JP4248017B2 - White blood cell classification and counting method and white blood cell classification and counting reagent kit - Google Patents

Description

  The present invention relates to a method for classifying and counting leukocytes by flow cytometry and a kit for classifying and counting reagents.

  In the field of clinical examination, information useful for diagnosis of diseases can be obtained by classifying and counting leukocytes. For example, in normal white blood cells in peripheral blood, five normal white blood cells such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils are present in a certain ratio. The ratio of these white blood cells may vary depending on the presence of the disease, and measuring the ratio of each white blood cell by classifying and counting these normal white blood cells is useful for obtaining information on the presence of the disease.

  On the other hand, depending on the disease, in addition to these normal leukocytes, peripheral blood is usually present in the bone marrow such as immature granulocytes such as myeloblasts, promyelocytes, myelocytes, and rear myelocytes, and erythroblasts. In some cases, immature leukocytes or immature red blood cells that are not present in the blood may appear in the peripheral blood. Furthermore, abnormal leukocytes such as lymphoblasts, atypical lymphocytes and abnormal lymphocytes may appear. Detection of the appearance of these cells, and further classification and counting, are extremely useful in the diagnosis of diseases.

  Conventionally, in order to classify white blood cells, it has been common to prepare blood smears, perform appropriate staining, and then perform classification and counting while observing with a microscope. On the other hand, in recent years, various fully automatic white blood cell classification and counting apparatuses that apply the principle of a flow cytometer have been provided. However, although these devices can classify normal white blood cells with high accuracy, the above-mentioned classification and counting of abnormal cells such as immature white blood cells cannot be performed simultaneously with the normal white blood cell classification and counting. .

  For example, a reagent and method for detecting the appearance of immature leukocytes with high accuracy using an RF / DC measurement principle are provided (Patent Document 1). The method described in Patent Document 1 performs electrical measurement using the property that immature leukocytes are less likely to be destroyed than normal leukocytes under specific conditions. It is also suggested that it can be measured by scattered light information. However, this method is intended only for the detection of immature leukocytes, and has excellent performance in the detection of immature leukocytes. However, normal leukocytes cannot be classified and counted simultaneously with the measurement of immature leukocytes. In order to classify and count normal white blood cells, it is necessary to use another method.

  In addition to this, there is provided a method for classifying and counting various immature leukocytes at the same time as classifying leukocytes into four using the fluorescence measurement principle (Patent Document 2). This method can only classify normal white blood cells into four at the same time, and in order to measure basophils, it is necessary to perform it separately by another method. In addition, it is necessary to measure a plurality of fluorescent signals, and there is a problem that the apparatus is complicated and expensive.

  Furthermore, a method of classifying and counting various immature leukocytes at the same time as classifying leukocytes into 5 is provided (Patent Document 3). In this method, a complex detector is required to classify leukocytes into five, and two pieces of fluorescence information are required, resulting in a problem that the apparatus is large and expensive.

JP-A-6-273413 JP-A-6-207942 JP-A-5-34251

  The present invention provides a method capable of classifying and counting abnormal cells such as immature leukocytes and abnormal leukocytes with high accuracy in a simple manner, and simultaneously performing normal leukocyte classification and leukocyte counting, and a reagent kit used in this method. For the purpose.

（式中、Ｒ₁及びＲ₄は、水素原子又はアルキル基を表し、かつ、いずれか一方が炭素数６〜１８のアルキル基であり、他方が水素原子又は炭素数６未満のアルキル基であり；Ｒ₂及びＲ₃は水素原子、メチル基、エチル基、メトキシ基又はエトキシ基であり；Ｚは硫黄、酸素又はメチル基を有する炭素であり；ｎは０，１又は２であり；Ｘ^-はアニオンである。）
２) 前記１）で調製した測定用試料をフローサイトメータで測定し、少なくとも１つの散乱光と、少なくとも１つの蛍光を測定する工程、および
３）前記２）で測定した散乱光と蛍光の強度差を用いて正常な白血球を多くとも５つに分類計数する工程、
を具備することを特徴とする白血球分類計数方法に関するものである。
That is, the present invention
1) Blood sample
A hemolytic agent comprising an aqueous solution having a pH of 4.5 to 11.0 containing a cationic surfactant, a nonionic surfactant and a buffer ;
A dye compound that specifically binds to RNA in cells represented by the following general formula (I) and increases the fluorescence intensity;
A step of fluorescently staining nucleated cells in a blood sample by mixing to prepare a measurement sample;

(In the formula, R ₁ and R ₄ represent a hydrogen atom or an alkyl group, and either one is an alkyl group having 6 to 18 carbon atoms, and the other is a hydrogen atom or an alkyl group having less than 6 carbon atoms. ; R ₂ and R ₃ is a hydrogen atom, a methyl group, an ethyl group, a methoxy group or an ethoxy group; Z is a carbon having sulfur, oxygen or a methyl group; n is 0, 1 or 2; X ^- Is an anion.)
2) a step of measuring at least one scattered light and at least one fluorescence by measuring the measurement sample prepared in 1) with a flow cytometer; and 3) intensity of the scattered light and fluorescence measured in 2) above. Classifying and counting normal white blood cells into at most 5 using the difference,
The present invention relates to a white blood cell classification and counting method.

  The blood sample referred to in the present invention refers to a body fluid sample containing leukocytes such as peripheral blood, bone marrow fluid, urine, blood sample collected by apheresis.

  An abnormal cell is an immature cell that is normally present in the bone marrow and not present in the peripheral blood, for example, immature granulocytes (IG) such as myeloblasts (Blast), promyelocytes, myelocytes, and rear myelocytes. Normal leukocytes such as atypical lymphocytes (A-Lymph), lymphoblasts (L-Blast), abnormal neutrophils (Ab-Neut), and erythroblasts (NRBC) A cell that does not exist in peripheral blood.

The hemolytic agent used in the present invention dissolves red blood cells in a blood sample to such an extent that does not hinder measurement, and makes normal or abnormal cells suitable for staining . That is, at least a dye compound in the cell membrane of white blood cells to be measured is one that open enough pores to penetrate. Such a hemolytic agent has a pH of 4.5-11.0, preferably a pH of 5.0-10. Which contains at least one cationic surfactant, at least one nonionic surfactant, and a buffer for keeping the pH constant. 0 aqueous solution .

〔Ｒ₁は炭素数６〜１８のアルキル又はアルケニル基、Ｒ₂およびＲ₃は炭素数１〜４のアルキル又はアルケニル基、Ｒ₄は炭素数１〜４のアルキルおよびアルケニル基又はベンジル基、Ｘはハロゲン原子である。〕
で表される全炭素数９〜３０の界面活性剤が挙げられる。Ｒ₁の炭素数６〜１８のアルキル又はアルケニル基としてはへキシル、オクチル、デシル、ドデシル、テトラデシル等を挙げることができるが、とりわけオクチル、デシル、ドデシル等の直鎖のアルキル基が好ましい。また、Ｒ₂およびＲ₃の炭素数１〜４のアルキル、アルケニル基としては、メチル、エチル、プロピル、ブチル等を挙げることができるが、とりわけ、メチル、エチル、プロピル等の炭素数１〜３のアルキル基が好ましい。さらに、Ｒ₄の炭素数１〜４のアルキルおよびアルケニル基としては、メチル、エチル、プロピル、ブチル等を挙げることができるが、とりわけ、メチル、エチル、プロピル等の炭素数１〜３のアルキル基が好ましい。 As the cationic surfactant, a quaternary ammonium salt type surfactant or a pyridinium salt type surfactant is preferable. Quaternary ammonium salt type and pyridinium salt type surfactants are:

[R ₁ is an alkyl or alkenyl group having 6 to 18 carbon atoms, R ₂ and R ₃ are alkyl or alkenyl groups having 1 to ₄ carbon atoms, R ₄ is an alkyl and alkenyl group having 1 to ₄ carbon atoms, or a benzyl group, X Is a halogen atom. ]
And a surfactant having a total carbon number of 9 to 30. Examples of the alkyl or alkenyl group having 6 to 18 carbon atoms of R ₁ include hexyl, octyl, decyl, dodecyl, tetradecyl and the like, and straight chain alkyl groups such as octyl, decyl and dodecyl are particularly preferable. Examples of the alkyl or alkenyl group having 1 to 4 carbon atoms of R ₂ and R ₃ include methyl, ethyl, propyl, butyl and the like. Are preferred. Furthermore, examples of the alkyl or alkenyl group having 1 to ₄ carbon atoms of R ₄ include methyl, ethyl, propyl, butyl, etc., among others, alkyl groups having 1 to 3 carbon atoms such as methyl, ethyl, propyl, etc. Is preferred.

またはＣＯＯ；ｎは１０〜５０の整数を表す。〕
で表されるポリオキシエチレン系ノニオン界面活性剤を用いるのが、好ましい。 As the nonionic surfactant, a polyoxyethylene nonionic surfactant represented by the following formula is preferred. That is, the general formula
R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H
Wherein R ₁ is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms; R ₂ is O,

Or COO; n represents an integer of 10 to 50. ]
It is preferable to use a polyoxyethylene nonionic surfactant represented by the formula:

  The composition of the hemolytic agent is not particularly limited. For example, the hemolytic agent described in JP-A-6-207942 or JP-A-7-181177 can be preferably used.

  Furthermore, it is preferable that the hemolytic agent contains an organic acid having at least one aromatic ring in the molecule or a salt thereof. This is because the scattered light intensity distribution of leukocytes can be adjusted to be more suitable for classification. As the organic acid or a salt thereof, benzoic acid, phthalic acid, hippuric acid, salicylic acid, p-aminobenzenesulfonic acid, benzenesulfonic acid or a salt thereof can be preferably used. By containing these organic acids, in particular, the scattered light intensity of eosinophils increases, and as a result, separation of neutrophils and eosinophils is improved. For example, four fractions of white blood cells can be obtained using substantially only side scattered light.

  The hemolytic agent has a pH of 4.5 to 11.0, preferably 5.0 to 10.0, and includes a buffer such as citrate, HEPES, and phosphate to keep the pH constant. . In addition, when the above-mentioned acid acts as a buffering agent, the buffering agent is not necessarily a necessary component. If the pH is too low, the separation of eosinophils and basophils will be poor, and it will be difficult to fractionate normal leukocytes into 5 fractions. However, it is possible to classify and count normal leukocytes into three fractions (lymphocytes, monocytes, granulocytes), immature leukocytes and abnormal leukocytes. On the other hand, if the pH is too high, leukocytes tend to be easily damaged.

（式中、Ｒ₁及びＲ₄は、水素原子又はアルキル基を表し、かつ、いずれか一方が炭素数６〜１８のアルキル基であり、他方が水素原子又は炭素数６未満のアルキル基であり；Ｒ₂及びＲ₃は水素原子、メチル基、エチル基、メトキシ基又はエトキシ基であり；Ｚは硫黄、酸素又はメチル基を有する炭素であり；ｎは０，１又は２であり；Ｘ^-はアニオンである。） As a dye compound that specifically binds to RNA in cells and increases fluorescence intensity, a dye compound that has low fluorescence intensity in an environment where RNA is not present and increases fluorescence intensity by binding to RNA can be used. In the present invention, as such a dye compound, a dye compound represented by the following general formula (I) is used.

(In the formula, R ₁ and R ₄ represent a hydrogen atom or an alkyl group, and either one is an alkyl group having 6 to 18 carbon atoms, and the other is a hydrogen atom or an alkyl group having less than 6 carbon atoms. ; R ₂ and R ₃ is a hydrogen atom, a methyl group, an ethyl group, a methoxy group or an ethoxy group; Z is a carbon having sulfur, oxygen or a methyl group; n is 0, 1 or 2; X ^- Is an anion.)

  Most abnormal cells such as immature leukocytes and abnormal leukocytes contain more RNA than normal leukocytes. This is because, for example, immature leukocytes are cells in the middle of maturation, and therefore various production activities (protein synthesis and the like) associated with cell maturation are performed, and therefore, it is considered that RNA is abundant. And the said pigment | dye compound has the property to which a fluorescence intensity increases remarkably by couple | bonding with RNA. More unexpectedly, the use of these dye compounds made it possible to classify basophils simultaneously.

The dye compound used in the present invention has less influence of binding to DNA and has increased specificity for RNA. That is, a dye compound having a low affinity (specificity) with DNA is used. For this reason, in general formula (I), either R ₁ or R ₄ is a long-chain (having 6 to 18 carbon atoms) alkyl group, and the other is a hydrogen atom or an alkyl group having less than 6 carbon atoms. It has become. When it has specificity for DNA, the fluorescence signal from DNA is superimposed on the fluorescence signal from RNA, and the difference in fluorescence intensity between normal leukocytes, immature leukocytes, and abnormal leukocytes becomes small, which is not preferable.

  Although the mechanism of action of the dye compound used in the present invention is not clear, it has a long-chain alkyl group (C6-C18 alkyl group) in the molecular structure, so it cannot penetrate the cell nuclear membrane, or because of steric hindrance Since binding to the DNA molecule is inhibited, there is little increase in fluorescence due to DNA, and it is considered that there is an advantage that fluorescence mainly composed of RNA can be measured. This significantly improves the separation of abnormal cells such as immature leukocytes and abnormal leukocytes from normal leukocytes.

The longer the chain length (carbon number) of a long-chain alkyl group (C18-C18 alkyl group), the greater the fluorescence intensity attributed to DNA, and the higher the RNA specificity, while the molecule tends to be hydrophobic. It becomes difficult to use in water, but is difficult to use. Taking these points into consideration, a dye compound in which the alkyl group of R ₁ or R ₄ has 6, 8, or 10 carbon atoms is particularly suitable.

R ₂ and R ₃ are a hydrogen atom, a methyl group, a methoxy group, an ethyl group or an ethoxy group.

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

  As Z, a carbon atom substituted with a sulfur atom, an oxygen atom, or a methyl group is used.

  The concentration of the dye compound varies depending on the dye compound used, but is generally 0.001 to 1000 ppm, preferably 0.01 to 100 ppm, and more preferably 0.1 to 10 ppm. This concentration is a concentration in a state where the hemolytic agent and the pigment compound are mixed. In the present invention, the measurement sample may be prepared by mixing the blood sample with the hemolytic agent and then mixing the pigment compound, or after mixing the hemolytic agent and the pigment compound in advance and measuring with the blood sample. A sample for use may be prepared. In either case, the concentration is in a state where the hemolytic agent and the pigment compound are mixed. Therefore, even in the case of a reagent kit in which a staining solution containing a dye compound and a hemolytic agent are combined separately, the concentration is in a state where the staining solution containing the dye compound and the hemolytic agent are mixed. The staining solution containing the dye compound used in the present invention is obtained by dissolving the dye compound in a water-soluble organic solvent such as ethylene glycol, methanol, DMSO.

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

  The scattered light referred to in the present invention is a scattered light that can be generally measured with a commercially available flow cytometer. Side scattered light, forward low-angle scattered light (light receiving angle of 0 to 5 degrees), forward high-angle scattered light (5 The scattering angle reflecting the size of white blood cells or internal structure information is selected. Of these, side scattered light is most preferred.

  Further, by using one or more scattered lights, the classification accuracy can be further increased. In particular, when forward low-angle scattered light is used, information on the cell size can be obtained. Therefore, the present invention is also applied to detection of abnormal spheres in which only the size of, for example, Hairy Cell Leukemia changes and the amount of RNA does not increase. It can be used and is useful. When forward high angle scattered light is used, intermediate information between the forward low angle scattered light and the side scattered light can be obtained. That is, information including both cell size information and cell internal structure information is obtained.

  Fluorescence is emitted when the dye compound used in the present invention binds to cell components such as RNA and DNA, and a suitable light receiving wavelength is selected depending on the dye compound used.

  The light source of the flow cytometer is not particularly limited, and a light source having a wavelength suitable for excitation of the dye compound is selected. For example, an argon ion laser, a He—Ne laser, a red semiconductor laser, a mercury arc lamp, or the like is used. In particular, a semiconductor laser is preferable because it is much cheaper and smaller than a gas laser, can greatly reduce the cost of the apparatus, and can be further downsized.

“The step of classifying and counting normal white blood cells into at most 5 using the difference in intensity between the measured scattered light and fluorescence” means (1) taking, for example, side scattered light on the X axis and red fluorescence on the Y axis. When a scattergram (two-dimensional distribution map) is drawn, for example, as shown in FIG. 1, each white blood cell forms a population for each cell; and (2) this population is analyzed with an appropriate analysis software. This means a step of calculating the number and ratio of each white blood cell. For example, the number and ratio of each white blood cell can be calculated by providing a window surrounding each cell population and calculating the number of cells therein.

When the method according to the present invention is used, not only normal white blood cells can be classified and counted into at most 5, but also abnormal cells can be classified and counted simultaneously. An abnormal cell suitable for classification and counting by the method according to the present invention is a cell having an increased amount of RNA compared to a normal cell, and is selected from, for example, immature granulocytes, myeloblasts, erythroblasts, and atypical lymphocytes. One or more.

The present invention further provides a reagent kit for preparing a measurement sample. This reagent kit includes 1) a hemolytic agent comprising an aqueous solution of pH 4.5 to 11.0 containing a cationic surfactant, a nonionic surfactant and a buffer , and 2) represented by the above general formula (I). It consists of a combination of staining solutions containing at least one dye compound that specifically binds to RNA and increases fluorescence intensity. Specific examples of the hemolytic agent used in the reagent kit include, as described above, pH 4.5-11 including at least one cationic surfactant, at least one nonionic surfactant, and a buffer for keeping the pH constant. 0.0, preferably an aqueous solution having a pH of 5.0 to 10.0. In addition, specific examples of the staining solution are obtained by dissolving the dye compound represented by the general formula (I) in a water-soluble organic solvent such as ethylene glycol, methanol, DMSO, as described above.

  As a method of preparing a measurement sample using this reagent kit, a blood sample may be mixed with a hemolyzing agent and then mixed with a staining solution, or a mixture solution of hemolytic agent and staining solution may be mixed. It may be prepared by mixing with a blood sample.

  According to the leukocyte classification and counting method of the present invention, a blood sample is mixed with a hemolytic agent, and simultaneously or thereafter, the nucleated cells in the blood sample are fluorescent by mixing with a dye compound represented by the general formula (I). By simply staining and measuring at least one scattered light and at least one fluorescence with a flow cytometer, it can easily and accurately classify and count immature leukocytes and abnormal cells of abnormal leukocytes, and at the same time classify and count normal leukocytes and leukocytes. It can be performed.

The present invention will be described in more detail with reference to the following examples, but 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. In the attached drawings, the following abbreviations mean the following:
Lymph: lymphocyte Eo: eosinophil
Mono: Monocyte Baso: Basophil
Neut: Neutrophil WBC: White blood cell
IG: Juvenile granulocyte Blast: Myeloblast
NRBC: erythroblast A-Lymph: atypical leukocyte

[Synthesis of Dye Compound A]
A dye compound A (R ₁ = methyl, R ₂ = R ₃ = H, R ₄ = n-octyl, n = 1, Z = S, X ⁻ = CF ₃ SO ₃ ⁻ ) was produced as follows.
1 equivalent of 3-methyl-2-methylbenzothiazolium methane sulfate and 3 equivalent of N, N-diphenylformamidine were heated and stirred in an acetic acid bath at 90 ° C. for 1.5 hours. The reaction solution was poured into hexane, and the red oil was further suspended and washed with hexane to remove acetic acid. The crude product was recrystallized from ethyl acetate-hexane (yield 48%). To this was added 1 equivalent of 1-octyllepidinium triflate and pyridine, and the mixture was heated and stirred on an oil bath at 90 ° C. for 3 hours. The reaction solution was concentrated, and the remaining blue crude product was purified with methanol-chloroform by flash chromatography (yield 62%).

As described above, a powder composed of the dye compound A was obtained, which was dark blue. Moreover, the characteristics of this dye compound A were as follows.
TLC (silica gel, 10% methanol-methylene chloride): Rf = 0.5
¹ H-NMR (CDCl ₃ ) δ ppm (TMS): 0.88 (t, 3H) 1.28 (bR, 10H) 1.64 (s, 2H) 1.82 (bR, 2H) 3.60 (s , 3H) 4.23 (t, 2H) 6.35 (d, 1H) 6.82-7.26 (m, 2H) 7.39-7.90 (m, 6H) 8.10-8.26 (Dd, 2H)
IR (cm ⁻¹ ): 1625, 1560, 1540, 1520, 1480, 1460, 1410, 1400, 1310, 1260, 1210, 1150, 1130, 740, 640
MASS (FAB positive) m / z = 429
TLC 95.7% (10% methanol-methylene chloride)
Maximum absorption spectrum 629nm (methanol)

[Synthesis of Dye Compound B]
A dye compound B (R ₁ = hexyl, R ₂ = R ₃ = H, R ₄ = methyl, n = 1, Z = S, X ⁻ = CF ₃ SO ₃ ⁻ ) was produced as follows.
1 equivalent of 3-hexyl-2-methylbenzothiazolium triflate and 4 equivalents of N, N-diphenylformamidine were heated and stirred in an acetic acid bath at 90 ° C. for 10 hours. The reaction solution was concentrated, and the remaining crude product was purified by flash chromatography with hexane-ethyl acetate (yield 27%). To this was added 1.3 equivalents of 1-methyllepidinium iodide and pyridine, and the mixture was heated and stirred on an oil bath at 90 ° C. for 3 hours. The reaction solution was concentrated, and the remaining blue crude product was purified with methanol-chloroform by flash chromatography (yield 75%).

As described above, a powder composed of the dye compound B was obtained, which was dark blue. Further, the characteristics of the dye compound B were as follows.
TLC (silica gel, 10% methanol-methylene chloride): Rf = 0.5
¹ H-NMR (CDCl ₃ ) δ ppm (TMS): 0.90 (t, 3H) 1.17 to 1.81 (m, 12H) 4.12 (s, 3H) 6.50 (m, 1H) 6 .96-8.26 (m, 10H)
IR (cm ⁻¹ ): 1620, 1560, 1530, 1510, 1480, 1460, 1410, 1380, 1310, 1250, 1210, 1150, 1100, 750, 640
MASS (FAB positive) m / z = 401
TLC 93.0% (10% methanol-methylene chloride)
Maximum absorption spectrum 629nm (methanol)

Example 1
A solution containing a hemolytic agent and dye compound A was prepared as follows.
HEPES 10 mM commercial product phthalic acid 2Na 20 mM commercial product BC30TX (polyoxyethylene (30) cetyl ether)
1500ppm Nikko Chemicals Lauryltrimethylammonium chloride 550ppm Commercial product Dye Compound A 0.5ppm
Adjust pH to 7.0 with NaOH 30 μl of healthy human blood (specimen No. 1) treated with anticoagulant was added to 1.0 ml of the above solution and reacted at 35 ° C. for 40 seconds to prepare a measurement sample.

  With a flow cytometer (FCM), side scattered light, forward low angle scattered light, and fluorescence were measured for this measurement sample. The light source used was a 633 nm red semiconductor laser. For fluorescence, fluorescence having a wavelength of 660 nm or more (red fluorescence) was measured.

  FIG. 1 shows a scattergram in which side scattered light is taken on the X axis and red fluorescence is taken on the Y axis. FIG. 2 shows a scattergram in which the forward low angle scattered light is taken on the X axis and red fluorescence is taken on the Y axis. Leukocytes are divided into five groups: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. A window is provided for each group, and the number of cells in the window is counted and the cell ratio is calculated.

  5 to 9 show correlation diagrams of white blood cell classification between the conventional method (Dongguan Medical Electronics SE-9000) and this method (FIG. 1). FIG. 10 shows a correlation diagram of the white blood cell count between the conventional method (Dongguan Medical Electronics SE-9000) and the present method (FIG. 1). All items have a good correlation with the conventional method.

Here, the measurement of the conventional method was performed using the following measuring reagents, and the operation was fully automatic with SE-9000.
- WBC: 1 cell pack -3D (II) ^TM (Toa Medical Electronics) and Stromatolyzer -EO (II) ^TM (Toa Medical Electronics) was measured by adding blood to 1 mixture.
Lymphocytes and monocytes: After hemolysis by the cell pack -3D (II) ^TM (Toa Medical Electronics) was measured by adding Stromatolyzer -3D (II) ^TM (Toa Medical Electronics).
Eosinophils: Measured using Stomatolyzer-EO (II) ^™ (Dongguan Medical Electronics).
Basophil: Measured using Stomatolyzer-BA (II) ^™ (Dongguan Medical Electronics).
Neutrophils: (white blood cell count) − {(lymphocyte count) + (monocyte count) + (eosinophil) + (basophil count)}

  For reference, FIG. 3 shows a scattergram with side scattered light on the X axis and forward low angle scattered light on the Y axis, and FIG. 4 shows a histogram of side scattered light intensity. Leukocytes are divided into four groups: lymphocytes, monocytes, neutrophils, and eosinophils.

Example 2
A scattergram having side scattered light on the X axis and red fluorescence on the Y axis was obtained in the same manner as in Example 1 except that the following specimens were used as blood samples.
(I) The scattergram shown in FIG. 11 was obtained for the specimen (specimen No. 2) in which immature granulocytes (postmyelocytes, myelocytes) appeared. In FIG. 11, it was counted as 4.0%. When this specimen was counted by the conventional method (visual observation), it was 4.5%.
(Ii) The scattergram shown in FIG. 12 was obtained for the specimen (specimen No. 3) in which myeloblasts appeared. In FIG. 12, it was counted as 85.1%. When this specimen was counted by the conventional method (visual observation), it was 81.5%.
(Iii) The scattergram shown in FIG. 13 was obtained for the specimen (sample No. 4) in which erythroblasts appeared. In FIG. 13, it was counted as 21.5%. When this sample was counted by the conventional method (visual observation), it was 25%.
(Iv) For the specimen (specimen No. 5) in which atypical lymphocytes and immature granulocytes appeared, the scattergram shown in FIG. 14 was obtained. In FIG. 14, atypical lymphocytes were counted as 1.89%, and immature granulocytes were counted as 2.17%. When this specimen was counted by a conventional method (visual observation), it was 2.0% for atypical lymphocytes and 2.0% for immature granulocytes.

  According to the method according to Example 2, any immature granulocytes and abnormal leukocytes can be clearly separated from normal leukocytes and classified and counted. In the above-described conventional method, the smear is double-stained by the May-Grunwald Giemsa staining method, and 200 white blood cells are classified and counted with a microscope (magnification: x1000).

Example 3
A scattergram having side scattered light on the X axis and red fluorescence on the Y axis was obtained in the same manner as in Example 1 except that a normal sample (Sample No. 6) was used as the blood sample. This is shown in FIG.

Example 4
A scatterer having side scattered light on the X-axis and red fluorescence on the Y-axis in the same manner as in Example 1 except that the specimen (specimen No. 7) in which immature granulocytes appeared was used as a blood sample. Got gram. This is shown in FIG.

Example 5
A solution containing the hemolytic agent and the dye compound B was prepared as follows.
HEPES 10 mM commercial product phthalic acid 2Na 20 mM commercial product BC30TX (polyoxyethylene (30) cetyl ether)
1500ppm Nikko Chemicals Lauryltrimethylammonium chloride 550ppm Commercial product Dye Compound B 0.3ppm
Adjust pH to 7.0 with NaOH Add 30 μl of normal sample (sample No. 6 used in Example 3) treated with anticoagulant to 1.0 ml of the above solution and react for 40 seconds at 35 ° C. Was prepared. Then, in the same manner as in Example 1, a scattergram was obtained in which side scattered light was taken on the X axis and red fluorescence was taken on the Y axis. This is shown in FIG.

Example 6
Except for using a specimen in which immature granulocytes appeared (specimen No. 7 used in Example 4) as a blood sample, side scattered light on the X axis and side scattered light on the Y axis in the same manner as in Example 5. A scattergram with red fluorescence was obtained. This is shown in FIG.

Comparative Example 1
A solution containing the hemolytic agent and the dye compound B was prepared as follows.
HEPES 10 mM commercial product phthalic acid 2Na 20 mM commercial product BC30TX (polyoxyethylene (30) cetyl ether)
1500 ppm Nikko Chemicals Lauryltrimethylammonium chloride 550 ppm Commercial product DQTCI (pigment compound) 0.1 ppm Lambda Physik Company Adjust pH to 7.0 with NaOH Here, DQTCI is R ₁ = R ₄ = Eye, R ₂ = R ₃ = H, n = 1, Z = S, X ⁻ = I ⁻ .
30 μl of a normal sample (sample No. 6 used in Example 3) treated with an anticoagulant was added to 1.0 ml of the above solution, and reacted at 35 ° C. for 40 seconds to prepare a measurement sample. Then, in the same manner as in Example 1, a scattergram was obtained in which side scattered light was taken on the X axis and red fluorescence was taken on the Y axis. This is shown in FIG.

Comparative Example 2
Except for using a specimen in which immature granulocytes appeared (specimen No. 7 used in Example 4) as the blood sample, side scattered light on the X axis and side scattered light on the Y axis in the same manner as Comparative Example 1. A scattergram with red fluorescence was obtained. This is shown in FIG.

As is apparent from FIGS. 15 to 20 showing the results of Examples 3 to 6 and Comparative Examples 1 and 2, as the dye compound, R ₁ and R _{4 in} the general formula (I) are both ethyl groups Is used (in the case of Comparative Examples 1 and 2), it is difficult to discriminate between normal neutrophils and immature granulocytes, but _one of R ₁ and R ₄ has 6 and 8 carbon atoms. When the pigment compound A or B into which an alkyl group is introduced and a methyl group is introduced on the other side (in the case of Examples 3 to 6), normal neutrophils and immature granulocytes can be clearly distinguished.

Example 7
In the same manner as in Example 1, except that citric acid is used instead of phthalic acid 2Na as a solution containing the hemolytic agent and dye compound A, and a normal specimen (specimen No. 8) is used as a blood sample, Scattergrams with side scattered light and red fluorescence on the Y axis were obtained. This is shown in FIG.

Example 8
The solution containing the hemolytic agent and the dye compound A is the same as in Example 1 except that phthalic acid is used in place of 2Na phthalate and a normal sample (sample No. 8 used in Example 8) is used as a blood sample. By the method, a scattergram was obtained in which side scattered light was taken on the X axis and red fluorescence was taken on the Y axis. This is shown in FIG.

  As is apparent from FIGS. 21 and 22 showing the results of Examples 7 and 8, when phthalic acid, which is an organic acid having an aromatic ring in the molecular structure, is used as the acid (in the case of Example 8), Separation of neutrophils and eosinophils is improved as compared with the case of using citric acid, which is an organic acid having no aromatic ring (in the case of Example 7).

2 is a side scatter-red fluorescence scattergram obtained by the method according to Example 1. FIG. It is a forward low angle scattering-red fluorescence scattergram obtained by the method according to Example 1. 2 is a side scatter-forward low angle scattergram obtained by the method according to Example 1. FIG. 2 is a side scatter histogram obtained by the method according to Example 1; It is a correlation diagram of the lymphocyte ratio by the conventional method (Dongguan medical electronic SE-9000) and the classification counting method which concerns on Example 1. FIG. FIG. 6 is a correlation diagram of monocyte ratios according to a conventional method (Dongguan Medical Electronics SE-9000) and a classification counting method according to Example 1. It is a correlation diagram of the neutrophil ratio by the conventional method (Dongguan Medical Electronics SE-9000) and the classification counting method according to Example 1. It is a correlation diagram of the eosinophil ratio by the classification method according to the conventional method (Dongguan Medical Electronics SE-9000) and Example 1. It is a correlation diagram of the basophil ratio by the conventional method (Dongguan Medical Electronics SE-9000) and the classification counting method according to Example 1. It is a correlation diagram of the white blood cell count by the classification method according to the conventional method (Dongguan Medical Electronics SE-9000) and Example 1. FIG. 5 is a side scatter-red fluorescence scattergram obtained by the method according to Example 2 for a specimen (specimen No. 2) in which immature granulocytes (post-myelocytes, myelocytes) appeared. 5 is a side scatter-red fluorescence scattergram obtained by the method according to Example 2 for a specimen in which myeloblasts appear (Specimen No. 3). 5 is a side scatter-red fluorescence scattergram obtained by the method according to Example 2 for a specimen in which erythroblasts appear (Specimen No. 4). 5 is a side scatter-red fluorescence scattergram obtained by the method according to Example 2 for a specimen (specimen No. 5) in which atypical lymphocytes and immature granulocytes appeared. It is a side scattering-fluorescence scattergram obtained by the method according to Comparative Example 1. It is a side scattering-fluorescence scattergram obtained by the method according to Comparative Example 2. 4 is a side scatter-fluorescence scattergram obtained by the method according to Example 3. FIG. 6 is a side scatter-fluorescence scattergram obtained by the method according to Example 4. FIG. 10 is a side scatter-fluorescence scattergram obtained by the method according to Example 5. FIG. 10 is a side scatter-fluorescence scattergram obtained by the method according to Example 6. FIG. 10 is a side scatter-fluorescence scattergram obtained by the method according to Example 7. FIG. 10 is a side scatter-fluorescence scattergram obtained by the method according to Example 8.

Claims (7)

1) Blood sample
A hemolytic agent comprising an aqueous solution having a pH of 4.5 to 11.0 containing a cationic surfactant, a nonionic surfactant and a buffer ;
A dye compound that specifically binds to RNA in cells represented by the following general formula (I) and increases the fluorescence intensity;
A step of fluorescently staining nucleated cells in a blood sample by mixing to prepare a measurement sample;

(In the formula, R ₁ and R ₄ represent a hydrogen atom or an alkyl group, and either one is an alkyl group having 6 to 18 carbon atoms, and the other is a hydrogen atom or an alkyl group having less than 6 carbon atoms. ; R ₂ and R ₃ is a hydrogen atom, a methyl group, an ethyl group, a methoxy group or an ethoxy group; Z is a carbon having sulfur, oxygen or a methyl group; n is 0, 1 or 2; X ^- Is an anion.)
2) a step of measuring at least one scattered light and at least one fluorescence by measuring the measurement sample prepared in 1) with a flow cytometer; and 3) intensity of the scattered light and fluorescence measured in 2) above. Classifying and counting normal white blood cells into at most 5 using the difference,
A white blood cell classification and counting method comprising: Hemolytic agent, leukocyte classification and counting method according to claim 1, further containing an organic acid or its salt having at least one aromatic ring.   The leukocyte classification and counting method according to claim 1, wherein the scattered light is at least one scattered light selected from the group consisting of forward low angle scattered light, forward high angle scattered light, and side scattered light.   The white blood cell classification and counting method according to claim 1, wherein the abnormal cells are classified and counted simultaneously. The leukocyte classification and counting method according to any one of claims 1 to 4 , wherein the abnormal cell is a cell having an RNA amount increased from that of a normal cell. 6. The leukocyte classification and counting method according to claim 5 , wherein the abnormal cell is at least one cell selected from the group consisting of immature granulocytes, myeloblasts, erythroblasts, and atypical lymphocytes. 1) a hemolytic agent comprising an aqueous solution having a pH of 4.5 to 11.0 containing a cationic surfactant, a nonionic surfactant and a buffer ; and 2) specific to RNA represented by the following general formula (I) Staining solution containing a dye compound that binds and increases fluorescence intensity

(In the formula, R ₁ and R ₄ represent a hydrogen atom or an alkyl group, and either one is an alkyl group having 6 to 18 carbon atoms, and the other is a hydrogen atom or an alkyl group having less than 6 carbon atoms. ; R ₂ and R ₃ is a hydrogen atom, a methyl group, an ethyl group, a methoxy group or an ethoxy group; Z is a carbon having sulfur, oxygen or a methyl group; n is 0, 1 or 2; X ^- Is an anion.)
A white blood cell classification and counting reagent kit comprising the combination of

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