, by using a reagent comprising a lysis reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye as disclosed in Patent No. 4212827, nucleated cells and lipid particles can be measured using a common lysis reagent and staining reagent.

[0136] Examples of the hemolytic agent in this universal reagent include surfactants represented by formula (1), (2), or (7), MEGA-8, sucrose decanoate, deoxy-BIGCHAP, n-octyl-β-D-thioglucoside, n-nonyl-β-D-thiomaltoside, n-heptyl-β-D-thioglucoside, n-octyl-β-D-thioglucoside, CHAPS, and CHAPSO. The hemolytic agent may be a single agent or two or more agents may be used.

[0137] Examples of fluorescent dyes used in this universal reagent include NK-2825, NK-1836, NK-1954, Oxazine 750, Cryptocyanin, NK-376, NK-382, NK-2711, NK-138, Oxazine 720, LDS 730, LD 700, Nile Blue A, Brilliant Green, Iodide Green, and Malachite Green. The fluorescent dye may be a single species or two or more species may be used.

[0138] (Preparation and measurement of test samples)

[0139] In this embodiment, a measurement sample can be prepared by mixing bone marrow fluid and a reagent. When the reagent contains a hemolytic agent, at least the bone marrow fluid and the hemolytic agent are mixed to prepare the measurement sample. Due to the action of the hemolytic agent in the reagent, the nucleated cells in the bone marrow fluid become in a state where they can be stained with a fluorescent dye. The state where they can be stained with a fluorescent dye refers to a state where the cell membrane of the cells is damaged to the extent that the fluorescent dye can pass through. When red blood cells are mixed into the bone marrow fluid, the red blood cells can be lysed due to the action of the hemolytic agent. Due to the action of the hemolytic agent, the cell membrane of the erythroblast-type cells will also be destroyed, just like the red blood cells, but the cell nucleus of the erythroblast-type cells will be maintained. Therefore, the erythroblast-type cells become in a state where they can be stained and can be classified and counted by FCM.

[0140] In a preferred embodiment, a fluorescent dye is further mixed in the preparation of the assay sample. When the bone marrow fluid is treated with a hemolytic agent, the fluorescent dye can enter the cell through the damaged cell membrane of the nucleated cells and stain the nucleic acid in the cell nucleus. At this time, the leukocytes are strongly stained and emit a strong fluorescence. The degree of staining of erythroblasts is weaker than that of leukocytes and they emit a weaker fluorescence. The mechanism of action that produces the difference in fluorescence intensity between leukocytes and erythroblasts is not yet clear. It may be because the nucleus (DNA) of erythroblasts is condensed, thus hindering the entry of fluorescent dyes into the cell nucleus. In addition, lipid particles are also weakly stained with fluorescent dyes and emit a weaker fluorescence. On the other hand, cells and particles that do not have a nucleus, such as red blood cells and red blood cell ghosts, are hardly stained.

[0141] When the reagent is a reagent containing a hemolytic agent or a reagent containing a hemolytic agent and a fluorescent dye, the mixing ratio of the bone marrow fluid to the reagent is generally 1:5-500, preferably 1:10-100, expressed as a volume ratio. When the reagent is a combination of a lysis reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye, the mixing ratio of the bone marrow fluid to the lysis reagent and the staining reagent is generally 1:5-500:1-10, preferably 1:10-100:2-5, expressed as a volume ratio. Preferably, after mixing the bone marrow fluid and the reagent, the mixture is incubated under certain conditions. Examples of the certain conditions include incubation at a temperature of 15-50°C, preferably 30-45°C, for 5-120 seconds, preferably 5-30 seconds. Preparation of the measurement sample can be performed manually or by an automated blood cell analyzer.

[0142] In the present embodiment, the measurement of nucleated cells and lipid particles in the measurement sample is preferably performed by FCM. In the measurement performed by FCM, the prepared measurement sample is irradiated with light, and optical information is obtained. Specifically, first, the measurement sample is introduced into the flow cell of FCM, and light is irradiated on the particles one by one in the sample as they pass through the flow cell. Then, the scattered light and fluorescence emitted from the particles are measured to obtain optical information. Here, particles refer to formed elements present in the measurement sample, such as cells, lipid particles, debris such as the remains of hemolyzed red blood cells (red blood cell ghosts), etc. When the reagent does not contain a fluorescent pigment, it is preferred to obtain scattered light information as optical information. When the reagent contains a fluorescent pigment, it is preferred to obtain scattered light information and fluorescence signal information as optical information.

[0143] It may sometimes be impossible to distinguish lipid particles from other blood cells well using only two of the fluorescence signal information, forward scattered light information, and side scattered light information. Therefore, in this embodiment, it is preferred to obtain the lipid particle count based on the fluorescence signal information, forward scattered light information, and side scattered light information obtained by flow cytometry.

[0144] Preferably, the scattered light information is forward scattered light information and side scattered light information. As mentioned above, the scattered light information can list the peak, pulse width and pulse area of ​​the pulses of forward scattered light (for example, the light receiving angle is near 0 to 20 degrees) and side scattered light (for example, the light receiving angle is near 80 to 100 degrees). It is known that forward scattered light reflects the size of the cell, and side scattered light reflects the internal information such as the nucleus and particles in the cell. In the present embodiment, it is preferred to obtain the forward scattered light intensity and / or the side scattered light intensity as the scattered light information. Fluorescent signal information can, for example, list fluorescence intensity, fluorescence pulse width and fluorescence pulse area. Among them, fluorescence intensity is preferred. The wavelength of the irradiated excitation light can be appropriately selected according to the fluorescent pigment.

[0145] The FCM is not particularly limited, and commercially available automatic blood cell analyzers can be used. Examples of such devices include the XN series from Sysmex Corporation. The light source for the FCM is not particularly limited, and a light source having a wavelength suitable for excitation of the fluorescent pigment can be appropriately selected. Examples of light sources include blue semiconductor lasers, red semiconductor lasers, argon lasers, helium-neon lasers, and mercury arc lamps.

[0146] The number of nucleated cells and lipid particles in the measurement sample can be determined based on the optical information obtained through FCM measurement. In this embodiment, it is preferred to create a scatter plot with two selected from forward scattered light information, side scattered light information, and fluorescence signal information as the X-axis and Y-axis, and analyze the resulting scatter plot using appropriate analysis software to determine the number of nucleated cells and lipid particles. Each particle measured by FCM is represented by a dot on the scatter plot.

[0147] For example, when measuring with reagents for nucleated red blood cells and white blood cells and creating a scatter plot with fluorescence intensity as the X-axis and forward scattered light intensity as the Y-axis, Figure 1A As shown in the figure, white blood cells and nucleated red blood cells form a cluster distribution. When the white blood cell classification reagent is used for measurement and a scatter plot is created with the side scattered light intensity as the X axis and the fluorescence intensity as the Y axis, as shown in the figure, Figure 1B As shown in the figure, white blood cells form five clusters: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Furthermore, lipid particles also form clusters in areas with little fluorescence intensity. When a scatter plot is created using a universal reagent with fluorescence intensity as the X-axis and side scattered light intensity as the Y-axis, as shown in the figure, Figure 1C As shown, nucleated cells and lipid particles form clusters. For example, the number of cells in each cluster can be obtained by counting the points in each cluster on the scatter plot using the analysis software installed in FCM. Figure 1A ˜C are examples of scatter plots, but the present invention is not limited thereto.

[0148] (Obtaining indicators related to bone marrow nucleated cell density)

[0149] In the analysis method of this embodiment, an index related to bone marrow nucleated cell density is obtained based on the nucleated cell count and lipid particle count. In a preferred embodiment, the index related to bone marrow nucleated cell density is information related to bone marrow nucleated cell density obtained using the nucleated cell count and lipid particle count obtained by FCM measurement. Furthermore, the index related to bone marrow nucleated cell density is not limited to the values ​​obtained using the nucleated cell count and lipid particle count per se; it may also be information indicating, for example, hyperplasia, normal development, or hypoplasia, after determination using these values.

[0150] In this embodiment, it is preferred that the bone marrow nucleated cell density-related index is a related value of the ratio of the number of nucleated cells to the number of lipid particles. The related values ​​of the ratio of the number of nucleated cells (NC) to the number of lipid particles (LP) can be listed, for example, the ratio of the number of lipid particles to the number of nucleated cells (LP / NC), the ratio of the number of nucleated cells to the number of lipid particles (NC / LP), the ratio of the number of lipid particles to the sum of the number of lipid particles and the number of nucleated cells (LP / (NC+LP)), the ratio of the number of nucleated cells to the sum of the number of lipid particles and the number of nucleated cells (NC / (NC+LP)), and the values ​​calculated from the above ratios. The values ​​calculated from the above ratios can be listed, for example, as the values ​​obtained by using arbitrary coefficients and / or constants when calculating the above ratios. For example, in order to make the value of LP / NC and the value of the bone marrow nucleated cell density of the smear specimen under microscopic observation be of the same order of magnitude, arbitrary coefficients and / or constants can be used. In addition, the ratio of the number of nucleated cells to the number of lipid particles can be multiplied by 100 to express the ratio as a percentage. In addition, as needed, the bone marrow nucleated cell density-related index can also be an arbitrary value added or subtracted from the above ratio. In a preferred embodiment, the value related to the ratio of nucleated cell number to lipid particle number is preferably the ratio of nucleated cell number to lipid particle number, and LP / NC is particularly preferred.

[0151] Indicators related to bone marrow nucleated cell density can be output to an output unit of a sample analyzer. The output unit is preferably comprised of a display such as a liquid crystal display, plasma display, or CRT (Cathode Ray Tube) monitor mounted on an FCM. Indicators related to bone marrow nucleated cell density can be used as information with equivalent value to the bone marrow nucleated cell density of a smear specimen observed under a microscope. Medical professionals such as physicians can use indicators related to bone marrow nucleated cell density for, for example, the identification of blood disorders. Furthermore, indicators related to bone marrow nucleated cell density are preferably used in combination with other test results and medical findings.

[0152] (Determination of bone marrow nucleated cell density)

[0153] In this technical field, it is determined whether the bone marrow nucleated cell density of a smear specimen under microscopic observation is hypoplastic, normally developed, or hyperplastic. As shown in the examples, the bone marrow nucleated cell density-related index obtained by the analysis method of this embodiment is well correlated with the bone marrow nucleated cell density of the smear specimen under microscopic observation. In the analysis method of this embodiment, the bone marrow state can be determined based on the number of nucleated cells and the number of lipid particles. Alternatively, the bone marrow state can be determined based on the bone marrow nucleated cell density-related index. For example, at least one determination selected from "whether the bone marrow state is hypoplastic," "whether the bone marrow state is normally developed," and "whether the bone marrow state is hyperplasia" can be made based on the bone marrow nucleated cell density-related index.

[0154] The above determination is preferably made based on a comparison of an indicator related to bone marrow nucleated cell density with a threshold corresponding to the indicator. For example, when using the ratio of LP / NC as an indicator related to bone marrow nucleated cell density to determine hypoplasia, the LP / NC value is compared with a first threshold. In this embodiment, when the LP / NC value is higher than the first threshold, the bone marrow is determined to be hypoplastic. When the LP / NC value is lower than the first threshold, the bone marrow is determined to be normally developed or hyperplastic.

[0155] In this embodiment, when the LP / NC value is equal to the first threshold, the bone marrow status can be determined as hypoplasia, or as normal development or hyperplasia. That is, when the LP / NC value is greater than the first threshold, the bone marrow status can be determined as hypoplasia. Alternatively, when the LP / NC value is less than the first threshold, the bone marrow status can be determined as normal development or hyperplasia.

[0156] For example, when using LP / NC as an indicator related to bone marrow nucleated cell density to determine hyperplasia, the LP / NC value is compared with a second threshold value. In this embodiment, when using LP / NC as an indicator related to bone marrow nucleated cell density, the second threshold value is a value lower than the first threshold value. When the LP / NC value is lower than the second threshold value, the bone marrow status is determined to be hyperplastic. When the LP / NC value is higher than the second threshold value, the bone marrow status is determined to be normal development or hypoplasia.

[0157] In this embodiment, when the LP / NC value is equal to the second threshold, the bone marrow status can be determined as hyperplasia, or as normal development or hypoplasia. That is, when the LP / NC value is below the second threshold, the bone marrow status can be determined as hyperplasia. Alternatively, when the LP / NC value is above the second threshold, the bone marrow status can be determined as normal development or hypoplasia.

[0158] For example, when using LP / NC as an indicator of bone marrow nucleated cell density to determine normal development, the LP / NC value is compared with a first threshold and a second threshold. If the LP / NC value is lower than the first threshold and higher than the second threshold, the bone marrow is considered to be developing normally.

[0159] In this embodiment, the bone marrow status can be determined to be normally developing when the LP / NC value is the same as the first threshold. Alternatively, the bone marrow status can be determined to be normally developing when the LP / NC value is the same as the second threshold. That is, the bone marrow status can be determined to be normally developing when the LP / NC value is below the first threshold and above the second threshold. Alternatively, the bone marrow status can be determined to be normally developing when the LP / NC value is below the first threshold and above the second threshold. Alternatively, the bone marrow status can be determined to be normally developing when the LP / NC value is below the first threshold and above the second threshold.

[0160] The determination result of the bone marrow status can be output to the output unit. In this way, the analysis method of this embodiment can provide medical personnel such as doctors with information that assists in determining the bone marrow status.

[0161] The threshold value is not particularly limited and can be set as appropriate. For example, the number of nucleated cells and lipid particles in the bone marrow fluid of healthy individuals and patients with various blood diseases can be measured, and the accumulated data can be used to empirically set the threshold value. Specifically, the threshold value can be set as follows.

[0162] First, bone marrow fluid is collected from subjects including several healthy normal people and patients with various blood diseases, and the bone marrow nucleated cell density of the smear specimen under microscope observation and the bone marrow nucleated cell density-related index obtained by FCM measurement are obtained. Next, based on the bone marrow nucleated cell density of the smear specimen under microscope observation, the subjects are classified into a hypoplastic group, a normally developed group, and a hyperplastic group. Then, for the bone marrow nucleated cell density-related index obtained by FCM measurement, a value that can distinguish each group with the highest accuracy is obtained, and this value is set as a certain threshold. In setting the threshold, it is preferred to consider sensitivity, specificity, positive predictive value, negative predictive value, etc.

[0163] The first threshold value can be set, for example, from a range higher than 0.3 to 0.75 or less, preferably from 0.4 to 0.5 or less. The second threshold value can be set, for example, from 0.05 to 0.3 or less, preferably from 0.08 to 0.25 or less.

[0164] [2. Sample analysis device]

[0165] An example of a sample analysis device according to this embodiment will be described below with reference to the drawings.

[0166] (Structure of Sample Analysis Device)

[0167] like Figure 2A As shown, the sample analyzer 1 includes a measuring unit 2 and an analyzing unit 3. The measuring unit 2 receives bone marrow fluid, prepares a measurement sample from the bone marrow fluid, and performs optical measurements on the measurement sample. The analyzing unit 3 processes the measurement data obtained by the measuring unit 2 and outputs the analysis results of the bone marrow fluid. However, this embodiment is not limited to this example; for example, a device in which the measuring unit 2 and analyzing unit 3 are integrally formed may be used.

[0168] The measuring unit 2 includes an aspirating unit 4, a sample preparing unit 5, a detecting unit 6, a signal processing circuit 81, a microcomputer 82, and a communication interface 83. The aspirating unit 4 includes a pipette 42. The aspirating unit 4 aspirates the bone marrow fluid contained in the test tube 41 using the pipette 42.

[0169] The sample preparation unit 5 has a reaction tank 54 and is connected to reagent containers 51, 52, and 53. The test tube 41 contains bone marrow fluid. The reagent container 51 contains a diluent. The diluent contained in the reagent container 51 is used as a sheath fluid in flow cytometry measurements. The reagent container 52 contains a lysis reagent containing a hemolytic agent. The reagent container 53 contains a staining reagent containing a fluorescent dye. The pipetting unit 4 moves the pipette 42 toward the top of the reaction tank 54 and discharges the bone marrow fluid aspirated from the test tube 41 into the reaction tank 54. The bone marrow fluid, the lysis reagent, and the staining reagent are mixed in the reaction tank 54 to prepare a measurement sample. In this embodiment, a single reagent containing both a hemolytic agent and a fluorescent dye can be mixed with the bone marrow fluid to prepare the measurement sample. The measurement sample is supplied to an optical measurement performed by flow cytometry. In addition, in this embodiment, the number of nucleated cells and the number of lipid particles in the bone marrow fluid are obtained by flow cytometry, but the present invention is not limited to this. For example, a bone marrow fluid smear can be imaged and analyzed to determine the number of nucleated cells and lipid particles. Alternatively, cells can be made to flow in a curved flow path and, by varying the amount of force applied to the particles, the flow path can be altered to target individual nucleated cells and lipid particles, separating the particles. The separated nucleated cells and lipid particles can then be counted separately to determine the number of nucleated cells and lipid particles.

[0170] like Figure 2B As shown, the sample analyzer of this embodiment may include two or more sample preparation units. Figure 2B The sample analyzer 1 is similar to the sample analyzer 1 in that the measuring unit 2 includes the sample preparation units 5a and 5b. Figure 2AThe sample analysis device 1 is the same as the sample preparation unit 5a. The sample preparation unit 5a has a reaction tank 54a connected to reagent containers 51a, 52a, and 53a. Test tube 41 contains bone marrow fluid. The sample preparation unit 5b has a reaction tank 54b connected to reagent containers 51b, 52b, and 53b. Reagent containers 51a and 51b contain a diluent. Reagent containers 52a and 52b contain a lysis reagent containing a hemolytic agent. The types of hemolytic agents in reagent containers 52a and 52b may be different. Reagent containers 53a and 53b contain a staining reagent containing a fluorescent dye. The types of fluorescent dyes in reagent containers 53a and 53b may be different. For example, reagent containers 52a and 53a may contain a lysis reagent for counting nucleated red blood cells and white blood cells, and a staining reagent, respectively, as a first reagent. Furthermore, reagent containers 52b and 53b may contain a lysis reagent for classifying white blood cells, and a staining reagent, respectively, as a second reagent. The aspirator 4 discharges the bone marrow fluid aspirated from the test tube 41 into reaction reservoirs 54a and 54b, respectively. The bone marrow fluid discharged into reaction reservoir 54a is called the first bone marrow fluid, and the bone marrow fluid discharged into reaction reservoir 54b is called the second bone marrow fluid. The first measurement sample is prepared in reaction reservoir 54a, and the second measurement sample is prepared in reaction reservoir 54b.

[0171] In this embodiment, the detection unit 6 is used for optical measurement of particles by flow cytometry. The detection unit 6 includes a flow chamber 61, a light source unit 62, and light receiving units 63 and 64. The diluent contained in the reagent container 51 and the measurement sample prepared by the sample preparation unit 5 are supplied to the flow chamber 61. The following describes a method for detecting particles in the detection unit 6 by flow cytometry and obtaining the number of nucleated cells and the number of lipid particles, but is not limited to this. The detection unit 6 can be designed to have a shooting unit for shooting a smear specimen of bone marrow fluid, and the number of nucleated cells and the number of lipid particles are obtained based on the particle image shot by the shooting unit.

[0172] The flow cell 61 is made of a light-transmitting material such as quartz, glass, or synthetic resin and is formed into a tubular shape. The interior of the flow cell 61 is a flow path through which the measurement sample and sheath fluid flow. Figure 3 The flow chamber 61 has an orifice 61a whose internal space is narrower than the rest of the chamber. Furthermore, the vicinity of the entrance to the orifice 61a has a double-tube structure, the inner portion of which serves as a sample nozzle 61b, through which the measurement sample prepared by the sample preparation unit 5 is supplied. The space outside the sample nozzle 61b serves as a flow path 61c through which sheath liquid flows. The sheath liquid is introduced into the orifice 61a through the flow path 61c. In this manner, the sheath liquid supplied to the flow chamber 61 flows so as to envelop the measurement sample discharged from the sample nozzle 61b. Since the measurement sample flow is narrowed by the orifice 61a, particles in the measurement sample encapsulated by the sheath liquid pass through the orifice 61a one by one.

[0173] The light source 62 is a semiconductor laser light source, for example, emitting red laser light with a wavelength of 633 nm toward the orifice 61a of the flow cell 61. When the sample flow in the flow cell 61 is illuminated by the light, the light receiving units 63, 64, and 65 detect the light emitted from the individual particles in the sample. Light receiving units 63, 64, and 65 can employ avalanche photodiodes (APDs), photodiodes, or photomultiplier tubes. Hereinafter, the direction connecting the light source 62 and the flow cell 61 is referred to as the "X direction," and the direction orthogonal to the X direction is referred to as the "Y direction." A dichroic mirror 66 is disposed on the Y side of the flow cell 61. The dichroic mirror 66 transmits fluorescence emitted by the individual particles in the sample and reflects side-scattered light emitted by the particles. The light receiving unit 63 is disposed on the Y side of the flow cell 61 and detects the fluorescence that passes through the dichroic mirror 66. The light receiving unit 65 detects the side-scattered light reflected from the dichroic mirror 66. The light receiving unit 64 is arranged on the X direction side of the flow cell 61. More specifically, the light receiving unit 64 is arranged on the opposite side of the light source unit 62 with the flow cell 61 interposed therebetween. The light receiving unit 64 can detect forward scattered light emitted from each particle of the measurement sample.

[0174] Side scattered light is not limited to light scattered in a direction (Y direction) of 90° relative to the optical axis direction (X direction) of the light source unit 62. For example, side scattered light may be light scattered in a direction of 80° to 100° relative to the X direction. Forward scattered light is not limited to light scattered in the optical axis direction (X direction) of the light source unit 62. For example, forward scattered light may be light scattered in a direction of -10° to 10° relative to the X direction.

[0175] In this embodiment, an irradiation lens system composed of a plurality of lenses (not shown) may be disposed between the light source unit 62 and the flow cell 61. The irradiation lens system can focus the parallel beam emitted from the semiconductor laser light source.

[0176] Light receiving units 63, 64, and 65 perform photoelectric conversion on the detected fluorescence, forward scattered light, and side scattered light, respectively, and output analog signals representing the intensity of the received light. Hereinafter, the analog signal output from light receiving unit 63 is referred to as a "fluorescence signal," the analog signal output from light receiving unit 64 is referred to as a "forward scattered light signal," and the analog signal output from light receiving unit 65 is referred to as a "side scattered light signal."

[0177] The signal processing circuit 81 processes the analog signals output by the light receiving units 63, 64, and 65. The signal processing circuit 81 extracts the peak values ​​of the pulses contained in the fluorescence signal, the forward scattered light signal, and the side scattered light signal as characteristic parameters. Hereinafter, the peak value of the fluorescence signal will be referred to as "fluorescence intensity," the peak value of the forward scattered light signal will be referred to as "forward scattered light intensity," and the peak value of the side scattered light signal will be referred to as "side scattered light intensity."

[0178] Microcomputer 82 controls the aspiration unit 4, sample preparation unit 5, detection unit 6, signal processing circuit 81, and communication interface 83. Communication interface 83 is connected to analysis unit 3 via a communication cable. Measurement unit 2 communicates data with analysis unit 3 via communication interface 83. Communication interface 83 transmits measurement data, including various characteristic parameters, to analysis unit 3.

[0179] Reference Figure 4 The structure of the analysis unit 3 is described below. The analysis unit 3 includes a main body 300, an input unit 309, and an output unit 310. The main body 300 includes a CPU (Central Processing Unit) 301, a ROM (Read Only Memory) 302, a RAM (Random Access Memory) 303, a hard disk 304, a reader 305, an input / output interface 306, an image output interface 307, and a communication interface 308. In this embodiment, a display that displays images is used as the output unit 310.

[0180] CPU301 executes the computer program 322 stored in ROM302 and the computer program loaded into RAM303. RAM303 is used to read out each computer program recorded in ROM302 and hard disk 304. When executing each computer program, RAM303 also serves as the work area of ​​CPU301. Hard disk 304 is installed with application 320, which is a computer program for analyzing the measurement data obtained from measurement unit 2 and outputting the analysis results. Computer program 322 includes BIOS (Basic Input Output System). Application 320 includes OS (Operating System), bone marrow fluid analysis program, and bone marrow status determination program. The bone marrow fluid analysis program refers to a program for obtaining the number of nucleated cells and lipid particles in the bone marrow fluid, and obtaining bone marrow nucleated cell density-related indicators based on the number of nucleated cells and lipid particles. The bone marrow status determination program refers to a program for determining the state of the bone marrow based on the bone marrow nucleated cell density-related indicators.

[0181] The reading device 305 is a CD-ROM drive, a DVD-ROM drive, a USB port, an SD card reader, a CF card reader, a memory stick reader, a solid-state drive, a floppy disk drive, or the like, and is capable of reading computer programs or data recorded on a removable recording medium 321. Furthermore, the removable recording medium 321 stores a computer program 320 that causes the computer to function as the analysis unit 3. The computer program 320 read from the removable recording medium 321 is installed on the hard disk 304.

[0182] The input unit 309 is connected to the input / output interface 306. The output unit 310 is connected to the image output interface 307. The communication interface 308 is connected to the communication interface 83 of the measurement unit 2.

[0183] (Operation of the Sample Analyzer)

[0184] Reference Figure 5 Next, the operation of the sample analyzer 1 will be described. First, the CPU 301 of the analysis unit 3 receives an instruction to execute measurement from the user via the input unit 309 (step S101).

[0185] Preferably, the input of a measurement execution instruction is accepted based on a sample type selection interface displayed on the output unit 310 of the sample analyzer 1. For example, if the sample analyzer 1 can measure both blood samples and body fluid samples including bone marrow fluid, a sample type selection interface for specifying the sample type can be displayed on the input interface for a new analysis instruction, allowing the user to select the sample type from blood and body fluid. After setting the sample type and measurement items, the user clicks "Start Measurement" displayed on the interface to instruct the measurement execution. The sample type selection is not limited to this example; a specific body fluid type, such as bone marrow fluid, cerebrospinal fluid, pleural effusion, or ascites, can also be specified.

[0186] As previously mentioned, bone marrow fluid collected from a subject may sometimes contain solid impurities such as bone fragments and blood cell aggregates that may interfere with cell measurement. Furthermore, the concentrations of blood cells in bone marrow fluid and blood differ. Therefore, when the sample analyzer 1 is capable of measuring both blood and bone marrow fluid, in order to suppress the effects of carryover contamination between blood and bone marrow fluid samples, an additional cleaning operation may be performed on the pipetting unit 4 and flow chamber 61 in addition to the usual cleaning operation, based on the sample type set when the measurement execution instruction is input. Specifically, when bone marrow fluid is set as the sample type, the aforementioned additional cleaning operation is performed before the pipetting unit 4 aspirates the bone marrow fluid.

[0187] After the analysis unit 3 receives the instruction to start measurement, the CPU 301 sends instruction data to the measurement unit 2 to start measurement (step S102). The measurement unit 2 receives the instruction data (step S103). The microcomputer 82 of the measurement unit 2 executes measurement sample preparation processing (step S104) and then performs measurement processing (step S105).

[0188] Reference Figure 6 , the measurement sample preparation process is described. The microcomputer 82 controls the pipetting unit 4 to supply a certain amount of bone marrow fluid to the reaction tank 54 (step S201). Next, the microcomputer 82 controls the sample preparation unit 5 to supply a certain amount of the first reagent from the reagent container 52 to the reaction tank 54, and to supply a certain amount of the second reagent from the reagent container 53 to the reaction tank 54 (step S202). The reaction tank 54 is heated to a certain temperature by the heater. In the heated state, the mixture in the reaction tank 54 is stirred (step S203). Through the actions of steps S201 to S203, the measurement sample is prepared in the reaction tank 54. The microcomputer 82 controls the sample preparation unit 5 to guide the measurement sample from the reaction tank 54 to the detection unit 6 (step S204). When a measurement unit having multiple reaction tanks is used, for example, when preparing multiple measurement samples such as a first measurement sample for nucleated cell measurement and a second measurement sample for lipid particle measurement, the above steps are repeated. After completing the processing of step S204, the microcomputer 82 returns to the main routine.

[0189] Refer again Figure 5 During the measurement process after sample preparation, the detection unit 6 performs measurement of the sample. The sample preparation unit 5 supplies both the sheath fluid and the sample to the flow cell 61. After the sample flows through the flow cell 61, the particles sequentially pass through the orifice 61a of the flow cell 61. The light source unit 62 irradiates the sample flowing through the flow cell 61 with light. More specifically, the light source unit 62 irradiates each particle passing through the orifice 61a of the flow cell 61 with light. Each time a particle is irradiated with light, fluorescence, forward scattered light, and side scattered light are emitted from the particle.

[0190] The fluorescence emitted by the particles is detected by light receiving unit 63. The forward scattered light emitted by the particles is detected by light receiving unit 64. The side scattered light emitted by the particles is detected by light receiving unit 65. Light receiving units 63, 64, and 65 output electrical signals corresponding to the light reception levels as a fluorescence signal, a forward scattered light signal, and a side scattered light signal, respectively. Signal processing circuit 81 extracts the fluorescence intensity from the fluorescence signal, the forward scattered light intensity from the forward scattered light signal, and the side scattered light intensity from the side scattered light signal. After the measurement process, microcomputer 82 transmits the measurement data including each characteristic parameter to analysis unit 3 (step S106), concluding the process.

[0191] The analysis unit 3 receives the measurement data (step S107 ). The CPU 301 then executes a measurement data analysis process to generate an analysis result of the bone marrow fluid and stores the analysis result in the hard disk 304 (step S108 ).

[0192] Reference Figure 7 The measurement data analysis process will be described. After the measurement data analysis process begins, the CPU 301 of the analysis unit 3 classifies nucleated cells and lipid particles based on the fluorescence intensity, forward scattered light intensity, and side scattered light intensity contained in the measurement data (step S301). The CPU 301 can use the data of fluorescence intensity, forward scattered light intensity, and side scattered light intensity to create a scatter plot. After measuring multiple measurement samples, a scatter plot can be created for each measurement sample based on the data of each measurement sample.

[0193] The processing of step S301 is described by taking the example of using universal reagents as the above-mentioned lysis reagent and staining reagent. However, the present invention is not limited to this example. In step S301, CPU 301 combines the particle size distribution of the side scattered light intensity and the particle size distribution of the fluorescence intensity to determine the group including leukocytes, the group including erythroblasts, the group including lipid particles, and the group including red blood cell ghosts. More specifically, for example, Figure 1C As shown, CPU 301 identifies a particle group with both high fluorescence intensity and side scattered light intensity as a group including leukocytes. CPU 301 identifies a group with the same side scattered light intensity as that of the group including leukocytes and a lower fluorescence intensity than that of the group including leukocytes as a group including erythroblasts. CPU 301 identifies a group with the same or higher side scattered light intensity as that of the group including leukocytes and a lower fluorescence intensity than that of the group including erythroblasts as a group including lipid particles. CPU 301 identifies a group with both lower side scattered light intensity and lower fluorescence intensity than that of the group including erythroblasts as a group including erythrocyte ghosts.

[0194] In step S302, CPU 301 counts the number of particles in the group of leukocytes and the group of erythroblasts classified in step S301 as the nucleated cell count, and also counts the number of particles in the group of lipid particles. CPU 301 then stores the nucleated cell count and the lipid particle count on hard disk 304. In step S303, CPU 301 obtains an index related to bone marrow nucleated cell density based on the nucleated cell count and lipid particle count. When obtaining the ratio of the lipid particle count to the nucleated cell count as this index, CPU 301 divides the lipid particle count by the nucleated cell count to obtain the ratio of the lipid particle count to the nucleated cell count. CPU 301 stores the obtained index related to bone marrow nucleated cell density on hard disk 304.

[0195] In step S304, when the CPU 301 receives an instruction from the user to start the determination via the input unit 309, the CPU 301 determines the bone marrow condition based on the bone marrow nucleated cell density-related index acquired in step S303. If the CPU 301 does not receive an instruction from the user to start the determination, the CPU 301 ends the measurement data analysis process and returns the process to the main routine. Figure 5 After the above-mentioned measurement data analysis process is completed, the CPU 301 outputs the analysis result to the output unit 310 (step S109), and the process ends. In addition, in this embodiment, the bone marrow status is determined based on the instruction from the user to start the determination execution, but it is not limited to this. A technical solution can also be used in which the determination is automatically performed even without the instruction from the user.

[0196] [3. Computer Program]

[0197] Reference Figure 8A , the determination process of whether the bone marrow state is hypoplastic is described. Here, the case where the value of the ratio of the number of lipid particles to the number of nucleated cells (LP / NC) is obtained as an indicator related to the density of nucleated cells in the bone marrow is described as an example. However, the present invention is not limited to this example. In step S401, CPU301 compares the obtained LP / NC value with the first threshold value stored in the hard disk 304. When the measured value is higher than a certain threshold value, the processing proceeds to step S402. CPU301 obtains the determination result that the bone marrow state is hypoplastic, and stores it in the hard disk 304. When the LP / NC value is below the first threshold value, the processing proceeds to step S403. CPU301 obtains the determination result that the bone marrow state is normal development or hyperplasia, and stores it in the hard disk 304. Above, CPU301 ends the determination process and returns the process to the main program. Refer to Figure 5 , CPU 301 outputs the determination result to output unit 310 (step S110 ), and ends the processing.

[0198] Reference Figure 8B , the determination process of whether the bone marrow state is hyperplasia is described. Here, the case where the LP / NC value is obtained as an indicator related to the density of nucleated cells in the bone marrow is described as an example. However, the present invention is not limited to this example. In step S501, CPU301 compares the obtained LP / NC value with the second threshold value stored in the hard disk 304. When the measured value is lower than a certain threshold value, the processing proceeds to step S502. CPU301 obtains the determination result that the bone marrow state is hyperplasia, and stores it in the hard disk 304. When the LP / NC value is above the second threshold value, the processing proceeds to step S503. CPU301 obtains the determination result that the bone marrow state is normal development or underdevelopment, and stores it in the hard disk 304. Above, CPU301 ends the determination process and returns the process to the main program. Refer to Figure 5, CPU 301 outputs the determination result to output unit 310 (step S110 ), and ends the processing.

[0199] Reference Figure 8C , the process of determining whether the bone marrow state is hypoplasia, normal development, or hyperplasia will be described. Here, the case where the LP / NC value is obtained as an indicator related to bone marrow nucleated cell density will be described as an example. However, the present invention is not limited to this example. In step S601, CPU 301 compares the obtained LP / NC value with a first threshold value stored in hard disk 304. If the measured value is higher than a certain threshold value, the process proceeds to step S602. CPU 301 obtains the result of determining that the bone marrow state is hypoplasia and stores it in hard disk 304. If the LP / NC value is below the first threshold value, the process proceeds to step S603.

[0200] In step S603, CPU301 compares the obtained LP / NC value with the second threshold value stored in the hard disk 304. When the measured value is lower than the certain threshold value, the processing proceeds to step S604. CPU301 obtains the determination result that the bone marrow state is hyperplasia and stores it in the hard disk 304. When the LP / NC value is above the second threshold value, the processing proceeds to step S605. CPU301 obtains the determination result that the bone marrow state is normal development and stores it in the hard disk 304. Above, CPU301 ends the determination processing and returns the processing to the main program. Figure 5 , CPU 301 outputs the determination result to output unit 310 (step S110 ), and ends the processing.

[0201] In this embodiment, the Figure 8A In Steps 1 to 3, the "determination process of whether it is hypoplasia", the "determination process of whether it is hyperplasia", and the "determination process of whether it is hypoplasia, normal development, or hyperplasia" are described separately, but the bone marrow status determination process (S305) only needs to execute part of them, or all of them can be performed.

[0202] Reference Figure 9 , an example of analysis results displayed on the output unit 310 will be described. However, the present invention is not limited to this example. The output unit 310 displays an analysis result interface 500. The analysis result interface 500 includes a sample information display area 510, a patient information display area 520, a measurement result display area 530, and a reference information display area 540.

[0203] Sample information display area 510 displays information about the measured bone marrow fluid. Patient information display area 520 displays information about the subject whose bone marrow fluid was collected. Measurement result display area 530 displays the measured values ​​of various items obtained through analysis of the measurement data. The measured values ​​displayed in measurement result display area 530 include the white blood cell count (WBC), nucleated red blood cell count (NRBC), and lipid particle count (LIPID). The LP / NC ratio is also displayed as an indicator of bone marrow nucleated cell density. Indicators related to bone marrow nucleated cell density are not limited to the LP / NC ratio; other values ​​may be displayed. Furthermore, the sum of the white blood cell count (WBC) and nucleated red blood cell count (NRBC) may be displayed as the nucleated cell count. Measurement result display area 530 displays scatter plots 531 and 532 used for counting the lipid particle count. Scatter plot 531 shows the distribution of particles in a coordinate space with side scattered light intensity and fluorescence intensity as axes, while scatter plot 532 shows the distribution of particles in a coordinate space with side scattered light intensity and forward scattered light intensity as axes. In addition, a scattergram 533 used for counting the number of nucleated cells is displayed in the measurement result display area 530 . The scattergram 533 shows the distribution of particles in a coordinate space having fluorescence intensity and forward scattered light intensity as coordinate axes.

[0204] When the bone marrow condition is determined based on the index related to the bone marrow nucleated cell density, the determination result is displayed in the reference information display area 540 . Figure 9 In this example, the output unit 310 displays a "Normocellular" mark as a result of the determination that the subject's bone marrow status is normal. Alternatively, if the bone marrow status is hyperplasia, for example, the output unit 310 may display a "Hypercellular" mark. Alternatively, if the bone marrow status is hypoplasia, for example, the output unit 310 may display a "Hypocellular" mark. However, the information indicating the determination result is not limited to this.

[0205] As described above, the sample analyzer and computer program of this embodiment can assist in the diagnosis and differentiation of blood diseases by providing the bone marrow nucleated cell density-related index and the determination results based on the bone marrow nucleated cell density-related index to physicians and the like.

[0206] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example

[0207] Example 1 (1) Reagents

[0208] In Example 1, a first reagent for measuring nucleated cells and a second reagent for measuring lipid particles were used. The first reagent was a reagent for counting nucleated red blood cells and white blood cells, consisting of a lysis reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye. The second reagent was a reagent for classifying white blood cells, consisting of a lysis reagent containing a hemolytic agent and a staining reagent containing a fluorescent dye. Each reagent was prepared as follows.

[0209] (1.1) First Reagent: Dissolving Reagent

[0210] Dodecyltrimethylammonium chloride (LTAC: Tokyo Chemical Industry Co., Ltd.), polyoxyethylene (20) polyoxypropylene (8) hexadecyl ether (PBC-44: Nikko Chemical Co., Ltd.), potassium hydrogen phthalate (Fujifilm Wako Pure Chemical Industries, Ltd.), DL-malic acid, and EDTA-2K (Chubu Chelest Co., Ltd.) were mixed in the following composition. Purified water was used as the solvent. The pH of the reagent was adjusted to 3.0 using sodium hydroxide.

[0211] [Composition of the dissolving reagent] 2000 ppm LTAC, 1000 ppm PBC-442 mM potassium hydrogen phthalate, 10 mM D-malic acid, 0.2 g / L EDTA-2K, 0.0324 g / L NaOH.

[0212] ・Staining reagents

[0213] Fluorescent dye NK-3383 (Hayashibara Co., Ltd.) was dissolved in ethylene glycol. The concentration of NK-3383 in the staining reagent was 51.1 mg / L.

[0214] (1.2) Second reagent, dissolving reagent

[0215] LTAC (Tokyo Chemical Industry Co., Ltd.), polyoxyethylene (30) hexadecyl ether (BC30TX: Nikko Chemical Co., Ltd.), potassium hydrogen phthalate (Fujifilm Wako Pure Chemical Industries, Ltd.), and EDTA-2K (Chubu Chelest Co., Ltd.) were mixed in the following composition. Purified water was used as the solvent. The pH of the reagent was adjusted to 6.0 using sodium hydroxide.

[0216] [Composition of the dissolving reagent] 685 ppm LTAC, 1750 ppm BC30TX, 40 mM potassium hydrogen phthalate, 0.2 g / LEDTA-2K, 1.431 g / L NaOH.

[0217] ・Staining reagents

[0218] As the staining reagent of the second reagent, Stromatolyser 4DS (Sysmex Corporation) was used.

[0219] (2) Measurement (2.1) Preparation of measurement samples

[0220] Bone marrow fluid (bone marrow fluid supplemented with EDTA-2K) collected from 18 subjects was used. A portion of the bone marrow fluid of each subject was taken out and stained according to a conventional method to prepare a smear specimen. The remaining bone marrow fluid was filtered through a nylon mesh with a pore size of 40 μm to remove bone fragments. The bone marrow fluid was measured as follows using an automatic blood cell analyzer XN-2000 (Sysmex Corporation) with FCM. First, the bone marrow fluid was divided into the first bone marrow fluid and the second bone marrow fluid. The dissolving reagent (50 μL) and the staining reagent (1 μL) of the first reagent were added to the first bone marrow fluid (1 μL), and the mixture was incubated at 40°C for 20 seconds to prepare the first measurement sample. The dissolving reagent (50 μL) and the staining reagent (1 μL) of the second reagent were added to the second bone marrow fluid (1 μL), and the mixture was incubated at 40°C for 20 seconds to prepare the second measurement sample.

[0221] (2.2) Measurement of test samples

[0222] The first measurement sample was irradiated with light, and optical signals emitted from each particle in the sample were obtained. Separately, the second measurement sample was irradiated with light, and optical information emitted from each particle in the sample was obtained. In Example 1, fluorescence intensity, side scattered light intensity, and forward scattered light intensity were obtained as optical information. Furthermore, a scatter plot was created based on the optical signals obtained from the measurement of each measurement sample. Furthermore, the distribution of bone marrow fluid, preparation of measurement samples, and measurement were performed automatically by the XN-2000.

[0223] An example of a scatter plot is shown in Figure 10A , B and C. Figure 10A This is a scatter plot of the measurement using the first reagent (reagent for counting nucleated red blood cells and white blood cells). Figure 10B A and C are scattergrams of measurements using the second reagent (reagent for leukocyte differentiation). Figure 10A In the figure, the X-axis is the fluorescence intensity and the Y-axis is the forward scattered light intensity. Figure 10B In the figure, the X-axis is the side scattered light intensity and the Y-axis is the fluorescence intensity. Figure 10C In the figure, the X-axis is the side scattered light intensity and the Y-axis is the forward scattered light intensity. Figure 10A In FIG, as shown in the area surrounded by an ellipse, a group of nucleated erythrocytes, a group of basophils, and a group of leukocytes other than basophils appear as a group of nucleated cells. Figure 10B In the figure, as shown by the elliptical area, white blood cells are divided into five groups: lymphocytes, monocytes, neutrophils and basophils, eosinophils, and immature granulocytes. In addition, the lipid particle group appears along the X-axis as a group in an area with almost no fluorescence intensity. Figure 10C In the scatter plot, white blood cells are divided into four groups: lymphocytes, monocytes, neutrophils and basophils, and eosinophils. Furthermore, lipid particle groups appear in the meandering region of the scatter plot. In Example 1, the number of nucleated cells was determined based on the fluorescence intensity and forward scattered light intensity obtained by measurements using reagents for counting nucleated red blood cells and white blood cells. Furthermore, the number of lipid particles was determined based on the fluorescence intensity, side scattered light intensity, and forward scattered light intensity obtained by measurements using a reagent for classifying white blood cells.

[0224] (3) Analysis and results

[0225] The smear specimens were observed under a microscope to obtain the density of nucleated cells in the bone marrow of each subject, and the state of the bone marrow was determined. The results showed that 7 subjects had hypoplasia, 5 subjects had normal development, and 6 subjects had hyperplasia. For the bone marrow fluid of each subject, the ratio of the number of lipid particles to the number of nucleated cells (lipid particle number / nucleated cell number) was calculated from the number of nucleated cells and lipid particles obtained by XN-2000. The distribution of the ratio of the number of lipid particles to the number of nucleated cells for each subject classified by the density of nucleated cells in the bone marrow observed under a microscope was investigated. The results are shown in Figure 11 .

[0226] Regarding the value of the ratio of the number of lipid particles to the number of nucleated cells, the critical value that is most suitable for distinguishing subjects with hypoplasia from the remaining subjects (normal development and hyperplasia) was obtained through ROC analysis. The result was that when the critical value of the ratio was 0.45, the sensitivity was 100.0% and the specificity was 85.7%. In addition, the critical value that is most suitable for distinguishing subjects with hyperplasia from the remaining subjects (normal development and hypoplasia) was also obtained. The result was that when the critical value of the ratio of the number of lipid particles to the number of nucleated cells was 0.17, the sensitivity was 94.1% and the specificity was 90.9%. The obtained ROC curve is as follows Figure 12A and B.

[0227] (4) Investigation

[0228] from Figure 11 It was found that the bone marrow nucleated cell density observed under a microscope and the ratio of lipid particle number to nucleated cell number obtained by an automated blood cell analyzer correlated well. These results demonstrate that by analyzing bone marrow fluid using FCM, the ratio of lipid particle number to nucleated cell number can be used to obtain information related to bone marrow nucleated cell density. Furthermore, this demonstrates that the ratio of lipid particle number to nucleated cell number can be used to accurately assess bone marrow status.

[0229] Number Description

[0230] 1. Sample analysis device

[0231] 2. Measurement unit

[0232] 3Analysis Department

[0233] 5. Sample preparation department

[0234] 6. Detection Department

[0235] 61 flow cells

[0236] 62 Light Source

[0237] 63 light receiving unit

[0238] 64 light receiving unit

[0239] 65 light receiving unit

[0240] 301CPU

[0241] 310 output unit

[0242] 320 Applications

[0243] 321 Removable recording media

Claims

1. A sample analysis device, characterized in that have: a sample preparation unit that prepares a first measurement sample and a second measurement sample from bone marrow fluid; a detection unit for detecting particles contained in the first and second measurement samples; a control unit that counts the number of nucleated cells in the first measurement sample and the number of lipid particles in the second measurement sample based on the information obtained by the detection unit; Output part, outputs bone marrow nucleated cell density related indicators; Wherein, the detection unit has: The flow chamber allows the first and second measurement samples prepared by the sample preparation unit to flow. a light source unit for irradiating light onto the first and second measurement samples flowing in the flow chamber; a light receiving unit that acquires first fluorescent signal information and first forward scattered light information obtained when the first measurement sample is irradiated with light, and acquires second fluorescent signal information, second forward scattered light information, and second side scattered light information obtained when the second measurement sample is irradiated with light, The control unit obtains the number of nucleated cells based on the first fluorescence signal information and the first forward scattered light information obtained by the light receiving unit, and obtains the number of lipid particles based on the second fluorescence signal information, the second forward scattered light information, and the second side scattered light information obtained by the light receiving unit. The control unit obtains the bone marrow nucleated cell density-related index based on the nucleated cell number and the lipid particle number, The bone marrow nucleated cell density-related index is a correlation value of the ratio of the nucleated cell number to the lipid particle number. The nucleated cell count is the total of the leukocyte cell count and the erythroblast cell count.

2. The sample analysis device according to claim 1, wherein: The control unit determines the bone marrow state based on the bone marrow nucleated cell density-related index.

3. The sample analysis device according to claim 2, characterized in that: The control unit determines at least one of aplasia, normal development, and hyperplasia in determining the bone marrow state.

4. The sample analysis device according to claim 1, wherein: The control unit compares the bone marrow nucleated cell density-related index with a certain threshold value to determine the bone marrow condition.

5. The sample analysis device according to claim 4, characterized in that: In determining the bone marrow status, the control unit When the bone marrow nucleated cell density-related index is the value obtained by dividing the number of lipid particles by the number of nucleated cells, When the bone marrow nucleated cell density-related index is higher than the first threshold, it is determined to be hypoplasia. When the bone marrow nucleated cell density-related index is lower than the first threshold and higher than the second threshold, it is determined to be normal development. When the index related to bone marrow nucleated cell density is lower than the second threshold, it is determined to be hyperplasia.

6. The sample analysis device according to claim 2 or 4, characterized in that: The control unit outputs the determined information about the bone marrow state to the output unit.

7. The sample analysis device according to claim 1, wherein: The sample preparation unit mixes at least the bone marrow fluid and a hemolytic agent to prepare the first and second measurement samples.

8. The sample analysis device according to claim 7, characterized in that: The sample preparation unit further mixes fluorescent dyes to prepare the first and second measurement samples.

9. The sample analysis device according to claim 1, wherein: The detection unit detects particles contained in a first measurement sample containing the bone marrow fluid and a first reagent, and particles contained in a second measurement sample containing the bone marrow fluid and a second reagent different from the first reagent. The control unit counts the number of nucleated cells in the first measurement sample and counts the number of lipid particles in the second measurement sample.

10. The sample analysis device according to claim 9, characterized in that: The first reagent includes a dissolving reagent having a pH of 2.0 to 4.5, and the second reagent includes a dissolving reagent having a pH of 5.5 to 7.0.

Images