Hemolytic agent for leukocyte classification detection and application

Abstract

The invention discloses a hemolytic agent for leukocyte classification detection and application. The hemolytic agent for classified detection of leukocytes comprises a surfactant, organic acid and water, the organic acid is selected from any one or more of aromatic organic acid and derivatives thereof; the surface active agent is formed by compounding a nonionic surface active agent and an anionic surface active agent, or compounding the nonionic surface active agent and a zwitterionic surface active agent, or compounding the nonionic surface active agent and a cationic surface active agent. The hemolytic agent disclosed by the invention can be mixed with AIE fluorescent dye for use, does not generate interference or interaction, can efficiently lyse red blood cells, maximally retains morphological characteristics and internal structures of white blood cells, has high response sensitivity to white blood cell DNA, realizes specific marking of white blood cell nucleuses, and has good application prospects. The treatment efficiency of blood samples in leukocyte classification detection is effectively improved, and the reliability of sample treatment and the accuracy of detection results are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of leukocyte classification detection, and in particular to a hemolytic agent for leukocyte classification detection and application. Background Art

[0002] The main components of peripheral blood are white blood cells, red blood cells and platelets. White blood cells are composed of five types of cells, namely neutrophils, eosinophils, basophils, monocytes and lymphocytes. The proportions and numbers of various white blood cells in the blood fluctuate within a stable range. When a person is sick, the number and proportion of white blood cells will change to varying degrees. Monitoring their changes can provide valuable information for clinical diagnosis. Therefore, in the field of routine blood testing, the accuracy of white blood cell classification is crucial for the rapid diagnosis and precise treatment of diseases.

[0003] At present, the classification and detection of white blood cells in the blood mostly use blood cell analyzers. This detection method requires the use of hemolytic agents and nucleic acid dyes to pre-treat the sample. The role of the hemolytic agent is to dissolve the large number of red blood cells in the blood, exposing the white blood cells for subsequent counting and classification; the nucleic acid dye can enter the cell through the cell membrane of the white blood cells, and emit fluorescence after combining with the nucleic acid substances in the cell. Since the cell size and intracellular structure of each subgroup of white blood cells are different, the fluorescence intensity emitted after the intracellular nucleic acid combines with the fluorescent dye is also different. Therefore, by detecting the scattered light and fluorescent signals of each cell, the white blood cells of different subgroups can be distinguished by using, for example, a scatter plot of two-dimensional detection information, so that classification and counting can be performed.

[0004] Sample processing is a key link, which directly affects the accuracy and reliability of the test results. The traditional blood sample processing method mainly uses a hemolytic agent to mix with the blood sample for a certain period of time, then adds a nucleic acid dye for staining to obtain a sample to be tested, and imports the sample to be tested into a blood analyzer for classification and counting analysis. This step-by-step processing method needs to control the first step of the hemolytic agent and the blood sample mixing processing time (the processing time is too short, the hemolytic agent and the blood sample are not fully reacted, affecting the subsequent staining effect, resulting in inaccurate white blood cell classification and counting; the processing time is too long, which may cause excessive damage to white blood cells, affect the activity of white blood cells, and cause inaccurate white blood cell classification and counting), so this method is relatively cumbersome in actual operation, especially when the number of samples is large, the inspection personnel's processing efficiency is difficult to improve, and the probability of introducing errors due to improper operation may be increased. Chinese patent application CN103460041A discloses a white blood cell classification reagent, which can first mix the hemolytic agent and the staining reagent, and then add it to the blood sample to obtain a sample to be tested, which simplifies the operation steps, can effectively improve the sample processing efficiency and ensure the reliability of sample processing. However, this sample processing method requires that the hemolytic agent and the staining reagent have good compatibility, the system has good stability after mixing and does not affect the hemolytic and staining effects, and it is difficult to apply other existing hemolytic agents and staining reagents.

[0005] Chinese patent CN117233067B discloses an AIE fluorescent dye for leukocyte detection, which has the characteristics of high detection sensitivity, good photostability, low single-use dose, and low fluorescence quenching phenomenon, and has great application advantages in leukocyte classification detection. However, the existing hemolytic agent has low compatibility with the AIE fluorescent dye, and the sample processing still needs to adopt the traditional step-by-step processing method of "first mixing the hemolytic agent with the blood sample for a certain period of time, and then adding nucleic acid dye for staining", which limits the market competitiveness of the AIE fluorescent dye to a certain extent. Therefore, developing a hemolytic agent adapted to the AIE fluorescent dye and simplifying the operating steps of blood sample processing are of great significance for broadening the application of AIE fluorescent dye in the field of leukocyte classification detection. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a hemolytic agent for leukocyte classification detection and its application; the hemolytic agent for leukocyte classification detection of the present invention is highly compatible with AIE fluorescent dyes (especially the AIE fluorescent dyes described in Chinese patent CN117233067B), can be mixed with AIE fluorescent dyes for use, can efficiently lyse red blood cells, while retaining the morphological characteristics and internal structure of leukocytes to the greatest extent, and has a high response sensitivity to leukocyte DNA, realizes specific labeling of leukocyte nuclei, effectively improves the processing efficiency of blood samples in leukocyte classification detection, and ensures the reliability of sample processing, and obtains accurate detection results.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a hemolytic agent for leukocyte classification detection, comprising a surfactant, an organic acid and water;

[0009] The organic acid is selected from any one or more of aromatic organic acids and their derivatives;

[0010] The surfactant is a compound of a nonionic surfactant and an anionic surfactant, or a compound of a nonionic surfactant and a zwitterionic surfactant, or a compound of a nonionic surfactant and a cationic surfactant;

[0011] When the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the total molar concentration of the surfactant is 1-20 mM, wherein the molar ratio of the nonionic surfactant to the anionic surfactant is 0.05-4; the molar concentration of the organic acid is 2-10 mM; and the pH of the hemolytic agent is 6.5-8.0;

[0012] When the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the total molar concentration of the surfactant is 1-10 mM, wherein the molar ratio of the nonionic surfactant to the zwitterionic surfactant is 0.1-1; the molar concentration of the organic acid is 2-10 mM; and the pH of the hemolytic agent is 6.5-8.0;

[0013] When the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the total molar concentration of the surfactant is 1-3.5 mM, wherein the molar ratio of the nonionic surfactant to the cationic surfactant is 0.3-1.5; the molar concentration of the organic acid is 30-50 mM; and the pH of the hemolytic agent is 6.5-8.0.

[0014] Preferably, the organic acid is selected from any one or more of salicylic acid and its salt derivatives, caffeic acid and its salt derivatives, phthalic acid and its salt derivatives;

[0015] Preferably, the nonionic surfactant is selected from any one or more of polyoxyethylene surfactants, alkyl alcohol amide surfactants, and polyethylene glycol surfactants;

[0016] Further preferably, the polyoxyethylene surfactant is selected from any one or more of polyoxyethylene lauryl ether-35 (abbreviated as Brij35), polyoxyethylene (20) oleyl ether (abbreviated as BrijO20), fatty alcohol polyoxyethylene ether-50 (abbreviated as O50), and Tween;

[0017] Further preferably, the alkyl alcohol amide surfactant is selected from any one or more of coconut acid diethanolamide, lauric acid diethanolamide, and myristic acid diethanolamide;

[0018] Further preferably, the polyethylene glycol surfactant is selected from any one or more of polyethylene glycol-4000, polyethylene glycol-6000, and polyethylene glycol-8000;

[0019] Preferably, the anionic surfactant is selected from any one or more of sulfonate surfactants and sulfate surfactants;

[0020] Further preferably, the sulfonate surfactant is selected from any one or more of sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, and sodium fatty alcohol polyoxyethylene ether sulfonate;

[0021] Further preferably, the sulfate surfactant is selected from at least one or more of sodium dodecyl sulfate (abbreviated as SDS), potassium dodecyl sulfate, and sodium polyoxyethylene fatty alcohol sulfate;

[0022] Preferably, the zwitterionic surfactant is selected from any one or more of amino acid surfactants and betaine surfactants;

[0023] Further preferably, the amino acid surfactant is selected from any one or more of sodium cocoyl sarcosinate, sodium N-lauroyl sarcosinate, sodium dodecyl glutamate and sodium palmitoleoyl glutamate;

[0024] Further preferably, the betaine surfactant is selected from any one or more of betaine, cocamidopropyl betaine, and lauramidopropyl betaine;

[0025] Preferably, the cationic surfactant is selected from any one or more of ammonium salt surfactants.

[0026] Further preferably, the ammonium salt surfactant includes any one or more of dodecyltrimethylammonium chloride (LTAC), tetradecyltrimethylammonium chloride (TTAC), hexadecyltrimethylammonium bromide (CTAB), and hydroxyethyl lauryldimethylammonium chloride.

[0027] Preferably, when the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the nonionic surfactant is selected from polyoxyethylene lauryl ether-35 (abbreviated as Brij35), the anionic surfactant is selected from sodium dodecyl sulfate (abbreviated as SDS), and the organic acid is selected from phthalic acid or its salt derivatives;

[0028] Preferably, when the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the nonionic surfactant is selected from fatty alcohol polyoxyethylene ether-50 (abbreviated as O50), the zwitterionic surfactant is selected from sodium N-lauroyl sarcosinate, and the organic acid is selected from phthalic acid or its salt derivatives;

[0029] Preferably, when the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the nonionic surfactant is selected from polyoxyethylene lauryl ether-35 (abbreviated as Brij35), the cationic surfactant is selected from dodecyltrimethylammonium chloride (LTAC); and the organic acid is selected from phthalic acid or its salt derivatives.

[0030] Preferably, when the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the pH of the hemolytic agent is 7.2-7.6;

[0031] Preferably, when the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the pH of the hemolytic agent is 7.2-7.6.

[0032] Preferably, when the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the pH of the hemolytic agent is 7.2-7.6;

[0033] Preferably, when the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the molar ratio of the nonionic surfactant to the cationic surfactant is 0.8-1.2.

[0034] The present invention provides a method for preparing the above-mentioned hemolytic agent for leukocyte classification detection, comprising the following steps:

[0035] The organic acid and the surfactant are completely dissolved in water according to the ratio, and the pH is adjusted to the required value to obtain a hemolytic agent for leukocyte classification detection.

[0036] Preferably, the desired pH is adjusted with hydrochloric acid solution or sodium hydroxide solution.

[0037] The present invention provides a leukocyte classification detection reagent, comprising the above-mentioned hemolytic agent for leukocyte classification detection and AIE leukocyte classification staining solution.

[0038] The present invention provides an application of the above-mentioned hemolytic agent for leukocyte classification detection or the above-mentioned leukocyte classification detection reagent in leukocyte classification and / or counting detection in the field of non-disease diagnosis.

[0039] The present invention provides a leukocyte classification detection method, comprising the following steps:

[0040] The above-mentioned hemolytic agent for leukocyte classification detection is fully mixed with the AIE leukocyte classification staining solution to obtain a mixture solution, and a blood sample is added and fully mixed to obtain a sample test solution, and then a flow cytometer is used to perform leukocyte classification and / or counting detection.

[0041] Preferably, the volume ratio of the hemolytic agent for leukocyte classification detection to the AIE leukocyte classification staining solution is 1000:(1-3);

[0042] Preferably, the concentration of the AIE leukocyte classification staining solution is 5-20 mM;

[0043] Preferably, the ratio of the blood sample to the mixture solution is 10-30 μL:1 mL;

[0044] Preferably, after the blood sample is added to the mixture solution, it is fully mixed for 30-90 seconds.

[0045] Preferably, the AIE leukocyte classification staining solution comprises an AIE fluorescent dye having any one structure of formula (1) to formula (4);

[0046]

[0047] In formula (1), R1 and R2 are each independently selected from one of the following structures:

[0048]

[0049] R3 is selected from one of the following structures:

[0050]

[0051] R8, R9, R 10 , R 12 Each is independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN; n1, n2, n3 are integers greater than or equal to 1;

[0052] R5 is H or one selected from the structures of R1;

[0053] R7 is one of -H, -CN, and -CH3;

[0054] R4 - 、R6 - , R 11 - , R 13 - It is a monovalent anion;

[0055] In formula (2), Ar1 is selected from one of the following structures:

[0056]

[0057] Ar2 is selected from one of the following structures:

[0058]

[0059] R' is the same or different, substituted or unsubstituted linear, branched or cyclic alkyl chain having 1 to 20 carbon atoms;

[0060] X1 and X2 are the same or different, substituted or unsubstituted linear, branched or cyclic alkyl chains having 1 to 20 carbon atoms;

[0061] X3, X4, and X5 are identical or different, substituted or unsubstituted linear, branched, or cyclic alkyl chains having 1 to 20 carbon atoms;

[0062] n is an integer greater than or equal to 1;

[0063] X6 - 、X7 - It is a monovalent anion;

[0064] In formula (3), Q1, Q2, Q3, and Q4 are each independently selected from one of the following structures:

[0065]

[0066] Q5, Q7, Q8 are each independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN;

[0067] n4 is an integer greater than or equal to 1;

[0068] Q6 - It is a monovalent anion;

[0069] In formula (4), K1 and K2 are each independently selected from one of the following structures:

[0070]

[0071] K3, K4, and K6 are each independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, and -CN;

[0072] K5 - It is a monovalent anion;

[0073] n5 is an integer greater than or equal to 1;

[0074] Here, * indicates the substitution position.

[0075] Further preferably, the monovalent anion is selected from F - , Cl - Br - ,I - 、NO2 - 、NO3 - 、BF4 - PF6 - 、SbF6 - CF3SO3 - 、ClO4 - One of them.

[0076] Further preferably, n, n1, n2, n3, n4, n5 are integers of 1-20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20);

[0077] More preferably, the substituent in the substituted or unsubstituted linear, branched or cyclic alkyl chain having 1 to 20 carbon atoms is halogen, hydroxyl, carboxyl, amino, aldehyde or cyano.

[0078] Further preferably, the structural formula of the AIE molecule is one of Chemical Formula I to Chemical Formula V;

[0079]

[0080] R4 - 、X6 - 、X7 - 、Q6 - 、K5 - It is a monovalent anion.

[0081] Further preferably, the structural formula of the AIE molecule is one of the following chemical formulas I to V;

[0082]

[0083] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0084] The hemolytic agent of the present invention is highly compatible with AIE fluorescent dyes (especially the AIE fluorescent dyes described in Chinese patent CN117233067B), can be mixed with AIE fluorescent dyes for use, and will not cause interference or interaction during the mixed use process. It can efficiently lyse red blood cells while retaining the morphological characteristics and internal structure of white blood cells to the greatest extent, and has a high response sensitivity to white blood cell DNA, achieving specific labeling of white blood cell nuclei, effectively improving the processing efficiency of blood samples in white blood cell classification detection, and ensuring the reliability of sample processing and the accuracy of detection results.

[0085] When the hemolytic agent of the present invention is used for leukocyte classification detection, it can be first mixed with the AIE fluorescent dye, and then added to the blood sample to prepare the test sample, which greatly simplifies the operation steps and significantly shortens the sample processing time. When the number of samples is large, it is easy to implement programmed processing, effectively improve the efficiency of leukocyte detection, and reduce the difficulty and cost of operation. The development of the hemolytic agent of the present invention is conducive to broadening the application of AIE fluorescent dye in the field of leukocyte classification detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 It is a diagram of leukocyte fluorescence imaging effects after the samples are treated with the hemolytic agent of Example 1-1, Example 2-1, Example 3-1, Comparative Example 1-1, and Comparative Example 4 and the AIE leukocyte classification staining solution.

[0087] Figures 2 to 4 It is a diagram showing the leukocyte classification effect after the samples are treated with the hemolytic agent and AIE leukocyte classification staining solution of the embodiment and the comparative example. DETAILED DESCRIPTION

[0088] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

[0089] The method for preparing the hemolytic agent in the following examples and comparative examples is as follows:

[0090] Weigh the organic acid and surfactant according to the ratio, dissolve them completely in water, and then adjust the pH to the required value with hydrochloric acid solution or sodium hydroxide solution.

[0091] Embodiment 1:

[0092] Prepare the hemolytic agent according to Table 1.

[0093] Take 1 μL of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 mL of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μL of fresh EDTA-K2 anticoagulated blood, add 1 mL of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0094] Table 1

[0095]

[0096] Embodiment 2:

[0097] Prepare the hemolytic agent according to Table 2.

[0098] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 mL of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0099] Table 2

[0100]

[0101] Embodiment 3:

[0102] Prepare the hemolytic agent according to Table 3.

[0103] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 mL of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0104] Table 3

[0105]

[0106] Comparative Example 1:

[0107] The hemolytic agent of the example was prepared according to Table 4.

[0108] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 ml of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0109] Table 4

[0110]

[0111] Comparative Example 2:

[0112] The hemolytic agent of the example was prepared according to Table 5 and Table 6.

[0113] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 ml of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0114] Table 5

[0115]

[0116]

[0117] Table 6

[0118]

[0119] Comparative Example 3:

[0120] The hemolytic agent of the example was prepared according to Table 7 and Table 8.

[0121] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO, add 1 ml of hemolytic agent, mix thoroughly to obtain a hemolytic agent mixture containing a staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolytic agent mixture containing a staining solution, mix thoroughly for 30-60 seconds, and obtain a sample test solution.

[0122] Table 7

[0123]

[0124] Table 8

[0125]

[0126]

[0127] Comparative Example 4:

[0128] Prepare a commercial hemolytic agent (RM-68LD hemolytic agent purchased from Jiangsu Rongsheng Jiamei Biological Reagents for blood cell analysis).

[0129] Take 1 μl of 10 mM AIE leukocyte classification staining solution (AIE molecule is The solvent is anhydrous DMSO), 1 ml of commercial hemolytic agent is added, and the mixture is fully mixed to obtain a hemolytic agent mixture containing a staining solution; 20 μl of fresh EDTA-K2 anticoagulated blood is added, 1 ml of the commercial hemolytic agent mixture containing a staining solution is added, and the mixture is fully mixed for 30-60 seconds to obtain a sample test solution.

[0130] Performance Test:

[0131] The morphological characteristics of leukocytes were observed by fluorescence microscopy on the sample test solutions of the embodiments and comparative examples. The fluorescence imaging effect diagrams of leukocytes of the sample test solutions of the embodiments 1-1, 2-1, 3-1, comparative example 1-1 and 4 are shown in FIG. Figure 1At the same time, the side scattered light intensity and fluorescence intensity of the leukocytes are detected to obtain a leukocyte classification scatter plot. The leukocyte classification effect diagram of the sample test solution of the embodiment is shown in FIG. Figure 2 The white blood cell classification effect diagram of the sample test solution of the comparative example is as follows: Figure 3 and Figure 4 The separation degree of the center of the particle group of neutrophils and lymphocytes and lymphocytes and monocytes was calculated according to the scatter plot, so as to evaluate the separation effect of the reagent on neutrophils and lymphocytes and lymphocytes and monocytes. The results are shown in Table 9.

[0132] Table 9 Separation effect of neutrophils and lymphocytes and lymphocytes and monocytes in the SSC direction of the embodiments and comparative examples

[0133]

[0134]

[0135] Data Analysis:

[0136] Figure 1 The results show that the hemolytic agents of Examples 1-1, 2-1, and 3-1 of the present invention have high efficiency in lysing red blood cells and less damage to the morphology of white blood cells, while the commercial hemolytic agent of Comparative Example 4 has greater damage to the morphology of white blood cells;

[0137] Figure 2 The results show that the hemolytic agent of the embodiment of the present invention combined with the flow cytometer and the AIE leukocyte classification staining solution can classify leukocytes into four particle groups: lymphocytes, monocytes, neutrophils, and eosinophils;

[0138] Figure 3 and Figure 4 The results showed that the hemolytic agent combined with the AIE leukocyte classification staining solution in the control ratio had a poor classification effect on leukocytes on the flow cytometer and could not significantly distinguish the leukocyte subpopulations.

[0139] The results in Table 9 show that the hemolytic agent of the embodiment of the present invention combined with the flow cytometer and the AIE leukocyte classification staining solution can effectively distinguish between neutrophil granule scatter point groups and lymphocyte granule scatter point groups and between lymphocyte granule scatter point groups and monocyte granule scatter point groups, while the comparison example hemolytic agent has a poor distinction effect and is not suitable for the AIE leukocyte classification staining solution.

Claims

1. A hemolytic agent for leukocyte classification detection, characterized in that: including surfactant, organic acid and water; The organic acid is selected from any one or more of aromatic organic acids and their derivatives; The surfactant is a compound of a nonionic surfactant and an anionic surfactant, or a compound of a nonionic surfactant and a zwitterionic surfactant, or a compound of a nonionic surfactant and a cationic surfactant; When the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the total molar concentration of the surfactant is 1-20 mM, wherein the molar ratio of the nonionic surfactant to the anionic surfactant is 0.05-4; the molar concentration of the organic acid is 2-10 mM; and the pH of the hemolytic agent is 6.5-8.0; When the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the total molar concentration of the surfactant is 1-10 mM, wherein the molar ratio of the nonionic surfactant to the zwitterionic surfactant is 0.1-1; the molar concentration of the organic acid is 2-10 mM; and the pH of the hemolytic agent is 6.5-8.0; When the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the total molar concentration of the surfactant is 1-3.5 mM, wherein the molar ratio of the nonionic surfactant to the cationic surfactant is 0.3-1.5; the molar concentration of the organic acid is 30-50 mM; and the pH of the hemolytic agent is 6.5-8.

0.

2. The hemolytic agent for leukocyte classification detection according to claim 1, characterized in that: The organic acid is selected from any one or more of salicylic acid and its salt derivatives, caffeic acid and its salt derivatives, phthalic acid and its salt derivatives; The nonionic surfactant is selected from any one or more of polyoxyethylene surfactants, alkyl alcohol amide surfactants, and polyethylene glycol surfactants; The anionic surfactant is selected from any one or more of sulfonate surfactants and sulfate surfactants; The zwitterionic surfactant is selected from any one or more of amino acid surfactants and betaine surfactants; The cationic surfactant is selected from any one or more of ammonium salt surfactants.

3. The hemolytic agent for leukocyte classification detection according to claim 2, characterized in that: The polyoxyethylene surfactant is selected from any one or more of polyoxyethylene lauryl ether-35, polyoxyethylene (20) oleyl ether, fatty alcohol polyoxyethylene ether-50, and Tween; The alkyl alcohol amide surfactant is selected from any one or more of coconut acid diethanolamide, lauric acid diethanolamide, and myristic acid diethanolamide; The polyethylene glycol surfactant is selected from any one or more of polyethylene glycol-4000, polyethylene glycol-6000, and polyethylene glycol-8000; The sulfonate surfactant is selected from any one or more of sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, and sodium fatty alcohol polyoxyethylene ether sulfonate; The sulfate surfactant is selected from at least one or more of sodium lauryl sulfate, potassium lauryl sulfate, and sodium polyoxyethylene fatty alcohol sulfate; The amino acid surfactant is selected from any one or more of sodium cocoyl sarcosinate, sodium N-lauroyl sarcosinate, sodium dodecyl glutamate and sodium palmitoleyl glutamate; The betaine surfactant is selected from any one or more of betaine, cocamidopropyl betaine, and lauramidopropyl betaine; The ammonium salt surfactant includes any one or more of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, and hydroxyethyl lauryldimethylammonium chloride.

4. The hemolytic agent for leukocyte classification detection according to claim 1, characterized in that: When the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the nonionic surfactant is selected from polyoxyethylene lauryl ether-35, the anionic surfactant is selected from sodium lauryl sulfate, and the organic acid is selected from phthalic acid or its salt derivatives; When the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the nonionic surfactant is selected from fatty alcohol polyoxyethylene ether-50, the zwitterionic surfactant is selected from sodium N-lauroyl sarcosinate, and the organic acid is selected from phthalic acid or its salt derivatives; When the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the nonionic surfactant is selected from polyoxyethylene lauryl ether-35, the cationic surfactant is selected from dodecyltrimethylammonium chloride; and the organic acid is selected from phthalic acid or its salt derivatives.

5. The hemolytic agent for leukocyte classification detection according to claim 1, characterized in that: When the surfactant is a compound of a nonionic surfactant and an anionic surfactant, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a compound of a nonionic surfactant and a zwitterionic surfactant, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a compound of a nonionic surfactant and a cationic surfactant, the molar ratio of the nonionic surfactant to the cationic surfactant is 0.8-1.

2.

6. The method for preparing the hemolytic agent for leukocyte classification detection according to any one of claims 1 to 5, characterized in that: The following steps are involved: The organic acid and the surfactant are completely dissolved in water according to the ratio, and the pH is adjusted to the required value to obtain a hemolytic agent for leukocyte classification detection.

7. A leukocyte classification detection reagent, characterized in that: It comprises the hemolytic agent for leukocyte classification detection and the AIE leukocyte classification staining solution as described in any one of claims 1 to 5.

8. Use of the hemolytic agent for leukocyte classification detection according to any one of claims 1 to 5 or the leukocyte classification detection reagent according to claim 7 in leukocyte classification and / or counting detection in the field of non-disease diagnosis.

9. A method for detecting leukocyte classification, characterized in that: The following steps are involved: The hemolytic agent for leukocyte classification detection according to any one of claims 1 to 5 is fully mixed with the AIE leukocyte classification staining solution to obtain a mixture solution, a blood sample is added and fully mixed to obtain a sample test solution, and then leukocyte classification and / or counting detection is performed using a flow cytometer.

10. The method for detecting leukocyte classification according to claim 9, characterized in that: The volume ratio of the hemolytic agent for leukocyte classification detection to the AIE leukocyte classification staining solution is 1000:(1-3); The concentration of the AIE leukocyte classification staining solution is 5-20 mM; The ratio of the blood sample to the mixture solution is 10-30 μL:1 mL; After the blood sample is added to the mixture solution, it is fully mixed for 30-90 seconds; The AIE leukocyte classification staining solution comprises an AIE fluorescent dye having any one structure of formula (1) to formula (4); In formula (1), R1 and R2 are each independently selected from one of the following structures: R3 is selected from one of the following structures: R8, R9, R 10 , R 12 Each is independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN; n1, n2, n3 are integers greater than or equal to 1; R5 is H or one selected from the structures of R1; R7 is one of -H, -CN, and -CH3; R4 - 、R6 - , R 11 - , R 13 - It is a monovalent anion; In formula (2), Ar1 is selected from one of the following structures: Ar2 is selected from one of the following structures: R' is the same or different, substituted or unsubstituted linear, branched or cyclic alkyl chain having 1 to 20 carbon atoms; X1 and X2 are the same or different, substituted or unsubstituted linear, branched or cyclic alkyl chains having 1 to 20 carbon atoms; X3, X4, and X5 are identical or different, substituted or unsubstituted linear, branched, or cyclic alkyl chains having 1 to 20 carbon atoms; n is an integer greater than or equal to 1; X6 - 、X7 - It is a monovalent anion; In formula (3), Q1, Q2, Q3, and Q4 are each independently selected from one of the following structures: Q5, Q7, Q8 are each independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN; n4 is an integer greater than or equal to 1; Q6 - It is a monovalent anion; In formula (4), K1 and K2 are each independently selected from one of the following structures: K3, K4, and K6 are each independently selected from one of -H, -CH3, -COOH, -OH, -NH2, -CHO, and -CN; K5 - It is a monovalent anion; n5 is an integer greater than or equal to 1; Here, * indicates the substitution position.

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