JPH10339729A - Reagent for classifying and counting erythroblast and method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately classify and measure erythroblasts by dissolving red blood cell, mixing a fluorescent coloring matter for generating the difference in fluorescent intensity between leukocytes and erythroblasts for dyeing and performing measurement using a flow cytometer. SOLUTION: A blood sample and a hemolyzing agent are mixed, thus hemolyzing a red blood cell that becomes an obstacle when measuring leukocytes and erythroblasts. A fluorescent coloring matter is used for generating a difference in fluorescent intensity between the leukocytes and erythroblasts. The leukocyte cell is strongly dyed and emits strong fluorescence when being measured by a flow cytometer. On the other hand, the erythroblast is weakly dyed and emits a weak fluorescence. At least one scattered beam and at least one fluorescence are measured by the flow cytometer. Then, erythroblasts are classified and counted by using the intensity difference between the measured scattered beam and the fluorescence. For example, when a scattergram is drawn with a forward low-angle scattered beam at X axis and fluorescence at Y axis, each cell forms a group and is distributed. By analyzing the group with a proper analysis software, the number and the ratio of erythroblasts are calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to classification and counting of erythroblasts by flow cytometry.

[0002]

2. Description of the Related Art In the field of clinical examination, classification and counting of erythroblasts can provide extremely useful information for diagnosing a disease.

[0003] Normally, erythroblasts are present in bone marrow and not in peripheral blood. The appearance of erythroblasts in peripheral blood indicates the possibility of the presence of diseases such as acute myeloid leukemia, hemolytic anemia, iron deficiency anemia, and pernicious anemia. It is very useful in diagnosing these diseases.

Conventionally, to classify and count erythroblasts, it has been common practice to prepare a smear of blood, perform appropriate staining, and then perform classification and counting while observing with a microscope.

On the other hand, in recent years, various fully automatic leukocyte classification / counting apparatuses utilizing the principle of a flow cytometer have been provided. However, these devices can accurately classify and count erythroblasts only by suggesting that erythroblasts may be present when abnormal erythroblasts appear, such as by an abnormal flag. Did not.

[0006] Separately, Japanese Unexamined Patent Publication No.
No. 3, U.S. Pat. No. 5,559,037 discloses a method for classifying and counting erythroblasts.

[0007] In any of these methods, only the cell membrane of erythroid cells is damaged by the appropriate hemolytic agent (to impart cell membrane permeability of pigment), and the cell membrane of leukocyte cells is not damaged (cell membrane permeability of pigment is decreased). After treatment with a lysing agent (not applied) (or at the same time), only erythroblasts whose cell membrane has been damaged with a fluorescent dye are stained, and their fluorescence intensity is measured to discriminate between erythroblasts and leukocytes. It is a method of measuring a sphere.

[0008] These methods enable accurate measurement when fresh blood is used immediately after blood collection. However, as time elapses after blood collection, not only erythroblasts but also cell membranes of white blood cells are easily damaged. Since the cell membrane is damaged before mixing with the hemolytic agent, a part of the white blood cells is stained by the fluorescent dye. In particular, when lymphoid cells are damaged, it is difficult to clearly distinguish damaged lymphocytes from erythroblasts, and there is a problem that erythroblasts cannot be accurately classified and counted. In addition, it is difficult to accurately classify and count erythroblasts even immediately after blood collection in some lymphoblast-appearing specimens or specimens in which the cell membrane of leukocyte cells has become susceptible to damage by a hemolytic agent due to chemotherapy or the like. is there.

[0009]

An object of the present invention is to provide a method for accurately classifying and counting erythroblasts with high accuracy even when a sample whose time has passed after blood collection or a white blood cell which is easily damaged exists. Aim.

[0010]

According to the present invention, there is provided an erythroid classification / counting reagent characterized by containing a fluorescent dye which causes a difference in fluorescence intensity between at least white blood cells and red blood cells, and the use of the reagent. Erythroid fraction counting method.

A preferred erythroblast fraction counting method of the present invention comprises the following steps: 1) dissolving a sample so that erythrocytes in a blood sample are not hindered in measurement, and staining leukocytes and erythroblasts; 2) mixing the sample prepared in 1) and at least a fluorescent dye that produces a difference in fluorescence intensity between leukocytes and erythroblasts, thereby mixing leukocytes and erythroblasts. 3) measuring the measurement sample prepared in 2) with a flow cytometer, measuring at least one scattered light, and measuring at least one fluorescence, and 4) measuring the scattered light measured in 3). A step of classifying and counting erythroblasts using the difference in fluorescence intensity;

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The blood sample referred to in the present invention refers to a body fluid sample containing leukocytes and erythroblasts, such as a sample collected by peripheral blood, bone marrow fluid, urine, apheresis and the like.

In the present invention, the step of lysing erythrocytes in a blood sample to such an extent that it does not interfere with the measurement and mixing with a hemolytic agent to render leukocytes and erythroblasts in a state suitable for staining comprises: This is a step of mixing various hemolytic agents. A suitable hemolytic agent is, for example, an osmotic pressure of 100 mOsm / kg or less, p
It is an aqueous solution of H 2.0 to 5.0.

[0014] The purpose of this step is usually to measure 100 white blood cells.
It is to lyse erythrocytes, which are present at a concentration of 0 times and hinder the measurement of leukocytes and erythroblasts, and to cause a difference in fluorescence intensity between erythroblasts and leukocytes.

[0015] Erythrocytes are usually 15
Osmotic pressure of 0 mOsm / kg or less creates pores in the cell membrane,
The hemoglobin inside the cells flows out and becomes optically clear (haemolyses). The red blood cells that have become optically clear will not interfere with the measurement. Hemolysis of red blood cells progresses faster as the osmotic pressure is lower and the pH is lower. In the hemolytic agent used in the present invention, 100 mOsm / kg in consideration of individual differences.
The following osmotic pressure is used. In order to achieve this osmotic pressure, the osmotic pressure can be adjusted by, for example, an electrolyte such as NaCl or KCl, a saccharide, or the concentration of a buffer described below.

If the pH is too low, not only red blood cells,
Since leukocytes and erythroblasts are also excessively damaged, it is difficult to obtain a difference in fluorescence intensity described later.

[0017] The pH of the solution is preferably on the acidic side, since the hemolysis of red blood cells is performed efficiently. Particularly preferably, a pH of from 2.0 to 5.0 is used. More preferably, a pH of 2.5 to 4.5 is selected.

In order to keep the pH constant, it is preferable to use a buffer, and a buffer having a pKa around the set pH ± 2.0 can be used. For example, a buffer such as citric acid, malic acid, diglycolic acid, and malonic acid can be included.

Furthermore, by containing at least one organic acid having at least one aromatic ring in the molecule or a salt thereof in the hemolytic agent, red blood cells can be more effectively (shortly) hemolyzed. . Preferred organic acids include, for example, salicylic acid, phthalic acid and the like.

Under these conditions, the cell membrane of erythroblasts also forms pores like erythrocytes and lyses, but the properties of erythroblast cell nuclei are maintained almost like living cells.

On the other hand, although damage to the cell membrane of white blood cells is not clear, observation with an optical microscope shows no significant difference from living cells.

The preferred hemolytic agent used in the present invention lyses erythroid cells under hypotonic osmotic pressure, and includes various surfactants such as those used in other hemolytic agents, saponins, alcohols and the like. Solubilizers and solubilizers are essentially unnecessary.

However, it may contain a solubilizing agent for a poorly soluble dye, a surfactant, alcohol, saponin, etc., added for the purpose of preventing erythrocyte ghost aggregation, ghost shrinkage, and promoting erythrocyte hemolysis.

However, the presence of a large amount of a surfactant is not preferable because it particularly changes the properties of nuclei of erythroblasts and reduces the difference in the fluorescence intensity between erythroblasts and leukocytes, which will be described later.

Therefore, unlike the conventional hemolytic agent, the hemolytic agent used in the present invention preferably does not contain components such as a surfactant that essentially dissolves cell components.

As a result, unexpectedly, a clear difference in fluorescence intensity between erythroblasts and leukocytes, which was considered impossible in the past, could be produced.

As the fluorescent dye which causes a difference in fluorescence intensity between at least leukocytes and erythroblasts, at least one of the following groups of dyes is used.

[0028]

Embedded image (Wherein, R ₁ and R ₂ are a hydrogen atom or an alkyl group or an alkynyl group or an alkyl group substituted with a hydroxyl group; Y and Z are sulfur or oxygen or nitrogen or a carbon having a lower alkyl group; n is 0, 1 Or 2; X ^- is an anion.)

Embedded image (Wherein R ₁ is a hydrogen atom or an alkyl group; R ₂ and R ₃
Is 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 a carbon having a lower alkyl group; n is 0,
1 or 2; X ^- is an anion. )

Embedded image (Wherein, R ₁ is a hydrogen atom or a dimethylamino group; R ₂ is an alkyl group, R ₃ is a hydrogen atom or a dimethylamino group; n is 1 or 2; X ⁻ is an anion.)

Embedded image (Wherein, R ₁ is a hydrogen atom or an alkyl group; R ₂ is a dimethylamino group; R ₃ is a hydrogen atom or an amino group; R ₄ is a hydrogen atom or an alkyl group or an amino group; R ₅ is a hydrogen atom or a dimethylamino group) ; X ^- is an anion; Y is sulfur or oxygen).

Embedded image (In the formula, R ₁ is a hydrogen atom or a hydroxyl group; R ₂ is a hydrogen atom or a sulfonic acid group; R ₃ is a hydrogen atom or a sulfonic acid group; Y ⁺ is an alkali metal ion.)

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Embedded image In the formula, the alkyl group bonded to the nitrogen atom of the hetero ring has 1-20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1 carbon atom.
-6 alkyl group, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl and the like. The lower alkyl group or lower alkoxy group is a linear or branched alkyl group or alkoxy group having 1 to 8 carbon atoms, and is preferably methyl, ethyl, methoxy or ethoxy. The acyl group preferably has 1 to 3 carbon atoms, for example, formyl, acetyl, and propionyl. Preferred ^{anions, F -, Cl -, Br} -, I - halogen ions, such as, and _{^{CF 3 SO 3 -, BF 4}} -, ClO 4 - , and the like.

Of the above-mentioned dyes, the NK series is a product of LDS730 and LD7 from Japan Photographic Dye Laboratories.
00 can be purchased from Exiton and others can be purchased commercially.

The fluorescent dye may be dissolved in a hemolytic agent and acted upon (mixed with) the blood at the same time as the hemolytic agent, or after the dissolution treatment (in the process), an appropriate solvent (water, lower alcohol, ethylene). Glycol, DMSO, etc.).

The concentration of the dye varies depending on the dye used, but is generally 0.01 to 100 mg / L, preferably 0.1 to 100 mg / L.
1-10 mg / L, more preferably 0.3-3.0 mg
/ L. This concentration is a concentration in a state where the hemolytic agent and the dye solution are mixed.

When blood cells treated with the above-described hemolytic agent are stained with the above-described dye, white blood cells are strongly stained, and emit strong fluorescence when measured with a flow cytometer. Erythroblasts, on the other hand, are weakly stained and emit weak fluorescence. Although the mechanism of action that causes a difference in the fluorescence intensity between leukocytes and erythroblasts is not clear,
Probably because the erythroblast nucleus (DNA) is condensed,
It is considered that the uptake of the dye into the cell nucleus was inhibited.

In a usual hemolytic agent, since a surfactant or the like is used, the aggregate structure of the cell nucleus of erythroblasts is loosened,
It shows the same degree of staining as white blood cells, and it is considered difficult to discriminate erythroblasts from white blood cells.

In a preferred embodiment of the present invention, an organic acid such as salicylic acid, a dye and, if desired, a surfactant such as a polyoxyethylene nonionic surfactant are dissolved in purified water, and the pH is adjusted using NaOH or the like. A reagent having a simple composition can be used. The reagent is mixed with the sample, and at 15-50 ° C, preferably 20-40 ° C, 5-1.
React for 20 seconds, preferably 10-40 seconds.

The measurement sample thus prepared is measured with a flow cytometer, and at least one scattered light
At least one fluorescence is measured.

The scattered light referred to in the present invention refers to scattered light that can be measured with a commercially available flow cytometer, and includes forward low-angle scattered light (around a light receiving angle of 0 to 5 degrees) and forward high-angle scattered light (a light receiving angle). (About 5 to 20 degrees), and a scattering angle that reflects the size information of white blood cells is selected. Of these, low angle forward scattered light is more preferred.

The fluorescence is emitted from the dye combined with the above-mentioned cell components, and a suitable light receiving wavelength is selected depending on the dye used. The fluorescent signal reflects the cytochemical properties.

The light source of the flow cytometer is not particularly limited, and a light source having a wavelength suitable for exciting the dye is selected. For example, an argon ion laser, a He-Ne laser, a red semiconductor laser, or the like is used. In particular, semiconductor lasers are very inexpensive compared to gas lasers, and can greatly reduce the cost of equipment.

Next, erythroblasts are classified and counted using the difference in intensity between the measured scattered light and fluorescence. "Step of classifying and counting erythroblasts using the difference in intensity between the measured scattered light and fluorescence"
(1) When, for example, a scattergram is drawn by taking forward low-angle scattered light on the X axis and fluorescence on the Y axis, for example, as shown in FIG. 1, each cell forms and distributes a cluster (cluster); (2) A step of calculating the number and ratio of erythroblasts by analyzing this population with appropriate analysis software.

The present invention will be described in more detail with reference to the following examples. However, various changes and modifications can be made to the present invention, and the scope of the present invention is not limited by the following examples.

[0041]

Example 1 A reagent having the following composition was prepared. Salicylic acid 10 mM Commercial product NK-2825 0.3 mg / L Japan Photosensitive Dye Laboratories Co., Ltd. BC-20TX (polyoxyethylene (20) cetyl ether) 3 g / L Nikko Chemicals Co., Ltd. Purified water 1 L NaOH with pH 3. Adjusted to 0 (osmotic pressure 30 mOsm / kg)

To 1.0 ml of the above reagent, 30 μl of the blood of a patient in which erythroblasts treated with an anticoagulant appeared in peripheral blood were added.
After reacting at 5 ° C for 10 seconds, use a flow cytometer.
Forward low angle scattered light and fluorescence were measured. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or more.

FIG. 2 shows a scattergram with the forward low-angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations of monocytes (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.

Example 2 A reagent having the following composition was prepared. Salicylic acid 10 mM Commercially available NK-321 0.3 mg / L Nippon Kosaku Dye Laboratories Co., Ltd. BO-16 (polyoxyethylene (16) oleyl ether) 3 g / L Nikko Chemicals Co., Ltd. Purified water 1 L NaOH at pH 3. Adjusted to 0 (osmotic pressure 32 mOsm / kg)

To 1.0 ml of the above reagent, 30 μl of the blood of a patient in which erythroblasts treated with an anticoagulant appeared in the peripheral blood were added.
After reacting at 5 ° C for 10 seconds, use a flow cytometer.
Forward low angle scattered light and fluorescence were measured. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or more.

FIG. 3 shows a scattergram with the forward low-angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations of monocytes (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.

Example 3 A reagent having the following composition was prepared. Salicylic acid 10 mM Commercial product NK-1836 3 mg / L Japan Photochromic Laboratories Ltd. Purified water 1 L Adjust pH to 3.0 with NaOH (Osmotic pressure 25 mOsm / kg)

To 1.0 ml of the above reagent, 30 μl of a patient's blood in which erythroblasts treated with an anticoagulant appeared in the peripheral blood were added.
After reacting at 5 ° C for 10 seconds, use a flow cytometer.
Forward low angle scattered light and fluorescence were measured. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or more.

FIG. 4 shows a scattergram with the forward low-angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations of monocytes (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.

Example 4 A reagent having the following composition was prepared. Salicylic acid 10 mM Commercial product DiIC1 (5) 3 mg / L Japan Photochromic Laboratories Co., Ltd. Purified water 1 L Adjust pH to 3.0 with NaOH (Osmotic pressure 25 mOsm / kg)

To 1.0 ml of the above reagent, 30 μl of a patient's blood in which erythroblasts treated with an anticoagulant appeared in the peripheral blood were added.
After reacting at 5 ° C for 10 seconds, use a flow cytometer.
Forward low angle scattered light and fluorescence were measured. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or more.

FIG. 5 shows a scattergram in which the X axis represents the forward low-angle scattered light intensity and the Y axis represents the red fluorescence intensity. Blood cells form three populations of monocytes (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.

A window is provided for each population, the number of cells in the window is counted, and the cell ratio is calculated.

Example 5 A reagent having the following composition was prepared. Salicylic acid 10 mM Commercial product Citric acid 10 mM Commercial product NK-1836 0.3 mg / L Japan Photochromic Research Laboratories Purified water 1 L Adjust pH to 3.0 with NaOH (osmotic pressure 40 mOsm / kg)

To 1.0 mL of the above reagent, 30 μl of the blood of a patient in which erythroblasts treated with an anticoagulant appeared in peripheral blood, were added.
After reacting at 5 ° C for 10 seconds, use a flow cytometer.
Anterior hypokeratosis and fluorescence were measured. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or more.

FIG. 6 shows a scattergram with the forward low-angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations: monocytes (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.

A window is provided for each population, the number of cells in the window is counted, and the cell ratio is calculated.

FIG. 7 shows a correlation diagram between the method of use (May-Grunwald-Giemsa staining, 500 counts) and the measurement results when this method was used.

[0059]

According to the method of the present invention, erythroblasts can be accurately classified and counted with high accuracy even when there is a sample whose blood has passed for a long time or a leukocyte which is easily damaged. In addition, the method of the present invention can efficiently classify and count erythroblasts in a short time by using a flow cytometer, and can provide useful information for diagnosis of various diseases.

[Brief description of the drawings]

FIG. 1 is an example of a forward low-angle scattering intensity-red fluorescence intensity scattergram of a blood sample measured using the method of the present invention.

FIG. 2 is a scattergram of forward low-angle scattering intensity-red fluorescence intensity of a blood sample of a patient in which erythroblasts appeared in peripheral blood, measured with the reagent of Example 1.

FIG. 3 is a scattergram of forward low-angle scattering intensity-red fluorescence intensity of a blood sample of a patient in which erythroblasts appeared in peripheral blood, measured with the reagent of Example 2.

FIG. 4 is a scattergram of forward low-angle scattering intensity-red fluorescence intensity of a blood sample of a patient in which erythroid cells measured in the reagent of Example 3 appeared in peripheral blood.

FIG. 5 is a scattergram of forward low-angle scattering intensity-red fluorescence intensity of a blood sample of a patient in which erythroblasts appeared in peripheral blood, measured with the reagent of Example 4.

FIG. 6 is a scattergram of forward low-angle scattering intensity-red fluorescence intensity of a blood sample of a patient in which erythroblasts appeared in peripheral blood, measured with the reagent of Example 5.

FIG. 7 shows a correlation diagram of measurement results obtained using the method of the present invention and the method of the present invention.

Claims (7)

[Claims] 1. A erythroblast classification and counting reagent comprising at least one fluorescent dye selected from the following group that produces a difference in fluorescence intensity between at least white blood cells and erythroblasts. Embedded image (Wherein, R ₁ and R ₂ are a hydrogen atom or an alkyl group or an alkynyl group or an alkyl group substituted with a hydroxyl group; Y and Z are sulfur or oxygen or nitrogen or a carbon having a lower alkyl group; n is 0, 1 Or 2; X ^- is an anion.) (Wherein R ₁ is a hydrogen atom or an alkyl group; R ₂ and R ₃
Is 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 a carbon having a lower alkyl group;
Or 2; X ^- is an anion. ) (Wherein, R ₁ is a hydrogen atom or a dimethylamino group; R ₂ is an alkyl group; R ₃ is a hydrogen atom or a dimethylamino group; n is 1 or 2; X ⁻ is an anion). (Wherein, R ₁ is a hydrogen atom or an alkyl group; R ₂ is a dimethylamino group; R ₃ is a hydrogen atom or an amino group; R ₄ is a hydrogen atom or an alkyl group or an amino group; R ₅ is a hydrogen atom or a dimethylamino group) ;. X ^- is an anion; Y is sulfur or oxygen) embedded image (In the formula, R ₁ is a hydrogen atom or a hydroxyl group; R ₂ is a hydrogen atom or a sulfonic acid group; R ₃ is a hydrogen atom or a sulfonic acid group; Y ⁺ is an alkali metal ion.) Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image 2. A hemolytic agent for lysing erythrocytes to such an extent that it does not interfere with the measurement, so as to render leukocytes and erythroblasts suitable for staining, and producing a difference in fluorescence intensity between leukocytes and erythroblasts. An erythroid classification and counting reagent comprising the fluorescent dye according to claim 1. 3. The erythroblast classification and counting reagent according to claim 2, wherein the hemolytic agent is an aqueous solution having an osmotic pressure of 100 mOsm / kg or less and a pH of 2.0 to 5.0. 4. A method for classifying and counting erythroblasts, characterized in that the measurement is performed using a reagent containing at least one fluorescent dye selected from the following group that produces a difference in fluorescence intensity between at least leukocytes and erythroblasts. . Embedded image (Wherein, R ₁ and R ₂ are a hydrogen atom or an alkyl group or an alkynyl group or an alkyl group substituted with a hydroxyl group; Y and Z are sulfur or oxygen or nitrogen or a carbon having a lower alkyl group; n is 0, 1 Or 2; X ^- is an anion.) (Wherein R ₁ is a hydrogen atom or an alkyl group; R ₂ and R ₃
Is 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 a carbon having a lower alkyl group;
Or 2; X ^- is an anion. ) (Wherein R ₁ is a hydrogen atom or a dimethylamino group; R ₂ is an alkyl group; R ₃ is a hydrogen atom or a dimethylamino group; n is 1 or 2; X ⁻ is an anion). (Wherein, R ₁ is a hydrogen atom or an alkyl group; R ₂ is a dimethylamino group; R ₃ is a hydrogen atom or an amino group; R ₄ is a hydrogen atom or an alkyl group or an amino group; R ₅ is a hydrogen atom or a dimethylamino group) ;. X ^- is an anion; Y is sulfur or oxygen) embedded image (Wherein, R ₁ is a hydrogen atom or a hydroxyl group; R ₂ is a hydrogen atom or a sulfonic acid group; R ₃ is a hydrogen atom or a sulfonic acid group; Y ⁺ is an alkali metal ion). Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image 5. A method for classifying and counting erythroblasts according to claim 4, comprising the following steps: 1) dissolving the sample to such an extent that the erythrocytes in the blood sample do not interfere with the measurement, and staining leukocytes and erythroblasts; 2) mixing the sample prepared in 1) and at least a fluorescent dye that produces a difference in fluorescence intensity between leukocytes and erythroblasts, thereby mixing leukocytes and erythroblasts. 3) measuring the measurement sample prepared in 2) with a flow cytometer, measuring at least one scattered light and at least one fluorescence, and 4) measuring the scattered light measured in 3). A step of classifying and counting erythroblasts using the difference in fluorescence intensity; 6. A hemolytic agent that lyses erythrocytes in a blood sample to such an extent that it does not interfere with the measurement and renders leukocytes and erythroblasts suitable for staining, has a pH of 2.0 to 5 with an osmotic pressure of 100 mOsm / kg or less. The erythroid classification and counting method according to claim 4, which is an aqueous solution of erythroblasts. 7. The light receiving angle of the scattered light is forward low-angle scattered light,
5. The erythroid classification and counting method according to claim 4, wherein the method is at least one selected from forward high-angle scattered light.