Fluorescent dye for blood cell analysis and blood cell dyeing method

Abstract

The invention provides a fluorescent dye for blood cell analysis. The fluorescent dye has the following structural general formula I, wherein X is CH2, C (CH3) 2, O, S or Se; y is-CH2-CH2 = CH-, or is selected from any one of the following structural formulas II, III and IV, and Z is an anion; r1 and R2 are respectively and independently selected from straight-chain or branched-chain C1-10 alkyl, benzyl and-(CH2) mNR3R4, the benzyl is optionally substituted by the following substituent groups: halogen, hydroxyl, sulfydryl and alkyl, and R1 and R2 are not benzyl at the same time; m is 1-10, and R3 and R4 are independently selected from H and straight-chain or branched-chain C1-6 alkyl groups respectively.

Description

Technical Field

[0001] This invention relates to a new class of fluorescent dyes in the field of fine chemicals, and in particular to a fluorescent dye for blood cell analysis and a method for staining blood cells. Background Technology

[0002] Nucleated red blood cells are rarely found in normal whole blood samples. Nucleated red blood cells are immature red blood cells formed during red blood cell development. These cells contain numerous organelles and nucleic acids and are an important indicator of the red blood cell production status in the human body. Normal whole blood should not contain nucleated red blood cells. The presence of nucleated red blood cells indicates abnormal hematopoietic function, such as proliferative anemia, erythrocyte disorders, erythroleukemia, bone marrow metastases, or extramedullary hematopoiesis.

[0003] In a normal human body, basophils are few in number, accounting for only 0-1% of the total number of white blood cells. However, in diseases such as heavy metal poisoning, Hodgkin's disease, and chronic myeloid leukemia, the number of basophils will be significantly increased. Therefore, basophil count is of great significance for the diagnosis of these diseases.

[0004] Therefore, it is of great significance to develop fluorescent dyes that can effectively fluoresce in nucleated erythrocytes and basophils, thereby enabling the effective detection of nucleated erythrocytes and basophils. Summary of the Invention

[0005] To achieve effective detection of nucleated red blood cells and basophils, this invention provides a fluorescent dye for blood cell analysis and a method for staining blood cells, which can achieve effective detection of nucleated red blood cells and basophils.

[0006] This invention provides a fluorescent dye for blood cell analysis, the fluorescent dye having the following general structural formula I:

[0007]

[0008] in:

[0009] X is CH2, C(CH3)2, O, S, or Se;

[0010] Y is -CH2-CH2=CH-, or selected from any one of the following structural formulas II, III, and IV.

[0011]

[0012] Z is an anion;

[0013] R1 and R2 are each independently selected from straight-chain or branched C1-10 alkyl, benzyl, -(CH2)mNR3R4, wherein the benzyl is optionally substituted by a substituent selected from the following: halogen, hydroxyl, mercapto, alkyl, and R1 and R2 are not both benzyl; m is 1-10, and R3 and R4 are each independently selected from H, straight-chain or branched C1-6 alkyl.

[0014] In some embodiments, R1 and R2 are each independently selected from straight-chain or branched C1-8 alkyl groups, or -(CH2)mNR3R4, where m is 1-6.

[0015] In some embodiments, X is C(CH3)2, S, or Se.

[0016] In some embodiments, Y is Or -CH2-CH2=CH-.

[0017] In some embodiments, Z is OH - Or halide ions.

[0018] The present invention provides a method for staining blood cells, the method comprising contacting the blood cells with the fluorescent dye described in any of the above embodiments.

[0019] The compounds provided in the embodiments of the present invention are used as fluorescent dyes and have effective fluorescent staining effects on nucleated erythrocytes and basophils, thereby achieving effective detection of nucleated erythrocytes and basophils. Attached Figure Description

[0020] Figure 1 This is a scatter plot of cell classification in Embodiment A of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity;

[0021] Figure 2 This is a scatter plot of cell classification in Embodiment B of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity.

[0022] Figure 3 This is a scatter plot of cell classification in Embodiment C of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity;

[0023] Figure 4 This is a scatter plot of cell classification in Embodiment D of the present invention, where the X-axis represents lateral fluorescence intensity and the Y-axis represents forward scattered light intensity. Detailed Implementation

[0024] Nucleated red blood cells should not be present in the whole blood of a normal person. If nucleated red blood cells are detected, it indicates abnormal hematopoietic function, such as proliferative anemia, erythrocyte disease, erythroleukemia, bone marrow metastases, extramedullary hematopoiesis, etc.

[0025] In a normal human body, basophils are few in number, accounting for only 0-1% of the total number of white blood cells. However, in diseases such as heavy metal poisoning, Hodgkin's disease, and chronic myeloid leukemia, the number of basophils will be significantly increased. Therefore, basophil count is of great significance for the diagnosis of these diseases.

[0026] Existing blood cell analyzers can detect nucleated red blood cells and basophils. The detection principle involves the combined action of reagents and fluorescent dyes on blood cells. Normal red blood cells are destroyed by hemolytic components in the reagents, while nucleated red blood cells and white blood cells, containing relatively intact organelles and cellular structures, can maintain a certain cellular state. The fluorescent dye in the reagents binds to the nucleic acid substances in these cells, producing a specific fluorescent signal under laser irradiation. Normal red blood cells are dissolved into blood shadows, nucleated red blood cells maintain a certain cellular structure and have a low fluorescence signal, while white blood cells maintain a certain cellular structure and have a high fluorescence signal. The optical information of the cells is collected by a flow cytometry detector and analyzed by an algorithm to further calculate the number and proportion of nucleated red blood cells and white blood cells.

[0027] Therefore, developing fluorescent dyes that can effectively fluoresce in nucleated erythrocytes and basophils, thereby enabling the effective detection of nucleated erythrocytes and basophils, is of great significance.

[0028] Unless otherwise stated, the terms used in this invention have the following meanings.

[0029] The term "alkyl" as used in this invention includes both straight-chain alkyl and branched-chain alkyl. When referring to a single alkyl group such as "propyl," it specifically refers to a straight-chain alkyl group; when referring to a single branched alkyl group such as "isopropyl," it specifically refers to a branched-chain alkyl group. For example, "C1-6 alkyl" includes C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Similar rules apply to other groups used in this specification.

[0030] The term "halogen" as used in this invention includes fluorine, chlorine, bromine, and iodine.

[0031] As used in this invention, the term "benzyl" refers to the -CH2-Ph group. When benzyl is modified with "optional substitution," it means that the benzyl group may exist in an unsubstituted form or may be substituted by a suitable substituent at any suitable position. Suitable substituents include, but are not limited to, H, C1-18 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-6 alkoxy, C1-6 alkylamino, C1-6 amide, halogen, or C1-6 haloalkyl, as long as the final compound has the properties desired by this invention. Preferably, the benzyl group is optionally substituted with a halogen, hydroxyl, mercapto, or alkyl group.

[0032] This invention provides a fluorescent dye for blood cell analysis, the fluorescent dye having the following general structural formula I:

[0033]

[0034] Where: X is CH2, C(CH3)2, O, S, or Se; Y is -CH2-CH2=CH-, or selected from any one of the following structural formulas II, III, and IV:

[0035]

[0036] Z is an anion; R1 and R2 are each independently selected from straight-chain or branched C1-10 alkyl, benzyl, ...

[0037] -(CH2)mNR3R4, wherein the benzyl group is optionally substituted by a substituent selected from the following: halogen, hydroxyl, mercapto, alkyl, and R1 and R2 are not both benzyl; m is 1-10, and R3 and R4 are each independently selected from H, straight-chain or branched C1-6 alkyl groups.

[0038] In some embodiments, R1 and R2 are each independently selected from straight-chain or branched C1-8 alkyl groups, or -(CH2)mNR3R4, where m is 1-6.

[0039] In some embodiments, X is C(CH3)2, S, or Se.

[0040] In some embodiments, Y is Or -CH2-CH2=CH-.

[0041] In some embodiments, Z is OH - Or halide ions.

[0042] This invention also provides a method for staining blood cells, the method comprising contacting the blood cells with the fluorescent dye described in any of the above embodiments. The term "contact" as used in the embodiments may include contact in a solution or a solid phase.

[0043] The compounds provided in the embodiments of the present invention are used as fluorescent dyes and have effective fluorescent staining effects on nucleated erythrocytes and basophils, thereby achieving effective detection of nucleated erythrocytes and basophils.

[0044] Example A

[0045] The composition of Example A of the present invention was prepared using the following components.

[0046]

[0047] The structure of the fluorescent dye is as follows:

[0048]

[0049] Blood samples containing nucleated red blood cells were added to the reagent mixture described above at a ratio of 1:50. The mixture was reacted at 40 degrees Celsius for 20 seconds. Nucleated red blood cells in the blood sample were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees were detected. The scatter plot results, which differentiated nucleated red blood cells from white blood cells based on the combined analysis of forward scattering and side fluorescence information, are shown below. Figure 1 As shown, the test results were 12.9% nucleated red blood cells and 0.5% basophils. The same specimen, confirmed by the Wright-Gimiza staining method recommended by the International Committee for Standardization in Hematology (ICSH), showed 13.0% nucleated red blood cells (13.0 NRBCs / 100 white blood cells) and 0.5% basophils (0.5 basophils / 100 white blood cells).

[0050] Example B

[0051] The composition of Example B of the present invention was prepared using the following components.

[0052]

[0053] The structure of the fluorescent dye is as follows:

[0054]

[0055] A blood sample containing nucleated red blood cells was added to the reagent mixture described above at a ratio of 1:50. The mixture was reacted at 41 degrees Celsius for 20 seconds. Nucleated red blood cells, lymphocytes, and basophils in the blood sample were detected using laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm). Forward scattering (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees were detected. Based on the combined analysis of the forward scattering and side fluorescence information, basophils and lymphocytes were separated from the white blood cells. The scatter plot results are shown below. Figure 2 As shown, the test results were 3.2% nucleated red blood cells and 1.3% basophils. The same specimen, confirmed by the Wright-Gimiza staining method recommended by the International Committee for Standardization in Hematology (ICSH), showed 3.5% nucleated red blood cells and 1.5% basophils.

[0056] Example C

[0057] The composition of Example C of the present invention was prepared using the following components.

[0058]

[0059] The structure of the fluorescent dye is as follows:

[0060]

[0061] Take 20 μL of clinical blood sample and mix it with 1 mL of the reagent prepared according to the above composition. React at 40°C for 15 seconds. Use laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm) to detect nucleated erythrocytes, basophils, and lymphocytes in the blood sample. Detect forward scatter (FS) information from 1 to 10 degrees and side fluorescence (SFL) information from 90 degrees. Based on the comprehensive analysis of the forward scatter and side fluorescence information, separate nucleated erythrocytes from leukocytes, and simultaneously separate basophils and lymphocytes from leukocytes. The scatter plot results are shown below. Figure 3 As shown, the test results were 0.8% nucleated red blood cells and 0.2% basophils. The same specimen was confirmed by the Wright-Gimza staining method recommended by the International Committee for Standardization in Hematology (ICSH), which showed a nucleated red blood cell percentage of 0.5% and a basophil percentage of 0%. Since the staining method involves manually counting nucleated red blood cells or white blood cell subtypes under a microscope, each count is 100 white blood cells to determine the final percentage. When the percentage of nucleated red blood cells or basophils is less than 1%, it is very likely that the percentage of the above cells will be 0. From this point of view, the test method of the present invention is more accurate in reporting the values ​​of various cells, especially for cells with low content in normal human blood, such as nucleated red blood cells and basophils.

[0062] Example D

[0063] The composition of Example D of the present invention was prepared using the following components.

[0064]

[0065] The structure of the fluorescent dye is as follows:

[0066]

[0067] Take 5 μL of blood sample containing nucleated erythrocytes and mix it with 1 mL of the reagent prepared above. React at 46°C for 15 s. Use laser flow cytometry (excitation source: semiconductor laser, excitation wavelength: 635 nm) to detect nucleated erythrocytes, lymphocytes, and basophils in the blood sample. Detect forward scatter (FS) information (1–10 degrees) and side fluorescence (SFL) information (90 degrees). Based on the combined analysis of forward scatter and side fluorescence information, nucleated erythrocytes are separated from the total white blood cells. Simultaneously, basophils and lymphocytes are also separated from the white blood cells. In the scatter plot, nucleated erythrocytes form an independent cell group located in the lower left corner of the scatter plot. The scatter plot result is shown below. Figure 4 As shown, the test results were 8.7% nucleated red blood cells and 0.3% basophils. The same specimen was confirmed by the Wright-Gimza staining method recommended by the International Committee for Standardization of Hematology (ICSH), and the percentage of nucleated red blood cells was 8.5% and the percentage of basophils was 0.5%.

[0068] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered within the scope of protection of the present invention. Using the compound as a fluorescent dye is one application of the novel compound of the present invention; it should not be construed that the compound of the present invention is only used for fluorescent dyes. For those skilled in the art, based on the same mechanism of action of the compound of the present invention as a fluorescent dye, several simple inferences can be made to derive other applications of the compound of the present invention, and all such applications should be considered within the scope of protection of the present invention.

Claims

1. A fluorescent dye for blood cell analysis, characterized in that, The fluorescent dye has the following general structural formula I: in: X is CH2, C(CH3)2, O, S, or Se; Y is -CH2-CH2=CH-, or selected from any one of the following structural formulas II, III, and IV. Z is an anion; R1 and R2 are each independently selected from straight-chain or branched C1-10 alkyl, benzyl, -(CH2)mNR3R4, wherein the benzyl is optionally substituted by a substituent selected from the following: halogen, hydroxyl, mercapto, alkyl, and R1 and R2 are not both benzyl; m is 1-10, and R3 and R4 are each independently selected from H, straight-chain or branched C1-6 alkyl.

2. The fluorescent dye for blood cell analysis as described in claim 1, characterized in that, R1 and R2 are each independently selected from straight-chain or branched C1-8 alkyl groups, or -(CH2)mNR3R4, where m is 1-6.

3. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, X is C(CH3)2, S, or Se.

4. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, The Y is Or -CH2-CH2=CH-.

5. The fluorescent dye for blood cell analysis according to claim 1, characterized in that, Z is OH - Or halide ions.

6. A method for staining blood cells, characterized in that, The method includes contacting blood cells with the fluorescent dye of any one of claims 1-5.

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