Method for analyzing blood cells, reagent for analyzing blood cells, kit for analyzing blood cells, and method for fluorescent staining of blood cells

Abstract

The purpose of the present invention is to provide a means for blood cell analysis that can be used in conjunction with the classification of leukocytes and the classification of reticulated erythrocytes and platelets, and that uses a reagent that can correspond to blood cell analysis by a plurality of lights. The problem is solved by staining blood cells in a test specimen using an anthocyanin-based fluorescent dye that can be excited by light having a wavelength of 315-600 nm and that can bind to a nucleic acid.

Description

[Technical field] The present invention relates to a blood cell analysis method. The present invention relates to a blood cell analysis reagent. The present invention relates to a blood cell analysis kit. The present invention relates to a method for fluorescently staining blood cells. [Background technology] Blood contains various blood cell components such as red blood cells, white blood cells, and platelets. By analyzing these blood cells, useful information for clinical diagnosis of various diseases can be provided. In the analysis of blood cells, a method using an automatic blood cell analyzer is known. In the analysis using an automatic blood cell analyzer, a sample prepared using a dedicated reagent corresponding to the type of blood cell is generally measured. However, from the perspective of simplifying the inspection and simplifying the device, it is desirable to use an analysis using a reagent that can be used in conjunction with a variety of blood cells. For example, Patent Document 1 describes the use of a fluorescent dye and a hemolytic reagent for the classification and counting of white blood cells, and the use of the same fluorescent dye and a diluting reagent for the classification and counting of reticulocytes and platelets. [Prior art literature] [Patent Literature] [Patent Document 1] International Publication No. 2022 / 100717 [Summary of the Invention] [Problems to be solved by the invention] In recent years, a method of analyzing blood cells by irradiating multiple lights of different wavelengths to blood cells using an automatic blood cell analyzer equipped with multiple light sources has been known. Therefore, a blood cell analysis method using a reagent that can also correspond to blood cell analysis using such multiple lights is desired. The present invention aims to provide a means of blood cell analysis that can be used together with classification of white blood cells and classification of reticulocytes and platelets and that uses a reagent that can also correspond to blood cell analysis using multiple lights.

Methods for solving the problem

[19] .

[0001] A blood cell analysis method comprising: A step of irradiating a measurement sample containing particles dyed with a fluorescent dye with light to obtain information on fluorescence and scattered light generated from the particles, and a step of classifying blood cells from particles in the measurement sample based on the fluorescence information and the scattered light information; The fluorescent dye is represented by the following formula (I):

Chemistry 1

[0002] The method described in [1] above, wherein in the above formula (I), when one of X and Y is an oxygen atom, the other is a sulfur atom, a selenium atom or C(CH3)2.

[0003] The method described in [1] or [2] above, wherein the fluorescent dye is at least one selected from 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]-6-methyl-benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-methyl-2-[(1,3,3-trimethyl-2-indolylidene)methyl]benzothiazolium and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolylidene)methyl]-3H-indolylium.

[0004] The method according to any one of [1] to [3] above, wherein The measurement sample is prepared by mixing the fluorescent dye, hemolysis reagent and whole blood. In the classification step, the leukocytes in the measurement sample are classified into subgroups.

[0005] The method described in [4] above, wherein The light is a light of a first wavelength of 315 nm to 600 nm. The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength.

[0006] The method described in [4] above, wherein The light is a first wavelength of light having a wavelength of 315 nm to 600 nm and a second wavelength of light having a wavelength of 610 nm to 750 nm. The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information includes information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength and information related to side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

[0007] The method described in [5] or [6] above, wherein The above-mentioned fluorescence information is the fluorescence intensity, and the above-mentioned scattered light information is the side scattered light intensity. In the classification step, the leukocytes in the measurement sample are classified into subgroups based on the fluorescence intensity and the side scattered light intensity.

[0008] The method described in [7] above, wherein a scattergram based on the fluorescence intensity and the side scattered light intensity is prepared, and the leukocytes are classified into subgroups in the scattergram.

[0009] The method described in [8] above, wherein the subpopulation includes a lymphocyte population, a monocyte population, and a granulocyte population.

[0010] The method described in [8] above, wherein the subpopulation includes a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population.

[0011] The method according to any one of [1] to [3] above, wherein The measurement sample is prepared by mixing the fluorescent dye, the diluent and whole blood. In the classification step, reticulocytes and platelets are classified from particles in the measurement sample.

[0012] The method described in

[11] above, wherein the measurement sample does not contain a hemolytic reagent.

[0013] The method described in

[11] or

[12] above, wherein The light is a first wavelength of light having a wavelength of 315 nm to 600 nm and a second wavelength of light having a wavelength of 610 nm to 750 nm. The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

[0014] The method described in

[13] above, wherein The above-mentioned fluorescence information is the fluorescence intensity, and the above-mentioned scattered light information is the forward scattered light intensity. In the classification step, reticulocytes, platelets, and mature erythrocytes are classified from particles in the measurement sample based on the fluorescence intensity and the forward scattered light intensity.

[0015] The method described in

[14] above, wherein a scatter plot is prepared based on the fluorescence intensity and the forward scattered light intensity, and in the scatter plot, the particles in the measurement sample are classified into a reticulocyte group, a platelet group, and a mature erythrocyte group.

[0016] The method described in any one of [1] to

[15] above, wherein the first wavelength is greater than 315 nm and less than 490 nm.

[0017] The blood cell analysis reagent used in the method described in any one of [1] to

[16] contains the fluorescent dye represented by the above formula (I).

[0018] The blood cell analysis kit used in the method described in any one of [1] to

[16] above comprises a staining reagent containing the fluorescent dye represented by the above formula (I) and a hemolysis reagent.

[0019] A method for fluorescently staining blood cells, comprising contacting a fluorescent dye represented by the following formula (I) with blood cells:

Chemistry 2

Figure 1

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Fig.11A

Fig. 11B

Fig.12

Fig.13

Fig.14

【 Fig.15A (A) is a schematic diagram showing the positions where RET clusters, mRBC clusters, and PLT clusters appear in the second scatter plot (logarithmic display) with the logarithm of the first fluorescence intensity (SFL-1 (log)) on the horizontal axis and the logarithm of the second forward scattered light intensity (FSC-2 (log)) on the vertical axis. (B) is a schematic diagram showing the positions where RET clusters, mRBC clusters, and PLT clusters appear in the second scatter plot (linear display) with the SFL-1 intensity on the horizontal axis and the FSC-2 intensity on the vertical axis.

Fig. 15B

Fig.16

【 Fig.17 (A) is a schematic diagram illustrating the region where immature platelets appear in the second scattergram (logarithmic display). (B) is a schematic diagram illustrating the region where large platelets appear in the second scattergram (logarithmic display).

Fig.18

Fig.19

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Fig.21

Chemistry 3

Chemistry 4

Chemistry 5

Table 1

Explanation of symbols

Claims

1. A blood cell analysis method comprising: a step of irradiating a measurement sample containing particles dyed with a fluorescent dye with light to obtain information on fluorescence and scattered light generated from the particles, and a step of classifying blood cells from particles in the measurement sample based on the fluorescence information and the scattered light information, The fluorescent dye is represented by the following formula (I): 【Chemistry 1】 In the formula, R1 and R2 are the same or different and are halogen, an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 3 to 20 carbon atoms, an alkyl group having 1 to 18 carbon atoms having a hydroxyl group, an alkyl group having 1 to 18 carbon atoms having a carboxyl group, an alkyl group having 1 to 18 carbon atoms having a sulfonic group, -(CH2) m -NR6R7, -(CH2) n -O-R8 or benzyl optionally having a substituent, R3 and R4 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, alkoxy having 1 to 6 carbon atoms, or -(CH2) m -O-R8, R5 is a hydrogen atom, a halogen, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and having a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and having a carboxyl group, an alkyl group having 1 to 6 carbon atoms and having a sulfonic group, a phenyl group optionally having a substituent, a benzyl group optionally having a substituent, -(CH2) n -O-R6, R6 and R7 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R8 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent, m is an integer from 1 to 18, n is an integer from 1 to 6, X and Y are the same or different and are sulfur atoms, oxygen atoms, selenium atoms or CR9R 10 , R9 and R 10 are the same or different and are alkyl groups having 1 to 3 carbon atoms, Z- is the counter ion.

2. The method according to claim 1, wherein in the formula (I), when one of X and Y is an oxygen atom, the other is a sulfur atom, a selenium atom or C(CH3)2.

3. The method of claim 1, wherein the fluorescent dye is at least one selected from the group consisting of: 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]-6-methyl-benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-methyl-2-[(1,3,3-trimethyl-2-indolylene)methyl]benzothiazolium, and 1,3,3-Trimethyl-2-[(3-methyl-2-benzoxazolidinylidene)methyl]-3H-indolium.

4. The method of claim 1, wherein The measurement sample is prepared by mixing the fluorescent dye, the hemolytic reagent and whole blood. In the classification step, the leukocytes in the measurement sample are classified into subgroups.

5. The method of claim 4, wherein The light is light of a first wavelength not less than 315 nm and not more than 600 nm, The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength.

6. The method of claim 4, wherein The light is a first wavelength of light between 315 nm and 600 nm, and a second wavelength of light between 610 nm and 750 nm. The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information includes information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength and information related to side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

7. The method of claim 5 or 6, wherein The fluorescence information is the fluorescence intensity, The scattered light information is the side scattered light intensity, In the classification step, the leukocytes in the measurement sample are classified into subgroups based on the fluorescence intensity and the side scattered light intensity.

8. The method according to claim 7, wherein a scattergram based on the fluorescence intensity and the side scattered light intensity is prepared, and the leukocytes are classified into subgroups in the scattergram.

9. The method of claim 8, wherein the subpopulation comprises a lymphocyte population, a monocyte population, and a granulocyte population.

10. The method of claim 8, wherein the subpopulation comprises a population of lymphocytes, a population of monocytes, a population of neutrophils, a population of eosinophils, and a population of basophils.

11. The method according to claim 1, wherein the measurement sample is prepared by mixing the fluorescent dye, a diluent and whole blood, In the classification step, reticulocytes and platelets are classified from particles in the measurement sample.

12. The method of claim 11, wherein the assay sample does not contain a hemolytic agent.

13. The method of claim 11, wherein The light is a first wavelength of light between 315 nm and 600 nm, and a second wavelength of light between 610 nm and 750 nm. The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength, The scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

14. The method of claim 13, wherein The fluorescence information is the fluorescence intensity, The scattered light information is the forward scattered light intensity, In the classification step, reticulocytes, platelets, and mature erythrocytes are classified from particles in the measurement sample based on the fluorescence intensity and the forward scattered light intensity.

15. The method of claim 14, wherein a scattergram is prepared based on the fluorescence intensity and the forward scattered light intensity, and in the scattergram, particles in the measurement sample are classified into a reticulocyte group, a platelet group, and a mature erythrocyte group. The method of claim 13 , wherein the first wavelength is greater than 315 nm and less than 490 nm.

17. A reagent for blood cell analysis used in the method according to claim 1, comprising a fluorescent dye represented by the formula (I).

18. A blood cell analysis kit for use in the method according to claim 1, comprising a staining reagent containing the fluorescent dye represented by the formula (I) and a hemolytic reagent.

19. A method for fluorescently staining blood cells, comprising contacting the blood cells with a fluorescent dye represented by the following formula (I): 【Chemistry 2】 In the formula, R1 and R2 are the same or different and are halogen, an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 3 to 20 carbon atoms, an alkyl group having 1 to 18 carbon atoms having a hydroxyl group, an alkyl group having 1 to 18 carbon atoms having a carboxyl group, an alkyl group having 1 to 18 carbon atoms having a sulfonic group, -(CH2) m -NR6R7, -(CH2) n -O-R8 or benzyl optionally having a substituent, R3 and R4 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, alkoxy having 1 to 6 carbon atoms, or -(CH2) m -O-R8, R5 is a hydrogen atom, a halogen, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and having a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and having a carboxyl group, an alkyl group having 1 to 6 carbon atoms and having a sulfonic group, a phenyl group optionally having a substituent, a benzyl group optionally having a substituent, -(CH2) n -O-R6, R6 and R7 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R8 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent, m is an integer from 1 to 18, n is an integer from 1 to 6, X and Y are the same or different and are sulfur atoms, oxygen atoms, selenium atoms or CR9R 10 , R9 and R 10 are the same or different and are alkyl groups having 1 to 3 carbon atoms, Z- is the counter ion.

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