JP4884049B2 - Megakaryocyte counting method and apparatus - Google Patents

Description

  The present invention relates to a megakaryocyte counting method.

  In the field of clinical examination, extremely useful information can be obtained in diagnosing diseases by classifying and counting megakaryocytes of bone marrow. For example, there are usually a certain number of megakaryocytes in normal bone marrow. Megakaryocytes are the mother cells of platelets, and the number of megakaryocytes may fluctuate due to the presence of diseases such as thrombocytopenia and platelet increase in peripheral blood. Useful for obtaining information about existence. For example, in idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), essential thrombocythemia, and chronic myeloid leukemia, the number of megakaryocytes increases and aplastic anemia occurs. Decreases the number of megakaryocytes.

  Conventionally, in order to calculate the number of megakaryocytes, the bone marrow aspiration fluid is appropriately stained, then transferred to a hemocytometer such as the Fuchs-Rosenthal calculator, and then counted and counted with a microscope. Met. The morphological features of megakaryocytes include (1) large cells, (2) multinuclear, (3) the cytoplasm looks dull, but the criteria are very ambiguous, Since the ratio of megakaryocytes in the bone marrow is very low, the visual method varies greatly depending on the measurer.

  In recent years, attempts have been made to classify and count megakaryocytes using the principle of a flow cytometer. For example, in Non-Patent Document 1, megakaryocytes are classified and counted by a two-color measurement method using a fluorescently labeled antiplatelet antibody and propidium iodide. Specifically, first, mononuclear cells are separated from the bone marrow aspirate. The obtained sample containing mononuclear cells contains megakaryocytes, and the megakaryocytes in this sample are labeled with a fluorescently labeled anti-platelet antibody. Next, the cells in the sample are fixed with 2% paraformaldehyde and then stained with propidium iodide. The samples thus obtained are measured with a flow cytometer to classify and count megakaryocytes. In this method, since mononuclear cells are separated, the operation becomes complicated and it takes time until measurement. In addition, some megakaryocytes may not be collected during the separation operation, and the number of megakaryocytes cannot be accurately classified and counted.

  On the other hand, Patent Document 1 describes a method of measuring megakaryocytes using an automatic blood cell counter. Specifically, first, Dami cells, which are megakaryocyte cell lines, are cultured to obtain samples containing megakaryocytes, or purified CD34 positive cells are cultured to obtain samples containing megakaryocytes. The obtained sample is measured with an automatic blood cell counter, and it is described that megakaryocytes appear in the obtained two-dimensional distribution map. In addition, by comparing a two-dimensional distribution chart obtained by measuring a sample containing purified megakaryocytes with a two-dimensional distribution chart obtained by measuring a sample not containing megakaryocytes, It is also described that the region where the sphere appears can be determined. However, bone marrow puncture fluid collected from a living body contains various cells in addition to megakaryocytes. In particular, plasma cells appearing in multiple myeloma are very similar to the location of megakaryocytes. Therefore, a method capable of measuring megakaryocytes more accurately by distinguishing megakaryocytes contained in the bone marrow aspirate from other cells such as plasma cells is required.

Tomer et al., Blood, 1988; 71: 1244-1252 JP 2005-024557 A

  It is an object of the present invention to provide a method and apparatus for counting megakaryocytes more accurately.

In view of the above problems, the present invention provides a step of lysing red blood cells in a bone marrow puncture solution containing megakaryocytes and preparing a measurement sample by staining a nucleic acid in the megakaryocyte with a fluorescent dye. A step of irradiating excitation light, a step of detecting forward scattered light, side scattered light and fluorescence emitted from a cell, a first cell population including megakaryocytes is identified and detected based on the detected forward scattered light Identifying a second cell population containing megakaryocytes based on side scattered light and fluorescence, identifying cells belonging to the first cell population and belonging to the second cell population as megakaryocytes , And a method for counting megakaryocytes, comprising counting the identified megakaryocytes.
The present invention further includes a step of lysing red blood cells in a bone marrow aspirate containing megakaryocytes and staining a nucleic acid in megakaryocytes with a fluorescent dye to prepare a measurement sample, and irradiating cells in the measurement sample with excitation light A step of detecting forward scattered light, side scattered light and fluorescence emitted from a cell, a first cell population including megakaryocytes is identified based on the detected forward scattered light, and detected side scattering Identifying a second cell population containing megakaryocytes from the first cell population based on light and fluorescence, identifying cells belonging to the second cell population as megakaryocytes, and identified megakaryocytes A method of counting megakaryocytes, comprising the step of counting.
The present invention further includes a step of lysing red blood cells in a bone marrow aspirate containing megakaryocytes and staining a nucleic acid in megakaryocytes with a fluorescent dye to prepare a measurement sample, and irradiating cells in the measurement sample with excitation light A step of detecting forward scattered light, side scattered light and fluorescence emitted from the cells, a second cell population containing megakaryocytes is identified and detected based on the detected side scattered light and fluorescence Identifying a first cell population containing megakaryocytes from the second cell population based on forward scattered light, identifying cells belonging to the first cell population as megakaryocytes, and identified megakaryocytes A method of counting megakaryocytes, comprising the step of counting.

The present invention also provides a measurement sample preparation unit for preparing a measurement sample by mixing a bone marrow puncture solution containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes. A light source for irradiating cells in the measurement sample with excitation light, a first scattered light detector for detecting forward scattered light emitted from the cells, and for detecting side scattered light emitted from the cells. A second scattered light detector, a fluorescence detector for detecting fluorescence emitted from the cells, and a side on which the first cell population including megakaryocytes is identified and detected based on the detected forward scattered light Identifying a second cell population containing megakaryocytes based on the scattered light and fluorescence, identifying cells belonging to the first cell population and belonging to the second cell population as megakaryocytes, and Megakaryocyte counter including an analysis unit for counting spheres To provide.
The present invention further includes a bone marrow puncture solution containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes to prepare a measurement sample preparation unit for preparing a measurement sample, A light source for irradiating the cells in the measurement sample with excitation light, a first scattered light detector for detecting forward scattered light emitted from the cells, and a first for detecting side scattered light emitted from the cells. 2 scattered light detectors, a fluorescence detector for detecting fluorescence emitted from the cells, and a first cell population containing megakaryocytes based on the detected forward scattered light, and the detected lateral side Based on the scattered light and fluorescence, a second cell population containing megakaryocytes is identified from the first cell population, cells belonging to the second cell population are identified as megakaryocytes, and the identified megakaryocytes are counted. A megakaryocyte counting device including an analysis unit for That.
The present invention further includes a bone marrow puncture solution containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes to prepare a measurement sample preparation unit for preparing a measurement sample, A light source for irradiating the cells in the measurement sample with excitation light, a first scattered light detector for detecting forward scattered light emitted from the cells, and a first for detecting side scattered light emitted from the cells. Two scattered light detectors, a fluorescence detector for detecting fluorescence emitted from the cells, and a second cell population containing megakaryocytes based on the detected side scattered light and fluorescence are identified and detected Based on the forward scattered light, the first cell population including megakaryocytes is identified from the second cell population, the cells belonging to the first cell population are identified as megakaryocytes, and the identified megakaryocytes are counted. A megakaryocyte counting device including an analysis unit for That.

  According to the present invention, megakaryocytes can be counted more accurately.

  Megakaryocytes are very large cells compared to other blood cells. Therefore, megakaryocytes and other blood cells can be distinguished based on the forward scattered light that reflects the size of the cells. However, blood cells (eg, plasma cells) that are similar in size to megakaryocytes are difficult to distinguish from megakaryocytes based on forward scattered light. Therefore, the inventors have focused on the following characteristics regarding the nucleus of the megakaryocyte. Meganuclear spheres have large nuclei, and are markedly irregular with overlapping nuclei or pits with a pronounced nuclei shape, and the amount of nucleic acids contained in the nuclei is large. The inventors then analyzed blood cells that are similar in size to megakaryocytes based on side scattered light that reflects the intracellular structure and fluorescence that reflects the amount of nucleic acid in cells stained with nucleic acid fluorescent dyes. A distinction was made to find a method for accurately counting megakaryocytes.

  Specifically, first, erythrocytes in a sample containing megakaryocytes are lysed, and nucleic acids in the megakaryocytes are stained with a fluorescent dye to prepare a measurement sample. Next, the cells in the measurement sample are irradiated with excitation light, and forward scattered light, side scattered light and fluorescence emitted from the cells are detected. A megakaryocyte is identified based on the detected forward scattered light, fluorescence and side scattered light. Then, the identified megakaryocytes are counted. Here, “megakaryocytes” and “cells different in size from megakaryocytes” are distinguished by forward scattered light, and “megakaryocytes” and “cells that are similar in size to megakaryocytes by side scattered light and fluorescence. "Is distinguished. Thereby, megakaryocytes contained in the sample can be distinguished from other cells such as plasma cells and more accurately counted.

  Examples of the sample containing megakaryocytes include bone marrow puncture fluid collected by bone marrow puncture from bone marrow in which blood cells are made. Bone marrow aspiration fluid contains megakaryocytes and other blood cells. When a bone marrow puncture solution is used as a sample, there are a large number of contaminants such as bone fragments and fibrin clots. Therefore, it is preferable to filter in advance with a nylon mesh having an appropriate diameter. By this operation, megakaryocytes pass through the nylon mesh, but impurities cannot pass through them, so that the contaminants can be removed from the sample.

  In order to prepare a measurement sample from the above-described sample, a hemolytic agent for lysing red blood cells in the sample and a nucleic acid fluorescent dye for staining a nucleic acid in a megakaryocyte with a fluorescent dye can be used.

  The hemolytic agent is used to lyse red blood cells in a sample to such an extent that it does not interfere with measurement, and to form pores sufficient for at least dye molecules to permeate the cell membrane of the megakaryocyte to be measured. The hemolytic agent used for this purpose is, for example, pH 4.5-11.0, preferably pH 5 comprising at least one cationic surfactant, at least one nonionic surfactant, and a buffer to keep the pH constant. An aqueous solution of 0.0-10.0 is mentioned.

  As the cationic surfactant, a quaternary ammonium salt type surfactant or a pyridinium salt type surfactant is preferable. Examples of the quaternary ammonium salt type surfactant include surfactants having a total carbon number of 9 to 30 represented by the following chemical formula 1.

  Examples of the pyridinium salt-type surfactant include surfactants having a total carbon number of 11 to 30 represented by the following chemical formula 2.

In Chemical Formulas 1 and 2, R ₁ is an alkyl group having 6 to 18 carbon atoms or an alkenyl group having 6 to 18 carbon atoms. In Chemical Formula 1, R ₂ and R ₃ are an alkyl group having 1 to 4 carbon atoms or an alkenyl group having 1 to 4 carbon atoms. In Chemical Formula 1, R ₄ is an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 1 to 4 carbon atoms, or a benzyl group. In Chemical Formulas 1 and 2, X is a halogen atom. Examples of the alkyl group or alkenyl group for R ₁ include hexyl, octyl, decyl, dodecyl, tetradecyl and the like, and straight chain alkyl groups such as octyl, decyl, dodecyl are particularly preferable. Examples of the alkyl group and alkenyl group for R ₂ and R ₃ include methyl, ethyl, propyl, butyl and the like, and an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl and propyl is particularly preferable. . Furthermore, examples of the alkyl group or alkenyl group for R ₄ include methyl, ethyl, propyl, butyl and the like, and an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl and propyl is particularly preferable. The concentration of the cationic surfactant is generally 200-1,500 ppm, preferably 500-1,000 ppm, more preferably 600-800 ppm.

As the nonionic surfactant, a polyoxyethylene nonionic surfactant having the following formula is preferred: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H [wherein R ₁ has 8 to 25 carbon atoms. alkyl group, an alkynyl group an alkenyl group or a C 8-25 8-25 carbon atoms; R ₂ is O,

またはＣＯＯ；ｎは１０〜５０の整数を表す。〕ノニオン性界面活性剤の濃度は、一般に500−3,000ppm、好ましくは、1,000−2,500ppm、さらに好ましくは1,500−2,000ppmである。

Or COO; n represents an integer of 10 to 50. The concentration of the nonionic surfactant is generally 500 to 3,000 ppm, preferably 1,000 to 2,500 ppm, and more preferably 1,500 to 2,000 ppm.

  Further, the hemolytic agent may contain at least one organic acid having at least one aromatic ring in the molecule or a salt thereof. For example, benzoic acid, phthalic acid, hippuric acid, salicylic acid, p-aminobenzenesulfonic acid, benzenesulfonic acid or a salt thereof can be preferably used. The concentration of the organic acid or salt thereof is generally 10-100 mM, preferably 15-50 mM, more preferably 20-30 mM.

  The pH of the hemolytic agent is 4.5-11.0, preferably 5.0-10.0, and contains a buffer such as citrate, HEPES, phosphate, etc. in order to keep the pH constant. The concentration of the buffer is generally 5-100 mM, preferably 10-50 mM. In addition, when the above-mentioned organic acid acts as a buffering agent, the buffering agent is not necessarily a necessary component.

  A hemolytic agent is added to the sample to dissolve the red blood cells in the sample, and the dye is allowed to pass through the cell membrane of megakaryocytes. Thereby, megakaryocytes can be stained with a nucleic acid fluorescent dye. For example, when using a bone marrow puncture solution as a sample, the mixing ratio of the bone marrow puncture solution and the hemolytic agent is generally 1: 20-1: 200, preferably 1: 30-1: 100, more preferably 1: 40-. 1:60.

  The nucleic acid fluorescent dye is a fluorescent dye that specifically stains intracellular nucleic acid. As a nucleic acid fluorescent dye, although it changes with wavelengths of the light source to be used, for example, a dye having a structure of the following chemical formula 4 can be used.

(Wherein R ₁ is a hydrogen atom or an alkyl group; R ₂ and R ₃ are a hydrogen atom, a lower alkyl group or a lower alkoxy group; R ₄ is a hydrogen atom, an acyl group or an alkyl group; Z is sulfur, oxygen, or Carbon having a lower alkyl group; n is 0, 1 or 2; X ⁻ is an anion).

Of the above dyes, dyes in which at least _{one of} R _{1 and} R ₄ is a long-chain (having 6 to 18 carbon atoms) alkyl group are preferable.

Particularly preferred are dyes in which the alkyl group of R ₁ or R ₄ has 6, 8, or 10 carbon atoms.

The lower alkyl group or lower alkoxy group in R ₂ and R ₃ refers to a linear alkyl group having 1 to 8 carbon atoms or a linear alkoxy group having 1 to 8 carbon atoms, preferably a methyl group, a methoxy group, An ethyl group and an ethoxy group.

Anions suitable as X ⁻ include halogen ions such as F ⁻ , Cl ⁻ , Br ⁻ and I ⁻ , and CF ₃ SO ₃ ⁻ and BF ₄ ^{− and the} like.

  Z is preferably a sulfur atom, an oxygen atom, or a carbon atom substituted with a lower alkyl group such as a methyl group, an ethyl group or an isopropyl group.

In the case where a red semiconductor laser is used as the light source, the nucleic acid fluorescent dye may be R ₁ = methyl, R ₂ = R ₃ = H, R ₄ = n-octyl, n = 1, Z = S, X = CF ₃ in chemical formula 4. A dye that is SO ₃ ^— is particularly preferred. Hereinafter, this nucleic acid fluorescent dye is referred to as Compound A.

  Examples of other nucleic acid fluorescent dyes include propidium iodide, N-methyl-4- (1-pyrene) vinyl-propidium iodide, ethidium bromide, TOTO-1, TOTO-3, YOYO-1, YOYO-3, BOBO-1, BOBO-3, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2), POPO-1, POPO-3, BO-PRO-1, YO-PRO-1, TO- PRO-1 etc. are mentioned.

  The nucleic acid fluorescent dye to be used can be appropriately selected depending on the wavelength of the light source used for the measurement.

  The concentration of the dye varies depending on the dye used, but is generally 0.0001-1000 ppm, preferably 0.01-100 ppm, more preferably 0.1-10 ppm. In addition, this density | concentration is a density | concentration when a hemolytic agent and a nucleic acid fluorescent dye are mixed.

  When the hemolytic agent and the nucleic acid fluorescent dye described above are used in the preparation of the measurement sample, for example, a measurement sample may be prepared by mixing a reagent composed of a hemolytic agent containing a nucleic acid fluorescent dye and the sample. Alternatively, a measurement sample may be prepared by mixing a first reagent composed of a hemolytic agent, a second reagent containing a nucleic acid fluorescent dye, and a sample. The order of mixing the first reagent composed of the hemolytic agent, the second reagent containing the nucleic acid fluorescent dye, and the sample is not particularly limited. For example, a measurement sample may be prepared by mixing a first reagent composed of a hemolytic agent and a second reagent containing a nucleic acid fluorescent dye, and mixing the mixture and the sample. Alternatively, a measurement sample may be prepared by mixing a first reagent made of a hemolytic agent and a sample, and mixing the mixed solution with a second reagent containing a nucleic acid fluorescent dye.

  The measurement sample prepared as described above is irradiated with excitation light. Then, forward scattered light, side scattered light and fluorescence emitted from the cells in the measurement sample are detected. For example, when a flow cytometer is used for measurement, the prepared measurement sample is guided to the flow cell of the flow cytometer. Then, excitation light is irradiated to the measurement sample flowing in the flow cell, and scattered light and fluorescence emitted from the cells in the measurement sample are detected.

  An example of a configuration diagram of the megakaryocyte counting device of the present embodiment is shown in FIG. The megakaryocyte counting device 100 includes a measurement sample preparation unit 101 capable of preparing a measurement sample from a sample, a detection unit 102 for measuring the measurement sample prepared by the measurement sample preparation unit 101, and a detection unit. An analysis unit 35 for analyzing the signal detected in 102 and a display unit 103 for displaying the result analyzed by the analysis unit 35 are configured.

  An example of an optical system of a flow cytometer used as the detection unit 102 is shown in FIG. In FIG. 2, excitation light emitted from a light source (for example, a laser diode) 21 irradiates the orifice portion of the sheath flow cell 23 via a collimator lens 22. The light source is not particularly limited, and an argon laser, a He—Ne laser, a semiconductor laser, or the like is used.

  The forward scattered light emitted from the cells discharged from the nozzle 6 and passing through the orifice part is incident on the forward scattered light detector (photodiode) 26 via the condenser lens 24 and the pinhole plate 25.

  On the other hand, the side scattered light emitted from the cell passing through the orifice part enters the side scattered light detector (photomultiplier tube) 29 via the condenser lens 27 and the dichroic mirror 28. Side fluorescence emitted from cells passing through the orifice part enters a side fluorescence detector (photomultiplier tube) 31 through a condenser lens 27, a dichroic mirror 28, a filter 29, and a pinhole plate 30. Since megakaryocytes are cells having a larger amount of nucleic acid than other blood cells, megakaryocytes stained with a nucleic acid fluorescent dye have high fluorescence intensity. Therefore, it is preferable to adjust so that megakaryocytes can be measured by lowering the sensitivity of the fluorescence detector than when measuring white blood cells.

  The forward scattered light signal output from the forward scattered light detector 26, the side scattered light signal output from the side scattered light detector 29, and the side fluorescent signal output from the side fluorescent detector 31 are as follows. Are amplified by the amplifiers 32, 33 and 34, respectively, and input to the analysis unit 35.

  Here, the analysis unit 35 performs an analysis for identifying a megakaryocyte based on the input forward scattered light signal, side scattered light signal, and fluorescent signal. For example, a first two-dimensional distribution map having two axes of forward scattered light intensity and side scattered light intensity is created, and the first megakaryocyte region is specified on the first two-dimensional distribution map. Further, a second two-dimensional distribution map having two axes of the side scattered light intensity and the fluorescence intensity is created, and the second megakaryocyte region is specified on the second two-dimensional distribution map. Then, the number of cells appearing in the first megakaryocyte region and appearing in the second megakaryocyte region is counted, a desired calculation is performed, and the counting result and the calculation result are displayed on the display unit 103.

  Since megakaryocytes are very large cells compared to other blood cells, the intensity of forward scattered light is increased. In addition, megakaryocytes have large nuclei, and their shapes are overlapped with marked depressions or lobes, resulting in significant irregularities, resulting in increased side scattered light intensity. In addition, since megakaryocytes have a very large amount of nucleic acid, the fluorescence intensity of megakaryocytes stained with a nucleic acid fluorescent dye increases.

  Therefore, a first two-dimensional distribution map having two axes of the forward scattered light intensity and the side scattered light intensity is created, and the forward scattered light intensity and the side scattered light are relatively compared on the first two-dimensional distribution map. The number of megakaryocytes can be counted by specifying a region having a high intensity as the first megakaryocyte region (for example, the MEG1 region in FIG. 3) and counting the number of cells appearing in this region. However, when the sample contains cells similar in size to megakaryocytes such as plasma cells, the number of megakaryocytes may become a pseudo high value due to the presence of plasma cells in the above region. Create a second two-dimensional distribution map with the two axes of the side scattered light intensity and the fluorescence intensity. On the second two-dimensional distribution map, select a region with a relatively large side scattered light intensity and a high fluorescence intensity. A second megakaryocyte region is identified (for example, the MEG2 region in FIG. 4). Next, cells that appear in the first megakaryocyte region and appear in the second megakaryocyte region are counted as megakaryocytes. Thereby, the number of megakaryocytes can be accurately counted.

  The number of megakaryocytes may be obtained by the following method. First, a first two-dimensional distribution diagram having two axes of the forward scattered light intensity and the side scattered light intensity is created, and the forward scattered light intensity and the side scattered light are relatively compared on the first two-dimensional distribution map. A region having a high intensity is identified as the first megakaryocyte region. And about the cell which appeared in the 1st megakaryocyte area | region, the 2nd two-dimensional distribution map which makes a side scattered light intensity | strength and fluorescence intensity two axes | shafts further is created, and on a 2nd two-dimensional distribution map, The second megakaryocyte region is specified as a region having relatively high side scattered light intensity and high fluorescence intensity, and the cells in the second megakaryocyte region are counted as megakaryocytes.

  Further, the megakaryocyte count may be obtained by the following method. First, a first two-dimensional distribution map having two sides of the side scattered light intensity and the fluorescence intensity is created. On the first two-dimensional distribution chart, the side scattered light intensity is relatively large and the fluorescence intensity is A large region is identified as the first megakaryocyte region. And about the cell which appeared in the 1st megakaryocyte area | region, the 2nd two-dimensional distribution map which makes a forward scattered light intensity | strength and a side scattered light intensity | strength 2 axis | shafts further is created, Then, the second megakaryocyte region is specified as a region where the forward scattered light intensity and the side scattered light intensity are relatively large, and the cells in the second megakaryocyte region are counted as megakaryocytes.

  In the method described above, a two-dimensional distribution diagram having two axes of the forward scattered light intensity and the side scattered light intensity is created, but the present invention is not limited to this. Instead of a two-dimensional distribution diagram having the forward scattered light intensity and the side scattered light intensity as two axes, a particle size distribution diagram in which the horizontal axis represents the forward scattered light intensity and the vertical axis represents the number of cells can be used. In this case, a predetermined threshold value is set for the forward scattered light intensity, and a cell showing the forward scattered light intensity equal to or higher than the threshold value can be identified as a megakaryocyte.

In short, in the present embodiment, megakaryocytes are identified based on the detected forward scattered light, fluorescence, and side scattered light. More specifically,
(1) Based on the forward scattered light that reflects the size of the cells, the first cell population containing megakaryocytes is identified,
(2) identifying a second cell population containing megakaryocytes based on side scattered light reflecting internal information of cells and fluorescence reflecting nucleic acid content;
(3) Cells belonging to the first cell population and belonging to the second cell population are identified as megakaryocytes.

  In addition, on a two-dimensional distribution diagram having the side scattered light intensity and the fluorescence intensity as two axes, a region having a predetermined scattered light intensity and fluorescence intensity is represented as a nucleated cell region (for example, the NC region in FIG. 4; NC region includes MEG2 region), count the number of cells in that region as the number of nucleated cells, and obtain the megakaryocyte ratio in nucleated cells from this and the number of megakaryocytes obtained above it can.

Example (Preparation of a sample containing megakaryocytes)
The bone marrow puncture solution was filtered through a nylon mesh having a pore diameter of 100 μm to remove contaminants such as bone fragments, and a megakaryocyte-containing sample was prepared.

(Hemolytic agent)
HEPES (commercially available) 10mM
Potassium hydrogen phthalate (commercially available) 20mM
BC30TX (POE (30) cetyl ether) (Nikko Chemicals) 1850ppm
Lauryltrimethylammonium chloride (commercially available) 650ppm
1L of purified water
Adjust pH to 7.0 with NaOH

(Staining solution)
Dye compound A 27.5ppm
Ethylene glycol 1L

(Measurement)
Add 20μL of megakaryocyte-containing sample to 980μL of hemolyzing agent and 20μL of the above staining solution, react at 40 ° C for 30 seconds, and then use a flow cytometer with a red semiconductor laser as the light source, side scattered light, forward scattered light The fluorescence was measured.

  Next, a first two-dimensional distribution map having two axes of the forward scattered light intensity and the side scattered light intensity is created, and the forward scattered light intensity and the side scattered light are relatively compared on the first two-dimensional distribution map. A region having a high light intensity was defined as a first megakaryocyte region (MEG1 region). In addition, a second two-dimensional distribution map having two sides of the side scattered light intensity and the fluorescence intensity is created, and the side scattered light intensity is relatively large on the second two-dimensional distribution chart. A region having a high intensity was defined as a second megakaryocyte region (MEG2 region). Cells appearing in the MEG1 region and MEG2 region were counted as megakaryocytes. On the other hand, as a control method, a megakaryocyte-containing sample was stained with Turku's solution, transferred to a Fuchs-Rosental hemocytometer, and megakaryocytes were counted by a visual method.

  3, 5, and 7 are first two-dimensional distribution diagrams of three samples out of 33 samples used for the measurement, respectively. In particular, FIG. 7 is a first two-dimensional distribution map of a specimen containing plasma cells. MEG1 in the figure indicates the first megakaryocyte region. On the other hand, FIG. 4 is a second two-dimensional distribution diagram for the same specimen as FIG. FIG. 6 is a second two-dimensional distribution diagram for the same specimen as FIG. FIG. 8 is a second two-dimensional distribution diagram for the same specimen as FIG. In the figure, MEG2 represents the second megakaryocyte region, and NC represents the nucleated cell region.

  Comparing FIG. 4, FIG. 6, and FIG. 8, in FIG. 8, a population of cells appeared at a position that was not seen in FIG. 4 or FIG. Since FIG. 8 shows data obtained by measurement using a specimen containing plasma cells, it was found that the cell population is a plasma cell. The cell population appeared at a position different from the region where megakaryocytes appear (MEG2). This indicates that the region where plasma cells appear and the region where megakaryocytes appear (MEG2) are different.

  Furthermore, the correlation between the number of megakaryocytes obtained by the method of the present embodiment and the number of megakaryocytes obtained by the visual method was examined for 33 samples used for measurement (FIG. 9). The regression line equation was y = 1.0211x + 19.034, the correlation coefficient was r = 0.79, and the correlation was good.

  According to the present invention, an automatic blood cell analyzer employing a flow cytometry method can easily and quickly accurately count megakaryocytes, which is useful for automating megakaryocyte tests that had so far had to rely on visual methods. It is.

It is a block diagram of the megakaryocyte counter of embodiment of this invention. It is an example of the optical system of the flow cytometer used as a detection part of the megakaryocyte counter of embodiment of this invention. It is the 1st two-dimensional distribution map which makes the front scattered light intensity and the side scattered light intensity obtained in the example two axes. It is the 2nd two-dimensional distribution map which makes the side scattered light intensity | strength and fluorescence intensity which were obtained in the Example 2 axes. It is the 1st two-dimensional distribution map which makes the front scattered light intensity and the side scattered light intensity obtained in the example two axes. It is the 2nd two-dimensional distribution map which makes the side scattered light intensity | strength and fluorescence intensity which were obtained in the Example 2 axes. It is the 1st two-dimensional distribution map which makes the front scattered light intensity and the side scattered light intensity obtained in the example two axes. It is the 2nd two-dimensional distribution map which makes the side scattered light intensity | strength and fluorescence intensity which were obtained in the Example 2 axes. It is the figure which showed the correlation with the megakaryocyte number obtained in the Example, and the megakaryocyte number obtained by the visual observation method.

Claims (15)

Giant Red blood cells were lysed bone marrow aspirate containing a mononuclear, to prepare a measurement sample to stain the nucleic acids of megakaryocytes with a fluorescent dye,
Irradiated with excitation light to cells measuring titration, the sample,
Forward scattered light emitted from the cells, and detecting the side scattered light and fluorescence,
Test the issued based on the forward scattered light to identify the first cell population comprising megakaryocytes, identifying a second cell population comprising megakaryocytes based on the detected side scattered light and fluorescence,
Identifying cells belonging to the first cell population and belonging to the second cell population as megakaryocytes ,
Counting the identification megakaryocyte,
A counting method for megakaryocytes. Lysis red blood cells in bone marrow puncture fluid containing megakaryocytes, prepare nucleic acid samples in megakaryocytes with fluorescent dyes, and prepare samples for measurement.
Irradiate the cells in the measurement sample with excitation light,
Detect forward scattered light, side scattered light and fluorescence emitted from cells,
A first cell population including megakaryocytes is identified based on the detected forward scattered light, and a second cell population including megakaryocytes from the first cell population is determined based on the detected side scattered light and fluorescence. Identify
Identifying cells belonging to the second cell population as megakaryocytes,
Counting the identified megakaryocytes,
A counting method for megakaryocytes. Lysis red blood cells in bone marrow puncture fluid containing megakaryocytes, prepare nucleic acid samples in megakaryocytes with fluorescent dyes, and prepare samples for measurement.
Irradiate the cells in the measurement sample with excitation light,
Detect forward scattered light, side scattered light and fluorescence emitted from cells,
A second cell population including megakaryocytes is identified based on the detected side scattered light and fluorescence, and a first cell population including megakaryocytes from the second cell population is determined based on the detected forward scattered light. Identify
Identifying cells belonging to the first cell population as megakaryocytes,
Counting the identified megakaryocytes,
A counting method for megakaryocytes. In the preparation step, bone marrow aspirate containing megakaryocytes, according to any one of claims 1 to 3 for preparing a measurement sample by mixing the hemolytic agent and a fluorescent dye to lyse red blood cells Megakaryocyte counting method. The method for counting megakaryocytes according to claim 4 , wherein, in the preparation step, a measurement sample is prepared by mixing a bone marrow puncture solution containing megakaryocytes and a hemolytic agent, and adding a fluorescent dye to the mixture. The meganucleus according to any one of claims 1 to 5, wherein, in the irradiation step, a measurement sample is introduced into a flow cell of a flow cytometer, and cells in the measurement sample flowing in the flow cell are irradiated with excitation light. Sphere counting method. The megakaryocyte counting method according to any one of claims 1 to 6, wherein, in the specifying step, the first cell population is further specified based on the detected side scattered light. Furthermore, the process of identifying a nucleated cell based on the detected fluorescence and side scattered light, and the process of counting the identified nucleated cell are included in any one of Claims 1-7. Of counting megakaryocytes. The method for counting megakaryocytes according to claim 8 , further comprising a step of calculating a megakaryocyte ratio from the number of megakaryocytes and the number of nucleated cells. Bone marrow puncture fluid containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes to prepare a measurement sample preparation unit for preparing a measurement sample,
A light source for irradiating the cells in the measurement sample with excitation light,
A first scattered light detector for detecting forward scattered light emitted from a cell;
A second scattered light detector for detecting side scattered light emitted from the cells;
A fluorescence detector for detecting fluorescence emitted from the cell, and a first cell population including megakaryocytes based on the detected forward scattered light, and a meganucleus based on the detected side scattered light and fluorescence An analysis unit for identifying a second cell population including spheres, identifying cells belonging to the first cell population and belonging to the second cell population as megakaryocytes, and counting the identified megakaryocytes;
A megakaryocyte counting device comprising: Bone marrow puncture fluid containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes to prepare a measurement sample preparation unit for preparing a measurement sample,
A light source for irradiating the cells in the measurement sample with excitation light,
A first scattered light detector for detecting forward scattered light emitted from a cell;
A second scattered light detector for detecting side scattered light emitted from the cells;
A fluorescence detector for detecting fluorescence emitted from the cells; and
A first cell population including megakaryocytes is identified based on the detected forward scattered light, and a second cell population including megakaryocytes from the first cell population is determined based on the detected side scattered light and fluorescence. Identifying the cells belonging to the second cell population as megakaryocytes and counting the identified megakaryocytes,
A megakaryocyte counting device comprising: Bone marrow puncture fluid containing megakaryocytes, a hemolytic agent for lysing red blood cells, and a nucleic acid fluorescent dye for staining megakaryocytes to prepare a measurement sample preparation unit for preparing a measurement sample,
A light source for irradiating the cells in the measurement sample with excitation light,
A first scattered light detector for detecting forward scattered light emitted from a cell;
A second scattered light detector for detecting side scattered light emitted from the cells;
A fluorescence detector for detecting fluorescence emitted from the cells; and
A second cell population including megakaryocytes is identified based on the detected side scattered light and fluorescence, and a first cell population including megakaryocytes from the second cell population is determined based on the detected forward scattered light. An analysis unit for identifying the cells belonging to the first cell population as megakaryocytes and counting the identified megakaryocytes,
A megakaryocyte counting device comprising: The measurement sample preparation section mixes a bone marrow puncture solution containing megakaryocytes and a hemolytic agent, and prepares a measurement sample by adding a solution containing a nucleic acid fluorescent dye to the mixed solution. The megakaryocyte counting device according to any one of claims 10 to 12 . 14. The method according to claim 10 , further comprising a flow cell for introducing a measurement sample, wherein the light source irradiates cells in the measurement sample flowing in the flow cell with excitation light. megakaryocyte counting apparatus according to One. The megakaryocyte counting device according to any one of claims 10 to 14, wherein the analysis unit further specifies a first cell population based on the detected side scattered light.

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