White blood cell differential reagents, red blood cell analysis reagents, kits, and analytical methods

Abstract

A white blood cell classification reagent, kit, and classification method; and a red blood cell analysis reagent, kit, and analytical method. Both the white blood cell classification reagent and the red blood cell analysis reagent contain a fluorescent dye with the general formula F. This dye can stain the nucleic acids of white blood cells and red blood cells, thus enabling accurate classification and / or counting of white blood cells and red blood cells in blood samples during blood analysis, especially the identification and / or counting of RETs. Furthermore, it simplifies the types of dyes used in blood cell analysis.

Description

Technical Field

[0001] This invention relates to reagents for analyzing red and white blood cells and methods of using them, specifically to reagents for automatically distinguishing different subgroups of white blood cells in blood using an analyzer, reagents for identifying red blood cells and their types in blood, and methods thereof. Background Technology

[0002] Blood cells are divided into three types: red blood cells (RBC), white blood cells (WBC), and platelets (PLT). A routine blood test often requires testing the white blood cell count, red blood cell count, and platelet count.

[0003] Normal peripheral blood leukocytes are typically classified into five categories: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Analyzing leukocyte populations in blood samples can provide valuable information for the clinical diagnosis of numerous diseases. For example, the proportions and numbers of different types of leukocytes in the blood may change during disease, and abnormal leukocytes such as abnormal lymphocytes and immature granulocytes may also be present. Therefore, classifying and measuring different types of normal and abnormal leukocytes can provide information about the latency, onset, and progression of the disease.

[0004] Normal peripheral blood red blood cells include mature red blood cells and a small number of reticulocytes (RETs). Reticulocytes (RETs) are transitional cells between late erythroblasts and mature red blood cells, slightly larger than mature red blood cells. Reticulocytes are nucleated red blood cells that have just lost their nuclei and are still not fully mature. Their cytoplasm still contains basophilic substances such as ribosomes and ribonucleic acid. After being stained with brilliant cresyl blue or neomethylene blue, blue or blue-green punctate or even reticular structures can be seen in the cytoplasm, hence the name reticulocyte. Current research suggests that reticulocytes, as an important stage in the red blood cell maturation process, should be given due attention.

[0005] Current fluorescence analysis methods in blood analyzers rely on different dyes to analyze red and white blood cells, which leads to complex instrument and liquid circuit designs. Therefore, a new dye is needed that can be used separately for the classification and counting of red and white blood cells, thereby simplifying the structure of blood analyzers. Summary of the Invention

[0006] The summary section introduces a series of simplified concepts, which will be further explained in detail in the detailed description section. The summary section of this invention is not intended to limit the key features and essential technical features of the claimed technical solution, nor is it intended to determine the scope of protection of the claimed technical solution.

[0007] To at least partially address the above-mentioned problems, a first aspect of this application provides a leukocyte classification reagent comprising a fluorescent dye having formula F:

[0008]

[0009] Among them, X is selected from the group composed of C(CH3)2, O, S and Se;

[0010] R1 and R2 are each independently chosen from H, C1-C 18 The group consisting of alkyl, phenyl, OR6 and halogens;

[0011] R3 and R4 are each independently selected from C1 to C2. 18 Alkyl, C1-C 18 Carboxyl group, C1-C 18 Hydroxyl group, C1-C 18 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 18 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl;

[0012] R5 and R6 are each independently selected from H and C1-C. 18 Groups composed of alkyl groups;

[0013] Y - It is a negative ion.

[0014] The fluorescent dye of formula F in this application belongs to the cyanine class of compounds and has good permeability to living cells. It can enter cells without damaging the cell membrane to stain nucleic acids. In blood analysis, it is used to stain leukocytes for classification and / or counting. In one embodiment, the excitation light of the dye is a short-wavelength blue-green light, which can identify tiny particles and improve the detection capability of small particles.

[0015] A second aspect of this application provides a white blood cell analysis kit, comprising the white blood cell classification reagent described in the first aspect. The white blood cell analysis kit of this application can classify and / or count white blood cells in blood analysis; classifying white blood cells into lymphocytes, monocytes, neutrophils, and eosinophils, and counting the classified cells.

[0016] A third aspect of this application provides a method for classifying white blood cells, the method comprising: mixing a blood sample, a fluorescent dye having formula F, and a erythrolysis agent to form a mixture; wherein, formula F is...

[0017]

[0018] Among them, X, R1, R2, R3, R4 and Y - As defined in the first aspect above in the leukocyte differential reagent;

[0019] The particles in the test sample are irradiated with light, and at least one light scattering characteristic and at least one fluorescence characteristic of the test sample are measured.

[0020] Leukocytes are classified and / or counted based on the scattered light and fluorescence characteristics.

[0021] A fourth aspect of this application provides a red blood cell analysis reagent, the reagent comprising a fluorescent dye having formula F; wherein, formula F is...

[0022]

[0023] Among them, X, R1, R2, R3, R4 and Y - As defined in the first aspect above in the white blood cell differential reagent. Red blood cell analysis reagents are used in blood analysis to stain red blood cells and obtain their parameters.

[0024] The fifth aspect of this application provides a red blood cell analysis kit, the kit comprising the red blood cell analysis reagents as described in the fourth aspect. The white blood cell analysis kit of this application can classify and / or count red blood cells in blood analysis; red blood cell classification includes distinguishing red blood cells into mature red blood cells and / or reticulocytes; red blood cell counting includes counting mature red blood cells and / or reticulocytes.

[0025] A sixth aspect of this application provides a method for red blood cell analysis, comprising: mixing a blood sample, a fluorescent dye having formula F, and a spheroidizing reagent to form a mixture; wherein, formula F is...

[0026]

[0027] Among them, X, R1, R2, R3, R4 and Y - As defined in the white blood cell differential reagent above;

[0028] The mixture is measured to have at least one light scattering property and at least one fluorescence property.

[0029] Red blood cell classification and / or counting are obtained based on the scattered light characteristics and fluorescence characteristics.

[0030] The white blood cell classification reagent and red blood cell analysis reagent in this application both contain a fluorescent dye with formula F. This dye can stain the nucleic acids of white blood cells and red blood cells, thus enabling accurate classification and / or counting of white blood cells and red blood cells in blood samples during blood analysis, especially the identification and / or counting of RETs. Furthermore, it simplifies the types of dyes used in blood cell analysis. Attached Figure Description

[0031] The following drawings, which are incorporated herein by reference as part of this invention, are provided for understanding the invention. The drawings illustrate embodiments of the invention and their descriptions, serving to explain the principles of the invention.

[0032] In the attached image:

[0033] Figure 1 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula I as a fluorescent dye for classifying leukocytes.

[0034] Figure 2 The fluorescence-forward scatter plot of a compound with chemical formula I as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0035] Figure 3 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula II as a fluorescent dye for classifying leukocytes.

[0036] Figure 4 The fluorescence-forward scatter plot of a compound with chemical formula II as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0037] Figure 5 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula III as a fluorescent dye for classifying leukocytes.

[0038] Figure 6 The fluorescence-forward scatter plot of a compound with chemical formula III as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0039] Figure 7 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula IV as a fluorescent dye for classifying leukocytes.

[0040] Figure 8 The fluorescence-forward scatter plot of a compound with chemical formula IV as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0041] Figure 9The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula V as a fluorescent dye for classifying leukocytes.

[0042] Figure 10 The fluorescence-forward scatter plot of a compound with the chemical formula V as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0043] Figure 11 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula VI as a fluorescent dye for classifying leukocytes.

[0044] Figure 12 The fluorescence-forward scatter plot of a compound with chemical formula VI as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0045] Figure 13 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula VII as a fluorescent dye for classifying leukocytes.

[0046] Figure 14 The fluorescence-forward scatter plot of a compound having chemical formula VII as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0047] Figure 15 The side-scattered light-fluorescence scatter plot shows the use of a compound with chemical formula VIII as a fluorescent dye for classifying leukocytes.

[0048] Figure 16 The fluorescence-forward scatter plot of a compound with chemical formula VIII as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0049] Figure 17 The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula IX as a fluorescent dye for classifying leukocytes.

[0050] Figure 18 The fluorescence-forward scatter plot of a compound with the chemical formula IX as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0051] Figure 19 The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula X as a fluorescent dye for classifying leukocytes.

[0052] Figure 20 The fluorescence-forward scatter plot of a compound with the chemical formula X as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0053] Figure 21The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula XI as a fluorescent dye for classifying leukocytes.

[0054] Figure 22 The fluorescence-forward scatter plot of a compound with the chemical formula XI as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0055] Figure 23 The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula XII as a fluorescent dye for classifying leukocytes.

[0056] Figure 24 The fluorescence-forward scatter plot of a compound with the chemical formula XII as a fluorescent dye for the detection of erythrocytes and platelets is shown.

[0057] Figure 25 The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula XIII as a fluorescent dye for classifying leukocytes.

[0058] Figure 26 The following is a fluorescence-forward scatter plot showing the use of a compound with the chemical formula XIII as a fluorescent dye for the detection of erythrocytes and platelets.

[0059] Figure 27 The side-scattered light-fluorescence scatter plot shows the use of a compound with the chemical formula XVIII as a fluorescent dye for classifying leukocytes.

[0060] Figure 28 The following is a fluorescence-forward scatter plot showing the use of a compound with the chemical formula XVIII as a fluorescent dye for the detection of erythrocytes and platelets. Detailed Implementation

[0061] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described in order to avoid obscuring the invention.

[0062] To fully understand the present invention, a detailed description will be set forth in the following description. It is obvious that the implementation of embodiments of the present invention is not limited to the specific details familiar to those skilled in the art. Preferred embodiments of the present invention are described in detail below; however, in addition to these detailed descriptions, the present invention may have other embodiments.

[0063] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof.

[0064] Unless otherwise stated, the terms “blood” or “blood sample” as used herein refer to a body fluid sample containing blood cells, such as peripheral blood, bone marrow fluid, etc.

[0065] In particular, as used throughout this application, the term alkyl can be understood in the broadest sense as any linear, branched, or cyclic alkyl substituent. As used herein, the term "C" is also used... 1-18 "Alkyl" generally refers to a saturated hydrocarbon group having 1 to 18 carbon atoms in its configuration. 1-18 Alkyl groups include, but are not limited to, C 1-12 Alkyl, C 1-6Alkyl groups, etc. Unless otherwise specified as “substituted alkyl,” the term “alkyl” generally refers to an unsubstituted alkyl group. Exemplarily, the term alkyl includes substituents such as methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), cyclopropyl, n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), tert-butyl (tBu), cyclobutyl, 2-methylbutyl, n-pentyl, sec-pentyl, tert-pentyl, 2-pentyl, neopentyl, cyclopentyl, n-hexyl, sec-hexyl, tert-hexyl, 2-hexyl, 3-hexyl, neohexyl, cyclohexyl, 1- Methylcyclopentyl, 2-methylpentyl, n-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, cycloheptyl, 1-methylcyclohexyl, n-octyl, 2-ethylhexyl, cyclooctyl, 1-bicyclo[2,2,2]octyl, 2-bicyclo[2,2,2]octyl, 2-(2,6-dimethyl)octyl, 3-(3,7-dimethyl)octyl, adamantyl, 2,2,2-trifluoroethyl, 1,1-dimethyl-n-hex-1-yl, 1,1-dimethyl- n-Hept-1-yl, 1,1-Dimethyl-n-oct-1-yl, 1,1-Dimethyl-n-dec-1-yl, 1,1-Dimethyl-n-dodecane-1-yl, 1,1-Dimethyl-n-tetradecane-1-yl, 1,1-Dimethyl-n-hexadecane-1-yl, 1,1-Dimethyl-n-octadecane-1-yl, 1,1-Diethyl-n-hexane-1-yl, 1,1-Diethyl-n-hept-1-yl, 1,1-Diethyl-n-octane-1-yl, 1,1- Diethyl-n-decyl-1-yl, 1,1-diethyl-n-dodecyl-1-yl, 1,1-diethyl-n-tetradecyl-1-yl, 1,1-diethyl-n-hexadecyl-1-yl, 1,1-diethyl-n-octadecyl-1-yl, 1-(n-propyl)-cyclohexyl-1-yl, 1-(n-butyl)-cyclohexyl-1-yl, 1-(n-hexyl)-cyclohexyl-1-yl, 1-(n-octyl)-cyclohexyl-1-yl and 1-(n-decyl)-cyclohexyl-1-yl, etc.

[0066] As used above and herein, the term carboxyl includes any linear, branched, or cyclic carboxyl substituent. The term C, as used herein, refers to... 1-18 The carboxyl group generally refers to a carboxyl substituent group having 1 to 18 carbon atoms in its configuration. 1-18 Carboxyl groups include, but are not limited to, C 1-12 Carboxyl group, C 1-6 Carboxyl group, etc. In particular, the term carboxyl group refers to a group having a carboxyl group attached to an alkyl group as defined above. The term carboxyl group includes, by example, methylcarboxyl, ethylcarboxyl, propionic carboxyl, butylcarboxyl, pentylcarboxyl, hexylcarboxyl, heptylcarboxyl, and octylcarboxyl, as well as their isomers, etc.

[0067] As used above and herein, the term hydroxyl includes any linear, branched, or cyclic hydroxyl substituent. As used herein, the term C... 1-18Hydroxyl groups generally refer to hydroxyl substituents having 1 to 18 carbon atoms in their configuration. 1-18 Hydroxyl groups include, but are not limited to, C 1-12 Hydroxyl group, C 1-6 Hydroxyl group, etc. In particular, the term hydroxyl refers to a group having a hydroxyl group attached to an alkyl group as defined above. The term hydroxyl exemplarily includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl, and hydroxyoctyl, as well as their isomers, etc.

[0068] As used above and herein, the term alkoxy includes any linear, branched, or cyclic alkoxy substituent. In particular, the term alkoxy refers to an alkoxy group linked to an alkyl group as defined above. Exemplary examples of the term alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and 2-methylbutoxy, etc.

[0069] As used above and here, the terms “halogen” and “halogenated” can be understood in the broadest sense as preferably fluorine, chlorine, bromine or iodine.

[0070] As used in this article, unless otherwise defined in a specific context, the wavelength of blue-green light is approximately in the range of 400-560 nm.

[0071] Fluorescent dyes

[0072] The fluorescent dye of this application belongs to the cyanine class of compounds and has good permeability to living cells, enabling it to enter cells without damaging the cell membrane and specifically stain nucleic acids (RNA, DNA). The dye is a blue-green light-excited fluorescent dye, which can be excited by blue or green laser light emitted from devices that provide blue or green light regions, such as blue or green semiconductor lasers. Therefore, the reagent of this invention can be used in blood analyzers or flow cytometers that use inexpensive equipment such as semiconductor lasers as light sources.

[0073] In one embodiment, the fluorescent dye has the structure shown in Formula F:

[0074]

[0075] Among them, X is selected from the group composed of C(CH3)2, O, S and Se;

[0076] R1 and R2 are each independently chosen from H, C1-C 18 The group consisting of alkyl, phenyl, OR6 and halogens;

[0077] R3 and R4 are each independently selected from C1 to C2. 18 Alkyl, C1-C 18 Carboxyl group, C1-C 18 Hydroxyl group, C1-C18 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 18 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl;

[0078] R5 and R6 are each independently selected from H and C1-C. 18 Groups composed of alkyl groups;

[0079] Y - It is a negative ion.

[0080] In one embodiment, X in formula F is selected from the group consisting of C(CH3)2 and S.

[0081] In one embodiment, R1 and R2 in equation F are each independently selected from H, C1-C 12 The group consisting of alkyl, phenyl, OR6 and halogens.

[0082] In one embodiment, R1 and R2 in Formula F are each independently selected from the group consisting of H, C1-C6 alkyl, phenyl, OR6 and halogen.

[0083] In one embodiment, R1 in formula F is selected from the group consisting of H, C1-C6 alkyl, phenyl and halogen.

[0084] In one embodiment, R2 in formula F is H.

[0085] In one embodiment, R3 and R4 in equation F are each independently selected from C1-C 12 Alkyl, C1-C 12 Carboxyl group, C1-C 12 Hydroxyl group, C1-C 12 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 12 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 acylamino, halogen and C1-C6 haloalkyl.

[0086] In one embodiment, R3 and R4 in Formula F are each independently selected from the group consisting of C1-C6 alkyl, C1-C6 carboxyl, C1-C6 hydroxy, C1-C6NR5R6, benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C1-C6 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl.

[0087] In one embodiment, R3 in formula F is selected from the group consisting of C1-C6 alkyl, C1-C6 hydroxy, C1-C6 carboxyl, C1-C6NR5R6 and benzyl.

[0088] In one embodiment, R4 in formula F is selected from the group consisting of C1-C6 alkyl, C1-C6 hydroxy, C1-C6 carboxyl, and benzyl.

[0089] In one embodiment, R5 and R6 in equation F are each independently selected from H and C1-C 12 A group composed of alkyl groups.

[0090] In one embodiment, R5 and R6 in Formula F are each independently selected from the group consisting of H and C1-C6 alkyl groups.

[0091] In one embodiment, R5 and R6 in formula F are each independently C1-C6 alkyl groups.

[0092] In one embodiment, Y in formula F - Choose free halide anions, ClO4 - PF6 - BF4 - CH3COO - or OTs - A group that is formed.

[0093] In one embodiment, the fluorescent dye comprises structures of chemical formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, and XVIII.

[0094]

[0095]

[0096]

[0097] The fluorescent dye of this application can be dissolved in glycerol, glycol, or ethylene glycol and stored as a stock solution, or it can be dissolved in other non-aqueous solvents. Ethylene glycol is preferably used as the solvent, and optionally, 5-10% methanol is included as a component of the diluent. Furthermore, to prevent aggregation of the fluorescent dye, the invention may optionally include a nonionic surfactant as a dispersant to maintain a certain solubility of the fluorescent dye and prevent aggregation. Examples of such nonionic surfactants are polyoxyethylene glycol (POE), polypropylene glycol (POP), polyoxyethylene glycol-polypropylene glycol (POE-POP), and Brij series nonionic surfactants. Brij 35 and Brij 56 are preferably used as dispersants for the fluorescent dye at a concentration of 0.02-2%.

[0098] Alternatively, the fluorescent dye of this application can be formulated together with a erythrolysis agent or a spheroidizing agent to form a single-component reagent.

[0099] Red blood cell lysing agent

[0100] The erythrolysis agent used may contain cationic surfactants, nonionic surfactants, anionic surfactants, or any combination thereof, as well as a buffer that maintains the pH of the assay system between 5 and 11.

[0101] Cationic surfactants, nonionic surfactants, anionic surfactants, or any combination thereof can be used as reagents for lysing red blood cells. They can dissolve red blood cells in blood samples and appropriately disrupt the membrane structure of different subsets of leukocytes, causing the different subsets of leukocytes in the blood to shrink to appropriate sizes, promoting different aggregations within the leukocytes, and resulting in differences in light scattering properties. Cationic surfactants can be octyltrimethylammonium bromide (OTAB), decyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride (LTAC), tetradecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium chloride (CTAC), etc., preferably LTAC, used at a concentration of 300-800 mg / L. Nonionic surfactants can be polyoxyethylene type nonionic surfactants, such as long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene esters, fatty amine polyoxyethylene ethers, etc., preferably polyoxyethylene-2,3-dodecyl ether (Brij35), used at a concentration of 1-5 g / L. The anionic surfactant is selected from dodecylbenzene sulfonic acid, sodium fatty alcohol acyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, and alcohol ether carboxylates, and is used at a concentration of 0.1-2 g / L.

[0102] The reagents of this application may contain a buffer solution to maintain pH and appropriately dilute blood samples. The buffer solution keeps the pH within a constant range, thereby stabilizing the staining effect of different leukocyte subgroups. The concentration of the buffer solution used is in the range of 0.01-200 mM. There is no particular limitation on the type of buffer solution, as long as it is suitable for the purposes of this invention, such as appropriate concentrations of carboxylates, phosphates, citrates, Tris-HCl, MOPS, and other organic buffers. The suitable pH in the reagents of this invention varies depending on the specific fluorescent dye selected, generally in the range of pH 5.0-11.0, with a preferred pH of 8.0-10.0.

[0103] The erythrolysis agent of the present invention may optionally contain an appropriate proportion of alcohol, such as methanol, to promote the aggregation of the internal structure of leukocytes.

[0104] White blood cell detection kit

[0105] In another aspect, the present invention also provides a kit for distinguishing and counting white blood cells in blood, said kit comprising a white blood cell classification reagent comprising a fluorescent dye having the structure shown in Formula F:

[0106]

[0107] Among them, X is selected from the group composed of C(CH3)2, O, S and Se;

[0108] R1 and R2 are each independently chosen from H, C1-C 18 The group consisting of alkyl, phenyl, OR6 and halogens;

[0109] R3 and R4 are each independently selected from C1 to C2. 18 Alkyl, C1-C 18 Carboxyl group, C1-C 18 Hydroxyl group, C1-C 18 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 18 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl;

[0110] R5 and R6 are each independently selected from H and C1-C. 18 Groups composed of alkyl groups;

[0111] Y - It is a negative ion.

[0112] In other embodiments, X, R1, R2, R3, R4 and Y -The choice of fluorescent dyes is as described in the previous section on fluorescent dyes.

[0113] Preferably, the fluorescent dye has a structure selected from chemical formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, and XVIII.

[0114]

[0115]

[0116] Preferably, the leukocyte classification reagent also includes the erythrocyte lysing agent described above.

[0117] In one embodiment, the fluorescent dye is present as a storage solution and stored in a separate container. The concentration of the fluorescent dye is 0.1-1000 mg / L, preferably 20-500 mg / L, and more preferably 50-200 mg / L.

[0118] In another embodiment, the fluorescent dye is prepared together with the aforementioned erythrolysis agent as a mixed reagent solution.

[0119] This kit preferably contains a fluorescent dye suitable for sealed storage in at least one container. The kit may also contain other white blood cell classification reagents required for classifying white blood cells in blood, as well as instructions for measuring white blood cells. The kit may also contain control samples or a series of control samples that can be measured and compared with the test sample. The components of the kit may be packaged in a single container, with all different containers, along with instructions, in a separate package. This kit is used for classifying and / or counting various types of white blood cells in blood.

[0120] Spheroidizing reagent

[0121] The spherification agent used may contain amphoteric surfactants, osmotic pressure regulators, buffers, and optionally cationic surfactants and / or preservatives.

[0122] The surfactant in the spheroidizing agent causes blood cells to swell and slightly damage the cell membrane to allow dye entry. The concentration range of the amphoteric surfactant is 100-2000 mg / L, preferably 100-500 mg / L. The amphoteric surfactant can be one or more of alkyl betaine, sulfonated betaine, cocamidopropyl betaine, etc. The cationic surfactant can be octyltrimethylammonium bromide (OTAB), decyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride (LTAC), tetradecyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium chloride (CTAC), etc., used at a concentration of 0-50 mg / L. The osmotic pressure regulator can be common salts such as sodium chloride and sodium sulfate, with an osmotic pressure range of 200 mOsm / kg-380 mOsm / kg. There are no particular limitations on the type of buffer solution, as long as they are suitable for the purposes of this invention, such as appropriate concentrations of carboxylates, phosphates, citrates, Tris-HCl, MOPS, and other organic buffers. The appropriate pH in the reagents of this invention varies depending on the specific fluorescent dye selected, and is generally in the range of pH 5.0-11.0, with the preferred pH being 8.0-10.0.

[0123] Red blood cell detection kit

[0124] On the other hand, the present invention also provides a kit for distinguishing and counting red blood cells in blood, particularly for the identification and counting of reticulocytes, the kit comprising a red blood cell analysis reagent comprising a fluorescent dye having the structure shown in Formula F:

[0125]

[0126] Among them, X is selected from the group composed of C(CH3)2, O, S and Se;

[0127] R1 and R2 are each independently chosen from H, C1-C 18 The group consisting of alkyl, phenyl, OR6 and halogens;

[0128] R3 and R4 are each independently selected from C1 to C2. 18 Alkyl, C1-C 18 Carboxyl group, C1-C 18 Hydroxyl group, C1-C 18 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 18 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl;

[0129] R5 and R6 are each independently selected from H and C1-C. 18 Groups composed of alkyl groups;

[0130] Y - It is a negative ion.

[0131] In other embodiments, X, R1, R2, R3, R4 and Y - The choice of fluorescent dyes is as described in the previous section on fluorescent dyes.

[0132] Preferably, the fluorescent dye has a structure selected from chemical formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII.

[0133]

[0134]

[0135] Preferably, the red blood cell analysis reagent also includes the spheroidizing reagent described herein.

[0136] In one embodiment, the fluorescent dye is present as a storage solution and stored in a separate container. The concentration of the fluorescent dye is 0.1-1000 mg / L, preferably 20-500 mg / L, and more preferably 50-200 mg / L.

[0137] In another embodiment, the fluorescent dye is prepared together with the above-mentioned spheroidizing agent to form a mixed reagent solution.

[0138] This kit preferably contains a fluorescent dye suitable for sealed storage in at least one container. The kit may also contain other red blood cell classification reagents required for classifying red blood cells in blood, as well as instructions for the method of determining red blood cells. The kit may also contain control samples or a series of control samples that can be measured and compared with the test sample. The components of the kit may be packaged in a single container, with all different containers, along with instructions, in separate packaging. This kit is used to classify and / or count various types of red blood cells in blood, such as mature red blood cells and / or reticulocytes.

[0139] How to use:

[0140] A: This application also provides a method for classifying and / or counting white blood cells in blood. Simply put, in the method of this application, a blood sample is mixed with a white blood cell classification reagent, followed by the determination of at least one light-scattering characteristic and at least one fluorescence characteristic of the sample, and the sample is classified and / or counted based on the light-scattering and fluorescence characteristics.

[0141] The blood sample used in this application's method can be whole blood or blood components. The blood sample is first mixed with a erythrolysis agent, causing the erythrocytes to dissolve and the leukocytes of different subgroups to shrink to varying degrees. This step simultaneously creates small pores in the cell membranes of the leukocytes to be tested, pores large enough to allow fluorescent dye molecules to pass through the cell membrane.

[0142] Subsequently, a fluorescent dye stock solution is added, causing the leukocytes to be fluorescently labeled. When mixing the blood sample with the reagents, the total volume of the blood sample and reagents should ensure a sufficient cell concentration to pass through the instrument's measuring cell. The reagent composition of this application dilutes the blood sample to any value within any range of 1:10, 1:50, or 1:100 or higher, as long as such dilution meets the requirements of practical use. Such adjustments are within the scope of those skilled in the art. Alternatively, the blood sample can be mixed simultaneously with the erythrolysis agent and the fluorescent dye.

[0143] The sample mixture can be incubated in an incubation chamber for no more than 60 seconds, preferably 30 seconds or 24 seconds; the incubation temperature can be any suitable temperature, such as 40°C. Leukocytes can be detected and analyzed using a blood analyzer or flow cytometer. The dissolved, diluted, and stained blood sample is introduced into the flow chamber of the measuring instrument. Light emitted from a light source illuminates the particles in the flow chamber, generating optical information. A detector collects this optical information, which includes at least one type of scattered light and at least one type of fluorescence.

[0144] The scattered light used in this application refers to scattered light detectable by a commercially available blood analyzer or similar flow cytometer. This scattered light includes, but is not limited to, side-scattered light, forward low-angle scattered light (receiving angle of approximately 0-5 degrees), and forward high-angle scattered light (receiving angle of approximately 5-20 degrees). Scattered light with these angles reflects information about the size or internal structure of white blood cells and is therefore used as the scattered light in this invention. Side-scattered light is preferred.

[0145] Fluorescent dyes that bind to nucleic acids in cells emit fluorescence. Fluorescence properties are parameters reflecting the amount of fluorescent dye within cells in a blood sample. Due to differences in intracellular metabolic activity among different subpopulations, resulting in variations in nucleic acid content, the fluorescence properties of different leukocyte subpopulations differ in certain aspects. The appropriate excitation wavelength is selected based on the specific dye used, and the emission light at the corresponding wavelength is monitored. In this application, for leukocyte detection, a green or blue semiconductor laser emitting a green or blue wavelength region laser can be used as the detection light source. There are no particular limitations on the green or blue wavelength light source, as long as it can emit green light (e.g., light with a wavelength of approximately 501-560 nm) or blue light (e.g., light with a wavelength of approximately 400-500 nm) near the excitation wavelength of the selected fluorescent dye. For example, the light source can be configured to emit light with a wavelength of approximately 450 nm or 520 nm. Such lasers are relatively inexpensive compared to other lasers, thus reducing equipment costs.

[0146] The characteristics of scattered light and fluorescence were used to identify different leukocyte subgroups, and the percentage of each subgroup was calculated by classifying and counting the scattered points. Scattered light reflects the degree of granulation within the cell. The degree of granulation within the cell is roughly as follows: eosinophils have bilobed nuclei and many fragile microparticles stained with acid dyes; neutrophils have nuclei (lobed or rod-shaped) with many internal granules; monocytes have a single macronucleus with fewer internal granules; and LYM cells have a single macronucleus with almost no granules. Therefore, under the same conditions, the order of scattered light intensity characteristics of different types of leukocytes is EO (eosinophils) > NEUT (neutrophils) > MONO (monocytes) > LYM (lymphocytes).

[0147] B: This application also provides a method for classifying and / or counting red blood cells in blood. Simply put, in the method of this application, a blood sample is mixed with a red blood cell analysis reagent, followed by the determination of at least one light-scattering characteristic and at least one fluorescence characteristic of the sample, and the sample is classified and / or counted based on the light-scattering and fluorescence characteristics.

[0148] The blood sample used in this application's method can be whole blood or blood components. The blood sample can be first mixed with a spheroidizing agent, causing red blood cells and white blood cells to swell and slightly damage their cell membranes, forming small pores on the cell membranes of red blood cells and white blood cells. These pores are large enough to allow fluorescent dye molecules to pass through the cell membrane.

[0149] Subsequently, a fluorescent dye stock solution is added, causing red blood cells and white blood cells to be fluorescently labeled. Because mature red blood cells, reticulocytes, and white blood cells have different nucleic acid contents, their fluorescence characteristics differ, allowing for differentiation between these three cell types. When mixing the blood sample with the reagents, the total volume of the blood sample and the reagents of this application should ensure a sufficient cell concentration to pass through the instrument's measuring cell. The reagent composition of this application dilutes the blood sample to any value within a range of 1:10, 1:50, or 1:100 or higher, as long as such dilution meets the requirements of practical use. Such adjustments are within the scope of those skilled in the art. Alternatively, the blood sample can be mixed simultaneously with the spheroidizing agent and the fluorescent dye.

[0150] The sample mixture can be incubated in an incubation chamber for no more than 60 seconds, preferably 30 seconds or 24 seconds; the incubation temperature can be any suitable temperature, such as 40°C. Red blood cells can be detected and analyzed using a blood analyzer or flow cytometer. The spheroidized and stained blood sample is introduced into the flow chamber of the measuring instrument. Light emitted from a light source illuminates the particles in the flow chamber, generating optical information. A detector collects this optical information, which includes at least one type of scattered light and at least one type of fluorescence.

[0151] The scattered light used in this application refers to scattered light detectable by a commercially available blood analyzer or similar flow cytometer. This scattered light includes, but is not limited to, side-scattered light, forward low-angle scattered light (receiving angle of approximately 0-5 degrees), and forward high-angle scattered light (receiving angle of approximately 5-20 degrees). Scattered light with these angles reflects information about cell size or internal structure and is therefore used as the scattered light in this invention. Forward scattered light is preferred.

[0152] When detecting red blood cells, this application can also use green or blue wavelength lasers emitted by green or blue semiconductor lasers as the detection light source.

[0153] Mature erythrocytes, reticulocytes, and platelets were identified using forward scattering and fluorescence properties. The clusters were then classified and counted to obtain erythrocyte (including mature erythrocytes and reticulocytes) counts, reticulocyte counts, and platelet counts. Forward scattering reflects cell size; platelets are smaller than erythrocytes, resulting in relatively weaker forward scattering. Mature erythrocytes and reticulocytes differ in their RNA content; reticulocytes have a relatively higher RNA content, leading to relatively higher fluorescence intensity.

[0154] Example 1

[0155] Synthesis of dyes

[0156] An exemplary fluorescent dye was synthesized by preparing compound A having the structure shown in formula I, in the following manner.

[0157]

[0158] In this embodiment, X is S, R1 is H, R2 is H, R3 is benzyl, and R4 is benzyl.

[0159] The specific preparation method of compound A is as follows:

[0160] The first step is to prepare 2-methylthiobenzothiazole according to the following reaction formula I (right side of reaction I).

[0161]

[0162] Measure 20 mL of DMF into a container, then add 10 mmol of 2-mercaptobenzothiazole (reaction formula I, left side), 11 mmol of methane, and 12 mmol of sodium carbonate. Stir the mixture for 8 hours under nitrogen protection and at 110 °C, then stop the reaction.

[0163] The resulting mixture was cooled to room temperature and then poured into a large volume of water. It was extracted three times with an appropriate amount of ethyl acetate, and the extracted organic phases were combined. The organic phase was washed twice with distilled water and then dried overnight with anhydrous magnesium sulfate.

[0164] The substance, after being dried overnight, was then purified by column chromatography to obtain approximately 8.5 mmol of an orange-yellow solid powder, which is 2-methylthiobenzothiazole. The yield of reaction I is approximately 85%.

[0165] The second step involves preparing 1-benzyl-4-methylpyridine quaternary ammonium salt according to reaction formula II below (right side of reaction formula II).

[0166]

[0167] Measure 50 mL of toluene into a container, and add 10 mmol of 4-methylpyridine (reaction II, left side) and 12 mmol of benzyl bromide into the container. Reflux and stir under nitrogen protection for 8 hours, then stop the reaction.

[0168] The mixture after the reaction was filtered, and the filter cake was washed three times with 50 mL of toluene to obtain the crude product.

[0169] The crude product was purified by column chromatography to obtain approximately 7.3 mmol of a white solid powder, which was 1-benzyl-4-methylpyridine quaternary ammonium salt. The yield of reaction II was approximately 73%.

[0170] The third step involves preparing 3-benzyl-2-thionone benzothiazole according to reaction formula III below (right side of reaction formula III).

[0171]

[0172] Measure 30 mL of toluene into a container, then add 3 mmol of 2-methylthiobenzothiazole obtained from the first step reaction (reaction III, left side), 4 mmol of benzyl bromide, and 5 mmol of potassium carbonate to the container. Stir the reaction under nitrogen protection at 110 °C for 8 hours.

[0173] The mixture after the reaction was filtered, and the filter cake was washed three times with 50 mL of toluene to obtain the crude product.

[0174] The crude product was purified by column chromatography to obtain approximately 2.1 mmol of white solid powder, which is 3-benzyl-2-thiononebenzothiazole. The yield of reaction III is approximately 70%.

[0175] Step four: Prepare 3-benzyl-2-ethylthiobenzothiazole according to reaction IV below (right side of reaction IV).

[0176]

[0177] Measure 30 mL of dichloromethane into a container, add 2 mmol of 3-benzyl-2-thionone benzothiazole obtained from the third step reaction, and 2 mmol of cynomolgus salt to the container. Reflux and stir for 24 hours under nitrogen protection.

[0178] The product was purified by column chromatography to obtain approximately 1.1 mmol of a white solid powder, which was 3-benzyl-2-ethylthiobenzothiazole. The yield of reaction IV was approximately 55%.

[0179] Step 5: Prepare compound A (right side of reaction V) according to the following reaction formula V.

[0180]

[0181] Measure 30 mL of ethanol into a container, weigh 1 mmol of 1-benzyl-4-methylpyridine quaternary ammonium salt obtained in the second step reaction, and weigh 1 mmol of 3-benzyl-2-ethylthiobenzothiazole obtained in the fourth step reaction. Add both to the container.

[0182] The mixture was refluxed and stirred under nitrogen protection for 24 hours. The product was purified by column chromatography to obtain approximately 0.3 mmol of an orange-yellow solid powder, which was compound A. The yield of reaction V was approximately 30%.

[0183] The results of nuclear magnetic resonance (NMR) testing of compound A are as follows.

[0184] 1H-NMR (500MHz, DMSO, TMS): δ4.00 (s, 3H), 5.59 (s, 2H), 6.39 (s, 1H), 7.26-7.31 (m, 4H), 7.35-7.38 (t , 2H), 7.40-7.42(d, 2H), 7.45-7.48(t, 1H), 7.52-7.54(d, 1H), 7.94-7.96(d, 1H), 8.33-8.35(d, 2H).

[0185] Example 2

[0186] Prepare compound J having the structure shown in chemical formula X.

[0187]

[0188] In this embodiment, X is S, R1 is H, R2 is H, R3 is carboxypentyl, and R4 is methyl.

[0189] The specific preparation method of compound J is as follows:

[0190] First, 10 mL of ethyl acetate was placed in a 50 mL round-bottom flask. Then, 5.37 mmol of 4-methylpyridine and 10.7 mmol of 6-bromohexanoic acid were added to the flask, and the mixture was refluxed at 80 °C for 8 h. The resulting mixture was recrystallized from the ethyl acetate and filtered, with thorough washing using ethyl acetate during filtration. The filtrate was dissolved in methanol and then evaporated to dryness. The final dried product was a yellow oily liquid, which was 1-(5-carboxypentyl)-4-methylpyridine, with a crude yield of approximately 91.07%.

[0191] In the second step, 10 mL of anhydrous tetrahydrofuran was placed in a 50 mL round-bottom flask. 11.84 mmol of 2-mercaptobenzothiazole was then added to the flask, followed by the slow addition of 14.21 mmol of iodomethane under stirring. The reaction was allowed to proceed overnight. A small amount of petroleum ether was then added to the resulting mixture, and the mixture was allowed to stand to precipitate. The mixture was then filtered, and the filtrate was washed with a large amount of petroleum ether. Finally, the filtrate was dried to give a white solid, which was 2-methylthiobenzothiazole, with a crude yield of approximately 98%.

[0192] In the third step, 10 mL of toluene was placed in a 50 mL round-bottom flask, and 10 mmol of 2-methylthiobenzothiazole was added to the flask. Then, under nitrogen protection, 12 mmol of iodomethane was slowly added dropwise to the flask, and the reaction was continued at 110 °C under reflux for 24 h. The resulting mixture was cooled to room temperature, filtered, and the filtrate was washed with dichloromethane. Finally, the filtrate was dried to obtain a pale yellow solid powder, which was 3-methyl-2-methylthiobenzothiazole, with a crude yield of approximately 30%.

[0193] In the fourth step, 5 mL of dichloromethane was added to a 25 mL round-bottom flask. 0.500 mmol of 3-methyl-2-methylthiobenzothiazole and 0.509 mmol of 1-(5-carboxypentyl)-4-methylpyridine were then added to the flask. The mixture was stirred for 10 min at room temperature and in the dark, and then 756 μL of triethylamine was slowly added dropwise. The reaction was continued for approximately 21 h at room temperature and in the dark. The resulting mixture was then evaporated to dryness, and the residue was separated by column chromatography using a mixture of dichloromethane and methanol as the eluent. The separated yellow fraction was collected and evaporated to dryness, yielding a brownish-yellow viscous substance, compound J, with a crude yield of 50%.

[0194] The NMR test of compound J yielded the following results.

[0195] 1 H-NMR (400MHz, DMSO): δ8.36 (s, 1H), 7.90 (d, J=7.6Hz, 1H), 7.58 (d, J=8.0Hz, 1H), 7.51 (t, J=7.4Hz, 1H), 7.40 (s, 1H ), 7.30 (t, J=7.1Hz, 1H), 6.25 (s, 1H), 4.21 (s, 1H), 3.72 (s, 3H), 2.10 (s, 1H), 1.82 (s, 1H), 1.53 (s, 1H), 1.28 (s, 1H).

[0196] Example 3:

[0197] Reagent preparation

[0198] Prepare a reagent system with the following composition.

[0199] A: Fluorescent dye storage solution:

[0200] 50mg of fluorescent dye (with chemical formula I)

[0201] 950ml of ethylene glycol

[0202] 50ml of methanol

[0203] B: Red blood cell lysing solution

[0204]

[0205] C: Spheroidizing reagent solution

[0206]

[0207] 1) Measurement and results of white blood cells in blood samples:

[0208] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0209] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 1) are as follows: Figure 1 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 1 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0210] 2) Measurement and results of red blood cells in blood samples:

[0211] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0212] Specifically, 1 ml of spheroidizing reagent is mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution is added. The mixture is then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer is equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample are measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample 1) are as follows: Figure 2 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 2The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0213] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0214] Using the same blood sample (blood sample number 1), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0215]

[0216]

[0217] As can be seen from the table, the test results of fluorescent dyes with chemical formula I on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0218] Example 4:

[0219] Reagent preparation

[0220] Prepare a reagent system with the following composition.

[0221] A: Fluorescent dye storage solution:

[0222] 50mg of fluorescent dye (with chemical formula II)

[0223] 950ml of ethylene glycol

[0224] 50ml of methanol

[0225] B: Red blood cell lysing solution

[0226]

[0227] C: Spheroidizing reagent solution

[0228]

[0229] 1) Measurement and results of white blood cells in blood samples:

[0230] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0231] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 2) are as follows: Figure 3 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 3 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0232] 2) Measurement and results of red blood cells in blood samples:

[0233] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0234] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample No. 2) are as follows: Figure 4 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 4 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0235] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0236] Using the same blood sample (blood sample number 2), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0237]

[0238]

[0239] As can be seen from the table, the test results of fluorescent dyes with chemical formula II on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0240] Example 5:

[0241] Reagent preparation

[0242] Prepare a reagent system with the following composition.

[0243] A: Fluorescent dye storage solution:

[0244] 50mg of fluorescent dye (with chemical formula III)

[0245] 950ml of ethylene glycol

[0246] 50ml of methanol

[0247] B: Red blood cell lysing solution

[0248]

[0249] C: Spheroidizing reagent solution

[0250]

[0251]

[0252] 1) Measurement and results of white blood cells in blood samples:

[0253] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0254] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample No. 3) are as follows: Figure 5 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 5 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0255] 2) Measurement and results of red blood cells in blood samples:

[0256] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0257] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample No. 3) are as follows: Figure 6 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 6 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0258] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0259] Using the same blood sample (blood sample number 3), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0260]

[0261] As can be seen from the table, the test results of fluorescent dyes with chemical formula III on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0262] Example 6:

[0263] Reagent preparation

[0264] Prepare a reagent system with the following composition.

[0265] A: Fluorescent dye storage solution:

[0266] 100mg of fluorescent dye (with chemical formula IV)

[0267] 950ml of ethylene glycol

[0268] 50ml of methanol

[0269] B: Red blood cell lysing solution

[0270]

[0271] C: Spheroidizing reagent solution

[0272]

[0273]

[0274] 1) Measurement and results of white blood cells in blood samples:

[0275] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0276] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 4) are as follows: Figure 7 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 7 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0277] 2) Measurement and results of red blood cells in blood samples:

[0278] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0279] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 4) are as follows: Figure 8 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 8The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0280] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0281] Using the same blood sample (blood sample number 4), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0282]

[0283] As can be seen from the table, the test results of fluorescent dyes with chemical formula IV on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0284] Example 7:

[0285] Reagent preparation

[0286] Prepare a reagent system with the following composition.

[0287] A: Fluorescent dye storage solution:

[0288] 500mg of fluorescent dye (with chemical formula V)

[0289] 950ml of ethylene glycol

[0290] 50ml of methanol

[0291] B: Red blood cell lysing solution

[0292]

[0293] C: Spheroidizing reagent solution

[0294]

[0295]

[0296] 1) Measurement and results of white blood cells in blood samples:

[0297] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0298] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 5) are as follows: Figure 9 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 9 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0299] 2) Measurement and results of red blood cells in blood samples:

[0300] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0301] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 5) are as follows: Figure 10 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 10 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0302] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0303] Using the same blood sample (blood sample number 5), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0304]

[0305] As can be seen from the table, the test results of fluorescent dyes with chemical formula V on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0306] Example 8:

[0307] Reagent preparation

[0308] Prepare a reagent system with the following composition.

[0309] A: Fluorescent dye storage solution:

[0310] 50mg of fluorescent dye (with chemical formula VI)

[0311] 950ml of ethylene glycol

[0312] 50ml of methanol

[0313] B: Red blood cell lysing solution

[0314]

[0315] C: Spheroidizing reagent solution

[0316]

[0317]

[0318] 1) Measurement and results of white blood cells in blood samples:

[0319] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0320] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 6) are as follows: Figure 11 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 11 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0321] 2) Measurement and results of red blood cells in blood samples:

[0322] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0323] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 6) are as follows: Figure 12 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 12 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0324] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0325] Using the same blood sample (blood sample number 6), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0326]

[0327] As can be seen from the table, the test results of fluorescent dyes with chemical formula VI on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0328] Example 9:

[0329] Reagent preparation

[0330] Prepare a reagent system with the following composition.

[0331] A: Fluorescent dye storage solution:

[0332] 250mg of fluorescent dye (with chemical formula VII)

[0333] 950ml of ethylene glycol

[0334] 50ml of methanol

[0335] B: Red blood cell lysing solution

[0336]

[0337] C: Spheroidizing reagent solution

[0338]

[0339] 1) Measurement and results of white blood cells in blood samples:

[0340] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0341] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 7) are as follows: Figure 13 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 13 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0342] 2) Measurement and results of red blood cells in blood samples:

[0343] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0344] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 7) are as follows: Figure 14 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 14 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0345] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0346] Using the same blood sample (blood sample number 7), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0347]

[0348] As can be seen from the table, the test results of fluorescent dyes with chemical formula VII on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0349] Example 10:

[0350] Reagent preparation

[0351] Prepare a reagent system with the following composition.

[0352] A: Fluorescent dye storage solution:

[0353] 50mg of fluorescent dye (with chemical formula VIII)

[0354] 950ml of ethylene glycol

[0355] 50ml of methanol

[0356] B: Red blood cell lysing solution

[0357]

[0358] C: Spheroidizing reagent solution

[0359]

[0360] 1) Measurement and results of white blood cells in blood samples:

[0361] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0362] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 8) are as follows: Figure 15 As shown, this application can achieve the classification and identification of five subpopulations of white blood cells. Figure 15 The five groups of particles marked in the text are lymphocytes, monocytes, neutrophils, eosinophils, and basophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0363] 2) Measurement and results of red blood cells in blood samples:

[0364] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0365] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 8) are as follows: Figure 16 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 16 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0366] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0367] Using the same blood sample (blood sample number 8), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0368]

[0369] As can be seen from the table, the test results of fluorescent dyes with chemical formula VIII on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0370] Example 11:

[0371] Reagent preparation

[0372] Prepare a reagent system with the following composition.

[0373] A: Fluorescent dye storage solution:

[0374] 50mg of fluorescent dye (with chemical formula IX)

[0375] 950ml of ethylene glycol

[0376] 50ml of methanol

[0377] B: Red blood cell lysing solution

[0378]

[0379]

[0380] C: Spheroidizing reagent solution

[0381]

[0382] 1) Measurement and results of white blood cells in blood samples:

[0383] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0384] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 9) are as follows: Figure 17 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 17 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0385] 2) Measurement and results of red blood cells in blood samples:

[0386] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0387] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 9) are as follows: Figure 18 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 18 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0388] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0389] Using the same blood sample (blood sample number 9), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0390]

[0391] As can be seen from the table, the test results of fluorescent dyes with the chemical formula IX on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0392] Example 12:

[0393] Reagent preparation

[0394] Prepare a reagent system with the following composition.

[0395] A: Fluorescent dye storage solution:

[0396] 50mg of fluorescent dye (with chemical formula X)

[0397] 950ml of ethylene glycol

[0398] 50ml of methanol

[0399] B: Red blood cell lysing solution

[0400]

[0401]

[0402] C: Spheroidizing reagent solution

[0403]

[0404] 1) Measurement and results of white blood cells in blood samples:

[0405] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0406] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 10) are as follows: Figure 19 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 19 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0407] 2) Measurement and results of red blood cells in blood samples:

[0408] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0409] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 10) are as follows: Figure 20 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 20The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0410] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0411] Using the same blood sample (blood sample number 10), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0412]

[0413] As can be seen from the table, the test results of fluorescent dyes with chemical formula X on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0414] Example 13:

[0415] Reagent preparation

[0416] Prepare a reagent system with the following composition.

[0417] A: Fluorescent dye storage solution:

[0418] 50mg of fluorescent dye (with chemical formula XI)

[0419] 950ml of ethylene glycol

[0420] 50ml of methanol

[0421] B: Red blood cell lysing solution

[0422]

[0423]

[0424] C: Spheroidizing reagent solution

[0425]

[0426] 1) Measurement and results of white blood cells in blood samples:

[0427] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0428] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 11) are as follows: Figure 21 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 21 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0429] 2) Measurement and results of red blood cells in blood samples:

[0430] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0431] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 11) are as follows: Figure 22 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 22 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0432] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0433] Using the same blood sample (blood sample number 11), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0434]

[0435] As can be seen from the table, the test results of fluorescent dyes with chemical formula XI on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0436] Example 14:

[0437] Reagent preparation

[0438] Prepare a reagent system with the following composition.

[0439] A: Fluorescent dye storage solution:

[0440] 1000mg of fluorescent dye (with chemical formula XII)

[0441] 950ml of ethylene glycol

[0442] 50ml of methanol

[0443] B: Red blood cell lysing solution

[0444]

[0445]

[0446] C: Spheroidizing reagent solution

[0447]

[0448] 1) Measurement and results of white blood cells in blood samples:

[0449] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0450] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 12) are as follows: Figure 23 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 23 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0451] 2) Measurement and results of red blood cells in blood samples:

[0452] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0453] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 12) are as follows: Figure 24 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 24 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0454] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0455] Using the same blood sample (blood sample number 12), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0456]

[0457] As can be seen from the table, the test results of fluorescent dyes with chemical formula XII on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0458] Example 15:

[0459] Reagent preparation

[0460] Prepare a reagent system with the following composition.

[0461] A: Fluorescent dye storage solution:

[0462] 50mg of fluorescent dye (with chemical formula XIII)

[0463] 950ml of ethylene glycol

[0464] 50ml of methanol

[0465] B: Red blood cell lysing solution

[0466]

[0467] C: Spheroidizing reagent solution

[0468]

[0469] 1) Measurement and results of white blood cells in blood samples:

[0470] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0471] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 13) are as follows: Figure 25 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 25 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0472] 2) Measurement and results of red blood cells in blood samples:

[0473] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0474] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 13) are as follows: Figure 26 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 26The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0475] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0476] Using the same blood sample (blood sample number 13), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0477]

[0478] As can be seen from the table, the test results of fluorescent dyes with chemical formula XIII on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0479] Example 16:

[0480] Reagent preparation

[0481] Prepare a reagent system with the following composition.

[0482] A: Fluorescent dye storage solution:

[0483] 50mg of fluorescent dye (with chemical formula XVIII)

[0484] 950ml of ethylene glycol

[0485] 50ml of methanol

[0486] B: Red blood cell lysing solution

[0487]

[0488] C: Spheroidizing reagent solution

[0489]

[0490] 1) Measurement and results of white blood cells in blood samples:

[0491] The sample testing process was performed automatically in Mindray's independently developed and improved BC-6000 blood analyzer. The sample aspiration volume was set to 20 μL, the injection volume of hemolysin solution B was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0492] Specifically, 1 ml of erythrolysis agent was mixed with 20 μL of blood treated with an anticoagulant, and 20 μL of fluorescent dye stock solution was immediately added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm to measure the side-scattered light intensity and fluorescence intensity of the test sample, obtaining a leukocyte scatter plot. The test results for the blood sample (blood sample number 13) are as follows: Figure 27 As shown, this application can achieve the classification and identification of four subpopulations of white blood cells. Figure 27 The four groups of particles marked in the text are lymphocytes, monocytes, neutrophils, and eosinophils, indicating that the white blood cell classification reagent of this application can effectively classify various white blood cell subgroups.

[0493] 2) Measurement and results of red blood cells in blood samples:

[0494] The sample testing process was carried out automatically in Mindray's self-developed blood analyzer. The sample aspiration volume was set to 4 μL, the injection volume of spheroidizing reagent solution C was 1 ml, the injection volume of fluorescent dye storage solution A was 20 μL, and the blood sample was anticoagulated blood.

[0495] Specifically, 1 ml of spheroidizing reagent was mixed with 20 μL of blood treated with an anticoagulant, and immediately 20 μL of fluorescent dye stock solution was added. The mixture was then incubated at 40°C for 30 seconds to form the test sample. The blood analyzer was equipped with a blue laser with an excitation wavelength of 450 nm. The forward scattered light intensity and fluorescence intensity of the test sample were measured to obtain a scatter plot containing red blood cells. The test results for the blood sample (blood sample number 13) are as follows: Figure 28 As shown, this application can achieve the classification and identification of red blood cells and platelets. Figure 28 The three groups of particles marked in the image are red blood cells (i.e., mature red blood cells), reticulocytes (RET), and platelets, indicating that the red blood cell detection reagent of this application can classify and identify red blood cells and platelets, especially RET.

[0496] 3) Measurement and results of white blood cells and red blood cells on the same blood sample using the Mindray BC-6800 instrument.

[0497] Using the same blood sample (blood sample number 13), the test was performed on the Mindray BC-6800 instrument in CDR mode, using the reagents provided with the BC-6800 instrument. The relevant parameters for white blood cells and red blood cells obtained are shown in the table below:

[0498]

[0499] As can be seen from the table, the test results of the fluorescent dye with chemical formula XVIII on leukocyte clustering and reticulocytes are largely consistent with the test results of BC-6800.

[0500] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many more variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A leukocyte classification reagent, said leukocyte classification reagent comprising a fluorescent dye having formula F: in, X is selected from the group consisting of C(CH3)2, O, S, and Se; R1 and R2 are each independently chosen from H, C1-C 18 The group consisting of alkyl, phenyl, OR6 and halogens; R3 and R4 are each independently selected from C1 to C2. 18 Alkyl, C1-C 18 Carboxyl group, C1-C 18 Hydroxyl group, C1-C 18 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 18 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl; R5 and R6 are each independently selected from H and C1-C. 18 Groups composed of alkyl groups; Y - It is a negative ion; The white blood cell classification reagent also includes a red blood cell lysing agent.

2. The leukocyte differential reagent according to claim 1, characterized in that, X is selected from the group consisting of C(CH3)2 and S.

3. The white blood cell differential reagent according to claim 1, characterized in that, R1 and R2 are each independently selected from H, C1-C 12 The group consisting of alkyl, phenyl, OR6 and halogens.

4. The leukocyte differential reagent according to claim 3, characterized in that, R1 and R2 are each independently selected from the group consisting of H, C1-C6 alkyl, phenyl, OR6 and halogen.

5. The leukocyte differential reagent according to claim 4, characterized in that, R1 is selected from the group consisting of H, C1-C6 alkyl, phenyl, and halogen. R2 is H.

6. The white blood cell differential reagent according to claim 1, characterized in that, R3 and R4 are each independently selected from C1-C 12 Alkyl, C1-C 12 Carboxyl group, C1-C 12 Hydroxyl group, C1-C 12 The group consisting of NR5R6, benzyl, and substituted benzyl groups, wherein the substituents of the substituted benzyl groups are selected from C1-C1. 12 The group consisting of alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 acylamino, halogen and C1-C6 haloalkyl.

7. The white blood cell differential reagent according to claim 6, characterized in that, R3 and R4 are each independently selected from the group consisting of C1-C6 alkyl, C1-C6 carboxyl, C1-C6 hydroxy, C1-C6NR5R6, benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C1-C6 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, halogen and C1-C6 haloalkyl.

8. The white blood cell differential reagent according to claim 7, characterized in that, R3 is selected from the group consisting of C1-C6 alkyl, C1-C6 hydroxy, C1-C6 carboxyl, C1-C6NR5R6 and benzyl; R4 is selected from the group consisting of C1-C6 alkyl, C1-C6 hydroxy, C1-C6 carboxyl and benzyl.

9. The white blood cell differential reagent according to claim 1, characterized in that, R5 and R6 are each independently selected from H and C1-C. 12 A group composed of alkyl groups.

10. The leukocyte differential reagent according to claim 9, characterized in that, R5 and R6 are each independently selected from the group consisting of H and C1-C6 alkyl groups.

11. The leukocyte differential reagent according to claim 10, characterized in that, R5 and R6 are each independently C1-C6 alkyl groups.

12. The leukocyte differential reagent according to claim 1, characterized in that, The Y - Choose free halide anions, ClO4 - PF6 - BF4 - CH3COO - or OTs - A group that is formed.

13. The leukocyte differential reagent according to claim 1, characterized in that, The fluorescent dye comprises at least one structure shown in the following formulas: Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, Formula XIV, Formula XV, Formula XVI, Formula XVII, and Formula XVIII.

14. The leukocyte differential reagent according to any one of claims 1-13, characterized in that, The concentration of the fluorescent dye is 0.1-1000 mg / L.

15. The leukocyte differential reagent according to claim 14, characterized in that... The concentration of the fluorescent dye is 20-500 mg / L.

16. The leukocyte differential reagent according to claim 14, characterized in that, The concentration of the fluorescent dye is 50-200 mg / L.

17. The leukocyte differential reagent according to claim 1, characterized in that, The erythrolysis agent comprises cationic surfactants, nonionic surfactants, anionic surfactants, or any combination thereof, and a buffer to maintain the pH of the assay system between 5 and 11.

18. A white blood cell analysis kit, wherein the white blood cell analysis kit comprises the white blood cell classification reagent according to any one of claims 1-17.

19. A method for classifying white blood cells, the method comprising: A blood sample, a fluorescent dye of formula F, and a erythrolysis agent are mixed to form a test sample; wherein, formula F is... Among them, X, R1, R2, R3, R4 and Y - As defined in any one of claims 1-12; The particles in the test sample are irradiated with light, and at least one light scattering characteristic and at least one fluorescence characteristic of the test sample are measured. Leukocytes are classified and / or counted based on the scattered light and fluorescence characteristics.

20. The method according to claim 19, wherein the fluorescent dye of formula F has the structure shown in at least one of the following formulas: formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, formula XVII, and formula XVIII.

21. The method according to any one of claims 19-20, characterized in that: The light irradiating the particles in the test sample is blue light, green light, or light with an emission wavelength range of 400-560nm.

22. A red blood cell analysis reagent, characterized in that, The red blood cell analysis reagent comprises a fluorescent dye having formula F; wherein, formula F is... Among them, X, R1, R2, R3, R4 and Y - As defined in any one of claims 1-12; The red blood cell analysis reagent also includes a spheroidizing reagent.

23. The red blood cell analysis reagent according to claim 22, characterized in that, The fluorescent dye comprises at least one structure shown in the following formulas: Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, Formula XIV, Formula XV, Formula XVI, Formula XVII, and Formula XVIII.

24. The red blood cell analysis reagent according to claim 22, characterized in that, The spheroidizing agent includes an amphoteric surfactant, an osmotic pressure regulator, and a buffer.

25. The erythrocyte analysis reagent according to any one of claims 22-24, characterized in that, The concentration of the fluorescent dye is 0.1-1000 mg / L.

26. The red blood cell analysis reagent according to claim 25, characterized in that, The concentration of the fluorescent dye is 20-500 mg / L.

27. The red blood cell analysis reagent according to claim 25, characterized in that, The concentration of the fluorescent dye is 50-200 mg / L.

28. A red blood cell analysis kit, said red blood cell analysis kit comprising the red blood cell analysis reagent according to any one of claims 22-27.

29. A method for analyzing red blood cells, comprising: The blood sample, a fluorescent dye with formula F, and a spheroidizing reagent are mixed to form the test sample; Where, equation F is Among them, X, R1, R2, R3, R4 and Y - As defined in any one of claims 1-12; The particles in the test sample are irradiated with light, and at least one light scattering characteristic and at least one fluorescence characteristic of the test sample are measured. Red blood cell classification and / or counting are obtained based on the scattered light characteristics and fluorescence characteristics.

30. The analytical method according to claim 29, characterized in that, The red blood cell classification includes: classifying red blood cells into mature red blood cells and / or reticulocytes; the red blood cell counting includes: counting mature red blood cells and / or reticulocytes.

31. The analytical method according to claim 30, characterized in that, Platelet counts can also be obtained based on the aforementioned light scattering and fluorescence properties.

32. The analytical method according to any one of claims 29-31, characterized in that, The fluorescent dye of formula F has the structure shown in at least one of the following formulas: I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, and XVIII.

33. The method according to any one of claims 29-31, characterized in that: The light irradiating the particles in the test sample is blue light, green light, or light with an emission wavelength range of 400-560nm.

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