Leukocyte classification reagent based on AIE fluorescent dye and preparation method and application thereof

Abstract

The invention discloses a leukocyte classification reagent based on AIE fluorescent dye as well as a preparation method and application of the leukocyte classification reagent. The leukocyte classification reagent comprises the following components: AIE fluorescent dye, a surfactant, aromatic organic acid and water, wherein the surface active agent comprises a non-ionic surface active agent and a cationic surface active agent in a mass ratio of (0.5-5): 1, or the non-ionic surface active agent and the anionic surface active agent in a mass ratio of (0.5-20): 1. The leukocyte classification reagent has both a red blood cell lysis function and a leukocyte fluorescent staining function, has good storage stability, can realize pretreatment of a blood sample only by adopting a single reagent, greatly simplifies a blood sample pretreatment operation process of leukocyte classification and counting analysis, effectively reduces test errors, and improves the detection accuracy. And the reliability of detection results is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and in particular relates to a leukocyte classification reagent based on AIE fluorescent dye, and a preparation method and application thereof. Background Art

[0002] White blood cells in human blood are an important part of the immune system. According to their morphology and function, they can be divided into five subtypes: monocytes, lymphocytes, and granulocytes (including eosinophils, neutrophils, and basophils). These white blood cells maintain a relatively constant ratio in a healthy state, but in a diseased state, their number and ratio will change, providing an important basis for the diagnosis of the disease. Therefore, the analysis of white blood cell classification and counting is of great clinical significance for evaluating the human immune function and the diagnosis and treatment of various diseases.

[0003] The classification and counting of white blood cells in blood samples using a blood cell analyzer usually requires the use of hemolytic agents and nucleic acid dyes to pre-treat the samples. Hemolytic agents are used to dissolve the large number of red blood cells in the blood, exposing the white blood cells for subsequent counting and classification; nucleic acid dyes can enter the cells through the cell membrane of white blood cells and emit fluorescence after combining with the nucleic acid substances in the cells. Since the cell size and intracellular structure of each subpopulation 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 subpopulations can be distinguished by using, for example, a scatter plot of two-dimensional detection information, so that classification and counting can be performed.

[0004] At present, hemolytic agents and nucleic acid dyes are used as independent reagents. Usually, the hemolytic agent is first mixed with the blood sample, and then the nucleic acid dye is added for staining, and then the sample is introduced into the blood analyzer for classification and counting analysis; the hemolytic agent and the nucleic acid dye can also be mixed first, and then added to the blood sample for mixing, but they need to be prepared and used immediately. This is mainly because the system is unstable after the existing nucleic acid dye is mixed with the hemolytic agent, which will reduce the hemolytic performance and nucleic acid staining performance of the reagent, affecting the accuracy of the test results. However, for a single white blood cell classification and counting project, the preparation and use of two independent reagents are required, which not only increases the production, transportation, and packaging costs of the reagents, but also because blood analysis has high requirements for precision and accuracy, multi-step processing operations on the sample are more likely to lead to deviations in the test results, which places higher requirements on the professionalism of the inspectors.

[0005] Therefore, exploring an integrated white blood cell classification reagent solution that combines hemolysis and staining functions into a single reagent to simplify the operating process, reduce costs, and significantly improve the stability and accuracy of detection will be an important technological breakthrough in the field of white blood cell analysis technology. Summary of the invention

[0006] In view of the above problems, the present invention provides a leukocyte classification reagent based on AIE fluorescent dye, and its preparation method and application; the leukocyte classification reagent of the present invention has both red blood cell lysis function and leukocyte fluorescent staining function, and has good storage stability. The reagent can be directly applied to blood samples, realizing one-step labeling of leukocytes, greatly simplifying the sample processing operation process, effectively reducing test errors, and improving the reliability of test results. Through the precise detection of flow cytometer, using the dual analysis of scattered light signals and fluorescent signals, leukocytes can be efficiently and accurately classified and / or counted, and abnormal leukocytes, such as primitive cells and immature granulocytes, can be identified.

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

[0008] A leukocyte classification reagent based on AIE fluorescent dye, comprising the following components: AIE fluorescent dye, surfactant, aromatic organic acid and water;

[0009] Wherein, the surfactant comprises a nonionic surfactant and a cationic surfactant in a mass ratio of 0.5 to 5:1, or a nonionic surfactant and an anionic surfactant in a mass ratio of 0.5 to 20:1;

[0010] The AIE fluorescent dye is selected from any one of formula (1) to formula (4):

[0011]

[0012]

[0013] Wherein, the R1 is selected from * indicates a replacement position;

[0014] R2, R3, R5, R 13 , R 14 , R 15 , R 16 Each is independently selected from any one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN;

[0015] R6 is selected from any one of -H, -CN, -CH3;

[0016] R7, R8, R9, R 10 , R 11 Each is independently selected from any one of a straight chain alkyl group or a branched chain alkyl group having 1 to 20 carbon atoms;

[0017] R4 - , R12 - , R 17 - , R 18 - It is an anion;

[0018] n1 and n2 are each independently a positive integer of 1 to 20.

[0019] Preferably, R7, R8, R9, R 10 , R 11 Each is independently selected from any one of a straight chain alkyl group or a branched chain alkyl group having 1 to 6 carbon atoms.

[0020] Preferably, the anion is a monovalent anion;

[0021] Further preferably, the monovalent anion is selected from F - , Cl - Br - ,I - 、NO2 - 、NO3 - 、BF4 - PF6 - 、SbF6 - 、ClO4 - One or more of .

[0022] Preferably, n1 and n2 are each independently a positive integer of 1 to 6.

[0023] Preferably, in the leukocyte classification reagent, the mass content of the AIE fluorescent dye is 0.4 mg / L to 50 mg / L.

[0024] Further preferably, in the leukocyte classification reagent, the mass content of the AIE fluorescent dye is 2 mg / L to 30 mg / L.

[0025] Preferably, the AIE fluorescent dye is selected from one of Formula I to Formula V;

[0026]

[0027] Preferably, the surfactant comprises a nonionic surfactant and a cationic surfactant in a mass ratio of 1.5 to 3:1, or a nonionic surfactant and an anionic surfactant in a mass ratio of 0.6 to 12:1;

[0028] Preferably, in the leukocyte classification reagent, the total mass content of the surfactant is 0.5 g / L to 10 g / L;

[0029] Preferably, the nonionic surfactant is selected from any one or more of cetearyl alcohol polyether-25, polyoxyethylene lauryl ether Brij35, fatty alcohol polyoxyethylene ether-5, and polyoxyethylene (20) oleyl ether;

[0030] Preferably, the cationic surfactant is selected from any one or more of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, and hexadecyltrimethylammonium bromide;

[0031] Preferably, the anionic surfactant is selected from any one or more of sodium lauryl sulfate and sodium N-lauroyl sarcosine.

[0032] Preferably, in the leukocyte classification reagent, the mass content of aromatic organic acid is 0.5 g / L to 15 g / L;

[0033] Preferably, the aromatic organic acid is selected from any one of salicylic acid, phthalic acid and caffeic acid.

[0034] More preferably, the aromatic organic acid is phthalic acid.

[0035] Preferably, the leukocyte classification reagent further comprises buffer salt;

[0036] Further preferably, the buffer salt is selected from any one or more of phosphate, Tris, and HEPES;

[0037] Further preferably, the mass content of the buffer salt is 0.05 g / L to 15 g / L;

[0038] Preferably, the pH of the leukocyte classification reagent is 6.0-8.0.

[0039] More preferably, the pH of the leukocyte classification reagent is 6.5-7.5.

[0040] The preparation method of the above-mentioned leukocyte classification reagent based on AIE fluorescent dye comprises the following steps:

[0041] The AIE fluorescent dye, a surfactant and an aromatic organic acid are dissolved in water, the pH value is adjusted, and the mixture is filtered to obtain a leukocyte classification reagent based on the AIE fluorescent dye.

[0042] The application of the above-mentioned leukocyte classification reagent based on AIE fluorescent dye in the classification and detection of leukocytes in blood is specifically used for the classification or counting of leukocytes.

[0043] A method for detecting leukocyte classification comprises the following steps:

[0044] The blood sample is mixed with the above-mentioned AIE fluorescent dye-based leukocyte classification reagent and incubated for 1-2 minutes to prepare a sample, and the sample is introduced into a flow cytometer to measure scattered light and fluorescent signals, and the leukocytes are classified and / or counted according to their scattered light and fluorescent signals.

[0045] Preferably, the blood sample is whole peripheral blood, venous blood or peripheral blood;

[0046] Preferably, the blood sample and the leukocyte classification reagent are mixed at a volume ratio of 1:25 to 1:50.

[0047] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0048] The present invention unexpectedly discovered during the research that a white blood cell classification reagent with a stable system can be prepared by mixing a specific AIE fluorescent dye with a surfactant and an aromatic organic acid, which has both red blood cell lysis function and white blood cell fluorescent staining function. The blood sample can be pretreated with only a single reagent, which greatly simplifies the blood sample pretreatment operation process for white blood cell classification and counting analysis, effectively reduces test errors, and improves the reliability of test results.

[0049] The present invention realizes an integrated leukocyte classification reagent that combines hemolysis and staining functions in a single reagent, which not only simplifies the operation process, but also can ensure storage stability (fluorescence intensity change is less than or equal to 15% after being placed at 37°C for 14 days, and pH change is less than or equal to 0.5), detection stability and accuracy, and at the same time greatly reduces the production, transportation and packaging costs of the reagent, which will be an important technological breakthrough in the field of leukocyte analysis technology.

[0050] The present invention directly mixes the leukocyte classification reagent with the blood sample, and then uses the precise detection of the flow cytometer and the dual analysis of the scattered light signal and the fluorescent signal to efficiently and accurately classify and / or count the leukocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the effect diagram and result statistics of AIE leukocyte classification reagent in Example 1-1;

[0052] Figure 2 This is the effect diagram and result statistics of AIE leukocyte classification reagent in Example 2-1;

[0053] Figure 3 This is the effect diagram and result statistics of the AIE leukocyte classification reagent in Example 3;

[0054] Figure 4 This is the effect diagram and result statistics of the AIE leukocyte classification reagent in Example 4;

[0055] Figure 5 This is the effect diagram and result statistics of the AIE leukocyte classification reagent in comparative example 1-1;

[0056] Figure 6 This is the effect diagram and result statistics of AIE leukocyte classification reagent in comparative example 2;

[0057] Figure 7 This is the effect diagram and result statistics of the AIE leukocyte classification reagent of comparative example 3. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with examples, but the implementation manner and protection scope of the present invention are not limited to the following examples.

[0059] Embodiment 1:

[0060] AIE leukocyte classification reagent: Prepare according to the composition in Table 1.

[0061] The AIE fluorescent dye, surfactant and aromatic organic acid are completely dissolved in water, the pH value of the solution is adjusted with sodium hydroxide solution and hydrochloric acid solution, the volume is made up to 100 mL with water, and finally the prepared solution is filtered once with a 0.2 μm filter membrane to obtain the AIE leukocyte classification reagent.

[0062] Table 1

[0063]

[0064]

[0065] Detection steps: clinically, take 20 μL of fresh normal blood sample and mix with 800 μL of AIE leukocyte classification reagent, incubate for 60-120 seconds to form the test sample. Use flow cytometer to measure its side scattered light and fluorescence signal.

[0066] After the AIE leukocyte classification reagent was placed in a shaker at 37°C for 14 days (according to the Arrhenius formula as the theoretical basis for reagent stability research, for reagents stored at 2-8°C, 14 days at 37°C is approximately equivalent to 13 months at 4°C, and the same applies to the following.), first observe whether the AIE leukocyte classification reagent has crystal precipitation or color change, and use a pH meter to measure its pH value, and then use the above detection steps to perform side scattered light and fluorescence signal detection to evaluate its leukocyte classification effect. The measurement results are as follows: Figure 1 And as shown in Table 2.

[0067] Table 2

[0068]

[0069] Embodiment 2:

[0070] AIE leukocyte classification reagent: Prepare according to the composition in Table 3.

[0071] The AIE fluorescent dye, surfactant, buffer salt (sodium dihydrogen phosphate) and aromatic organic acid are completely dissolved in water, the pH value of the solution is adjusted with sodium hydroxide solution and hydrochloric acid solution, the volume is made up to 100 mL with water, and finally the prepared solution is filtered once with a 0.2 μm filter membrane to obtain the AIE leukocyte classification reagent.

[0072] Table 3

[0073]

[0074]

[0075] Detection steps: clinically, take 20 μL of fresh normal blood sample and mix with 800 μL of AIE leukocyte classification reagent, incubate for 60-120 seconds to form the test sample. Use flow cytometer to measure its side scattered light and fluorescence signal.

[0076] After the AIE leukocyte classification reagent was placed in a shaker at 37°C for 14 days, the AIE leukocyte classification reagent was first observed for crystal precipitation or color change, and its pH value was measured with a pH meter. Then, the above detection steps were applied to perform side scattered light and fluorescence signal detection to evaluate its leukocyte classification effect. The measurement results were as follows: Figure 2 And as shown in Table 4.

[0077] Table 4

[0078]

[0079] Embodiment 3:

[0080] Preparation of AIE leukocyte classification reagent: 53 mg of dodecyltrimethylammonium chloride LTAC, 120 mg of polyoxyethylene lauryl ether Brij35, 664.53 mg of phthalic acid, and 1550 μg of fluorescent dye formula IV are completely dissolved in water, the pH value of the solution is adjusted to 7.5 with sodium hydroxide solution and hydrochloric acid solution, the volume is made up to 100 mL with water, and finally the prepared solution is filtered once with a 0.2 μm filter membrane to obtain the AIE leukocyte classification reagent.

[0081] Detection steps: clinically, take 20 μL of fresh normal blood sample and mix with 800 μL of AIE leukocyte classification reagent, incubate for 60-120 seconds to form the test sample. Use flow cytometer to measure its side scattered light and fluorescence signal.

[0082] After the AIE leukocyte classification reagent was placed in a shaker at 37°C for 14 days, the AIE leukocyte classification reagent was first observed for crystal precipitation or color change, and its pH value was measured with a pH meter. Then, the above detection steps were applied to perform side scattered light and fluorescence signal detection to evaluate its leukocyte classification effect. The measurement results were as follows: Figure 3 And as shown in Table 5.

[0083] Table 5

[0084]

[0085] Embodiment 4:

[0086] Preparation of AIE leukocyte classification reagent: 200 mg of fatty alcohol polyoxyethylene ether-5, 300 mg of N-lauroyl sarcosine sodium salt, 83 mg of phthalic acid, and 734 μg of fluorescent dye formula V are completely dissolved in water, the pH value of the solution is adjusted to 8.0 with sodium hydroxide solution and hydrochloric acid solution, the volume is made up to 100 mL with water, and finally the prepared solution is filtered once with a 0.2 μm filter membrane to obtain the leukocyte classification reagent.

[0087] Detection steps: clinically, take 20 μL of fresh normal blood sample and mix with 800 μL of AIE leukocyte classification reagent, incubate for 60-120 seconds to form the test sample. Use flow cytometer to measure its side scattered light and fluorescence signal.

[0088] After the AIE leukocyte classification reagent was placed in a shaker at 37°C for 14 days, the AIE leukocyte classification reagent was first observed for crystal precipitation or color change, and its pH value was measured with a pH meter. Then, the above detection steps were applied to perform side scattered light and fluorescence signal detection to evaluate its leukocyte classification effect. The measurement results were as follows: Figure 4 and as shown in Table 6.

[0089] Table 6

[0090]

[0091] Comparative Example 1:

[0092] AIE leukocyte classification reagent: Prepare according to the composition of Table 7-1 and Table 7-2 respectively.

[0093] The AIE fluorescent dye, surfactant and organic acid are completely dissolved in water, the pH value of the solution is adjusted with sodium hydroxide solution and hydrochloric acid solution, the volume is made up to 100 mL with water, and finally the prepared solution is filtered once with a 0.2 μm filter membrane to obtain the AIE leukocyte classification reagent.

[0094] Table 7-1

[0095] Components Comparative Example 1-1 Comparative Example 1-2 Comparative Examples 1-3 Comparative Examples 1-4 Acetic acid 0.2mL 0 0 0 Phthalic acid 0 0 664.53mg 664.53mg Dodecyltrimethylammonium chloride LTAC 53mg 53mg 20mg 133mg Polyoxyethylene lauryl ether Brij35 120mg 120mg 153mg 40mg Fluorescent dye formula Ⅲ 485 μg 485 μg 485 μg 485 μg pH 7.5 7.5 7.5 7.5 Dissolve in water to make up to 100mL 100mL 100mL 100mL

[0096] Table 7-2

[0097]

[0098]

[0099] Detection steps: Mix 20 μL of fresh normal blood sample with 800 μL of AIE leukocyte classification reagent and incubate for 60-120 seconds to form a test sample. Measure the side scattered light and fluorescence signal using a flow cytometer.

[0100] After the AIE leukocyte classification reagent was placed in a shaker at 37°C for 14 days, the AIE leukocyte classification reagent was first observed for crystal precipitation or color change, and its pH value was measured with a pH meter. Then, the above detection steps were applied to perform side scattered light and fluorescence signal detection to evaluate its leukocyte classification effect. The measurement results were as follows: Figure 5 and as shown in Table 8.

[0101] Table 8

[0102]

[0103] Comparative Example 2:

[0104] AIE leukocyte classification reagent preparation:

[0105] 53 mg of dodecyl trimethyl ammonium chloride LTAC, 120 mg of polyoxyethylene lauryl ether Brij35, 664.53 mg of phthalic acid, and fluorescent dye formula VI were added. 400 μg was completely dissolved in water, the pH value of the solution was adjusted to 7.5 with sodium hydroxide solution and hydrochloric acid solution, the volume was made up to 100 mL with water, and finally the prepared solution was filtered once with a 0.2 μm filter membrane to obtain the AIE leukocyte classification reagent.

[0106] Detection steps: clinically, take 20μL of fresh normal blood sample and mix with 800μL of AIE leukocyte classification reagent, incubate for 60-120s to form the test sample. Use flow cytometer to measure its side scattered light and fluorescence signal. The test results are as follows Figure 6 shown.

[0107] Comparative Example 3:

[0108] Preparation of white blood cell classification reagent:

[0109] Completely dissolve 53 mg of dodecyltrimethylammonium chloride LTAC, 120 mg of polyoxyethylene lauryl ether Brij35, 664.53 mg of phthalic acid, and 656 μg of Cy5 in water, adjust the pH value of the solution to 7.5 with sodium hydroxide solution and hydrochloric acid solution, make up to 100 mL with water, and finally filter the prepared solution once with a 0.2 μm filter membrane to obtain a leukocyte classification reagent.

[0110] Detection steps: Clinically, 20 μL of fresh normal blood sample is mixed with 800 μL of leukocyte classification reagent and incubated for 60-120 seconds to form the test sample. The side scattered light and fluorescence signal are measured by flow cytometry.

[0111] After the leukocyte classification reagent is placed in a shaker at 37°C for 14 days, observe whether there is crystal precipitation or color change in the leukocyte classification reagent, measure its pH value with a pH meter, and then apply the above detection steps to perform side scattered light and fluorescence signal detection to evaluate its effect on leukocyte classification. The measurement results are as follows: Figure 7 and as shown in Table 9.

[0112] Table 9

[0113]

[0114] Data Analysis:

[0115] The test results of the above embodiments show that the AIE leukocyte classification reagent of the present invention can realize the classification of leukocyte subpopulations, dividing them into lymphocytes, monocytes, neutrophils and eosinophils, and the reagent has good storage stability. After the accelerated stability test, the reagent system is stable and can still realize the classification of leukocyte subpopulations. From the comparison of Example 1-1, Example 1-2, and Example 1-3, it can be seen that the aromatic organic acid selects phthalic acid to ensure the detection accuracy while optimizing the storage stability. The organic acid of Comparative Example 1-1 uses acetic acid, and no organic acid is added to Comparative Example 1-2. The prepared reagent cannot separate the monocytes and lymphocytes of leukocytes, and has poor storage stability; the results of Comparative Examples 1-3 to Comparative Examples 1-8 show that only the appropriate mass ratio of surfactants can achieve leukocyte tetrazoning and good storage stability, and the performance is poor if it is too high or too low; Comparative Example 2 uses AIE fluorescent dye The classification of leukocyte subpopulations cannot be achieved; Comparative Example 3 uses Cy5 fluorescent dye, and the reagent system is unstable. After the accelerated stability test, the fluorescence intensity decreases significantly, and the leukocyte subpopulations cannot be classified.

[0116] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A leukocyte classification reagent based on AIE fluorescent dye, characterized in that: The method comprises the following components: an AIE fluorescent dye, a surfactant, an aromatic organic acid and water; Wherein, the surfactant comprises a nonionic surfactant and a cationic surfactant in a mass ratio of 0.5 to 5:1, or a nonionic surfactant and an anionic surfactant in a mass ratio of 0.5 to 20:1; The AIE fluorescent dye is selected from any one of formula (1) to formula (4): Wherein, the R1 is selected from * indicates a replacement position; R2, R3, R5, R 13 , R 14 , R 15 , R 16 Each is independently selected from any one of -H, -CH3, -COOH, -OH, -NH2, -CHO, -CN; R6 is selected from any one of -H, -CN, -CH3; R7, R8, R9, R 10 , R 11 Each is independently selected from any one of a straight chain alkyl group or a branched chain alkyl group having 1 to 20 carbon atoms; R4 - , R 12 - , R 17 - , R 18 - It is an anion; n1 and n2 are each independently a positive integer of 1 to 20.

2. The leukocyte classification reagent based on AIE fluorescent dye according to claim 1, characterized in that: In the leukocyte classification reagent, the mass content of the AIE fluorescent dye is 0.4 mg / L to 50 mg / L.

3. The leukocyte classification reagent based on AIE fluorescent dye according to claim 1, characterized in that: The AIE fluorescent dye is selected from one of Formula I to Formula V; 4. The leukocyte classification reagent based on AIE fluorescent dye according to claim 1, characterized in that: In the leukocyte classification reagent, the total mass content of the surfactant is 0.5 g / L to 10 g / L; The nonionic surfactant is selected from any one or more of cetearyl alcohol polyether-25, polyoxyethylene lauryl ether Brij35, fatty alcohol polyoxyethylene ether-5, and polyoxyethylene (20) oleyl ether; The cationic surfactant is selected from any one or more of dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, and hexadecyltrimethylammonium bromide; The anionic surfactant is selected from any one or more of sodium lauryl sulfate and sodium N-lauroyl sarcosine.

5. The leukocyte classification reagent based on AIE fluorescent dye according to claim 1, characterized in that: In the leukocyte classification reagent, the mass content of aromatic organic acid is 0.5 g / L to 15 g / L; The aromatic organic acid is selected from any one of salicylic acid, phthalic acid and caffeic acid.

6. The leukocyte classification reagent based on AIE fluorescent dye according to claim 1, characterized in that: The leukocyte classification reagent further includes buffer salt; The buffer salt is selected from any one or more of phosphate, Tris, and HEPES; The mass content of the buffer salt is 0.05 g / L to 15 g / L; The pH of the leukocyte classification reagent is 6.0-8.

0.

7. The method for preparing the leukocyte classification reagent based on AIE fluorescent dye according to any one of claims 1 to 6, characterized in that: The following steps are involved: The AIE fluorescent dye, a surfactant and an aromatic organic acid are dissolved in water, the pH value is adjusted, and the mixture is filtered to obtain a leukocyte classification reagent based on the AIE fluorescent dye.

8. Use of the leukocyte classification reagent based on AIE fluorescent dye according to any one of claims 1 to 6 in the detection of leukocyte classification in blood.

9. A method for detecting leukocyte classification, characterized in that: The following steps are involved: A blood sample is mixed with the leukocyte classification reagent based on the AIE fluorescent dye according to any one of claims 1 to 6, and incubated for 1 to 2 minutes to prepare a sample, and the sample is introduced into a flow cytometer to measure scattered light and fluorescence signals, and the leukocytes are classified and / or counted according to their scattered light and fluorescence signals.

10. The method for detecting leukocyte classification according to claim 9, characterized in that: The blood sample is whole peripheral blood, venous blood or peripheral blood; The blood sample and the leukocyte classification reagent are mixed at a volume ratio of 1:25 to 1:50.

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