Bone marrow fluid analysis method, sample analysis device, and computer program

Abstract

To provide a bone marrow fluid analysis method, analysis device, and computer program, which enable screening for hematopoietic tumors with performance similar to that of bone marrow image examination.SOLUTION: The above challenge is cleared by measuring a sample containing a bone marrow fluid and a fluorescent pigment capable of staining nucleic acids using a flow cytometer, acquiring optical information including fluorescent signal information on particles in the sample, counting particles with fluorescent signal information that is equal to or greater than a threshold as target cells, and obtaining a screening index for hematopoietic tumors based on the number of target cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for analyzing bone marrow fluid, a sample analyzer, and a computer program for analyzing bone marrow fluid. [Background technology]

[0002] Non-Patent Document 1 discloses that a bone marrow fluid test method using an automated blood cell counter XE-2100 (Sysmex Corporation) was investigated. Non-Patent Document 1 discloses that bone marrow fluid is pretreated using Stromatolyser IM (hereinafter also referred to as "IM reagent"), which is a reagent for measuring immature cells. The IM reagent damages the cell membrane to expose the nucleus, but immature cells are less damaged than mature white blood cells. Therefore, when cells in bone marrow fluid treated with the IM reagent are stained with a nucleic acid staining reagent, the exposed nuclei of mature white blood cells are strongly stained and emit strong fluorescent intensity. On the other hand, in immature cells such as myeloblasts and immature granulocytes, the nucleic acid staining dye is difficult to penetrate, so the fluorescent intensity is weak (see FIG. 1). In Non-Patent Document 1, by utilizing such a difference in staining property, bone marrow cells and mature white blood cells are counted by distinguishing them using XE-2100. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Mori Y. et al., Automation of Bone Marrow Aspirate Examination Using the XE-2100 Automated Hematology Analyzer.Cytometry Part B(Clinical Cytometry),2003,vol.58B,pp.25-31 Summary of the Invention [Problem to be solved by the invention]

[0004] The bone marrow fluid analysis method disclosed in Non-Patent Document 1 has room for improvement in terms of screening accuracy for hematopoietic tumors. The present invention aims to provide a bone marrow fluid analysis method, a sample analyzer, and a computer program that improve the screening accuracy for hematopoietic tumors. [Means for solving the problem]

[0005] The present inventors have noticed that the bone marrow fluid of patients with hematopoietic tumors contains an increased number of cells with high proliferation and protein production capabilities, and that these cells contain a relatively large amount of nucleic acid. The present inventors have come up with the idea of ​​staining cells in the bone marrow fluid with a fluorescent dye capable of staining nucleic acid, measuring the cells by flow cytometry, and detecting bone marrow fluid suspected of hematopoietic tumors based on the obtained fluorescent signal information. The present inventors have analyzed bone marrow fluid without abnormalities and bone marrow fluid with hematopoietic tumors or symptoms thereof, and have found that the value based on the number of particles with fluorescent signal information equal to or greater than a threshold value can distinguish bone marrow fluid with hematopoietic tumors from other bone marrow fluids. Furthermore, they have found that this discrimination result correlates well with the discrimination result of bone marrow fluid based on microscopic observation.

[0006] The present invention provides a method for analyzing bone marrow fluid, comprising the steps of measuring a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acid by flow cytometry to obtain optical information including fluorescent signal information about particles in the sample, counting particles whose fluorescent signal information is equal to or greater than a threshold as target cells, and obtaining an indicator for screening hematopoietic tumors based on the number of target cells.

[0007] The present invention provides a sample analysis device comprising a sample preparation unit that prepares a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acid, a detection unit that acquires optical information including fluorescent signal information about particles in the sample, and a control unit that counts particles whose optical signal information is equal to or greater than a threshold as target cells, wherein the control unit acquires an indicator for screening hematopoietic tumors based on the number of target cells.

[0008] The present invention provides a computer program for analyzing bone marrow fluid, which causes a computer to execute the steps of acquiring optical information including fluorescent signal information about particles in a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acid, counting particles having a fluorescent signal intensity equal to or greater than a threshold as target cells based on the optical information, and acquiring an indicator for screening hematopoietic tumors based on the number of target cells. Effect of the Invention

[0009] According to the present invention, it is possible to provide information that assists in screening for hematopoietic tumors, determining abnormalities in bone marrow fluid, and the like. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 is a schematic diagram of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by a flow cytometer (FCM). [Diagram 2] FIG. 1 is a schematic diagram of a scattergram obtained by measuring a control sample containing peripheral blood from a healthy subject and a fluorescent dye by FCM. [Figure 3A] FIG. 1 is a schematic diagram of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 3B] FIG. 1 is a schematic diagram of a scattergram obtained by measuring a control sample containing peripheral blood from a healthy subject and a fluorescent dye by FCM. [Figure 4A] FIG. 1 is a schematic diagram of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 4B] FIG. 1 is a schematic diagram of a scattergram obtained by measuring a control sample containing peripheral blood from a healthy subject and a fluorescent dye by FCM. [Figure 4C] FIG. 1 is a schematic diagram of a scattergram obtained by measuring a control sample containing peripheral blood from a healthy subject and a fluorescent dye by FCM. [Figure 5A] FIG. 1 is a schematic diagram of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 5B]FIG. 1 is a schematic diagram of a scattergram obtained by measuring a control sample containing peripheral blood from a healthy subject and a fluorescent dye by FCM. [Figure 6] 1 is a schematic diagram showing the configuration of a sample analyzer of the present embodiment. [Figure 7] FIG. 2 is a perspective view showing a configuration of a flow cell. [Figure 8] FIG. 2 is a block diagram showing a configuration of an analysis unit. [Figure 9] 4 is a flowchart showing the flow of operations of the sample analyzer of the present embodiment. [Figure 10] 1 is a flowchart showing the procedure of a sample preparation process. [Figure 11A] 11 is a flowchart showing a procedure for analyzing measurement data. [Figure 11B] 11 is a flowchart showing a procedure for analyzing measurement data. [Figure 11C] 11 is a flowchart showing a procedure for analyzing measurement data. [Figure 11D] 11 is a flowchart showing a procedure for analyzing measurement data. [Figure 11E] 11 is a flowchart showing a procedure for analyzing measurement data. [Figure 12A] 13 is a flowchart of a procedure for a determination process based on a first ratio. [Figure 12B] 13 is a flowchart of a procedure for a determination process based on a second ratio. [Figure 12C] 13 is a flowchart of a procedure for a determination process based on a third ratio. [Figure 12D] 13 is a flowchart of a procedure for a determination process based on a fifth ratio. [Figure 12E] 10 is a flowchart of a procedure for a determination process based on a sixth ratio. [Figure 13] 1 is an example of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 14] 1 shows a graph plotting the first ratio obtained by the analyzer and the ratio obtained by microscopic examination. [Figure 15]1 is an example of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 16] 1 shows a graph plotting the second ratio obtained by the analyzer and the ratio obtained by microscopic examination. [Figure 17] 1 is an example of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. [Figure 18] 13 is an example of a histogram based on the forward scattered light intensity of particles contained in a third region. [Figure 19] A graph is shown plotting the third ratio obtained by the analyzer and the ratio obtained by microscopic examination. [Figure 20] 1 is an example of a scattergram obtained when a sample containing bone marrow fluid and a fluorescent dye is measured by FCM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [1. Analysis method of bone marrow fluid] In the bone marrow fluid analysis method of this embodiment (hereinafter also referred to as the "analysis method of this embodiment"), first, a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acid is measured by flow cytometry, and optical information including fluorescent signal information about particles in the sample is obtained.

[0012] (Bone marrow fluid and the cells it contains) "Bone marrow fluid" refers to bone marrow fluid collected from a subject by bone marrow aspiration or bone marrow biopsy, and a sample containing the bone marrow fluid. When the bone marrow fluid collected from a subject contains solid impurities that may impede cell measurement, such as bone fragments or blood cell aggregates, the bone marrow fluid may be filtered using a mesh or the like. A chelating agent and / or an anticoagulant may be added to the bone marrow fluid as necessary. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) salts. Examples of anticoagulants include heparin, citric acid, and citrate salts.

[0013] Bone marrow fluid contains various nucleated cells. Examples of nucleated cells in bone marrow fluid include leukocyte cells, erythroid cells, megakaryoblasts, promegakaryocytes, myelomegakaryocytes, mature megakaryocytes, and plasma cells. Examples of "leukocyte cells" include myeloblasts, promyelocytes, myelocytes, metamyelocytes, monoblasts, promonocytes, band cells, segmented cells, immature eosinophils, immature basophils, monocytes, eosinophils, basophils, and lymphocytes. Band cells are mature neutrophils, and segmented cells are neutrophils that have further matured from band cells. "Erythroid cells" are also called nucleated red blood cells, and examples include proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and normochromatic erythroblasts.

[0014] Hereinafter, band cells and segmented cells are collectively referred to as "neutrophils". Monocytes, lymphocytes, neutrophils, eosinophils, and basophils are collectively referred to as "mature leukocytes". Monocytes and lymphocytes are collectively referred to as "mononuclear leukocytes". Neutrophils, eosinophils, and basophils are collectively referred to as "mature granulocytes".

[0015] Leukocyte cells and erythroblast cells may include tumorigenic cells that appear and increase due to hematopoietic tumors such as various leukemias and malignant lymphomas. For example, blast cells increase in acute leukemia, and plasma cells increase in plasmacytoma (also called myeloma). "Blast cells" refer to myeloblasts, monoblasts, and promonocytes.

[0016] In this specification, promyelocytes and myelocytes are also called "early differentiated immature granulocytes", and metamyelocytes are also called "late differentiated immature granulocytes". Proerythroblasts and basophilic erythroblasts are also called "early differentiated erythroblasts", and polychromatic erythroblasts and normochromatic erythroblasts are also called "late differentiated erythroblasts". Megakaryoblasts, promegakaryocytes, and bone marrow megakaryocytes are also called "megakaryocytic cells". Early differentiated immature granulocytes, early differentiated erythroblasts, blasts, megakaryocytic cells, and plasma cells contain more nucleic acids, especially ribonucleic acid, than other nucleated cells. As described above, in hematopoietic tumors, blasts and plasma cells increase in the bone marrow. In the analysis method of this embodiment, in order to selectively detect nucleated cells with a large amount of nucleic acids in the bone marrow fluid, the above sample is measured by FCM to obtain optical information including fluorescent signal information.

[0017] "Particles in a sample" refers to formed elements present in a sample. Particles include not only cells but also non-cellular particles such as remnants of lysed red blood cells (hereinafter also referred to as "red blood cell ghosts"), platelet aggregates, and lipid particles.

[0018] (Fluorescent dye) A "fluorescent dye capable of staining nucleic acid" is a fluorescent substance capable of staining the nucleic acid of a cell. A fluorescent dye capable of staining nucleic acid is also referred to simply as a "fluorescent dye" below. A preferred fluorescent dye is a fluorescent substance capable of staining ribonucleic acid (RNA) of a cell. Examples of fluorescent dyes include propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylenebis[[3-[[4-[[(3-methylbenzothiazol-3-ium)-2-yl]methylene]-1,4-dihydroquinolin]-1-yl]propyl]dimethylaminium] tetraiodide (TOTO-1), 4-[(3-methylbenzothiazol-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)propyl]quinolinium diiodide, and the like. Examples of the fluorescent dye include N,N,N',N'-tetramethyl-N,N'-bis[3-[4-[3-[(3-methylbenzothiazol-3-ium)-2-yl]-2-propenylidene]-1,4-dihydroquinolin-1-yl]propyl]-1,3-propanediaminium tetraiodide (TOTO-3) or 2-[3-[[1-[3-(trimethylaminio)propyl]-1,4-dihydroquinolin]-4-ylidene]-1-propenyl]-3-methylbenzothiazol-3-ium diiodide (TOPRO-3), fluorescent dyes represented by the following formula (I), and combinations thereof. The fluorescent dye contained in the sample may be one type or two or more types.

[0019] [ka]

[0020] In formula (I), R1 and R 4 R is a hydrogen atom, a methyl group, an ethyl group, or an alkyl group having 6 to 18 carbon atoms, and when one of them is an alkyl group having 6 to 18 carbon atoms, the other is a hydrogen atom, a methyl group, or an ethyl group. 2 and R 3 are the same or different and are a methyl group, an ethyl group, a methoxy group, or an ethoxy group. Z is a carbon atom having a sulfur atom, an oxygen atom, or a methyl group. n is 0, 1, 2, or 3. X - is an anion.

[0021] In formula (I), the alkyl group having 6 to 18 carbon atoms may be either linear or branched. Among the alkyl groups having 6 to 18 carbon atoms, alkyl groups having 6, 8 or 10 carbon atoms are preferred.

[0022] In formula (I), R 1 and R 4 Examples of the substituent on the benzyl group 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 these, a methyl group or an ethyl group is particularly preferred.

[0023] In formula (I), R 2 and R 3 Examples of the alkenyl group include alkenyl groups having 2 to 20 carbon atoms. 2 and R 3 The alkoxy group includes an alkoxy group having a carbon number of 1 to 20. Among them, a methoxy group or an ethoxy group is particularly preferable.

[0024] In formula (I), the anion X - As, F - , Cl - , Br - and I - Halogen ions such as CF3SO3 - , BF4 - , ClO4 - etc.

[0025] As the fluorescent dye represented by the above formula (I), a fluorescent dye represented by the following formula (II) is preferred.

[0026] [ka]

[0027] (Staining Reagents) In the analysis method of the present embodiment, it is preferable to use a staining reagent containing a solution of a fluorescent dye. The solvent is not particularly limited as long as it can dissolve the above-mentioned fluorescent dye. Examples include water, organic solvents, and mixtures thereof. As the organic solvent, a solvent that can be mixed with water is preferable, and examples thereof include alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, dimethyl sulfoxide (DMSO), and the like. The concentration of the fluorescent dye in the staining reagent can be appropriately determined depending on the type of the fluorescent dye, and is, for example, 0.01 mg / L or more. The concentration of the fluorescent dye in the staining reagent is preferably 0.1 mg / L or more, and more preferably 0.2 mg / L or more. In addition, the concentration of the fluorescent dye in the staining reagent is 100 mg / L or less, preferably 90 mg / L or less, and more preferably 80 mg / L or less.

[0028] Commercially available staining reagents containing a single fluorescent dye may also be used, such as Fluorocell WDF (Sysmex Corporation) and Stromatolyser 4DS (Sysmex Corporation).

[0029] (Surfactant) In the analysis method of this embodiment, it is preferable that the sample further contains a cationic surfactant. The cationic surfactant can lyse red blood cells in the bone marrow fluid and damage the cell membrane of nucleated cells to an extent that allows the fluorescent dye to penetrate. Examples of the cationic surfactant include quaternary ammonium salt type surfactants, pyridium salt type surfactants, and combinations thereof. The cationic surfactant contained in the sample may be one type or two or more types. As the quaternary ammonium salt type surfactant, for example, a surfactant represented by the following formula (III) having a total carbon number of 9 to 30 is preferable.

[0030] [ka]

[0031] In formula (III), R 1 is an alkyl or alkenyl group having 6 to 18 carbon atoms; R 2 and R 3 are the same or different and each is an alkyl or alkenyl group having 1 to 4 carbon atoms; R 4 is an alkyl group or an alkenyl group having 1 to 4 carbon atoms, or a benzyl group; X - is a halogen ion.

[0032] In formula (III), R 1 is preferably an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and is particularly preferably a linear alkyl group. 1 Examples of R include an octyl group, a decyl group, and a dodecyl group. 2 and R 3 are preferably the same or different and each is a methyl group, an ethyl group, or a propyl group. 4 is preferably a methyl group, an ethyl group, or a propyl group.

[0033] An example of the pyridinium salt surfactant is a surfactant represented by the formula (IV).

[0034] [ka]

[0035] In formula (IV), R 1 is an alkyl or alkenyl group having 6 to 18 carbon atoms; X - is a halogen ion.

[0036] In formula (IV), R 1 is preferably an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and is particularly preferably a linear alkyl group. 1 Examples of the alkyl group include octyl, decyl and dodecyl groups.

[0037] (Hemolysis Reagent) In the analysis method of the present embodiment, it is preferable to use a hemolysis reagent containing a solution of a cationic surfactant. The solvent is not particularly limited as long as it can dissolve the cationic surfactant. Examples include water, organic solvents, and mixtures thereof. As the organic solvent, a solvent that can be mixed with water is preferable, and examples thereof include alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, DMSO, and the like. The concentration of the cationic surfactant in the hemolysis reagent can be appropriately determined depending on the type of the cationic surfactant, and is, for example, 10 ppm or more. The concentration of the cationic surfactant is preferably 400 ppm or more, more preferably 500 ppm or more, and even more preferably 600 ppm or more. In addition, the concentration of the cationic surfactant in the hemolysis reagent is 10000 ppm or less. The concentration of the cationic surfactant in the hemolysis reagent is preferably 1000 ppm or less, more preferably 800 ppm or less, and even more preferably 700 ppm or less.

[0038] The sample and the hemolysis reagent preferably contain a nonionic surfactant in addition to the cationic surfactant. By containing both the cationic surfactant and the nonionic surfactant, excessive damage to nucleated cells caused by the cationic surfactant can be suppressed. Examples of nonionic surfactants include those represented by the following formula (V).

[0039] R 1 -R 2 -(CH2CH2O) n -H (V) (In formula (V), R 1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms; R 2 represents an oxygen atom, -(COO)-, or the following formula (VI):

[0040] [ka] and n is equal to or greater than 23 and equal to or less than 25, or equal to 30.

[0041] In formula (V), n is preferably 23 or 25, more preferably 23. When n is 23 or more and 25 or less, the concentration of the nonionic surfactant represented by formula (V) in the hemolysis reagent is 1700 ppm or more, preferably 1750 ppm or more. When n is 23 or more and 25 or less, the concentration of the nonionic surfactant represented by formula (I) in the sample is 2300 ppm or less, preferably 2200 ppm or less.

[0042] When n is 30, the concentration of the nonionic surfactant represented by formula (V) in the hemolysis reagent is 1900 ppm or more, preferably 2000 ppm or more, more preferably 2100 ppm or more. When n is 30, the concentration of the nonionic surfactant represented by formula (V) in the sample is 2300 ppm or less, preferably 2200 ppm.

[0043] Specific examples of the nonionic surfactant represented by formula (V) include polyoxyethylene alkyl ether, polyoxyethylene sterol, polyoxyethylene castor oil, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene alkylamine, polyoxyethylene polyoxypropylene alkyl ether, and combinations thereof. Among these, polyoxyethylene alkyl ether is preferred. Examples of polyoxyethylene alkyl ether include polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, and polyoxyethylene (30) cetyl ether. More preferred are polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, and combinations thereof, and even more preferred is polyoxyethylene (23) cetyl ether. The sample and the hemolysis reagent may contain one type of nonionic surfactant or two or more types. The hemolysis reagent may further contain a nonionic surfactant other than the nonionic surfactant represented by formula (V).

[0044] The sample and the hemolysis reagent may contain a buffer substance to keep the pH constant. Examples of the buffer include inorganic acid salts, organic acid salts, Good's buffers, and combinations thereof. Examples of the inorganic acid salts include phosphate, borate, and combinations thereof. Examples of the organic acid salts include citrate, malate, and combinations thereof. Examples of the Good's buffer include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, and combinations thereof.

[0045] The sample and the hemolysis reagent may further contain an aromatic organic acid. In this specification, the term "aromatic organic acid" refers to an acid having at least one aromatic ring in the molecule and its salt. Examples of aromatic organic acids include aromatic carboxylic acids and aromatic sulfonic acids. Examples of aromatic carboxylic acids include phthalic acid, benzoic acid, salicylic acid, hippuric acid, salts thereof, and combinations thereof. Examples of aromatic sulfonic acids include p-aminobenzenesulfonic acid, benzenesulfonic acid, salts thereof, and combinations thereof. The sample and the hemolysis reagent may contain one type of aromatic organic acid or two or more types of aromatic organic acids. Aromatic organic acids may exhibit a buffering effect. When an aromatic organic acid exhibiting a buffering effect is used, the addition of a buffering agent is optional and may be combined with the above-mentioned buffering agent.

[0046] The concentration of the aromatic organic acid in the hemolysis reagent is 20 mM or more, preferably 25 mM or more, and 50 mM or less, preferably 45 mM or less.

[0047] The pH of the hemolysis reagent is not particularly limited, but is, for example, 5.5 or more. The pH of the hemolysis reagent is preferably 5.7 or more, more preferably 5.9 or more. The pH of the hemolysis reagent is, for example, 7.2 or less, preferably 6.9 or less, more preferably 6.6 or less. The pH can be adjusted using a known base (such as sodium hydroxide) or acid (such as hydrochloric acid).

[0048] The osmotic pressure of the hemolysis reagent is not particularly limited, but is preferably 150 mOsm / kg or less, more preferably 130 mOsm / kg or less, and most preferably 110 mOsm / kg or less, from the viewpoint of the hemolysis efficiency of red blood cells. A suitable osmotic pressure regulator may be added to adjust the osmotic pressure. Examples of the osmotic pressure regulator include sugars, amino acids, organic solvents, sodium chloride, and combinations thereof.

[0049] The hemolysis reagent may be a commercially available hemolysis reagent containing a cationic surfactant, such as Lyzacell WDF (Sysmex Corporation) or Lyzacell WDFII (Sysmex Corporation).

[0050] (sample) The sample can be prepared by mixing the bone marrow fluid with a staining reagent containing a fluorescent dye. Preferably, the sample is prepared by mixing the bone marrow fluid with a staining reagent containing a fluorescent dye and a hemolytic reagent containing a cationic surfactant. When a hemolytic reagent is used, the action of the cationic surfactant makes the nucleated cells in the bone marrow fluid stainable with the fluorescent dye. The state in which the cells can be stained with the fluorescent dye refers to a state in which the cell membrane of the cells is damaged to an extent that the fluorescent dye can penetrate. If red blood cells are mixed into the bone marrow fluid, the red blood cells are lysed by the action of the cationic surfactant. The action of the cationic surfactant destroys the cell membrane of the erythroblastic cells as well as the red blood cells, but the cell nuclei of the erythroblastic cells are preserved. Therefore, the erythroblastic cells are stainable with the fluorescent dye.

[0051] The fluorescent dye can enter the cell through the damaged cell membrane of the nucleated cell and stain the nucleic acid in the cell nucleus. The stained nucleated cell can emit fluorescence, and the fluorescent signal information can be obtained by measurement by FCM. As described above, early differentiation immature granulocytes, early differentiation erythroblasts, blast cells, megakaryocytic cells, and plasma cells contain a lot of nucleic acid, so they are strongly stained and emit strong fluorescence. Compared to these nucleated cells, late differentiation immature granulocytes, late differentiation erythroblasts, mature leukocytes, and mature megakaryocytes are weakly stained. Cells and particles that have nuclei such as red blood cells, blood cell ghosts, and lipid particles are hardly stained.

[0052] When one reagent containing a fluorescent dye and a cationic surfactant is used, the mixing ratio of the bone marrow fluid to the reagent is usually 1:5-500, preferably 1:10-100, expressed by volume ratio. When a staining reagent containing a fluorescent dye and a hemolysis reagent containing a cationic surfactant are used, the mixing ratio of the bone marrow fluid to the staining reagent and the hemolysis reagent is usually 1:1-10:5-500, preferably 1:1-5:10-100, expressed by volume ratio. After mixing the bone marrow fluid with each reagent, it is preferable to incubate the mixture under a predetermined condition. The predetermined condition includes, for example, a condition of incubation at a temperature of 15-50°C, preferably 30-45°C, for 5-120 seconds, preferably 5-30 seconds. The sample may be prepared manually or by an analyzer equipped with a sample preparation unit.

[0053] (FCM measurement) In the measurement by FCM, the sample is irradiated with light to obtain optical information including fluorescent signal information about particles in the sample. Specifically, the sample is first introduced into a flow cell of the FCM, and light is irradiated to each particle in the sample as it passes through the flow cell. Then, the light emitted from each particle is measured to obtain optical information. The light emitted from the particles is fluorescence derived from the fluorescent dye and scattered light. Examples of the scattered light include forward scattered light (for example, scattered light with a light receiving angle of 0 degrees to about 20 degrees) and side scattered light (for example, scattered light with a light receiving angle of about 20 degrees to about 90 degrees). As the optical information, it is preferable to obtain fluorescent signal information and scattered light information. As the scattered light information, it is preferable to obtain at least one of side scattered light information and forward scattered light information, and it is particularly preferable to obtain both side scattered light information and forward scattered light information.

[0054] Examples of the fluorescent signal information and the scattered light information include the peak values ​​(height of the pulse peak), pulse area, pulse width, transmittance, Stokes shift, ratio, change over time, and values ​​correlated therewith. The fluorescent signal information is not particularly limited as long as it is information that reflects the amount of the fluorescent dye that stained the nucleic acid in the nucleated cells. The fluorescent information is preferably the peak value of the fluorescent signal (hereinafter also referred to as "fluorescent signal intensity"). The side scattered light information is not particularly limited as long as it is information that reflects internal information such as the complexity of the cell structure, granular characteristics, nuclear structure, and degree of lobulation. The forward scattered light information is not particularly limited as long as it is information that reflects the size of the cell. The side scattered light information is preferably the peak value of the side scattered light signal (hereinafter also referred to as "side scattered light intensity"). The forward scattered light information is preferably the peak value of the forward scattered light signal (hereinafter also referred to as "forward scattered light intensity").

[0055] A scattergram may be created based on the optical information acquired by FCM measurement of bone marrow fluid, in which each measured particle is displayed as a dot on a two-dimensional plane, for example, with side-scattered light information on the horizontal axis and fluorescent signal information on the vertical axis.

[0056] The FCM is not particularly limited, and a commercially available automatic blood cell analyzer may be used. Examples of such analyzers include the XR series from Sysmex Corporation. The light source of the FCM is not particularly limited, and a light source with a wavelength suitable for exciting the fluorescent dye can be appropriately selected. Examples of light sources that can be used include a blue semiconductor laser, a red semiconductor laser, an argon laser, a He-Ne laser, and a mercury arc lamp.

[0057] In the analysis method of the present embodiment, it is preferable to count nucleated cells. In this specification, the "number of nucleated cells" refers to the total number of leukocyte cells, erythroblast cells, megakaryocytic cells, mature megakaryocytes, and plasma cells. The method of counting nucleated cells itself is known, and the number of nucleated cells can be obtained based on optical information obtained by measuring a sample with an FCM. For example, nucleated cells can be counted by excluding cells and particles that do not have a nucleus, such as red blood cells, red blood cell ghosts, and lipid particles, from particles in a sample. Since the fluorescent signal intensity of cells and particles that do not have a nucleus is very low, these can be excluded based on the fluorescent signal intensity. The counting of nucleated cells can be performed by analysis software installed in the FCM. Such analysis software is known, and an example of such software is Flowjo (trademark) (BD Biosciences).

[0058] (First embodiment) In the analysis method of this embodiment, particles whose fluorescent signal information is equal to or greater than a threshold value are counted as target cells. Then, an index for screening hematopoietic tumors is obtained based on the number of target cells. An embodiment in which target cells are counted and an index for screening hematopoietic tumors is obtained based on the counted number is also referred to as the "first embodiment." In this specification, "target cells" refers to early differentiation immature granulocytes, early differentiation erythroblasts, blast cells, megakaryocytic cells, and plasma cells. As described above, these nucleated cells have a higher amount of nucleic acid than other nucleated cells. The threshold value corresponding to the fluorescent signal information is a value that allows the above target cells to be distinguished from other particles. The relationship between the target cells and the threshold value corresponding to the fluorescent signal information will be described below with reference to FIG. 1.

[0059] FIG. 1 is an example of a scattergram based on the fluorescent signal information and the scattered light information acquired by FCM measurement of bone marrow fluid. In FIG. 1, the horizontal axis of the scattergram is the side scattered light intensity, and the vertical axis is the fluorescent signal intensity. In FIG. 1, the mature white blood cells are classified into five subpopulations, namely, a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population, but the present invention is not limited thereto. The mature white blood cells may be classified into three subpopulations, namely, a lymphocyte population, a monocyte population, and a mature granulocyte population, or into four subpopulations, namely, a lymphocyte population, a monocyte population, a neutrophil and basophil population, and an eosinophil population. The classification of each cell population in the scattergram can be performed by analysis software installed in the FCM. In FIG. 1, a straight line (hereinafter also referred to as a "horizontal line") crossing the two-dimensional plane of the scattergram indicates a threshold value for the fluorescent signal intensity. The target cells, early differentiation immature granulocytes, early differentiation erythroblasts, blasts, megakaryocytic cells, and plasma cells, have a large amount of nucleic acid, and therefore the fluorescent signal intensity is high. Therefore, these cells form and distribute in the region of high fluorescent signal intensity in the scattergram. The blast cell group and the early differentiation erythroblast group overlap in the region where they appear. On the other hand, the late differentiation erythroblasts, late differentiation immature granulocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, and immature eosinophils have a relatively small amount of nucleic acid, and therefore the fluorescent signal intensity is low. Therefore, these cells form and distribute in the region of low fluorescent signal intensity in the scattergram. The lymphocyte and monocyte groups and the late differentiation erythroblast group overlap in the region where they appear. Here, as shown by the horizontal line in FIG. 1, if the two-dimensional plane of the scattergram is divided by a threshold value corresponding to the fluorescent signal intensity, the target cell group and the non-target cell group can be separated. The threshold value corresponding to the fluorescent signal information may be a fixed value, or may be variably set for each sample.

[0060] The threshold value corresponding to the fluorescent signal information can be defined as, for example, a value that can exclude 95% or more of mature leukocytes in the bone marrow fluid. An example of such a threshold value is the horizontal line on the scattergram shown in FIG. 1. In FIG. 1, for the purpose of explanation, a group of early differentiation immature granulocytes and a group of late differentiation immature granulocytes are shown separately. In an actual scattergram of bone marrow fluid, these groups of immature granulocytes appear continuously and may not be clearly separated. This is also true for the group of early differentiation erythroblasts and the group of late differentiation erythroblasts. Therefore, it is preferable to use a fixed value as the threshold value corresponding to the fluorescent signal information. Here, the FCM A / D converts the analog signals of the fluorescent light and the scattered light at a predetermined sampling rate to generate a digital signal. At this time, when the digital signal is represented by 8-bit data, the fluorescent signal intensity and the side scattered light intensity are each displayed in 256 gradations of 0 to 255 channels. A channel is a unit of intensity of a digital signal. When the fluorescence intensity is expressed in 256 gradations from 0 to 255 channels, a fluorescence signal intensity of, for example, 100ch can be set as a threshold value that can exclude 95% or more of mature white blood cells in bone marrow fluid. "ch" represents the channel. In other words, a fluorescence signal intensity of 100ch can be set as a threshold value corresponding to the fluorescence signal information.

[0061] Alternatively, the threshold value corresponding to the fluorescent signal information may be defined as a value represented by a horizontal line that intersects with the monocyte population but does not intersect with the lymphocyte population in the sample when the distribution of particles in the sample is plotted on a two-dimensional plane based on the fluorescent signal information and side scattered light information, as shown in Figure 1. The threshold value corresponding to the fluorescent signal information in this case is, for example, a value that satisfies at least the following two conditions: (1) greater than the maximum fluorescent signal intensity of the lymphocyte population; (2) It is smaller than the maximum value of the fluorescent signal intensity of the monocyte population. More preferably, the threshold value corresponding to the fluorescent signal information is a value that also satisfies the condition (3) that it is greater than the statistical representative value of the monocyte population. In this case, the statistical representative value is, for example, the mean, median, or mode. Instead of the maximum value in the above condition, a representative value of a subpopulation with a high fluorescent signal intensity of each cell population may be used. For example, it may be the mean, median, or mode of the top 5% of the fluorescent signal intensities of each cell population.

[0062] Alternatively, the threshold value corresponding to the fluorescent signal information may be defined by, for example, the ratio of a population Y of nucleated cells having fluorescent signal information equal to or greater than a given value to a population X of nucleated cells in peripheral blood of a healthy person. Specifically, any value of the fluorescent signal information that makes the ratio fall within a predetermined range, for example, 0.1% to 10%, more preferably 1% to 9%, and even more preferably 2.5% to 5%, may be defined as the threshold value corresponding to the fluorescent signal information. In this definition, the threshold value corresponding to the fluorescent signal information may be determined as follows based on the results of measuring peripheral blood of a healthy person (hereinafter also referred to as "normal blood") by FCM. First, a control sample containing normal blood and the above-mentioned fluorescent dye is measured by FCM in the same manner as bone marrow fluid to obtain fluorescent signal information and side scattered light information. Normal blood is easier to obtain and easier to measure by FCM than bone marrow fluid, and is therefore suitable as a control sample. It is preferable to use a plurality of control samples. For example, at least 20 control samples, preferably 20 to 40 control samples, are measured by FCM. A plurality of control samples are prepared from a plurality of normal blood samples (e.g., 20 samples). The control sample preferably further contains the cationic surfactant. More preferably, the control sample is prepared by mixing peripheral blood from a healthy person, the staining reagent, and the hemolysis reagent. The fluorescent signal information is preferably the fluorescent signal intensity. The side scattered light information is preferably the scattered light intensity. Next, nucleated cells are selected based on the optical information, and the number of nucleated cells is obtained. Furthermore, a scattergram may be created based on the optical information, and mature white blood cells may be classified into the above three to five subpopulations. Here, an arbitrary fluorescent signal intensity is set as a provisional threshold. The provisional threshold can be selected from values ​​higher than the fluorescent signal intensity of a monocyte population, for example. Here, the fluorescent signal intensity of a cell population on a scattergram can be a statistically representative value of the fluorescent signal intensity of cells contained in the population. Examples of statistically representative values ​​include the mean, median, mode, and quartile. Such statistically representative values ​​can be obtained by analysis software installed in the FCM. After setting the provisional threshold, cells having a fluorescent signal intensity equal to or greater than the provisional threshold are counted for each of a plurality of measurement samples.Then, the ratio of cells having a fluorescent signal intensity equal to or greater than the provisional threshold is calculated relative to the number of nucleated cells. The median of the ratios in a plurality of measurement samples is obtained. The median can be obtained using a known spreadsheet software such as Excel (registered trademark). When the median is, for example, 2.5% or more and 5% or less, the provisional threshold can be set as a threshold corresponding to the fluorescent signal information in the FCM measurement of the bone marrow fluid. Alternatively, after setting the provisional threshold, the threshold may be set as follows. For each of the plurality of measurement samples, the first and third quartiles of the side fluorescent intensity of the monocyte population are obtained, and the first and third quartiles are averaged in the measurements of the plurality of specimens to calculate the interquartile range, and the interquartile range (interval from the first quartile to the third quartile: IQR) including the median is obtained. Any provisional threshold included in the averaged interquartile range of the monocyte population set in this way may be set as a predetermined threshold corresponding to the fluorescent signal information in the FCM measurement of the bone marrow fluid.

[0063] The target cells are counted by extracting particles that are equal to or greater than the threshold value corresponding to the fluorescent signal information from among all particles in the sample measured by FCM and counting the number of particles. When a scattergram is created with side scattered light information on the horizontal axis and fluorescent signal information on the vertical axis, particles included in a region (hereinafter also referred to as the "first region") that is equal to or greater than the threshold value corresponding to the fluorescent signal information may be identified as target cells and the number of particles may be counted. For example, in the scattergram of FIG. 1, the first region is the upper region of a two-dimensional plane separated by a horizontal line indicating the threshold value corresponding to the fluorescent signal information. The target cells can be counted, for example, by analysis software installed in the FCM.

[0064] Alternatively, the first region may be specified by the following steps, and a threshold value corresponding to the fluorescent signal information may be determined. Specifically, the lowest fluorescent signal intensity in the first region may be set as the threshold value for the fluorescent signal information. Each step of specifying the first region will be described. First, a control sample containing normal blood and the above-mentioned fluorescent dye is measured by FCM in the same manner as bone marrow fluid to obtain fluorescent signal information and side scattered light information. There may be one or more control samples. Preferably, multiple control samples are measured. As the fluorescent signal information, fluorescent signal intensity is preferable. As the side scattered light information, scattered light intensity is preferable. Next, a scattergram is created based on the acquired fluorescent signal information and side scattered light information. In the created scattergram, a region where 95% or more of mature white blood cells are excluded and the fluorescent signal intensity is equal to or greater than the fluorescent signal intensity of a group of monocytes is specified as the first region. The first region specified in this way is, for example, the region surrounded by the dashed line shown in FIG. 2. Referring to Fig. 2, as long as 95% or more of mature white blood cells are excluded from the first region, some monocytes may appear in the first region. When the first region determined based on the FCM measurement of normal blood is applied to a scattergram created based on the FCM measurement of bone marrow fluid, the first region may include or overlap the region in which the above-mentioned target cells appear. The lowest fluorescent signal intensity in the first region, indicated by the arrow in Fig. 2, can be set as a threshold corresponding to the fluorescent signal information in the FCM measurement of bone marrow fluid.

[0065] As used herein, "peripheral blood from a healthy subject" refers to peripheral blood collected from a healthy subject, to which EDTA-2K has been added, and in which the blood cell counts and hemoglobin concentration satisfy the following criteria: ·White blood cell count 2500 / μL or more and 18000 / μL or less Hemoglobin level 10g / dL or higher ·Platelet count 60000 / μL or more and 600000 / μL or less ·Neutrophil count 1000 / μL or more and 11000 / μL or less Lymphocyte count: ≥800 / μL and ≤4000 / μL ·Monocyte count 1000 / μL or less ·Eosinophil count 700 / μL or less ·Basophil count 200 / μL or less

[0066] In the first embodiment, an index for screening hematopoietic tumors is obtained based on the number of target cells. The hematopoietic tumors are not particularly limited, and examples include acute leukemia, plasmacytoma, and mature lymphoma. Among these, acute leukemia is particularly important because it is a tumor that progresses quickly and is fatal. The index for screening hematopoietic tumors is, for example, a numerical value that indicates whether a subject has or is suspected of having a hematopoietic tumor, or whether there is a sign that leads to the development of a hematopoietic tumor. The number of target cells itself may be used as an index for screening hematopoietic tumors. Preferably, a ratio of the number of target cells to the number of nucleated cells is obtained as an index for screening hematopoietic tumors. This ratio is also referred to as the "first ratio" below. The number of nucleated cells is the number counted based on optical information acquired by FCM measurement of the sample, as described above. The first ratio is a value calculated from the following formula (A).

[0067] (First ratio) = [(number of target cells) / (number of nucleated cells)] × 100 (A)

[0068] The first ratio also includes values ​​calculated from the above formula (A) as long as they indicate a trend in the change in the number of target cells based on the number of nucleated cells in the sample. Examples of values ​​calculated from the above formula (A) include values ​​obtained by using any coefficient and / or constant in formula (A). The value calculated from formula (A) may be divided by 100 to show the percentage as a real number. Any constant may be added to or subtracted from the value calculated from formula (A).

[0069] In the first embodiment, the first ratio may be compared with a threshold value to determine whether or not there is an abnormality in the bone marrow fluid. When the first ratio is a value calculated from the above formula (A), the threshold value corresponding to the first ratio may be, for example, any value between 20% and 40%, preferably any value between 25% and 35%, and more preferably 30%. For example, when the first ratio is compared with the threshold value and the first ratio is equal to or greater than the threshold value, it is determined that there is an abnormality in the bone marrow fluid. In this case, it is suggested that the bone marrow fluid of the subject has an increased number of blast cells or plasma cells compared to normal bone marrow fluid. This indicates that acute leukemia or plasmacytoma has occurred or is suspected. On the other hand, when the first ratio is lower than the threshold value, it is determined that there is no abnormality in the bone marrow fluid. In this case, it is suggested that there is no suspicion of hematopoietic tumor in the subject.

[0070] In the first embodiment, when the first ratio is equal to or greater than a threshold, a distinguishable display may be provided. The distinguishable display is preferably displayed on a screen of a display device mounted on the FCM or communicably connected thereto. Examples of the display device include displays such as a liquid crystal display, a plasma display, and a CRT display. On the screen of the display device, for example, the first ratio itself and / or the fact that the ratio is equal to or greater than a threshold is displayed as a distinguishable display in a format that can be distinguished from other characters and numbers. For example, the numerical value of the first ratio may be displayed on the screen of the display device in a size or color different from other numerical values, or the first ratio may be highlighted with an underline or a flag. Alternatively, the fact that the first ratio is equal to or greater than a threshold may be displayed on the screen of the display device in a pop-up message, or in an area showing special notes. When the first ratio is lower than the threshold, the first ratio may be displayed normally. For example, the numerical value of the first ratio may be displayed on the screen of the display device in the same manner as other numerical values.

[0071] The significance and effect of such identifiable labeling is that it can provide medical professionals, such as doctors and examiners, who see the labeling with information that helps them determine whether or not there is an abnormality in the bone marrow fluid. For example, medical professionals who see the identifiable labeling can recognize the bone marrow fluid as a sample suspected of acute leukemia or plasmacytoma. Furthermore, if necessary, they can perform additional tests on the bone marrow fluid or start the process of treating the subject, hospitalizing the subject, or referring the subject to a specialized hospital.

[0072] When the first ratio is equal to or greater than the threshold, the identifiable indication may indicate that the bone marrow fluid is a sample that requires a test for hematopoietic malignancies as a priority. Examples of tests include bone marrow imaging, genetic testing, and antibody panel testing. Among these, bone marrow imaging is particularly preferred. As described above, when the first ratio is equal to or greater than the threshold, acute leukemia or plasmacytoma is suspected. The significance and effect of such an identifiable indication is that a doctor, examiner, etc. can arrange to test the bone marrow fluid in priority over other samples.

[0073] Second Embodiment It is known that in acute leukemia, cancerous blast cells proliferate abnormally. In addition, in plasmacytoma, an increase in plasma cells is observed in bone marrow fluid. Both blast cells and plasma cells are mononuclear cells. Therefore, in a further embodiment, an index for screening hematopoietic tumors may be obtained based on the number of mononuclear cells among the target cells. This embodiment is also called the "second embodiment." The target cells that are mononuclear cells are also called "target mononuclear cells" hereinafter. As described above, the target cells are particles whose fluorescent signal information is equal to or greater than a threshold value. Therefore, in the second embodiment, in the target cell counting step, mononuclear cells whose fluorescent signal information is equal to or greater than a threshold value are counted as target mononuclear cells. Here, among nucleated cells, the mononuclear cells are early differentiation erythroblasts, blast cells, plasma cells, monocytes, lymphocytes, and late differentiation erythroblasts. Therefore, the target mononuclear cells are early differentiation erythroblasts, blast cells, and plasma cells. Among nucleated cells, early differentiation immature granulocytes, megakaryocytic cells, mature megakaryocytes, late differentiation immature granulocytes, mature granulocytes, immature eosinophils, and immature basophils are polymorphonuclear cells.

[0074] In the second embodiment, the target mononuclear cells may be counted based on the fluorescent signal information and side scattered light information of particles in a sample, or after the target cells are counted, the target mononuclear cells may be counted based on the side scattered light information of the target cells.

[0075] In FCM measurement, it is known that side scattered light reflects internal information such as nuclei and granules in cells. Mononuclear cells tend to have lower side scattered light intensity than polymorphonuclear cells. Therefore, a mononuclear cell population and a polymorphonuclear cell population can be separated by an auxiliary line based on side scattered light information in a scattergram created based on fluorescent signal information and side scattered light information. This will be described with reference to FIG. 3A. In FIG. 3A, an auxiliary line based on side scattered light intensity is drawn on the scattergram of FIG. 1 as shown by the diagonal line. In FIG. 3, the horizontal line indicates a threshold value corresponding to the fluorescent signal intensity, as in FIG. 1. As can be seen from FIG. 3A, in the region above the horizontal line, the diagonal line separates a population of blast cells and early differentiation erythroblasts from a population of early differentiation immature granulocytes. In the region below the horizontal line, the diagonal lines separate the population of monocytes and late differentiating erythroblasts from the population of late differentiating immature granulocytes, neutrophils and basophils.

[0076] As shown in FIG. 3A, the two-dimensional plane of the scattergram is divided into four regions by a diagonal line that divides nucleated cells in the bone marrow into mononuclear cells and polymorphonuclear cells, and a horizontal line that indicates a threshold value corresponding to a fluorescent signal intensity that can exclude 95% or more of mature white blood cells in the bone marrow. In the second embodiment, particles appearing in the region above the horizontal line and to the left of the diagonal line (region surrounded by a dashed line) in the scattergram of FIG. 3A are counted. In this region, mononuclear cells with fluorescent signal information equal to or greater than a threshold value, i.e., blast cells, early differentiation erythroblasts, and plasma cells, are distributed in their respective groups. In the second embodiment, based on the fluorescent signal information and side scattered light information of particles in the sample, particles included in the region where blast cells, early differentiation erythroblasts, and plasma cells appear (hereinafter also referred to as the "second region") can be identified as target mononuclear cells. The target mononuclear cells can be counted, for example, by analysis software installed in the FCM.

[0077] In FIG. 3A, for the sake of explanation, the population of early differentiation erythroblasts and blasts and the population of early differentiation immature granulocytes are shown separately. In an actual scattergram of bone marrow fluid, these populations may appear continuously and may not be clearly separated. This is also true for the population of monocytes and late differentiation erythroblasts and the population of late differentiation immature granulocytes, neutrophils, and basophils. The second region may be a preset region or may be variably set for each sample. For example, the second region may be empirically set by accumulating data of a scattergram based on fluorescent signal information and side scattered light information obtained by FCM measurement of normal bone marrow fluid or bone marrow fluid of hematopoietic tumors such as acute leukemia. Alternatively, the region may be set from predetermined coordinates of a scattergram based on the fluorescent signal intensity and the side scattered light intensity. As described above, the FCM A / D converts the analog signals of the fluorescent light and the scattered light at a predetermined sampling rate to generate a digital signal. In this case, when the digital signal is represented by 8-bit data, the ranges of the fluorescence signal intensity and the side scattered light intensity in the scattergram are 0 to 255ch, respectively. The predetermined coordinates can be the following four coordinates (X:Y) = (0ch:100ch), (135ch:100ch), (0ch:255ch) and (180ch:255ch) in a scattergram with the side scattered light intensity on the X axis and the fluorescence signal intensity on the Y axis. The area surrounded by these four coordinates can be set as the second area.

[0078] Alternatively, the four coordinates of the second region may be set based on the analysis results of a plurality of control samples containing normal blood and the above-mentioned fluorescent dye. The number of control samples is at least 20, and preferably 20 to 40. For example, referring to FIG. 3B, the region surrounded by points A, B, C, and D is set as the second region. Here, FIG. 3B is a scattergram of normal blood with the side scattered light intensity on the X-axis and the fluorescent signal intensity on the Y-axis. In FIG. 3B, the straight line that crosses the two-dimensional plane of the scattergram is a straight line indicating the threshold value corresponding to the fluorescent signal intensity. This straight line can be set based on the analysis results of the control sample, as described in the first embodiment. The X-coordinate of point A can be determined to be, for example, any value between the average value of the median value on the X-axis of the monocyte population and the average value of the median value on the X-axis of the neutrophil population of each specimen. Since point A is a point on the straight line indicating the predetermined threshold value for the fluorescent signal intensity, the Y-coordinate of point A is the same as the predetermined threshold value for the fluorescent signal intensity. The X coordinate of point B can be determined to be any value between the average value of the X-axis median of the neutrophil population and the average value of the X-axis median of the eosinophil population of each sample. The Y coordinate of point B is the maximum value of the fluorescent signal intensity in the scattergram as shown in FIG. 3B. As for point C, as shown in FIG. 3B, the X coordinate is 0, and the Y coordinate is the maximum value of the fluorescent signal intensity in the scattergram. Like point A, point D is a point on a line indicating a predetermined threshold value for the fluorescent signal intensity. Therefore, as shown in FIG. 3B, the X coordinate of point D is 0, and the Y coordinate is the same as the predetermined threshold value for the fluorescent signal intensity. When the second region specified by the four coordinates determined in this way is applied to a scattergram created based on the FCM measurement of the bone marrow fluid, the second region may include or overlap the region in which the above-mentioned target mononuclear cells appear.

[0079] Alternatively, the second region may be set based on the analysis results of a control sample containing normal blood and the above-mentioned fluorescent dye, similarly to the first region. First, as described in the first embodiment, the control sample is measured by FCM to obtain fluorescent signal information and side scattered light information. Details of the control sample are as described above. As the fluorescent signal information, fluorescent signal intensity is preferable. As the side scattered light information, scattered light intensity is preferable. Next, a scattergram is created based on the acquired fluorescent signal information and side scattered light information. In the created scattergram, a region in which 95% or more of mature white blood cells are excluded, the fluorescent signal intensity is equal to or greater than the fluorescent signal intensity of a group of monocytes, and the side scattered light intensity is equal to or less than the side scattered light intensity of a group of neutrophils is specified as the second region. The fluorescent signal intensity and side scattered light intensity of a cell group on the scattergram may be statistically representative values ​​of the fluorescent signal intensity and side scattered light intensity of cells contained in the group. The statistically representative values ​​are as described above. When the second region identified based on FCM measurement of peripheral blood from a healthy individual is applied to a scattergram created based on FCM measurement of bone marrow fluid, the second region may encompass or overlap the region in which the above-mentioned target mononuclear cells appear.

[0080] In the second embodiment, an indicator for screening hematopoietic tumors is obtained based on the number of target mononuclear cells. Hematopoietic tumors and the indicator for screening thereof are as described above. The number of target mononuclear cells itself may be used as an indicator for screening hematopoietic tumors. Preferably, the ratio of the number of target mononuclear cells to the number of nucleated cells is obtained as an indicator for screening hematopoietic tumors. This ratio is also referred to as the "second ratio" below. The number of nucleated cells is the number counted based on optical information acquired by FCM measurement of the sample, as described above. The second ratio is a value calculated from the following formula (B).

[0081] (Second ratio) = [(number of target mononuclear cells) / (number of nucleated cells)] × 100 (B)

[0082] The second ratio also includes values ​​calculated from the above formula (B) as long as they indicate a trend in the change in the number of target mononuclear cells based on the number of nucleated cells in the sample. Examples of values ​​calculated from the above formula (B) are the same as those based on the above formula (A).

[0083] In the second embodiment, the second ratio may be compared with a threshold value to determine whether or not there is an abnormality in the bone marrow fluid. When the second ratio is a value calculated from the above formula (B), the threshold value corresponding to the second ratio may be, for example, any value between 10% and 30%, preferably any value between 15% and 25%, and more preferably 20%. For example, the second ratio is compared with a corresponding predetermined threshold value, and when the second ratio is equal to or greater than the threshold value, it is determined that there is an abnormality in the bone marrow fluid. In this case, it is suggested that the bone marrow fluid of the subject has an increased number of blast cells or plasma cells compared to normal bone marrow fluid. This indicates that acute leukemia or plasmacytoma has occurred or is suspected to have occurred. On the other hand, when the second ratio is lower than the threshold value, it is determined that there is no abnormality in the bone marrow fluid.

[0084] In the second embodiment, when the second ratio is equal to or greater than a threshold value, a distinguishable display may be provided. The distinguishable display is preferably displayed on the screen of the display device, similar to the first ratio. For example, the second ratio itself and / or the fact that the ratio is equal to or greater than a threshold value is displayed on the screen of the display device in a format that can be distinguished from other characters and numbers. The display device and the display on the screen are the same as those described for the first embodiment. When the second ratio is equal to or greater than a threshold value, the distinguishable display may indicate that the bone marrow fluid is a sample that requires a test for hematopoietic tumors as a priority. The test is as described above. The significance and effect of the distinguishable display are also the same as those described for the first embodiment.

[0085] (Third embodiment) As described above, in acute leukemia, cancerous blast cells proliferate abnormally. In a further embodiment, an index for screening hematopoietic tumors (particularly acute leukemia) may be obtained based on the number of blast cells among the target cells. This embodiment is also called "third embodiment." In the third embodiment, blast cells are counted in the step of counting the target cells.

[0086] In the third embodiment, the blast cells may be counted based on the optical information of the particles in the sample. Alternatively, the target cells may be counted, and then the blast cells may be counted based on the optical information of the target cells. Preferably, the blast cells are counted based on the optical information of the particles in the sample. In the third embodiment, the optical information is fluorescent signal information, side scattered light information, and forward scattered light information. For example, first, particles containing blast cells are selected based on the fluorescent signal information and side scattered light information of the particles in the sample. The particles containing blast cells may include early differentiated erythroblasts and plasma cells in addition to blast cells. The selection of blast cells from particles containing blast cells is performed based on the forward scattered light information of the particles. This point will be described later.

[0087] The above-mentioned selection of particles containing blast cells will be described with reference to FIG. 4A. In FIG. 4A, the region in which particles containing blast cells appear in the scattergram in FIG. 1 (hereinafter, also referred to as the "third region") is indicated by a dashed line. In FIG. 4A, the horizontal line indicates a threshold value corresponding to the fluorescent signal intensity, as in FIG. 1. In the third embodiment, particles appearing in the region surrounded by the dashed line in the scattergram in FIG. 4A can be identified as particles containing blast cells. In FIG. 4A, the region surrounded by the dashed line includes a part of the region below the horizontal line. This is because, in bone marrow fluid in which blast cells have proliferated, such as in a specimen of acute leukemia, a group of blast cells may be distributed up to the region in which a group of monocytes appears. The selection of particles containing blast cells can be performed, for example, by analysis software installed in the FCM.

[0088] The third region may be a preset region or may be variably set for each specimen. For example, the third region may be empirically set by accumulating data of a scattergram based on fluorescent signal information and side scattered light information obtained by FCM measurement of normal bone marrow fluid or bone marrow fluid of hematopoietic tumor such as acute leukemia. Alternatively, the third region may be determined based on a scattergram in which the fluorescent signal intensity and the side scattered light intensity are displayed in the range of 0 to 255 channels. Specifically, in the scattergram, the region surrounded by the following four coordinates (X:Y)=(80ch:100ch), (130ch:80ch), (180ch:255ch) and (130ch:255ch) can be set as the third region. X is the side scattered light intensity, and Y is the fluorescent signal intensity.

[0089] Alternatively, the four coordinates of the third region may be set as follows based on the analysis results of a plurality of control samples containing normal blood and the above-mentioned fluorescent dye. The number of control samples is at least 20, and preferably 20 to 40. For example, referring to FIG. 4B, the region surrounded by points B, E, F, and G is set as the third region. Here, FIG. 4B is a scattergram of normal blood with the side scattered light intensity on the X axis and the fluorescent signal intensity on the Y axis. In FIG. 4B, the straight line that crosses the two-dimensional plane of the scattergram is a straight line indicating the threshold corresponding to the fluorescent signal intensity. This straight line can be set based on the analysis results of the control sample, as described in the first embodiment. Point B in FIG. 4B is the same as point B in FIG. 3B, so the coordinates of point B are the same as those described in the second embodiment. The X coordinate of point E can be determined to be, for example, any value between the average value of the median on the X axis of the monocyte population and the average value of the median on the X axis of the neutrophil population of each specimen. The Y coordinate of point E is the maximum value of the fluorescent signal intensity in the scattergram as shown in FIG. 4B. The X coordinate of point F can be determined to be any value between the average value of the median of the X axis of the lymphocyte population of each specimen and the average value of the median of the X axis of the monocyte population. Since point F is a point on a line indicating the threshold value corresponding to the fluorescent signal intensity, the Y coordinate of point F is the same as the threshold value corresponding to the fluorescent signal intensity. The X coordinate of point G can be the same as the X coordinate of point E. The Y coordinate of point G can be determined to be any value between the 0th percentile value and the first quartile (25th percentile value) of the fluorescent signal intensity of the monocyte population of each specimen. When the third region specified by the four coordinates determined in this way is applied to a scattergram created based on the FCM measurement of bone marrow fluid, the third region may include or overlap the region where the above-mentioned blast cells appear.

[0090] Alternatively, the third region may be set based on the analysis results of a control sample containing normal blood and the above-mentioned fluorescent dye, as in the first region. First, as described in the first embodiment, the control sample is measured by FCM to obtain fluorescent signal information and side scattered light information. Details of the control sample are as described above. As the fluorescent signal information, fluorescent signal intensity is preferable. As the side scattered light information, scattered light intensity is preferable. Next, a scattergram is created based on the acquired fluorescent signal information and side scattered light information. In the created scattergram, a region in which 95% or more of mature white blood cells are excluded, the fluorescent signal intensity is equal to or greater than the fluorescent signal intensity of a group of monocytes, and the side scattered light intensity is equal to or greater than the side scattered light intensity of a group of lymphocytes and equal to or less than the side scattered light intensity of a group of neutrophils is specified as the third region. The fluorescent signal intensity and side scattered light intensity of a cell group on the scattergram may be statistically representative values ​​of the fluorescent signal intensity and side scattered light intensity of cells contained in the group. The statistically representative values ​​are as described above. When the third region identified based on FCM measurement of peripheral blood from a healthy individual is applied to a scattergram created based on FCM measurement of bone marrow fluid, the third region may encompass or overlap the region in which particles containing the above-mentioned blast cells appear.

[0091] As described above, particles containing blasts may include early differentiation erythroblasts and plasma cells in addition to blasts. Therefore, in the third embodiment, blasts are selected from particles containing blasts based on forward scattered light information of the particles. Here, it is known that forward scattered light reflects the size of cells in FCM measurement. It is also known that the size of blasts is 10 to 20 μm. For example, a histogram of forward scattered light intensity is created for particles containing blasts selected based on fluorescent signal information and side scattered light information, and a group corresponding to the size of the blasts can be selected based on the forward scattered light intensity. Here, when the FCM expresses the digital signal of the forward scattered light as 8-bit data, the range of the forward scattered light intensity is 0 to 255 channels. In this case, particles with 50ch or more and 100ch or less in the histogram of forward scattered light intensity may be selected as blasts.

[0092] In the third embodiment, an indicator for screening hematopoietic tumors is obtained based on the number of selected blast cells. Hematopoietic tumors and the indicator for screening thereof are as described above. The number of blast cells itself may be used as an indicator for screening hematopoietic tumors. Preferably, the ratio of the number of blast cells to the number of nucleated cells is obtained as an indicator for screening hematopoietic tumors. This ratio is also referred to as the "third ratio" below. The number of nucleated cells is the number counted based on optical information obtained by FCM measurement of the sample, as described above. The third ratio is a value calculated from the following formula (C).

[0093] (Third ratio) = [(number of blasts) / (number of nucleated cells)] × 100 (C)

[0094] The third ratio also includes values ​​calculated from the above formula (C) as long as they indicate a trend in the change in the number of blast cells based on the number of nucleated cells in the sample. Examples of values ​​calculated from the above formula (C) are the same as those of values ​​calculated from the above formula (A).

[0095] In the third embodiment, the third ratio may be compared with a threshold value to determine whether or not there is an abnormality in the bone marrow fluid. When the third ratio is a value calculated from the above formula (C), the threshold value corresponding to the third ratio may be, for example, any value between 5% and 25%, and may preferably be 10%. For example, the third ratio is compared with the threshold value, and when the third ratio is equal to or greater than the threshold value, it is determined that there is an abnormality in the bone marrow fluid. When the third ratio is equal to or greater than 10%, it is suggested that the bone marrow fluid of the subject has an increased number of blast cells compared to normal bone marrow fluid. This indicates that there is a sign that may lead to the development of acute leukemia, or that acute leukemia has developed or is suspected. On the other hand, when the third ratio is lower than the threshold value, it is determined that there is no abnormality in the bone marrow fluid. In this case, it is suggested that there is no sign that may lead to the development of acute leukemia, and there is no suspicion of acute leukemia.

[0096] Furthermore, the threshold value corresponding to the third ratio may be, for example, 20%. When the third ratio is 20% or more, it is suggested that there is a significant increase in blast cells in the bone marrow fluid of the subject, which indicates that acute leukemia has occurred or is suspected. On the other hand, when the third ratio is lower than 20%, there is no significant increase in blast cells in the bone marrow fluid, which indicates that there is no suspicion of acute leukemia.

[0097] In determining whether bone marrow fluid is abnormal based on the third ratio, two thresholds may be used. When the third ratio is a value calculated from the above formula (C), for example, the first threshold is set to 10% and the second threshold is set to 20% to perform the determination. Specifically, the third ratio is first compared with the first threshold. When the third ratio is lower than the first threshold, it is determined that there is no abnormality in the bone marrow fluid. When the third ratio is equal to or higher than the first threshold, it is compared with the second threshold. When the third ratio is lower than the second threshold, it is determined that there is an abnormality in the bone marrow fluid, and that there is a suspicion of an increase in blast cells. When the third ratio is equal to or higher than the second threshold, it is determined that there is an abnormality in the bone marrow fluid, and that there is a suspicion of acute leukemia.

[0098] In the third embodiment, when the third ratio is equal to or greater than a threshold value, a distinguishable display may be provided. The distinguishable display is preferably displayed on the screen of the display device, similar to the first ratio. For example, the third ratio itself and / or the fact that the ratio is equal to or greater than a threshold value is displayed on the screen of the display device in a format that can be distinguished from other characters and numbers. The display device and the display on the screen are the same as those described for the first embodiment. When the third ratio is equal to or greater than a threshold value, the distinguishable display may indicate that the bone marrow fluid is a sample that requires a test for hematopoietic tumors, particularly acute leukemia, as a priority. The test is as described above. In the third embodiment, blasts are particularly detected, so when the third ratio is equal to or greater than a threshold value, a sign suggesting an increase in blasts, such as "Blastosis?", may be displayed on the screen of the display device. The significance and effect of the distinguishable display are the same as those described for the first embodiment.

[0099] (Fourth embodiment) It is known that in mature lymphoma, the number of lymphoid tumor cells increases in bone marrow fluid compared to the number of mononuclear cells in the early stage of differentiation. In a further embodiment, an index for screening mature lymphoma may be obtained based on the number of target mononuclear cells and the number of mononuclear leukocytes and late differentiated erythroblasts. This embodiment is also called "fourth embodiment". In the fourth embodiment, in the target cell counting step, the target mononuclear cells, mononuclear leukocytes, and late differentiated erythroblasts are counted. The counting can be performed based on fluorescent signal information and side scattered light information. The mononuclear leukocytes and late differentiated erythroblasts are mononuclear cells whose fluorescent signal information is lower than a threshold value. Referring to FIG. 3A, in the scattergram, the mononuclear leukocytes and late differentiated erythroblasts can be selected as particles contained in the region below the horizontal line and to the left of the diagonal line. By counting the particles contained in the region, the mononuclear leukocytes and late differentiated erythroblasts can be counted. The mononuclear leukocytes and late differentiated erythroblasts may be counted separately. Alternatively, they may be counted as one cell group including mononuclear leukocytes and late differentiated erythroblasts. As described above, the target mononuclear cells can be counted as particles contained in the second region. The counting can be performed, for example, by analysis software installed in the FCM.

[0100] In the scattergram, the region where mononuclear leukocytes and late differentiated erythroblasts appear (hereinafter also referred to as the "fourth region") may be empirically set by accumulating scattergram data based on fluorescent signal information and side scattered light information obtained by FCM measurement of normal bone marrow fluid and bone marrow fluid of hematopoietic tumors such as acute leukemia, similarly to the second region. Alternatively, the fourth region may be determined based on a scattergram in which the fluorescent signal intensity and the side scattered light intensity are displayed in the range of 0 to 255 channels. Specifically, in the scattergram, the region surrounded by the following four coordinates (X:Y) = (0ch:0ch), (0ch:100ch), (135ch:100ch) and (0ch:100ch) can be set as the fourth region. X is the side scattered light intensity, and Y is the fluorescent signal intensity.

[0101] Alternatively, the four coordinates of the fourth region may be set based on the analysis results of a plurality of control samples containing normal blood and the above-mentioned fluorescent dye. The number of control samples is at least 20, and preferably 20 to 40. For example, referring to FIG. 4C, the region surrounded by points A, D, the origin, and H is set as the fourth region. Here, FIG. 4C is a scattergram of normal blood with the side scattered light intensity on the X axis and the fluorescent signal intensity on the Y axis. In FIG. 4C, the straight line that crosses the two-dimensional plane of the scattergram is a straight line indicating a threshold corresponding to the fluorescent signal intensity. This straight line can be set based on the analysis results of the control sample as described in the first embodiment. Points A and D in FIG. 4C are the same as points A and D in FIG. 3B, so the coordinates of these points are the same as those described in the second embodiment. The X coordinate of point H can be determined as the intersection of the line extending the straight line BA toward the X axis and the X axis. The Y coordinate of point H is 0 as shown in FIG. 4C. When the fourth region identified by the four coordinates determined in this manner is applied to a scattergram created based on FCM measurement of bone marrow fluid, the fourth region may encompass or overlap the region in which the above-mentioned mononuclear leukocytes and late-differentiated erythroblasts appear.

[0102] Alternatively, the fourth region may be set from a scattergram of a control sample containing peripheral blood of a healthy person and the above-mentioned fluorescent dye, similarly to the second region. In a scattergram based on the fluorescent signal information and the side scattered light information, a region containing 95% or more of mononuclear leukocytes, having a fluorescent signal intensity equal to or lower than that of a monocyte group, and having a side scattered light intensity equal to or lower than that of a neutrophil group is specified as the fourth region. When the fourth region specified based on the FCM measurement of peripheral blood of a healthy person is applied to a scattergram created based on the FCM measurement of bone marrow fluid, the fourth region may include or overlap the region in which mononuclear leukocytes and late-differentiated erythroblasts appear.

[0103] In order to obtain an index for screening mature lymphoma, it is preferable to obtain the ratio of the number of mononuclear leukocytes and late differentiated erythroblasts to the number of nucleated cells. This ratio is also referred to as the "fourth ratio" below. The number of nucleated cells is the number counted based on the optical information obtained by FCM measurement of the sample as described above. The fourth ratio is a value calculated from the following formula (D).

[0104] (4th ratio) = [(number of mononuclear leukocytes and late differentiated erythroblasts) / (number of nucleated cells)] × 100 (D)

[0105] As long as the number of nucleated cells in the sample is used as a reference and the increasing trend of the number of mononuclear leukocytes and differentiated late erythroblasts can be known, the value based on the value calculated from the above formula (D) is also included in the fourth ratio. Examples of the value based on the value calculated from the above formula (D) are the same as those of the value calculated from the above formula (A).

[0106] In the fourth embodiment, the second ratio and the fourth ratio may be obtained as an index for screening mature lymphoma. From these ratios, it is possible to know which of the target mononuclear cells, mononuclear leukocytes, and late differentiated erythroblasts tends to increase. Preferably, the ratio of the number of target mononuclear cells to the number of mononuclear leukocytes and late differentiated erythroblasts is obtained as an index for screening mature lymphoma. This ratio is also referred to as the "fifth ratio" below. The fifth ratio is the ratio of the value of the second ratio to the value of the fourth ratio, and is calculated from the following formula (E). The second ratio is a value calculated from the above formula (B).

[0107] (5th ratio) = [(2nd ratio) / (4th ratio)] × 100 (E)

[0108] The fifth ratio can also be obtained by dividing the number of target mononuclear cells by the number of mononuclear leukocytes and differentiated late erythroblasts to calculate the percentage. When the above formula (E) is used, the fourth ratio is required, which is preferable because it allows not only the fifth ratio but also the second and fourth ratios to be obtained.

[0109] In the fourth embodiment, the fifth ratio may be compared with a threshold value to determine whether or not there is an abnormality in the bone marrow fluid. When the fifth ratio is a value calculated from the above formula (E), the threshold value corresponding to the fifth ratio may be, for example, any value between 5% and 15%, and may preferably be 10%. For example, when the fifth ratio is compared with the threshold value and the fifth ratio is lower than the threshold value, it is determined that there is an abnormality in the bone marrow fluid. In this case, it is suggested that there is an increase in lymphatic tumor cells in the bone marrow fluid of the subject. This indicates that mature lymphoma has occurred or is suspected. On the other hand, when the fifth ratio is equal to or higher than the threshold value, it is determined that there is no abnormality in the bone marrow fluid.

[0110] In the fourth embodiment, when the fifth ratio is lower than the threshold, a distinguishable display may be provided. The distinguishable display is preferably displayed on the screen of the display device, similar to the first ratio. For example, the fifth ratio itself and / or the fact that the fifth ratio is lower than the threshold is displayed on the screen of the display device in a format that can be distinguished from other characters and numbers. The second ratio and the fourth ratio may also be displayed. The display device and the display on the screen are the same as those described in the first embodiment. When the fifth ratio is lower than the threshold, the distinguishable display may indicate that the bone marrow fluid is a sample that requires a test for hematopoietic tumors, particularly mature lymphoma, as a priority. The test is as described above. In the fourth embodiment, since the focus is particularly on mature lymphoma, when the fifth ratio is lower than the threshold, a sign suggesting an increase in lymphocytes, such as "Mature Lymphocytosis?", may be displayed on the screen of the display device. The significance and effect of the distinguishable display are the same as those described in the first embodiment.

[0111] Fifth embodiment In plasmacytoma, the ratio of plasma cells to nucleated cells in bone marrow fluid increases. Plasma cells are antibody-producing cells, and are known to have a higher amount of nucleic acid than blast cells and early differentiated erythroblasts. In a further embodiment, an index for screening plasmacytoma may be obtained based on the number of plasma cells. This embodiment is also referred to as the "fifth embodiment." In the fifth embodiment, plasma cells are counted in the target cell counting step.

[0112] As described above, plasma cells have a higher amount of nucleic acid than blast cells and early differentiation erythroblasts. Therefore, in order to count plasma cells, it is preferable to set the threshold corresponding to the fluorescent signal information in the counting step of the target cells higher than that in the first embodiment. For example, the threshold corresponding to the fluorescent signal information can be set to a fluorescent signal intensity higher than that of a group of blast cells or a group of early differentiation erythroblasts. Here, the FCM A / D converts the analog signals of the fluorescent light and the scattered light at a predetermined sampling rate to generate a digital signal. As described above, when a digital signal is represented by 8-bit data, the ranges of the fluorescent signal intensity and the side scattered light intensity in the scattergram are 0 to 255ch, respectively. If the analog signal of the fluorescent light emitted from the particle has an intensity within the range of 0 to 255ch after A / D conversion, the analog signal is converted into a digital signal of 0 to 255ch. However, if the analog signal of the fluorescent light emitted from the particle has an intensity exceeding 255ch after A / D conversion, it is converted into a digital signal of 255ch regardless of the intensity. In the scattergram, even if a particle has a fluorescent signal intensity exceeding 255ch, the fluorescent signal intensity is plotted on the coordinate of 255ch. The fluorescent signal intensity of blast cells and early differentiation erythroblasts is usually within the range of 100 to 255, while the fluorescent signal intensity of plasma cells usually exceeds 255. Therefore, in a scattergram displayed in the range of 0 to 255ch, it is difficult to clearly distinguish plasma cells from blast cells and early differentiation erythroblasts. Therefore, in the fifth embodiment, it is preferable to represent a digital signal of fluorescence with, for example, 10 to 16 bit data, preferably 10 bit data. When a digital signal is represented with 10 bit data, the fluorescent signal intensity is represented with 1024 gradations of 0 to 1023ch.

[0113] Since plasma cells are mononuclear cells, they can be distinguished from megakaryocyte cells and early differentiation immature granulocytes based on side scattered light information. Therefore, in the fifth embodiment, plasma cells can be selected based on the fluorescent signal information and side scattered light information of particles in a sample. Alternatively, after selecting target cells, plasma cells may be selected based on the side scattered light information of the target cells.

[0114] The counting of plasma cells will be described with reference to FIG. 5A. In FIG. 5A, the vertical axis (fluorescence signal intensity) of the scattergram in FIG. 1 is displayed as 0 to 1023 ch, and the horizontal axis (side scattered light intensity) is displayed as 0 to 255 ch. The region where plasma cells appear (hereinafter also referred to as the "fifth region") is indicated by a dashed line. In the fifth embodiment, particles contained in the region surrounded by the dashed line in the scattergram in FIG. 5A can be identified as plasma cells. The counting of plasma cells can be performed, for example, by analysis software installed in the FCM.

[0115] The fifth region may be a preset region, or may be variably set for each specimen. For example, the fifth region may be empirically set by accumulating data of a scattergram based on fluorescent signal information and side scattered light information obtained by FCM measurement of normal bone marrow fluid or bone marrow fluid of hematopoietic tumor such as acute leukemia. Alternatively, the fifth region may be determined based on a scattergram in which the fluorescent signal intensity is displayed in 0 to 1023ch and the side scattered light intensity is displayed in 0 to 255ch. Specifically, in the scattergram, the region surrounded by the following four coordinates (X:Y)=(0ch:1023ch), (170ch:1023ch), (170ch:296ch) and (0ch:296ch) can be set as the fifth region. X is the side scattered light intensity, and Y is the fluorescent signal intensity.

[0116] Alternatively, the four coordinates of the fifth region may be set based on the analysis results of a plurality of control samples containing normal blood and the above-mentioned fluorescent dye. The number of control samples is at least 20, and preferably 20 to 40. For example, referring to FIG. 5B, the region surrounded by points I, J, K, and L is set as the fifth region. Here, FIG. 5B is a scattergram of normal blood with the side scattered light intensity on the X axis and the fluorescent signal intensity on the Y axis. In this scattergram, the fluorescent signal intensity is displayed in 0 to 1023 ch, and the side scattered light intensity is displayed in 0 to 255 ch. As for point I, as shown in FIG. 5B, the X coordinate is 0, and the Y coordinate is the maximum value of the fluorescent signal intensity in the scattergram. As shown in FIG. 5B, the X coordinate of point J is 0. The Y coordinate of point J can be determined to be, for example, 15±1 times higher than the average value of the IQR (interquartile range) of the fluorescent signal intensity of nucleated cells in multiple control samples (i.e., 14 to 16×IQR). The X coordinate of point K can be determined to be, for example, any value between the average value of the median of the X axis of the neutrophil population and the average value of the median of the X axis of the eosinophil population in each specimen. The Y coordinate of point K is the same as the Y coordinate of point J. For point L, the X coordinate is the same as the X coordinate of point K, and the Y coordinate is the maximum value of the fluorescent signal intensity in the scattergram. When the fifth region specified by the four coordinates determined in this way is applied to a scattergram created based on the FCM measurement of bone marrow fluid, the fifth region may include or overlap the region where plasma cells appear.

[0117] In the fifth embodiment, an index for screening plasmacytoma is obtained based on the number of plasma cells. The number of plasma cells itself may be used as an index for screening plasmacytoma. Preferably, the ratio of the number of plasma cells to the number of nucleated cells is obtained as an index for screening plasmacytoma. This ratio is also referred to as the "sixth ratio" below. The number of nucleated cells is the number counted based on optical information acquired by FCM measurement of the sample, as described above. The sixth ratio is a value calculated from the following formula (F).

[0118] (6th ratio) = [(number of plasma cells) / (number of nucleated cells)] × 100 (F)

[0119] The sixth ratio also includes values ​​calculated from the above formula (F) as long as they indicate a trend in the change in the number of plasma cells based on the number of nucleated cells in the sample. Examples of values ​​calculated from the above formula (F) are the same as those based on the above formula (A).

[0120] In the fifth embodiment, whether or not there is an abnormality in the bone marrow fluid may be determined by comparing the sixth ratio with a threshold value. When the sixth ratio is a value calculated from the above formula (F), the threshold value corresponding to the sixth ratio may be, for example, any value between 5% and 15%, and may preferably be 5%. For example, when the sixth ratio is compared with the threshold value and the sixth ratio is equal to or greater than the threshold value, it is determined that there is an abnormality in the bone marrow fluid. In this case, it is suggested that there is an increase in plasma cells in the bone marrow fluid of the subject compared to normal bone marrow fluid. This indicates that plasmacytoma has occurred or is suspected to have occurred. On the other hand, when the sixth ratio is lower than the threshold value, it is determined that there is no abnormality in the bone marrow fluid.

[0121] In the fifth embodiment, when the sixth ratio is equal to or greater than a threshold, a distinguishable display may be provided. The distinguishable display is preferably displayed on the screen of the display device, similar to the first ratio. For example, the sixth ratio itself and / or the fact that the ratio is equal to or greater than a threshold is displayed on the screen of the display device in a format that can be distinguished from other characters and numbers. The display device and the display on the screen are the same as those described for the first embodiment. When the sixth ratio is equal to or greater than a threshold, the distinguishable display may indicate that the bone marrow fluid is a sample that requires a test for hematopoietic tumors, particularly plasmacytoma, as a priority. The test is as described above. In the fifth embodiment, since attention is particularly focused on plasmacytoma, when the sixth ratio is equal to or greater than a threshold, a sign suggesting an increase in plasma cells, such as "Plasmacytosis?", may be displayed on the screen of the display device. The significance and effect of the distinguishable display are the same as those described for the first embodiment.

[0122] In this way, the analysis method of the present embodiment makes it possible to provide medical professionals such as doctors with information that assists in screening for hematopoietic tumors and determining abnormalities in bone marrow fluid. Medical professionals who obtain the information can decide whether or not to prioritize testing for hematopoietic tumors on the analyzed bone marrow fluid. In addition, from the obtained information, they can perform bone marrow imaging tests by assuming suspected hematopoietic tumors and cells that should be noted.

[0123] [2. Sample analysis equipment] An example of the sample analyzer of this embodiment will now be described with reference to the drawings.

[0124] (Configuration of sample analyzer) As shown in Fig. 6, the sample analyzer 10 includes a measurement unit 20 and an analysis unit 30. The measurement unit 20 takes in bone marrow fluid, prepares a sample from the bone marrow fluid, and optically measures the sample. The analysis unit 30 processes measurement data obtained by the measurement of the measurement unit 20, and outputs the analysis results of the bone marrow fluid. However, this embodiment is not limited to this example, and may be, for example, an apparatus in which the measurement unit 20 and the analysis unit 30 are integrally configured.

[0125] The measurement unit 20 includes an aspirating unit 40, a sample preparing unit 50, a detecting unit 60, a signal processing circuit 81, a microcomputer 82, and a communication interface 83. The aspirating unit 40 has an aspirating tube 42. The aspirating unit 40 aspirates bone marrow fluid contained in a test tube 41 through the aspirating tube 42.

[0126] The sample preparation section 50 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 liquid in the measurement by the flow cytometry method. The reagent container 52 contains a hemolysis reagent containing a hemolytic agent. The reagent container 53 contains a staining reagent containing a fluorescent dye. The suction section 40 moves the suction tube 42 above 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 hemolysis reagent, and the staining reagent are mixed in the reaction tank 54 to prepare a sample. Instead of using the staining reagent and the hemolysis reagent, one reagent containing both the fluorescent dye and the cationic surfactant may be used. The sample is subjected to optical measurement by the flow cytometry method.

[0127] The detection unit 60 is used for optical measurement of particles by flow cytometry. The detection unit 60 includes a flow cell 61, a light source unit 62, and light receiving units 63 and 64. The flow cell 61 is supplied with a diluent contained in a reagent container 51 and a sample prepared by the sample preparation unit 50. A method for the detection unit 60 to obtain optical information including fluorescent signal information about particles in a sample will be described below, but is not limited to this description.

[0128] The flow cell 61 is tubular and made of a material such as quartz, glass, or synthetic resin having optical transparency. The inside of the flow cell 61 is a flow path through which the sample and sheath liquid flow. Referring to FIG. 7, the flow cell 61 is provided with an orifice 61a whose inner space is narrower than other parts. The vicinity of the inlet of the orifice 61a has a double-tube structure, and the inner tube part is a sample nozzle 61b, through which the sample prepared by the sample preparation unit 50 is supplied. The space outside the sample nozzle 61b is a flow path 61c through which the sheath liquid flows. The sheath liquid is introduced into the orifice 61a through the flow path 61c. In this way, the sheath liquid supplied to the flow cell 61 flows so as to envelop the sample discharged from the sample nozzle 61b. The flow of the sample is narrowed by the orifice 61a, and the particles in the sample enveloping the sheath liquid pass through the orifice 61a one by one.

[0129] The light source unit 62 is a semiconductor laser light source, and irradiates, for example, a red laser light having a wavelength of 633 nm to the orifice 61a of the flow cell 61. The wavelength of the laser is not particularly limited, and a laser light source having a wavelength suitable for exciting a fluorescent dye can be appropriately selected. The light receiving units 63, 64, and 65 detect light emitted from each particle in the sample when the sample flow in the flow cell 61 is irradiated with light. An avalanche photodiode, a photodiode, or a photomultiplier tube can be used for the light receiving units 63, 64, and 65. Hereinafter, the direction connecting the light source unit 62 and the flow cell 61 is referred to as the "X direction", and the direction perpendicular to the X direction is referred to as the "Y direction". A dichroic mirror 66 is disposed on the Y direction side from the flow cell 61. The dichroic mirror 66 transmits the fluorescence emitted from each particle and reflects the side scattered light emitted from each particle. The light receiving unit 63 is disposed on the Y direction side from the flow cell 61, and can detect fluorescence transmitted through the dichroic mirror 66. The light receiving unit 64 is disposed on the X direction side from the flow cell 61. More specifically, the light receiving unit 64 is disposed on the opposite side of the flow cell 61 from the light source unit 62. The light receiving unit 64 can detect forward scattered light emitted from each particle. The light receiving unit 65 can detect side scattered light reflected from the dichroic mirror 66.

[0130] The side scattered light is not limited to light scattered in a direction (Y direction) at an angle of 90° with respect to the optical axis direction (X direction) of the light source unit 62. The side scattered light may be, for example, light scattered in a direction between 80° and 100° with respect to the X direction. The forward scattered light is not limited to light scattered in the optical axis direction (X direction) of the light source unit 62. The forward scattered light may be, for example, light scattered in a direction between -10° and 10° with respect to the X direction.

[0131] In this embodiment, an illumination lens system made up of a plurality of lenses (not shown) may be disposed between the light source unit 62 and the flow cell 61. The illumination lens system can focus the parallel beam emitted from the semiconductor laser light source into a beam spot.

[0132] The light-receiving units 63, 64, and 65 receive fluorescence, forward scattered light, and side scattered light, respectively, and output analog electrical signals of different voltages to the signal processing circuit 81 according to the amount of received light. That is, the light-receiving units 63, 64, and 65 output analog signals indicating the intensity of received light. Hereinafter, the analog signal output from the light-receiving unit 63 will be referred to as the "fluorescence signal," the analog signal output from the light-receiving unit 64 will be referred to as the "forward scattered light signal," and the analog signal output from the light-receiving unit 65 will be referred to as the "side scattered light signal." These analog signals are input to the signal processing circuit 81 as waveform signals whose voltage changes in a waveform as particles pass through the flow cell 61.

[0133] The signal processing circuit 81 A / D converts the analog signals output by the light receiving units 63, 64, 65 at a predetermined sampling rate to generate a fluorescent signal, a forward scattered light signal, and a side scattered light signal, which are digital signals. The signal processing circuit 81 extracts the peak value of the fluorescent signal corresponding to the waveform of one particle as the "fluorescent signal intensity" of that particle. Similarly, the signal processing circuit 81 extracts the peak value of the forward scattered light signal as the "forward scattered light intensity" and the peak value of the side scattered light signal as the "side scattered light intensity."

[0134] When the digital signal generated by the signal processing circuit 81 is expressed by, for example, 8-bit data, the range of the fluorescence signal intensity is, for example, 0 to 255ch. When the digital signal is expressed by, for example, 10-bit data, the range of the fluorescence signal intensity is, for example, 0 to 1023ch. When the digital signal is expressed by, for example, 16-bit data, the fluorescence signal intensity is expressed within the range of, for example, 0 to 65535. When particles detected by FCM measurement are plotted on a scattergram whose vertical axis represents the fluorescence signal intensity of 256 channels, particles having a fluorescence intensity of 0 to 255 are plotted as is on the coordinates of the fluorescence intensity of 0 to 255 channels. On the other hand, particles having a fluorescence signal intensity of 256 or more (for example, 500) are plotted on the coordinates of the 255 channel. However, when plotted on a scattergram whose vertical axis represents the fluorescence signal intensity of 1023 channels, particles having a fluorescence signal intensity of 500 are plotted on the coordinates of the 500 channel.

[0135] The microcomputer 82 controls the aspirating unit 40, the sample preparing unit 50, the detecting unit 60, the signal processing circuit 81, and the communication interface 83. The communication interface 83 is connected to the analyzing unit 30 by a communication cable. The measuring unit 20 performs data communication with the analyzing unit 30 through the communication interface 83. The communication interface 83 transmits measurement data including each characteristic parameter to the analyzing unit 30.

[0136] The configuration of the analysis unit 30 will be described with reference to Fig. 8. The analysis unit 30 includes a main body 300, an input unit 309, and a display unit 310. The main body 300 includes a CPU 301, a ROM 302, a RAM 303, a solid state drive (SSD) 304, a readout device 305, an input / output interface 306, an image output interface 307, and a communication interface 308. The main body 300 may include a hard disk instead of the SSD. In Fig. 5, a display that displays images is used as the display unit 310.

[0137] The CPU 301 executes a computer program 322 stored in the ROM 302 and a computer program loaded in the RAM 303. The RAM 303 is used to read out each computer program recorded in the ROM 302 and the SSD 304. The RAM 303 is also used as a working area for the CPU 301 when executing each computer program. The SSD 304 is installed with an application program 320, which is a computer program for analyzing measurement data provided from the measurement unit 20 and outputting the analysis results. The computer program 322 includes a basic input / output system (BIOS). The application program 320 includes an OS, a program for analyzing bone marrow fluid, and a program for determining abnormality in bone marrow fluid. The program for analyzing bone marrow fluid refers to a program for acquiring the number of target cells based on optical information and acquiring an index for screening hematopoietic tumors based on the number of target cells. The program for determining abnormality in bone marrow fluid refers to a program for determining whether or not there is an abnormality in bone marrow fluid based on an index for screening hematopoietic tumors.

[0138] The reading device 305 is a USB port, an SD card reader, a CF card reader, a memory stick reader, a CD-ROM drive, a DVD-ROM drive, a flexible disk drive, or the like, and can read out a computer program or data recorded in a portable recording medium 321. The portable recording medium 321 stores a computer program 320 for causing a computer to function as the analysis unit 30. The computer program 320 read out from the portable recording medium 321 is installed in the SSD 304.

[0139] The input unit 309 is connected to the input / output interface 306. The display 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 20. Instead of the input unit 309 and the display unit 310, a touch panel with an input unit arranged on the surface of the display unit may be provided as the display input unit. Examples of the touch panel include well-known touch panels such as a capacitance type.

[0140] (Operation of the sample analyzer) The operation of the sample analyzer 10 will now be described with reference to Figure 9. First, the CPU 301 of the analysis section 30 receives an instruction to perform measurement from the user via the input section 309 (step S101).

[0141] When the analysis unit 30 receives an instruction to perform measurement, the CPU 301 transmits instruction data to the measurement unit 20 to instruct the measurement unit 20 to start measurement (step S102), and the measurement unit 20 receives the instruction data (step S103). The microcomputer 82 of the measurement unit 20 executes a sample preparation process (step S104) and executes a measurement process (step S105).

[0142] The sample preparation process will be described with reference to FIGS. 6 and 10. The microcomputer 82 controls the aspirator 40 to supply a predetermined amount of bone marrow fluid to the reaction tank 54 (step S201). Next, the microcomputer 82 controls the sample preparation unit 50 to supply a predetermined amount of hemolysis reagent from the reagent container 52 to the reaction tank 54, and supply a predetermined amount of staining reagent from the reagent container 53 to the reaction tank 54 (step S202). The reaction tank 54 is heated by a heater to a predetermined temperature (e.g., 30 to 45° C.). In the heated state, the mixture in the reaction tank 54 is stirred (step S203). A sample is prepared in the reaction tank 54 by the operations of steps S201 to S203. The microcomputer 82 controls the sample preparation unit 50 to guide the sample from the reaction tank 54 to the detection unit 60 (step S204). When the process of step S204 is completed, the microcomputer 82 returns the process to the main routine.

[0143] When the sample passes through the flow cell 61 of the detection unit 60, the light source unit 62 irradiates the particles in the sample with laser light. Fluorescence emitted from the particles is detected by the light receiving unit 63. Forward scattered light emitted from the particles is detected by the light receiving unit 64. Side scattered light emitted from the particles is detected by the light receiving unit 65. Each of the light receiving units 63, 64, and 65 outputs an electric signal according to the light receiving level as a fluorescent signal, a forward scattered light signal, and a side scattered light signal. The signal processing circuit 81 extracts the fluorescent intensity from the fluorescent signal, extracts the forward scattered light intensity from the forward scattered light signal, and extracts the side scattered light intensity from the side scattered light signal. With reference to FIG. 9, after the measurement process, the microcomputer 82 transmits the measurement data including each characteristic parameter to the analysis unit 3 (step S106) and ends the process. The analysis unit 3 receives the measurement data (step S107). Thereafter, the CPU 301 executes a measurement data analysis process, generates an analysis result of the bone marrow fluid, and stores the analysis result in the SSD 304 (step S108).

[0144] [3. Computer Programs] An example of the measurement data analysis process will be described below. Referring to FIG. 11A, in the measurement data analysis process of the first embodiment, when the measurement data analysis process is started, the CPU 301 of the analysis unit 3 selects target cells and nucleated cells based on the fluorescence intensity included in the measurement data (step S301). The CPU 301 selects nucleated cells by excluding cells and particles without nuclei from the particles in the sample based on the fluorescence signal intensity. The CPU 301 also selects target cells by extracting particles with a fluorescence signal intensity equal to or greater than a threshold from the particles in the sample. The CPU 301 may create a scattergram using data on the fluorescence signal intensity and the side scattered light intensity.

[0145] In step S302, CPU301 counts the target cells and nucleated cells selected in step S301, and stores the numbers in SSD304. In step S303, CPU301 acquires an index for screening hematopoietic tumors based on the number of target cells and the number of nucleated cells. When acquiring a first ratio as the index, CPU301 performs the calculation shown in formula (A) above to acquire the first ratio. CPU301 stores the acquired index for screening hematopoietic tumors in SSD304. In step S304, CPU301 determines abnormality in bone marrow fluid based on the index for screening hematopoietic tumors.

[0146] The measurement data analysis process of the second embodiment will be described with reference to Fig. 11B. When the measurement data analysis process is started, the CPU 301 of the analysis unit 3 selects target mononuclear cells and nucleated cells based on the fluorescence intensity and side scattered light intensity contained in the measurement data (step S311). The selection of nucleated cells is similar to the analysis process of the first embodiment. The CPU 301 selects target mononuclear cells by extracting particles contained in a second region from particles in a sample based on the fluorescence signal intensity and side scattered light intensity. The CPU 301 may create a scattergram using the data of the fluorescence signal intensity and the side scattered light intensity.

[0147] In step S312, CPU301 counts the target mononuclear cells and nucleated cells selected in step S311, and stores the numbers in SSD304. In step S313, CPU301 acquires an index for screening hematopoietic tumors based on the number of target mononuclear cells and the number of nucleated cells. When acquiring a second ratio as the index, CPU301 performs the calculation shown in the above formula (B) to acquire the second ratio. CPU301 stores the acquired index for screening hematopoietic tumors in SSD304. Step S314 is the same as step S304. That is, CPU301 determines abnormality of bone marrow fluid in step S315.

[0148] The analysis process of the measurement data of the third embodiment will be described with reference to FIG. 11C. In this analysis process, an index for screening acute leukemia is obtained as an index for screening hematopoietic tumors. When the analysis process of the measurement data is started, the CPU 301 of the analysis unit 3 selects blast cells and nucleated cells based on the fluorescence intensity, side scattered light intensity, and forward scattered light intensity included in the measurement data (step S321). The selection of nucleated cells is similar to the analysis process of the first embodiment. The CPU 301 extracts particles included in the third region from particles in the sample based on the fluorescence signal intensity and side scattered light intensity. The CPU 301 may create a scattergram using the data of the fluorescence signal intensity and the side scattered light intensity. The blast cells are selected by extracting particles corresponding to the size of the blast cells based on the forward scattered light intensity of the extracted particles. The CPU 301 may create a histogram using the data of the forward scattered light intensity.

[0149] In step S322, CPU 301 counts the blasts and nucleated cells selected in step S321, and stores the numbers in SSD 304. In step S323, CPU 301 acquires an index for screening acute leukemia based on the number of blasts and the number of nucleated cells. When acquiring a third ratio as the index, CPU 301 performs the calculation shown in the above formula (C) to acquire the third ratio. CPU 301 stores the acquired index for screening acute leukemia in SSD 304. Step S324 is the same as step S304. That is, CPU 301 determines abnormality of bone marrow fluid in step S325.

[0150] The analysis process of the measurement data of the fourth embodiment will be described with reference to FIG. 11D. In this analysis process, an index for screening mature lymphoma is obtained as an index for screening hematopoietic tumors. When the analysis process of the measurement data is started, the CPU 301 of the analysis unit 3 selects blast cells and nucleated cells based on the fluorescence intensity, side scattered light intensity, and forward scattered light intensity included in the measurement data (step S331). The selection of nucleated cells is the same as the analysis process of the first embodiment. The CPU 301 selects target mononuclear cells by extracting particles included in the second region from particles in the sample based on the fluorescence signal intensity and side scattered light intensity. In addition, the CPU 301 selects mononuclear leukocytes and late differentiated erythroblasts by extracting particles included in the fourth region. The CPU 301 may create a scattergram using the data of the fluorescence signal intensity and the side scattered light intensity.

[0151] In step S332, the CPU 301 counts the number of target mononuclear cells, mononuclear leukocytes, late differentiation erythroblasts, and nucleated cells selected in step S331, and stores the numbers in the SSD 304. In step S333, the CPU 301 acquires an index for screening mature lymphoma based on the number of target mononuclear cells, the number of mononuclear leukocytes and late differentiation erythroblasts, and the number of nucleated cells. When acquiring the fifth ratio as the index, the CPU 301 performs the calculations shown in the above formulas (B) and (D) to acquire the second ratio and the fourth ratio. Furthermore, the CPU 301 performs the calculations shown in the above formula (E) to acquire the fifth ratio. The CPU 301 stores the acquired index for screening mature lymphoma in the SSD 304. Step S334 is the same as step S304. That is, the CPU 301 judges abnormality of the bone marrow fluid in step 335.

[0152] The analysis process of the measurement data of the fifth embodiment will be described with reference to FIG. 11E. In this analysis process, an index for screening of plasmacytoma is obtained as an index for screening of hematopoietic tumor. When the analysis process of the measurement data is started, the CPU 301 of the analysis unit 3 selects plasma cells and nucleated cells based on the fluorescence intensity and side scattered light intensity included in the measurement data (step S341). In this analysis process, the digital signal for fluorescence is represented by 10-bit data, and the digital signal for side scattering is represented by 8-bit data. Therefore, the range of the fluorescence signal intensity is 0 to 1023ch, and the range of the side scattered light intensity is 0 to 255ch. The selection of nucleated cells is the same as the analysis process of the first embodiment. The CPU 301 selects plasma cells by extracting particles included in the fifth region from particles in the sample based on the fluorescence signal intensity and the side scattered light intensity. The CPU 301 may create a scattergram using the data of the fluorescence signal intensity and the side scattered light intensity.

[0153] In step S342, CPU 301 counts the target mononuclear cells and nucleated cells selected in step S341, and stores the numbers in SSD 304. In step S343, CPU 301 acquires an index for screening plasmacytoma based on the number of plasma cells and the number of nucleated cells. When acquiring a sixth ratio as the index, CPU 301 performs the calculation shown in the above formula (F) to acquire the sixth ratio. CPU 301 stores the acquired index for screening plasmacytoma in SSD 304. Step S344 is the same as step S304. That is, CPU 301 determines abnormality of bone marrow fluid in step S345.

[0154] 9, when the above-mentioned measurement data analysis process is completed, in step S109, CPU 301 outputs the analysis result to display unit 310, and the process ends.

[0155] As described above, the analysis process of the measurement data shown in each of Figs. 11A to 11E has been described individually. However, it is sufficient to carry out only a part of the analysis process of the measurement data in Fig. 9 (step S108), or all of the analysis process may be carried out. For example, referring to Fig. 11A, in addition to the target cells and nucleated cells, target mononuclear cells, blast cells, mononuclear leukocytes, differentiated late erythroblasts, and plasma cells may be selected in step S301, and counted in step S302. In addition, in step S303, as an index for screening hematopoietic tumors, an index for screening acute leukemia, mature lymphoma, and plasmacytoma may also be obtained.

[0156] Hereinafter, as an example of the process of determining abnormality in bone marrow fluid, a case of determining whether or not a subject is suspected of hematopoietic tumor will be described. With reference to FIG. 12A, in the determination process of the first embodiment, a first ratio calculated from the above formula (A) is obtained as an index for screening for hematopoietic tumor, and a determination is made based on this ratio. However, the present invention is not limited to this. In step S401, the CPU 301 compares the first ratio with a threshold value (e.g., 30%) corresponding to the first ratio. This threshold value is stored in the SSD 304. When the first ratio is lower than the predetermined threshold value, the process proceeds to step S402. The CPU 301 obtains a determination result that there is no suspicion of hematopoietic tumor, and stores it in the SSD 304. When the first ratio is equal to or greater than the predetermined threshold, the process proceeds to step S403. The CPU 301 obtains a determination result that there is a suspicion of hematopoietic tumor, and stores it in the SSD 304. With the above, the CPU 301 ends the determination process and returns the process to the main routine.

[0157] Referring to FIG. 12B, in the judgment process of the second embodiment, the second ratio calculated from the above formula (B) is obtained as an index for screening for hematopoietic tumors, and judgment is made based on this ratio. However, the present invention is not limited to this. In step S411, CPU301 compares the second ratio with a threshold value corresponding to the second ratio (e.g., 20%). This threshold value is stored in SSD304. When the second ratio is lower than the predetermined threshold, the process proceeds to step S412. CPU301 obtains a judgment result that there is no suspicion of hematopoietic tumors and stores it in SSD304. When the second ratio is equal to or greater than the predetermined threshold, the process proceeds to step S413. CPU301 obtains a judgment result that there is a suspicion of hematopoietic tumors and stores it in SSD304. With the above, CPU301 ends the judgment process and returns the process to the main routine.

[0158] Referring to FIG. 12C, in the judgment process of the third embodiment, the third ratio calculated from the above formula (C) is obtained as an index for screening for acute leukemia, and judgment is made based on this ratio. In the judgment, a first threshold and a second threshold higher than the first threshold are used as predetermined thresholds corresponding to the third ratio. However, the present invention is not limited to this. In step S421, the CPU 301 compares the third ratio with the first threshold (e.g., 10%). The first threshold is stored in the SSD 304. When the third ratio is lower than the first threshold, the process proceeds to step S422. The CPU 301 obtains a judgment result that there is no suspicion of acute leukemia and stores it in the SSD 304. When the third ratio is equal to or higher than the first threshold, the process proceeds to step S423. In step S423, the CPU 301 compares the third ratio with the second threshold (e.g., 20%). The second threshold is stored in SSD 304. When the third ratio is lower than the second threshold, the process proceeds to step S424. CPU 301 obtains a determination result that there is a suspicion of an increase in blast cells, and stores the result in SSD 304. When the third ratio is equal to or greater than the second threshold, the process proceeds to step S425. CPU 301 obtains a determination result that there is a suspicion of acute leukemia, and stores the result in SSD 304. With this, CPU 301 ends the determination process, and returns the process to the main routine.

[0159] Referring to FIG. 12D, in the judgment process of the fourth embodiment, the fifth ratio calculated from the above formula (E) is obtained as an index for screening mature lymphoma, and judgment is made based on this ratio. However, the present invention is not limited to this. In step S431, CPU301 compares the fifth ratio with a threshold value corresponding to the fifth ratio (for example, 10%). This threshold value is stored in SSD304. When the fifth ratio is lower than the predetermined threshold, the process proceeds to step S432. CPU301 obtains a judgment result that there is a suspicion of mature lymphoma, and stores it in SSD304. When the fifth ratio is equal to or greater than the predetermined threshold, the process proceeds to step S433. CPU301 obtains a judgment result that there is no suspicion of mature lymphoma, and stores it in SSD304. With the above, CPU301 ends the judgment process and returns the process to the main routine.

[0160] Referring to FIG. 12E, in the determination process of the fifth embodiment, the sixth ratio calculated from the above formula (F) is obtained as an index for screening for plasmacytoma, and a determination is made based on this ratio. However, the present invention is not limited to this. In step S441, CPU301 compares the sixth ratio with a threshold value corresponding to the sixth ratio (for example, 5%). This threshold value is stored in SSD304. When the sixth ratio is lower than the predetermined threshold, the process proceeds to step S442. CPU301 obtains a determination result that there is no suspicion of plasmacytoma and stores it in SSD304. When the sixth ratio is equal to or greater than the predetermined threshold, the process proceeds to step S443. CPU301 obtains a determination result that there is a suspicion of plasmacytoma and stores it in SSD304. With the above, CPU301 ends the determination process and returns the process to the main routine.

[0161] 9, when the above-mentioned determination process is completed, in step S109, CPU 301 outputs the determination result as the analysis result to display unit 310, and ends the process. As described above, the "determination process of whether or not there is a suspicion of hematopoietic tumor," the "determination process of whether or not there is a suspicion of acute leukemia," the "determination process of whether or not there is a suspicion of mature lymphoma," and the "determination process of whether or not there is a suspicion of plasmacytoma" have been individually explained using FIGS. 12A to 12E. However, it is sufficient to carry out any part of the determination process of abnormality in bone marrow fluid, or all of the determination processes may be carried out.

[0162] The analysis result screen output to the display unit 310 may include, for example, the number of nucleated cells (TNC) and a ratio (RATIO) as an index for screening for hematopoietic tumors. The screen may also include a scattergram created based on optical information in addition to these pieces of information. Furthermore, the screen may include the results of a determination of abnormalities in bone marrow fluid based on the index for screening for hematopoietic tumors.

[0163] In this way, the sample analyzer and computer program of this embodiment can provide medical professionals such as doctors with information to assist in screening for hematopoietic tumors and determining abnormalities in bone marrow fluid. Medical professionals who obtain the information can decide whether or not to prioritize testing for hematopoietic tumors on the analyzed bone marrow fluid. In addition, they can perform bone marrow imaging tests by assuming suspected hematopoietic tumors and cells of interest based on the obtained information.

[0164] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0165] Example 1: Screening based on the number of cells with a fluorescent signal intensity equal to or greater than a given threshold In acute leukemia, an increase in early differentiation immature cells in the bone marrow is a sign. In plasmacytoma (myeloma), an increase in plasma cells in the bone marrow is a sign. Early differentiation immature cells and plasma cells have a higher amount of nucleic acid than mature cells or late differentiation cells. Therefore, we analyzed cells in bone marrow fluid by FCM and investigated whether it is possible to screen for hematopoietic tumors based on the amount of nucleic acid in cells.

[0166] (1) Sample The specimens used were bone marrow fluid from the following cases (38 cases in total) confirmed by bone marrow imaging (microscopic examination) at the hospital. - 2 cases of acute leukemia (blast count to nucleated cell count ratio of 20% or more) -1 case of plasmacytoma (ratio of plasma cells to nucleated cells is 10% or more) -One case of moderate increase in blasts (ratio of blasts to nucleated cells is 10% to less than 20%) -Three cases of mild increase in blast cells (ratio of blast cells to nucleated cells: 5% to less than 10%) - 31 cases in which no increase in blast cells or plasma cells was observed

[0167] (2) Reagents and analytical equipment The hemolysis reagent used was Lysacell WDFII (Sysmex Corporation). The staining reagent used was Fluorocell WDF (Sysmex Corporation). The analyzer used was an automated blood cell analyzer equipped with an FCM, XR-1000 (Sysmex Corporation).

[0168] (3) Measurement (3.1) Preparation of smear samples and microscopic examination To compare with the analytical results obtained by the analyzer, the above bone marrow fluid was subjected to microscopic examination. A portion was taken from each specimen and stained according to standard methods to prepare smears. The smears were observed under a microscope to count the number of nucleated cells. The presence or absence of the following target cells was also confirmed. If target cells were detected, the number of cells was counted. The target cells in the microscopic examination were blast cells, proerythroblasts, polychromatic erythroblasts, plasma cells, promonocytes, immature neutrophils (promyelocytes and myelocytes), and megakaryocytic cells. The cell count was the sum of these target cells.

[0169] (3.2) Preparation and measurement of samples using analytical equipment Each specimen was filtered through a nylon mesh with a pore size of 40 μm to remove bone fragments, and then set in the automatic blood cell counter XR-1000. Sample preparation and measurement were performed automatically by the analyzer. Specifically, a hemolysis reagent (50 μL) and a staining reagent (1 μL) were added to bone marrow fluid (1 μL), and the sample was prepared by incubating at 40°C for 20 seconds. The sample was irradiated with light to obtain optical information emitted from each particle in the sample. As optical information, fluorescent signal intensity and side scattered light intensity were obtained. In addition, based on the obtained optical information, a scattergram was created with the side scattered light intensity on the horizontal axis and the fluorescent signal intensity on the vertical axis. The scattergram was created with the minimum value of the side scattered light intensity and the fluorescent signal intensity set to 0ch and the maximum value to 255ch. The time required from setting the specimen in the automatic blood cell counter to creating the scattergram was within 3 minutes in each case.

[0170] (4) Analysis and Results An example of the scattergram created is shown in Figure 13. For each sample measured by the analyzer, nucleated cells were counted based on the optical information. In addition, a predetermined threshold for the fluorescent signal intensity was set to 100ch, and particles with a fluorescent signal intensity of 100ch or more were counted. In Figure 13, particles with a fluorescent signal intensity of 100ch or more appeared in the area surrounded by a dashed line. This area was the upper area when the two-dimensional plane of the scattergram was divided by a fluorescent signal intensity of 100ch (hereinafter also referred to as the "area with a fluorescent signal intensity of 100ch or more").

[0171] The 100ch fluorescent signal intensity was a value previously determined as a predetermined threshold. Specifically, it was determined as follows. First, 20 control samples were prepared from peripheral blood (20 specimens) of healthy subjects using the above staining reagent and hemolysis reagent, and FCM measurement was performed using the above analyzer. Nucleated cells were selected and counted based on the fluorescent signal intensity and side scattered light intensity. Next, the 100ch fluorescent signal intensity was set as a provisional threshold, and cells having a fluorescent signal intensity of 100ch or more were counted. Then, the ratio of the number of cells having a fluorescent signal intensity of 100ch or more to the number of nucleated cells was calculated. The median, 10th percentile, and 90th percentile of the ratio in the 20 control samples were calculated. As a result, the median was 3.89%, the 10th percentile was 2.52%, and the 90th percentile was 5.07%. This result suggests that the fluorescence intensity of 100ch is a value that can effectively distinguish between mature leukocytes and cells in the late differentiation stage, which have relatively low amounts of nucleic acid, and control cells, which have relatively high amounts of nucleic acid. Therefore, the fluorescence intensity of 100ch was determined as the predetermined threshold. In fact, in FIG. 13, mature leukocytes (lymphocytes, monocytes, neutrophils, and eosinophils) in bone marrow fluid appeared in the lower region (hereinafter also referred to as the "region with a fluorescence signal intensity of less than 100ch") when the two-dimensional plane of the scattergram was divided by the fluorescence signal intensity of 100ch. Note that the diagonal lines in FIG. 13 indicate the boundary between the region where mononuclear cells appear and the region where polymorphonuclear cells appear.

[0172] For each specimen, the ratio of the number of particles with a fluorescent signal intensity of 100ch or more to the number of nucleated cells was calculated as the first ratio. For the nucleated cells and target cells counted by microscopic examination, the ratio of the number of target cells to the number of nucleated cells was also calculated. For each specimen, the first ratio obtained by the analyzer and the ratio obtained by microscopic examination were plotted to obtain a regression line (y=0.62x+8.69). The correlation coefficient (r) was 0.88. This graph is shown in FIG. 14. As can be seen from FIG. 14, there was a good correlation between the first ratio obtained by the analyzer and the ratio obtained by microscopic examination. In addition, specimens in which an increase in blasts or plasma cells was observed tended to have a high first ratio. In particular, it was found that when the threshold value of the first ratio was set to 30%, specimens of acute leukemia and plasmacytoma could be distinguished from other specimens. Therefore, it was suggested that rapid screening for acute leukemia and plasmacytoma can be performed by using an analytical device equipped with FCM to count nucleated cells and particles in bone marrow fluid whose fluorescent signal intensity is equal to or greater than a predetermined threshold and obtain a first ratio.

[0173] Example 2: Screening based on the number of mononuclear cells with a fluorescent signal intensity equal to or greater than a given threshold Blast cells and plasma cells are mononuclear cells. Therefore, mononuclear cells were further selected from the particles selected in Example 1. We investigated whether screening for hematopoietic tumors is possible by analyzing the selected mononuclear cells.

[0174] (1) Samples, reagents and analytical equipment The samples, reagents and analytical equipment were the same as in Example 1.

[0175] (2) Measurement data and analysis The counting results of Example 1 were used for the number of cells in the smear. In Example 2, the target cells for the microscopic examination were mononuclear cells, namely blast cells, proerythroblasts, polychromatic erythroblasts, plasma cells, and promonocytes. The sum of the numbers of these cells was obtained from the counting results of Example 1 and used as the number of target cells. The optical information of each specimen obtained in Example 1 was used as the measurement data of each specimen by the analyzer. With reference to FIG. 15, in order to select mononuclear cells from particles with a fluorescent signal intensity of 100ch or more, a region surrounded by points C, D, F, and E (hereinafter also referred to as "region CDFE") was specified among the regions with a fluorescent signal intensity of 100ch or more. In FIG. 15, the region CDFE is indicated by a dashed line. The coordinates of points C, D, E, and F were as follows, when the horizontal axis indicating the side scattered light intensity is called the X-axis and the vertical axis indicating the fluorescent signal intensity is called the Y-axis. Point C (X:Y=0ch:100ch), point D (X:Y=135ch:100ch), point E (X:Y=0ch:255ch) and point F (X:Y=180ch:255ch). In FIG. 15, the diagonal line passing through points F and D indicates the boundary between the area where mononuclear cells appear and the area where polymorphonuclear cells appear. In FIG. 15, point A is the origin (X:Y=0ch:0ch), and point B is the intersection of the diagonal line and the X-axis (X:Y=100ch:0ch).

[0176] As shown in FIG. 15, the region CDFE was the region to the left of the diagonal line among the regions with a fluorescent signal intensity of 100ch or more. For each sample, the particles that appeared in the region CDFE were counted, and the number of mononuclear cells with a fluorescent signal intensity of 100ch or more was obtained. As a second ratio, the ratio of the number of mononuclear cells with a fluorescent signal intensity of 100ch or more to the number of nucleated cells was calculated. For the nucleated cells and target cells counted by microscopic examination, the ratio of the number of target cells to the number of nucleated cells was also calculated.

[0177] For each sample, the second ratio obtained by the analyzer and the ratio obtained by microscopic examination were plotted to obtain a regression line (y=0.64x+5.07). The correlation coefficient (r) was 0.95. This graph is shown in FIG. 16. As can be seen from FIG. 16, there was a good correlation between the second ratio obtained by the analyzer and the ratio obtained by microscopic examination. In addition, samples in which an increase in blast cells or plasma cells was observed tended to have a high second ratio. In particular, it was found that when the threshold value of the second ratio was set to 20%, samples of acute leukemia and plasmacytoma could be distinguished from other samples. Therefore, it was suggested that the analysis device equipped with FCM can be used to count nucleated cells and mononuclear cells with a fluorescent signal intensity equal to or greater than a predetermined threshold in bone marrow fluid to obtain the second ratio, thereby quickly screening for acute leukemia and plasmacytoma.

[0178] Example 3: Screening based on blast counts In acute leukemia, neoplastic blast cells that do not have the ability to differentiate proliferate. Therefore, blast cells were further selected from the particles selected in Example 1. By analyzing the selected blast cells, it was examined whether it is possible to screen for acute leukemia.

[0179] (1) Samples, reagents and analytical equipment The samples, reagents and analytical equipment were the same as in Example 1.

[0180] (2) Measurement data and analysis The counting results of Example 1 were used for the number of cells in the smear. In Example 3, the target cells for the microscopic examination were blasts. However, the blasts were not distinguished as benign or malignant. Based on the counting results of Example 1, the number of blasts was used as the target cell number. The optical information of each specimen obtained in Example 1 was used as the measurement data of each specimen by the analyzer. With reference to FIG. 17, in order to select blasts from particles with a fluorescent signal intensity of 100ch or more, a region surrounded by points H, D, F, and I was specified among the regions with a fluorescent signal intensity of 100ch or more. In addition, in order to count blasts more accurately, a region surrounded by points D, G, and H was specified in the scattergram in FIG. 17. Therefore, a region surrounded by points H, G, F, and I (hereinafter also referred to as "region HGFI") was specified as a region in which blasts appear in the scattergram. The coordinates of points D, F, G, H, and I were as follows. Point D (X:Y = 135ch:100ch), point F (X:Y = 180ch:255ch), point G (X:Y = 130ch:80ch), point H (X:Y = 80ch:100ch) and point I (X:Y = 130ch:255ch).

[0181] As shown in FIG. 17, the region HGFI was the region where blast cells appeared in the scattergram based on the fluorescent signal intensity and the side scattered light intensity. For each specimen, the particles that appeared in the region HGFI were counted. Referring to FIG. 18, in order to count blast cells more accurately, particles that appeared in the region HGFI and had a forward scattered light intensity of 50ch or more and 100ch or less were selected. The number of selected particles was obtained as the number of blast cells. As the third ratio, the ratio of the number of blast cells to the number of nucleated cells was calculated. For the nucleated cells and blast cells counted by microscopic examination, the ratio of the number of blast cells to the number of nucleated cells was also calculated.

[0182] For each sample, the third ratio obtained by the analyzer and the ratio obtained by microscopic examination were plotted to obtain a regression line (y=0.60x+5.26). The correlation coefficient (r) was 0.921. This graph is shown in FIG. 19. As can be seen from FIG. 19, there was a good correlation between the third ratio obtained by the analyzer and the ratio obtained by microscopic examination. Furthermore, samples in which the ratio of the number of blast cells to the number of nucleated cells was 10% or more tended to have a high third ratio. In particular, it was found that when the threshold value of the third ratio was set to 20%, it was possible to distinguish between samples with acute leukemia and other samples. Therefore, it was suggested that acute leukemia screening could be performed quickly by counting the nucleated cells and blast cells in bone marrow fluid and obtaining the third ratio using an analyzer equipped with FCM.

[0183] Example 4: Indication of the appearance of neoplastic cells The performance of the analytical device to discriminate samples suspected of hematopoietic tumors was examined in comparison with that of samples examined by microscopy.

[0184] (1) Sample The specimens used were bone marrow fluid from the following cases (38 cases in total) that were confirmed by microscopic examination at the hospital. - 2 cases of acute leukemia (blast count to nucleated cell count ratio of 20% or more) -1 case of plasmacytoma (ratio of plasma cells to nucleated cells is 10% or more) -One case of moderate increase in blasts (ratio of blasts to nucleated cells is 10% to less than 20%) -Three cases of mild increase in blast cells (ratio of blast cells to nucleated cells: 5% to less than 10%) - 5 cases of bone marrow metastasis of mature lymphoma (ratio of mature lymphoma cells to nucleated cells is 5% or more) - 26 cases in which no increase in blast cells or plasma cells was observed

[0185] (2) Reagents, analytical equipment and measurements The reagents and the analytical device were the same as those in Example 1. Using these, the above samples were subjected to measurement by the analytical device and microscopic examination in the same manner as in Example 1.

[0186] (3) Analysis and Results (3.1) Identification of specimens with increased blast counts The smear of each sample was observed under a microscope to obtain the number of nucleated cells and the number of blast cells. Then, the ratio of the number of blast cells to the number of nucleated cells was calculated. Samples with a calculated ratio of 10% or more were determined to be samples suspected of having an increase in blast cells. Furthermore, based on the optical information of each sample obtained by the analyzer, analysis was performed in the same manner as in Example 3 to obtain the number of nucleated cells and the number of blast cells. Then, a third ratio was calculated. Samples with a third ratio of 10% or more were determined to be samples suspected of having an increase in blast cells. The results of the comparison between the determination by the analyzer and the determination by microscopy are shown in Table 1. In the table, "Positive" indicates a sample suspected of having an increase in blast cells, and "Negative" indicates a sample in which an increase in blast cells was not observed.

[0187] [Table 1]

[0188] As shown in Table 1, the agreement rate between the discrimination results by the analyzer and the discrimination results by microscopic examination was 84.2%. In addition, the sensitivity of discrimination by the analyzer was 100%, and the specificity was 82.9%. Thus, the agreement rate between the discrimination results by the analyzer and the microscopic examination was high, indicating that the discrimination performance of the analyzer for samples with increased blasts is good. When the analyzer obtains a discrimination result that the third ratio of the sample is 10% or more, the analyzer may output a sign such as "Blastosis?" on the display screen of the analysis result of the sample. This can provide the user with an indicator for screening for acute leukemia.

[0189] (3.2) Identification of mature lymphoma specimens The smear of each specimen was observed under a microscope to obtain the number of nucleated cells and the number of mature lymphoma cells. Then, the ratio of the number of mature lymphoma cells to the number of nucleated cells was calculated. Specimens with a calculated ratio of 10% or more were determined to be specimens suspected of having mature lymphoma. Furthermore, based on the optical information of each specimen obtained by the analyzer, analysis was performed in the same manner as in Example 2 to obtain the number of nucleated cells and the number of mononuclear cells with a fluorescent signal intensity of 100ch or more. Also, a second ratio was calculated. Furthermore, based on the optical information of each specimen, the number of mononuclear cells with a fluorescent signal intensity of less than 100ch was obtained. Mononuclear cells with a fluorescent signal intensity less than a predetermined threshold appeared in the region surrounded by points A, B, D, and C with reference to FIG. 15 (hereinafter, also referred to as "region ABDC") in the scattergram of each specimen. As a fourth ratio, the ratio of the number of mononuclear cells with a fluorescent signal intensity of less than 100ch to the number of nucleated cells was calculated. Then, a fifth ratio was calculated by the above formula (E).

[0190] Samples in which the fifth ratio was less than 10% were determined to be samples suspected of having mature lymphoma. The results of comparing the determination by the analyzer with the determination by microscopic examination are shown in Table 2. In the table, "Positive" indicates samples suspected of having mature lymphoma, and "Negative" indicates samples determined not to have mature lymphoma.

[0191] [Table 2]

[0192] As shown in Table 2, the agreement rate between the results of discrimination by the analyzer and the results of discrimination by microscopy was 92.1%. In addition, the sensitivity of discrimination by the analyzer was 75.0%, and the specificity was 94.1%. Thus, the agreement rate between the results of discrimination by the analyzer and the microscopy was high, demonstrating that the discrimination performance of the analyzer for mature lymphoma samples is good. When the analyzer obtains a discrimination result that the fifth ratio of the sample is less than 10%, the analyzer may output a sign such as "Mature Lymphocytosis?" on the display screen of the analysis result of the sample. This can provide the user with an indicator for screening for mature lymphoma.

[0193] (3.3) Identification of Plasmacytoma Samples The smear of each specimen was observed under a microscope to obtain the number of nucleated cells and the number of plasma cells. The ratio of the number of plasma cells to the number of nucleated cells was then calculated. Specimens with a calculated ratio of 10% or more were determined to be specimens suspected of plasmacytoma. In addition, based on the optical information of each specimen obtained by the analyzer, a scattergram was created with the side scattered light intensity on the horizontal axis and the fluorescence signal intensity on the vertical axis. In the scattergram, the minimum value of the fluorescence signal intensity was set to 0ch and the maximum value to 1023ch, and the minimum value of the side scattered light intensity was set to 0ch and the maximum value to 255ch. An example of the created scattergram is shown in FIG. 20. In the figure, plasma cells appeared in the area surrounded by an ellipse. In order to identify plasma cells, the area surrounded by points J, K, L, and M (hereinafter also referred to as "area JKLM") was identified with reference to FIG. 20. In FIG. 20, the area JKLM is shown by a dashed line. The coordinates of points J, K, L and M, where the horizontal axis indicating the side scattered light intensity is called the X-axis and the vertical axis indicating the fluorescent signal intensity is called the Y-axis, were as follows: point J (X:Y=0ch:1023ch), point K (X:Y=170ch:1023ch), point L (X:Y=170ch:296ch) and point M (X:Y=0ch:296ch).

[0194] Referring to FIG. 20, region JKLM was a region containing plasma cells in a scattergram based on the fluorescent signal intensity and the side scattered light intensity. For each sample, the particles that appeared in region JKLM were counted and obtained as the number of plasma cells. The ratio of the number of plasma cells to the number of nucleated cells was calculated as the sixth ratio. Samples with a sixth ratio of 5% or more were determined to be samples suspected of plasmacytoma. The results of comparing the determination by the analyzer with the determination by microscopic examination are shown in Table 3. In the table, "Positive" indicates samples suspected of plasmacytoma, and "Negative" indicates samples determined not to be plasmacytoma.

[0195] [Table 3]

[0196] As shown in Table 3, the agreement rate between the discrimination results by the analyzer and the discrimination results by microscopy was 100%. In addition, the sensitivity and specificity of discrimination by the analyzer were both 100%. Thus, the agreement rate between the discrimination results by the analyzer and the microscopy was high, demonstrating that the discrimination performance of the analyzer for plasmacytoma samples is good. When the analyzer obtains a discrimination result that the sixth ratio of the sample is 10% or more, it may output a sign such as "Plasmacytosis?" on the display screen of the analysis result of the sample. This can provide the user with an indicator for screening for plasmacytoma.

[0197] According to this embodiment, discrimination results highly correlated with visual inspection can be obtained within 3 minutes from setting the specimen on the device. Therefore, screening for hematopoietic tumors, which was previously performed by visual inspection, can be performed automatically and quickly, and for example, acute myeloid leukemia patients who require early treatment can be identified and subsequent detailed examinations can be performed promptly. [Explanation of symbols]

[0198] 10. Sample analysis equipment 20 Measuring part 30 Analysis Department 40 Suction part 50 Sample Preparation Section 51, 52 and 53 Reagent containers 54 Reactor 60 Detection unit 61 Flow Cell 62 Light source section 63, 64 and 65 Light receiving unit 66 Dichroic Mirror 300 Body 309 Input section 310 Display section 321 Portable recording media

Claims

1. Measuring a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acids by flow cytometry, and obtaining optical information including fluorescence signal information about particles in the sample; Counting particles with fluorescence signal information equal to or higher than a threshold value as target cells; Obtaining an index for screening hematopoietic tumors based on the number of the target cells. A method for analyzing bone marrow fluid, comprising these steps.

2. The analysis method according to claim 1, wherein the optical information further includes at least one of side scatter light information and forward scatter light information.

3. The analysis method according to claim 2, wherein in the step of obtaining the number of the target cells, the number of nucleated cells is further obtained based on the optical information.

4. The analysis method according to claim 3, wherein in the step of obtaining the index, a first ratio, which is the ratio of the number of the target cells to the number of the nucleated cells, is obtained.

5. The analysis method according to claim 4, further comprising the step of providing a distinguishable display when the first ratio is equal to or higher than a threshold value corresponding to the first ratio.

6. The analysis method according to claim 5, wherein the threshold value corresponding to the first ratio is any value between 20% and 40%.

7. The analysis method according to claim 5, wherein the threshold value corresponding to the first ratio is 30%.

8. The analysis method according to claim 1, wherein the threshold value corresponding to the fluorescence signal information is a value capable of excluding 95% or more of mature white blood cells.

9. The analysis method according to claim 2, wherein the threshold value corresponding to the fluorescence signal information is represented by a straight line that intersects the monocyte population in the sample but does not intersect the lymphocyte population when the distribution of particles in the sample is drawn on a two-dimensional plane based on the fluorescence signal information and the side scatter light information.

10. The analysis method according to claim 1, wherein the threshold value corresponding to the fluorescence signal information is a value satisfying the following conditions: Measuring a plurality of control samples prepared from peripheral blood of at least 20 healthy subjects and the fluorescent dye by flow cytometry, and obtaining fluorescence signal information and side scatter light information about the plurality of control samples; Obtaining the number of nucleated cells in the plurality of control samples based on the fluorescence signal information and the side scatter light information; Setting an arbitrary fluorescence signal intensity as a provisional threshold value, and obtaining the number of cells having a fluorescence signal intensity equal to or higher than the provisional threshold value; Calculating the ratio of the number of cells having a fluorescence signal intensity equal to or higher than the provisional threshold value to the number of nucleated cells. When calculating the median of the ratios in the plurality of control samples, the median is 2.5% or more and 5% or less.

11. The optical information includes fluorescence signal information and side scatter light information, In the step of counting the target cells, monocytes with fluorescence signal information equal to or greater than a threshold are counted, In the step of obtaining the index, a second ratio that is the ratio of the number of monocytes to the number of nucleated cells is obtained. The analysis method according to claim 3.

12. When the second ratio is equal to or greater than a threshold corresponding to the second ratio, the method further includes the step of providing a distinguishable display. The analysis method according to claim 11.

13. The threshold corresponding to the second ratio is any value between 10% and 30%. The analysis method according to claim 12.

14. The threshold corresponding to the second ratio is 20%. The analysis method according to claim 12.

15. The optical information includes fluorescence signal information, side scatter light information, and forward scatter light information, In the step of counting the target cells, blast cells are counted, In the step of obtaining the index, a third ratio that is the ratio of the number of blast cells to the number of nucleated cells is obtained. The analysis method according to claim 3.

16. When the third ratio is equal to or greater than a threshold corresponding to the third ratio, the method further includes the step of providing a distinguishable display. The analysis method according to claim 15.

17. The threshold corresponding to the third ratio is any value between 5% and 25%. The analysis method according to claim 16.

18. The threshold corresponding to the third ratio is 10%. The analysis method according to claim 17.

19. The threshold corresponding to the third ratio is 20%. The analysis method according to claim 17.

20. In the step of obtaining the number of target cells, monocytes with fluorescence signal information lower than the threshold are counted, In the step of obtaining the index, a fourth ratio that is the ratio of the number of monocytes to the number of nucleated cells is obtained, and a fifth ratio that is the ratio of the value of the second ratio to the value of the fourth ratio is obtained. The analysis method according to claim 11.

21. When the fifth ratio is less than or equal to a threshold corresponding to the fifth ratio, the method further includes the step of providing a distinguishable display. The analysis method according to claim 20.

22. The threshold corresponding to the fifth ratio is any value between 5% and 15%. The analysis method according to claim 21.

23. The analysis method according to claim 21, wherein the threshold value corresponding to the fifth ratio is 10%.

24. wherein the optical information includes fluorescence signal information and side scatter light information, in the step of counting the target cells, plasma cells are counted, The analysis method according to claim 3, wherein in the step of obtaining the index, a sixth ratio that is the ratio of the number of plasma cells to the number of nucleated cells is obtained.

25. The analysis method according to claim 24, further comprising the step of providing an identifiable display when the sixth ratio is equal to or greater than a threshold value corresponding to the sixth ratio.

26. The analysis method according to claim 25, wherein the threshold value corresponding to the sixth ratio is any value between 5% and 15%.

27. The analysis method according to claim 25, wherein the threshold value corresponding to the sixth ratio is 5%.

28. The analysis method according to claim 5, 12, 16, 21 or 25, wherein the identifiable display indicates that the bone marrow fluid is a specimen that needs to be preferentially tested for hematopoietic tumors.

29. The analysis method according to claim 1, wherein the sample further comprises a cationic surfactant.

30. The analysis method according to claim 29, wherein the sample is prepared by mixing bone marrow fluid, a staining reagent containing a fluorescent dye, and a hemolytic reagent containing a cationic surfactant.

31. A sample preparation unit that prepares a sample containing bone marrow fluid and a fluorescent dye capable of staining nucleic acids, a detection unit that acquires optical information including fluorescence signal information about particles in the sample, a control unit that counts particles having a fluorescence signal information equal to or greater than a threshold value as target cells, and the control unit acquires an index for screening hematopoietic tumors based on the number of the target cells, Sample analyzer.

32. The detection unit is a flow cell configured to flow the sample prepared by the sample preparation unit, a light source unit that irradiates light onto particles in the sample flowing in the flow cell, a light receiving unit that acquires the optical information obtained when the particles are irradiated with light, The sample analyzer according to claim 31, comprising:

33. A computer program for analyzing bone marrow fluid, obtaining optical information including fluorescence signal information about particles in a sample containing the bone marrow fluid and a fluorescent dye capable of staining nucleic acids, counting, based on the optical information, particles having a fluorescence signal intensity equal to or greater than a threshold value as target cells, A step of obtaining an index for screening hematopoietic tumors based on the number of the target cells; To be executed by a computer, A computer program.

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