A hemolytic agent for leukocyte differential detection and application thereof

Abstract

The application discloses a hemolytic agent for white blood cell differential detection and application; the hemolytic agent for white blood cell differential detection comprises a surfactant, an organic acid and water; the organic acid is selected from any one or more of aromatic organic acids and derivatives thereof; the surfactant is a complex of non-ionic surfactant and anionic surfactant, or a complex of non-ionic surfactant and zwitterionic surfactant, or a complex of non-ionic surfactant and cationic surfactant. The hemolytic agent can be mixed with AIE fluorescent dye, does not interfere or interact, can efficiently lyse red blood cells, can maximize the morphological characteristics and internal structure of white blood cells, has high response sensitivity to white blood cell DNA, realizes specific labeling of white blood cell nuclei, effectively improves the processing efficiency of blood samples in white blood cell differential detection, and guarantees the reliability of sample processing and the accuracy of detection results.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of leukocyte differential detection, and particularly relates to a hemolytic agent for leukocyte differential detection and application. BACKGROUND

[0002] The main components of peripheral blood are leukocytes, erythrocytes and platelets, and leukocytes are composed of five kinds of cells, namely neutrophils, eosinophils, basophils, monocytes and lymphocytes. The proportion and quantity of various leukocytes in blood fluctuate within a stable range, and the quantity and proportion of leukocytes change to different degrees when a person is ill, and monitoring the change can provide valuable information for clinical diagnosis. Therefore, in the field of blood routine detection, the accuracy of leukocyte differential is crucial for rapid and accurate diagnosis and treatment of diseases.

[0003] At present, the classification detection of leukocytes in blood mostly adopts a blood cell analyzer, and the detection method requires the use of a hemolytic agent and a nucleic acid dye for pretreatment of the sample. The function of the hemolytic agent is to dissolve a large number of erythrocytes in blood, expose leukocytes, and facilitate subsequent counting and classification; the nucleic acid dye can enter the cell interior through the cell membrane of leukocytes, and emit fluorescence after combining with intracellular nucleic acid substances. Due to the differences in cell size and intracellular structure of each subpopulation of leukocytes, the fluorescence intensity emitted after the intracellular nucleic acid combines with the fluorescent dye is also different, and thus the scattered light and fluorescence signal of each cell can be detected, and the leukocytes of different subpopulations can be distinguished by using, for example, a scatter plot of two-dimensional detection information, so as to perform classification counting.

[0004] Sample processing is a key link, which directly affects the accuracy and reliability of the detection result. The traditional blood sample processing method mainly adopts the following steps: first, mixing the hemolytic agent with the blood sample for a certain time, then adding the nucleic acid dye for staining to prepare the test sample, and finally introducing the test sample into the blood analyzer for classification and counting analysis. This step-by-step processing method needs to control the mixing time of the hemolytic agent and the blood sample in the first step (if the mixing time is too short, the hemolytic agent and the blood sample do not react sufficiently, which affects the subsequent staining effect, resulting in inaccurate white blood cell classification and counting; if the mixing time is too long, the white blood cells may be excessively damaged, affecting the activity of the white blood cells, which also results in inaccurate white blood cell classification and counting). Therefore, this method is relatively cumbersome in actual operation, especially when the number of samples is large, the processing efficiency of the laboratory personnel 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 mix the hemolytic agent and the staining reagent first, and then add the blood sample to prepare the test sample. This method simplifies the operation steps, effectively improves the sample processing efficiency, and ensures the reliability of sample processing. However, this sample processing method requires that the hemolytic agent and the staining reagent have good adaptability, the system after mixing has good stability, and does not affect the hemolysis and staining effects, which is difficult to apply to other existing hemolytic agents and staining reagents.

[0005] Chinese Patent CN117233067B discloses an AIE fluorescent dye for white blood cell detection, which has the characteristics of high detection sensitivity, good light stability, low single-use dosage, and low probability of fluorescence quenching phenomenon, and has great application advantages in white blood cell classification detection. However, the existing hemolytic agent has low adaptability with the AIE fluorescent dye, and the traditional step-by-step processing method of "first mixing the hemolytic agent with the blood sample for a certain time, and then adding the nucleic acid dye for staining" is still needed for sample processing, which to some extent limits the market competitiveness of the AIE fluorescent dye. Therefore, it is of great significance to develop a hemolytic agent suitable for the AIE fluorescent dye and simplify the operation steps of blood sample processing, in order to broaden the application of the AIE fluorescent dye in the field of white blood cell classification detection. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a hemolytic agent for white blood cell classification detection and application. The hemolytic agent for white blood cell classification detection of the present application has high adaptability with the AIE fluorescent dye (especially the AIE fluorescent dye described in Chinese Patent CN117233067B), can be used in combination with the AIE fluorescent dye, can efficiently lyse red blood cells, and at the same time, can maximize the preservation of the morphological characteristics and internal structure of white blood cells, has high response sensitivity to white blood cell DNA, realizes specific labeling of white blood cell nuclei, effectively improves the processing efficiency of blood samples in white blood cell classification detection, ensures the reliability of sample processing, and obtains accurate detection results.

[0007] The technical scheme of the present application is as follows:

[0008] The present application provides a hemolytic agent for white blood cell differential 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 derivatives thereof;

[0010] The surfactant is a combination of non-ionic surfactant and anionic surfactant, or a combination of non-ionic surfactant and zwitterionic surfactant, or a combination of non-ionic surfactant and cationic surfactant;

[0011] When the surfactant is a combination of non-ionic surfactant and anionic surfactant, the total molar concentration of the surfactant is 1-20 mM, wherein the molar ratio of non-ionic surfactant to 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 combination of non-ionic surfactant and zwitterionic surfactant, the total molar concentration of the surfactant is 1-10 mM, wherein the molar ratio of non-ionic surfactant to 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 combination of non-ionic surfactant and cationic surfactant, the total molar concentration of the surfactant is 1-3.5 mM, wherein the molar ratio of non-ionic surfactant to 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 salt derivatives thereof, caffeic acid and salt derivatives thereof, and phthalic acid and salt derivatives thereof;

[0015] Preferably, the non-ionic 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 Brij 35), polyoxyethylene (20) oil ether (abbreviated as Brij O20), fatty alcohol polyoxyethylene ether-50 (abbreviated as O50), and Tween;

[0017] Further preferably, the alkylolamide surfactant is selected from any one or more of cocodiethanolamide, lauric diethanolamide, myristyl diethanolamide;

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

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

[0020] Further preferably, the sulfonate surfactant is selected from any one or more of sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, 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 SDS), potassium dodecyl sulfate, sodium fatty alcohol polyoxyethylene ether sulfate;

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

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

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

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

[0026] Further preferably, the ammonium salt surfactant includes any one or more of dodecyl trimethyl ammonium chloride (LTAC), tetradecyl trimethyl ammonium chloride (TTAC), cetyl trimethyl ammonium bromide (CTAB), hydroxyethyl lauryl dimethyl ammonium chloride.

[0027] Preferably, when the surfactant is a combination of non-ionic surfactant and anionic surfactant, the non-ionic surfactant is selected from polyoxyethylene lauryl ether-35 (abbreviated Brij35), the anionic surfactant is selected from sodium dodecyl sulfate (abbreviated SDS), and the organic acid is selected from phthalic acid or its salt derivative;

[0028] Preferably, when the surfactant is a mixture of non-ionic surfactant and zwitterionic surfactant, the non-ionic surfactant is selected from fatty alcohol polyoxyethylene ether-50 (abbreviated as O50), the zwitterionic surfactant is selected from sodium N-lauroyl glutamate, and the organic acid is selected from phthalic acid or a salt derivative thereof.

[0029] Preferably, when the surfactant is a mixture of non-ionic surfactant and cationic surfactant, the non-ionic 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 a salt derivative thereof.

[0030] Preferably, when the surfactant is a mixture of non-ionic surfactant and anionic surfactant, the pH of the hemolytic agent is 7.2-7.6.

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

[0032] Preferably, when the surfactant is a mixture of non-ionic surfactant and cationic surfactant, the pH of the hemolytic agent is 7.2-7.6.

[0033] Preferably, when the surfactant is a mixture of non-ionic surfactant and cationic surfactant, the molar ratio of the non-ionic surfactant and the cationic surfactant is 0.8-1.2.

[0034] The present application provides a preparation method of the above-mentioned hemolytic agent for white blood cell differential detection, comprising the following steps:

[0035] The organic acid and the surfactant are completely dissolved in water according to the ratio, and adjusted to the required pH to obtain the hemolytic agent for white blood cell differential detection.

[0036] Preferably, the required pH is adjusted by using a hydrochloric acid solution or a sodium hydroxide solution.

[0037] The present application provides a white blood cell differential detection reagent, comprising the above-mentioned hemolytic agent for white blood cell differential detection and an AIE white blood cell differential staining solution.

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

[0039] The present application provides a white blood cell differential detection method, comprising the following steps:

[0040] The leukocyte differential detection hemolytic agent and the AIE leukocyte differential staining solution are mixed to obtain a mixture solution, the blood sample is added and mixed to obtain a sample to be tested, and then the leukocyte differential detection and / or counting are performed by using a flow cytometer.

[0041] Preferably, the leukocyte differential detection hemolytic agent and the AIE leukocyte differential staining solution are mixed in a volume ratio of 1000: (1-3).

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

[0043] Preferably, the blood sample and the mixture solution are mixed in a volume ratio of 10-30 μL: 1 mL.

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

[0045] Preferably, the AIE leukocyte differential staining solution comprises an AIE fluorescent dye having any one of the structures 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 are each independently selected from -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN; n1, n2, n3 are integers greater than or equal to 1.

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

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

[0054] R4 - , R6 - , R 11 - , R 13 - 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 1-20 carbon atoms;

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

[0061] X3, X4, X5 are the same or different, substituted or unsubstituted, linear, branched or cyclic alkyl chain having 1-20 carbon atoms;

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

[0063] X6 - , X7 - is a monovalent anion;

[0064] In formula (3), Q1, Q2, Q3, 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 - is a monovalent anion;

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

[0070]

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

[0072] K5 - is a monovalent anion;

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

[0074] wherein * indicates the substitution position.

[0075] Further preferably, the monovalent anion is selected from one of F - - - - - - - - - - -

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

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

[0078] Further preferably, the structure formula of the AIE molecule is one of the following chemical formula I-V:

[0079]

[0080] R4 - - - - - is a monovalent anion.

[0081] Further preferably, the structure formula of the AIE molecule is one of the following chemical formula I-V:

[0082]

[0083] Compared with the prior art, the application has the following advantages and benefits:

[0084] The hemolytic agent of the application is highly compatible with the AIE fluorescent dye (especially the AIE fluorescent dye described in Chinese patent CN117233067B), can be used in mixture with the AIE fluorescent dye, does not produce interference or interaction during the mixed use, can efficiently lyse red blood cells, maximally retains the morphological characteristics and internal structure of white blood cells, has high response sensitivity to white blood cell DNA, realizes white blood cell nucleus-specific labeling, effectively improves the processing efficiency of blood samples in white blood cell classification detection, and guarantees the reliability of sample processing and the accuracy of detection results. ​​​​​​​​​​​​​​​

[0085] The hemolytic agent of the present application can be mixed with the AIE fluorescent dye first, and then added to the blood sample to prepare the sample to be tested, which greatly simplifies the operation steps, significantly shortens the sample processing time, and is easy to realize programmed processing when the number of samples is large, effectively improves the white blood cell detection efficiency, and reduces the operation difficulty and cost. The development of the hemolytic agent of the present application is beneficial to broaden the application of AIE fluorescent dye in the field of white blood cell differential detection. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 is the white blood cell fluorescence imaging effect diagram after the sample is treated by the hemolytic agent and AIE white blood cell differential staining solution of Example 1-1, Example 2-1, Example 3-1, Comparative Example 1-1 and Comparative Example 4.

[0087] Figures 2 to 4 is the white blood cell differential effect diagram after the sample is treated by the hemolytic agent and AIE white blood cell differential staining solution of the examples and comparative examples. DETAILED DESCRIPTION

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

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

[0090] The organic acid and the surfactant are weighed according to the ratio, and then completely dissolved in water, and then adjusted to the required pH with hydrochloric acid solution or sodium hydroxide solution.

[0091] Example 1:

[0092] The hemolytic agent was prepared according to Table 1.

[0093] 1 μL of 10 mM AIE white blood cell differential staining solution (AIE molecule is The solvent is anhydrous DMSO) was added to 1 mL of hemolytic agent, and mixed thoroughly to obtain a hemolytic agent mixture containing the staining solution; 20 μL of fresh EDTA-K2 anticoagulated blood was added to 1 mL of the hemolytic agent mixture containing the staining solution, and mixed thoroughly for 30-60 seconds to obtain the sample to be tested.

[0094] Table 1

[0095]

[0096] Example 2:

[0097] The hemolytic agent was prepared according to Table 2.

[0098] 1 μL of 10 mM AIE white blood cell differential staining solution (AIE molecule is 1 mL of the hemolytic agent, mixed well, to obtain a hemolytic agent mixture containing the staining solution; 20 μΐ of fresh EDTA-K2 anticoagulated blood was taken, added to 1 ml of the hemolytic agent mixture containing the staining solution, mixed well for 30-60 seconds, to obtain the sample to be tested.

[0099] Table 2

[0100]

[0101] Example 3:

[0102] The hemolytic agent was prepared according to Table 3.

[0103] 1 μΐ of 10 mM AIE leukocyte differential staining solution (AIE molecule is 1 mL of the hemolytic agent, mixed well, to obtain a hemolytic agent mixture containing the staining solution; 20 μΐ of fresh EDTA-K2 anticoagulated blood was taken, added to 1 ml of the hemolytic agent mixture containing the staining solution, mixed well for 30-60 seconds, to obtain the sample to be tested.

[0104] Table 3

[0105]

[0106] Comparative Example 1:

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

[0108] 1 μΐ of 10 mM AIE leukocyte differential staining solution (AIE molecule is 1 mL of the hemolytic agent, mixed well, to obtain a hemolytic agent mixture containing the staining solution; 20 μΐ of fresh EDTA-K2 anticoagulated blood was taken, added to 1 ml of the hemolytic agent mixture containing the staining solution, mixed well for 30-60 seconds, to obtain the sample to be tested.

[0109] Table 4

[0110]

[0111] Comparative Example 2:

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

[0113] 1 μΐ of 10 mM AIE leukocyte differential staining solution (AIE molecule is 1 mL of the hemolytic agent, mixed well, to obtain a hemolytic agent mixture containing the staining solution; 20 μΐ of fresh EDTA-K2 anticoagulated blood was taken, added to 1 ml of the hemolytic agent mixture containing the staining solution, mixed well for 30-60 seconds, to obtain the sample to be tested.

[0114] Table 5

[0115]

[0116]

[0117] Table 6

[0118]

[0119] Comparative Example 3:

[0120] Hemolytic agents were prepared according to the examples in Tables 7 and 8.

[0121] Take 1 μl of 10 mM AIE leukocyte differential staining solution (AIE molecular weight is...). Add 1 ml of hemolysin (in anhydrous DMSO) to the solvent and mix thoroughly to obtain a hemolysin mixture containing the staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of the hemolysin mixture containing the staining solution, and mix thoroughly for 30-60 seconds to obtain the sample test solution.

[0122] Table 7

[0123]

[0124] Table 8

[0125]

[0126]

[0127] Comparative Example 4:

[0128] Prepare a commercially available hemolytic agent (purchased from Jiangsu Rongsheng Jiamei Biological Reagent Blood Cell Analysis Hemolytic Agent RM-68LD).

[0129] Take 1 μl of 10 mM AIE leukocyte differential staining solution (AIE molecular weight is...). Add 1 ml of commercially available hemolysin (in anhydrous DMSO) to the solvent and mix thoroughly to obtain a hemolysin mixture containing the staining solution; take 20 μl of fresh EDTA-K2 anticoagulated blood, add 1 ml of commercially available hemolysin mixture containing the staining solution, and mix thoroughly for 30-60 seconds to obtain the sample test solution.

[0130] Performance testing:

[0131] The morphological characteristics of leukocytes in the sample test solutions of the examples and comparative examples were observed using a fluorescence microscope. The fluorescence imaging results of leukocytes in the sample test solutions of Examples 1-1, 2-1, 3-1, Comparative Example 1-1, and Comparative Example 4 are shown in the figure below. Figure 1Meanwhile, the white blood cell classification scatter diagram is obtained by detecting the side scattering light intensity and the fluorescence intensity of the white blood cells, and the white blood cell classification effect diagram of the sample to be tested of the sample of the embodiment is as shown in Figure 2 The white blood cell classification effect diagram of the sample to be tested of the sample of the comparative example is as shown in Figure 3 and Figure 4 The separation degree of the granular clusters of the neutrophil granulocytes and the lymphocytes and the lymphocytes and the monocyte granulocytes is calculated according to the scatter diagram, so as to evaluate the separation effect of the reagent on the neutrophil granulocytes and the lymphocytes and the monocyte granulocytes, and the results are shown in Table 9.

[0132] Table 9: Separation effect of the neutrophil granulocytes and the lymphocytes and the lymphocytes and the monocyte granulocytes in the SSC direction of the sample of the embodiment and the comparative example

[0133]

[0134]

[0135] Data analysis:

[0136] Figure 1 The results show that the hemolytic agents of the embodiments 1-1, 2-1 and 3-1 have higher erythrocyte lysis efficiency and smaller morphological damage to the white blood cells, and the commercial hemolytic agent of the comparative example 4 has larger morphological damage to the white blood cells;

[0137] Figure 2 The results show that the hemolytic agent of the embodiment of the present application can combine the flow cytometer and the AIE white blood cell classification staining solution to divide the white blood cells into four granular clusters of the lymphocytes, the monocytes, the neutrophil granulocytes and the eosinophil granulocytes;

[0138] Figure 3 and Figure 4 The results show that the hemolytic agent of the comparative example combined with the AIE white blood cell classification staining solution has poor classification effect on the white blood cells on the flow cytometer, and cannot significantly distinguish the subgroups of the white blood cells;

[0139] The results in Table 9 show that the hemolytic agent of the embodiment of the present application combined with the flow cytometer and the AIE white blood cell classification staining solution can effectively distinguish the neutrophil granulocyte granular scatter cluster and the lymphocyte granular scatter cluster and the lymphocyte granular scatter cluster and the monocyte granular scatter cluster, and the hemolytic agent of the comparative example has poor distinguishing effect and is not suitable for the AIE white blood cell classification staining solution.

Claims

1. A hemolytic agent for white blood cell differential detection, characterized in that, Including surfactants, organic acids, and water; The organic acid is selected from any one or more aromatic organic acids and their derivatives; The surfactant is a mixture of nonionic surfactant and anionic surfactant, or a mixture of nonionic surfactant and amphoteric surfactant, or a mixture of nonionic surfactant and cationic surfactant; When the surfactant is a mixture of nonionic and anionic surfactants, the total molar concentration of the surfactants is 1-20 mM, wherein the molar ratio of nonionic to anionic surfactants 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 mixture of nonionic and amphoteric surfactants, the total molar concentration of the surfactants is 1-10 mM, wherein the molar ratio of nonionic surfactants to amphoteric surfactants 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 combination of nonionic and cationic surfactants, the total molar concentration of the surfactants is 1-3.5 mM, wherein the molar ratio of nonionic to cationic surfactants is 0.3-1.5; the molar concentration of the organic acid is 30-50 mM; the pH of the hemolytic agent is 6.5-8.0; and the organic acid is selected from any one or more of salicylic acid and its salt derivatives, caffeic acid and its salt derivatives, and phthalic acid and its salt derivatives. The nonionic surfactant is selected from any one or more of polyoxyethylene surfactants, alkylolamide 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.

2. The hemolytic agent for white blood cell differential detection according to claim 1, characterized in that, The polyoxyethylene surfactant is selected from any one or more of polyoxyethylene lauryl ether-35, polyoxyethylene 20 oil ether, fatty alcohol polyoxyethylene ether-50, and Tween. The alkylolamide surfactant is selected from any one or more of coconut oil diethanolamide, laurate diethanolamide, and myristyl 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 one or more of sodium dodecyl sulfate, potassium dodecyl sulfate, and sodium fatty alcohol polyoxyethylene ether 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 palmitoleoyl glutamate. The betaine surfactant is selected from any one or more of betaine, cocamidopropyl betaine, and lauramide propyl betaine; The ammonium salt surfactants include any one or more of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, and hydroxyethyl lauryl dimethylammonium chloride.

3. The hemolytic agent for white blood cell differential detection according to claim 1, characterized in that, When the surfactant is a combination of a nonionic surfactant and anionic surfactant, the nonionic surfactant is selected from polyoxyethylene lauryl ether-35, the anionic surfactant is selected from sodium dodecyl sulfate, and the organic acid is selected from phthalic acid or its salt derivatives. When the surfactant is a combination of a nonionic surfactant and an amphoteric surfactant, the nonionic surfactant is selected from fatty alcohol polyoxyethylene ether-50, the amphoteric 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 combination 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.

4. The hemolytic agent for white blood cell differential detection according to claim 1, characterized in that, When the surfactant is a combination of nonionic and anionic surfactants, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a combination of a nonionic surfactant and an amphoteric surfactant, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a combination of a nonionic surfactant and a cationic surfactant, the pH of the hemolytic agent is 7.2-7.6; When the surfactant is a mixture of nonionic and cationic surfactants, the molar ratio of the nonionic and cationic surfactants is 0.8-1.

2.

5. The method for preparing the hemolysin for white blood cell differential detection according to any one of claims 1-4, characterized in that, Includes the following steps: The organic acid and surfactant are completely dissolved in water according to the specified ratio, and the pH is adjusted to the required level to obtain a hemolytic agent for white blood cell differential detection.

6. A white blood cell differential detection reagent, characterized in that, Includes the hemolytic agent for white blood cell differential detection and the AIE white blood cell differential staining solution as described in any one of claims 1-5.

7. The use of the hemolysin for white blood cell differential detection according to any one of claims 1-5 or the white blood cell differential detection reagent according to claim 6 in white blood cell differential and / or counting detection in non-disease diagnostic fields.

8. A method for detecting white blood cell differential count, characterized in that, Includes the following steps: The hemolysin for white blood cell classification detection according to any one of claims 1-5 is thoroughly mixed with AIE white blood cell classification staining solution to obtain a mixture solution. A blood sample is added and thoroughly mixed to obtain a sample test solution. Then, white blood cell classification and / or counting detection is performed using a flow cytometer.

9. The white blood cell classification detection method according to claim 8, characterized in that, The volume ratio of the hemolysin used for white blood cell differential detection to the AIE white blood cell differential staining solution is 1000:(1~3). The concentration of the AIE leukocyte differential staining solution is 5-20 mM; The ratio of blood sample to mixture solution is 10~30μL:1mL; After the blood sample is added to the mixture solution, it is mixed thoroughly for 30-90 seconds. The fluorescent dye structure of the AIE leukocyte classification staining solution is as follows: 。

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