Detection kits and methods for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia

Abstract

This invention discloses a detection kit and method for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia. The detection kit includes a first reagent and a second reagent. The first reagent includes a hemolysin and a staining agent. The second reagent includes a hemolysin, a staining agent, and an antibody treatment agent, including CD13 and CD34 antibody treatment agents. The hemolysin, based on a total volume of 1L, includes the following components: 0.1-10g castor oil polyoxyethylene ether nonionic surfactant, 0.01-10g cationic surfactant, 0.5-15g buffer, 0.01-8g preservative, with the remainder being water. The pH value of the hemolysin is 5-9. The staining agent, based on a total volume of 1L, includes the following components: 0.001-0.2g nucleic acid dye, with the remainder being a solvent. The detection method includes: obtaining a blood sample to be tested, preparing a first sample, testing the first sample, preparing a second sample, and testing the second sample. This invention can accurately detect abnormal lymphocytes, myeloid blast cells, and diagnose acute promyelocytic leukemia.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a detection kit and detection method for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia. Background Technology

[0002] Currently, the gold standard for detecting blast cells is bone marrow aspiration and biopsy, which are invasive procedures with certain drawbacks. Detection of peripheral blood blast cells and abnormal lymphocytes is relatively convenient, and methods for this include peripheral blood smears, flow cytometry, and hematology analyzers. However, peripheral blood smears are susceptible to errors due to subjective judgment by the tester. Flow cytometry uses specific antibodies to label the surface antigens of blast cells and abnormal lymphocytes, but using monoclonal antibodies is time-consuming, costly, and complex. Hematology analyzers can rapidly analyze abnormal cells, but current technology primarily distinguishes between normal and immature leukocytes, and this method only performs differential staining of nucleic acid substances, which can easily lead to false negatives or false positives.

[0003] Furthermore, an increase in blast cells is a symptom of acute myeloid leukemia (AML), the most common type of acute leukemia in adults. Acute promyelocytic leukemia (APL) is a particularly important subtype (M3 AML), accounting for 10% to 15% of all AML patients. Early diagnosis of APL is crucial because this type of leukemia has unique molecular mechanisms and clinical manifestations. This requires physicians to have a deep understanding of it and to accurately diagnose and treat APL by observing the patient's clinical presentation and laboratory characteristics, thereby improving the cure rate and quality of life for patients. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a detection kit and detection method for abnormal lymphocytes, myeloid blast cells and acute promyelocytic leukemia.

[0005] The technical solution adopted by this invention to solve its technical problem is: a detection kit for abnormal lymphocytes, myeloid blast cells and acute promyelocytic leukemia, comprising a first reagent and a second reagent; the first reagent comprises a hemolytic agent and a staining agent; the second reagent comprises a hemolytic agent, a staining agent and an antibody treatment agent, the antibody treatment agent comprising a CD13 antibody treatment agent and a CD34 antibody treatment agent;

[0006] The hemolytic agent, based on a total volume of 1L, comprises the following components: 0.1-10g nonionic surfactant, 0.01-10g cationic surfactant, 0.5-15g buffer, 0.01-8g preservative, with the remainder being water. The pH value of the hemolytic agent is 5-9. The nonionic surfactant is a castor oil polyoxyethylene ether nonionic surfactant with the structural formula: Ricinoleic Acid-O-(CH2CH2O). n In the formula, n = 10 to 90; the staining agent, based on a total volume of 1L, includes the following components: 0.001-0.2g nucleic acid dye, the remainder being solvent; the CD13 antibody treatment agent, based on a total volume of 1L, includes the following components: 1-50mg fluorescently labeled CD13 antibody, the remainder being antibody diluent; the CD34 antibody treatment agent, based on a total volume of 1L, includes the following components: 1-100mg fluorescently labeled CD34 antibody, the remainder being antibody diluent; the antibody diluent, based on a total volume of 1L, includes the following components: 0.5-15g buffer, 0.01-8g preservative, the remainder being water.

[0007] In some embodiments, the cationic surfactant includes at least one alkyl quaternary ammonium salt, wherein the alkyl quaternary ammonium salt is decaalkyltrimethylammonium bromide, dodecyltrimethylammonium bromide, or hexadecyltrimethylammonium chloride.

[0008] In some embodiments, the nucleic acid dye is a compound with the following structure: In the formula, R1 and R2 are C1-C6 alkyl, C1-C6 alkoxy, acyl or hydrogen atoms, and X is a halogen atom, boron halide or phosphorus compound.

[0009] In some embodiments, the nucleic acid dye is prepared from raw material A, compound R1X, raw material B, compound R2X, and N,N-dimethylformamide, wherein raw material A is a compound with the following structure: Raw material B is a compound with the following structure: R1 and R2 are both C1-C6 alkyl groups, C1-C6 alkoxy groups, acyl groups, or hydrogen atoms, and X is a halogen atom, boron halide, or phosphorus compound.

[0010] In some embodiments, both the fluorescently labeled CD13 antibody and the fluorescently labeled CD34 antibody contain a fluorescently labeled dye, which is fluorescein isothiocyanate, a cyanine fluorescent dye, or an Alexa Fluor series dye. The cyanine fluorescent dye is Cy3 dye, Cy5 dye, or Cy7 dye.

[0011] In some embodiments, the buffer is HEPES buffer, MOPS buffer, citrate buffer, or phosphate buffer.

[0012] In some embodiments, the preservative is benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol, or benzyl alcohol.

[0013] In some embodiments, the hemolytic agent further includes 0-20g of an osmotic pressure regulator, which is an inorganic salt, a sugar, or an amino acid; the sugar is at least one of glucose, fructose, and mannitol; the inorganic salt includes at least one of sodium sulfate, sodium chloride, and potassium chloride; and the amino acid includes at least one of glycine, valine, and alanine.

[0014] In some embodiments, the hemolytic agent further includes 0.01-50g of a chelating agent, which is sodium ethylenediaminetetraacetate or potassium ethylenediaminetetraacetate.

[0015] This invention also proposes a method for detecting abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia. The method uses the aforementioned detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia, and includes the following steps:

[0016] S1. Obtaining the blood sample to be tested: Blood is collected from the subject and processed to obtain the blood sample to be tested;

[0017] S2. Preparation of the first sample: Mix the blood sample to be tested with the hemolysing agent and staining agent in the first reagent, and incubate at 35-39℃ for 40-60s to obtain the first sample; the mixing ratio of the blood sample to be tested to the first reagent is 1:(10-1000), and the mixing ratio of the hemolysing agent to the staining agent is (10-1000):1.

[0018] S3. Detect the first sample: Perform scattered light detection and fluorescence detection on the first sample to obtain the forward scattered light intensity and the lateral fluorescence intensity, and count the abnormal lymphocytes in the first sample.

[0019] S4. Preparation of the second sample: Mix the blood sample to be tested with the hemolysing agent, staining agent and antibody treatment agent in the second reagent, and incubate at 35-39℃ for 40-60s to obtain the second sample; the mixing ratio of the blood sample to be tested and the second reagent is 1:(10-1000), and the mixing ratio of the hemolysing agent, staining agent and antibody treatment agent is (10-1000):1:1;

[0020] S5. Detect the second sample: Perform scattered light detection and fluorescence detection on the second sample to obtain the forward scattered light intensity, side scattered light intensity, side fluorescence intensity and antibody fluorescence labeling intensity, count the myeloid blast cells in the second sample, and determine whether the subject has acute promyelocytic leukemia.

[0021] The beneficial effects of this invention are:

[0022] In the detection kit for abnormal lymphocytes, myeloid blasts, and acute promyelocytic leukemia of the present invention, a hemolytic agent containing nonionic and cationic surfactants is used to differentially treat the cell membranes of blasts and mature leukocytes. Abnormal lymphocytes and normal mature leukocytes are distinguished by the nucleic acid dye of the staining agent. An antibody treatment agent is used to differentially bind to the CD13 antigen strongly expressed on the surface of myeloid blasts, thereby identifying and counting myeloid blasts. The hemolytic agent and the antibody treatment agent can analyze the type of myeloid blasts, and the expression of CD13 and CD34 antibodies can determine whether it is acute promyelocytic leukemia.

[0023] In the detection method for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia of the present invention, the blood sample to be tested is processed using the detection kit of the present invention, and then subjected to scattered light detection and fluorescence detection. Based on the detection results, abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia can be accurately detected. The detection method of the present invention is simple to operate and has high detection accuracy. Attached Figure Description

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. In the accompanying drawings:

[0025] Figure 1 This is a two-dimensional scatter plot of FSC-SFL for the detection method of the present invention for normal blood samples and blood samples containing abnormal lymphocytes;

[0026] Figure 2 The present invention provides two-dimensional scatter plots of FSC-SFL and SSC-CD13 for the detection of normal blood samples and blood samples containing myeloid primitive cells without abnormal lymphocytes.

[0027] Figure 3 This is a two-dimensional scatter plot of CD13-CD34 in normal blood samples, blood samples from APL patients, and blood samples from other subtypes of AML patients, obtained by the detection method of this invention. Detailed Implementation

[0028] To provide a clearer understanding of the technical features, objectives, and effects of this invention, the invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the invention and do not constitute a limitation on the scope of protection of this invention.

[0029] This invention proposes a detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia. The kit includes a first reagent and a second reagent. The first reagent includes a hemolysin and a staining agent, and the second reagent includes a hemolysin, a staining agent, and an antibody treatment agent. The antibody treatment agent includes a CD13 antibody treatment agent and a CD34 antibody treatment agent. In the first reagent, the hemolysin and staining agent can be used as separate reagents and mixed only when processing the blood sample to be tested. In the second reagent, the hemolysin, staining agent, and antibody treatment agent can be used as separate reagents and mixed only when processing the blood sample to be tested.

[0030] Hemolytic agents utilize the differences in cell membranes between mature leukocytes and primitive cells to induce differential treatment between them. A 1L volume of hemolytic agent comprises the following components: 0.1-10g nonionic surfactant, 0.01-10g cationic surfactant, 0.5-15g buffer, 0.01-8g preservative, with the remainder being water. The pH of the hemolytic agent is 5-9. Understandably, the content of nonionic surfactant can be 0.1g, 2g, 5g, 7g, 10g, etc.; the content of cationic surfactant can be 0.01g, 0.1g, 1g, 5g, 8g, 10g, etc.; the content of buffer can be 0.5g, 1g, 5g, 10g, 15g, etc.; the content of preservative can be 0.01g, 0.5g, 2g, 5g, 6g, 8g, etc.; and the pH of the hemolytic agent can be 5, 6, 7, 8, 9, etc. The osmotic pressure of the hemolytic agent is preferably 10-300 mOsm / Kg, specifically 10 mOsm / Kg, 50 mOsm / Kg, 100 mOsm / Kg, 150 mOsm / Kg, 200 mOsm / Kg, 300 mOsm / Kg, etc.

[0031] Nonionic surfactants are used to disrupt the cell membranes of red blood cells and damage white blood cells. These nonionic surfactants are castor oil polyoxyethylene ether-based nonionic surfactants, with the structural formula: Ricinoleic Acid-O-(CH2CH2O). n In the formula, n = 10 to 90. The nonionic surfactant can specifically be EL-10, EL-20, EL-40, EL-60, EL-80, EL-90, or hydrogenated castor oil polyoxyethylene ether, all of which are commercially available products. The content of the nonionic surfactant can be adjusted appropriately according to its composition. Based on a total volume of 1L, the preferred content of the nonionic surfactant in the hemolytic agent is 0.1-10g.

[0032] Cationic surfactants (co-solvents) are used to shrink damaged red blood cells. Cationic surfactants include at least one alkyl quaternary ammonium salt, which may be decaalkyltrimethylammonium bromide, dodecyltrimethylammonium bromide, or hexadecyltrimethylammonium chloride. Cationic surfactants may include one alkyl quaternary ammonium salt, or two alkyl quaternary ammonium salts, such as decaalkyltrimethylammonium bromide and dodecyltrimethylammonium bromide; or three alkyl quaternary ammonium salts, such as decaalkyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride. The content of the cationic surfactant can be adjusted appropriately according to its composition and should be compatible with the content of the nonionic surfactant. Based on a total volume of 1L, the preferred content of the cationic surfactant in the hemolytic agent is 0.01-10g.

[0033] Buffers are used to maintain the stability of hemolysins, adjusting their pH value and osmotic pressure by regulating their concentration. Buffers can be HEPES (hydroxyethylpiperazine ethanethioic acid) buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, citrate buffer, or phosphate buffer. Additionally, pH adjusters, such as sodium hydroxide or hydrogen chloride, can be used to adjust the pH of the hemolysin.

[0034] The preservatives are benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol, or benzyl alcohol. The content of the preservatives varies depending on the components. For the hemolysin, based on a total volume of 1L, the preferred content of isothiazolinone is 0.01-0.5g, the preferred content of phenoxyethanol is 0.5-5.5g, the preferred content of sodium hydroxymethylglycinate is 0.1-3.5g, and the preferred content of benzyl alcohol is 0.2-7.5g.

[0035] In some embodiments, an osmotic pressure regulator may be used to adjust the osmotic pressure of the hemolytic agent. The hemolytic agent, based on a total volume of 1 L, further includes 0-20 g of the osmotic pressure regulator, with the content of the osmotic pressure regulator being 0.5 g, 2 g, 5 g, 10 g, 15 g, 20 g, etc. The osmotic pressure regulator may be an inorganic salt, a sugar, or an amino acid. The sugar is at least one of glucose, fructose, and mannitol. The inorganic salt includes at least one of sodium sulfate, sodium chloride, and potassium chloride. The amino acid includes at least one of glycine, valine, and alanine. A sugar is preferred as the osmotic pressure regulator.

[0036] The hemolytic agent, based on a total volume of 1L, also includes 0.01-50g of chelating agent. The content of the chelating agent can be 0.01g, 0.1g, 5g, 10g, 30g, or 50g. The chelating agent is sodium ethylenediaminetetraacetate (EDTA sodium salt) or potassium ethylenediaminetetraacetate (EDTA potassium salt), such as disodium EDTA.

[0037] In some embodiments, when preparing the hemolytic agent, a nonionic surfactant, a cationic surfactant, a buffer, a preservative, water (as well as a chelating agent and an osmotic pressure regulator) are mixed, and an appropriate amount of pH adjuster is added to adjust the pH of the solution to 5-9. The mixture is then stirred evenly in an environment with a temperature of 20-30°C and a humidity of 40-60%, and then filtered to obtain the hemolytic agent. The filtration can be performed using a filter membrane with a pore size of 0.1-0.3 μm, such as 0.22 μm.

[0038] The staining agent can stain the nucleic acids of mature leukocytes, primitive cells, and abnormal lymphocytes. The staining agent, with a total volume of 1L, includes the following components: 0.001-0.2g of nucleic acid dye, with the remainder being solvent. Understandably, the content of the nucleic acid dye can be 0.001g, 0.01g, 0.05g, 0.1g, 0.2g, etc. The nucleic acid dye is a fluorescent dye used for nucleic acid staining, and the nucleic acid dye can be a compound with the following structure: In the formula, R1 and R2 are C1-C6 (straight-chain or branched) alkyl, C1-C6 alkoxy, acyl or hydrogen atoms, and X is a halogen atom, boron halide or phosphorus compound. - Specifically, these can be fluoride ions, chloride ions, bromide ions, iodide ions, boron halide ions, phosphorus compound ions, etc., with iodide ions being preferred. Nucleic acid dyes can be compounds with the following structures: (Dye A) or (Dye B). The solvent may be at least one of water, methanol, ethanol or ethylene glycol.

[0039] In some embodiments, the nucleic acid dye is prepared from raw material A, compound R1X, raw material B, compound R2X, and N,N-dimethylformamide, wherein raw material A is a compound with the following structure: Raw material B is a compound with the following structure: R1 and R2 are both C1-C6 alkyl groups, C1-C6 alkoxy groups, acyl groups, or hydrogen atoms, and X is a halogen atom, boron halide, or phosphorus compound.

[0040] Furthermore, the preparation method of nucleic acid dyes includes the following steps:

[0041] S1. Reacting raw material A with compound R1X in reaction I produces a thiophene ring intermediate with the following structure: In this reaction, the molar ratio of raw material A to compound R1X is 1:(1-10), and reaction reagents are added to provide the reaction environment. The reaction atmosphere for reaction I is argon, the reaction temperature is 50-200℃, and the reaction time is 2-40 hours. After the reaction, the precipitate is collected by filtration and vacuum dried to obtain a pale yellow powder, which is the thiophene ring intermediate. The molar ratio of raw material A to compound R1X can be 1:1, 1:3, 1:5, 1:8, 1:10, etc., the reaction atmosphere can be other inert protective gases, the reaction temperature can be 50℃, 100℃, 130℃, 150℃, 200℃, etc., and the reaction time can be 2h, 10h, 20h, 30h, 40h. Understandably, the process parameters for reaction I can be selected according to actual needs and are not specifically limited.

[0042] S2. Reacting raw material B with compound R2X in reaction II, a quinoline ring intermediate is generated, the structure of which is: In this reaction, the molar ratio of raw material B to compound R2X is 1:(1-10), and reaction reagents are added. The reaction atmosphere for reaction II is argon, and the reaction time is 2-20 hours. After heating to the boiling point of the reaction reagents, the mixture is refluxed. After the reaction is complete, the precipitate is collected by filtration and vacuum dried to obtain a pale yellow powder, which is the quinoline ring intermediate. The molar ratio of raw material B to compound R2X can be 1:1, 1:3, 1:5, 1:8, 1:10, etc., and other inert protective gases can be selected for the reaction atmosphere. The reaction time can be 2h, 6h, 10h, 15h, or 20h. The boiling point range of the reaction reagents is 40-180℃, and the specific heating temperature is selected according to the boiling point of the reaction reagents. Understandably, the process parameters for reaction II can be selected according to actual needs and are not specifically limited.

[0043] S3. The quinoline ring intermediate generated in reaction II is reacted with N,N-dimethylformamidin in reaction III (melt reaction) to obtain product A with the following structure: In this reaction, the molar ratio of the quinoline ring intermediate to N,N-dimethylformamide is 1:(1-10). Reagents are added, and the mixture is heated in an oil bath at 70-300℃ with stirring. The reaction is allowed to proceed for 10-150 minutes. The resulting product is washed, recrystallized, filtered, and vacuum dried. Washing involves repeatedly washing the reddish-brown solid with diethyl ether, followed by recrystallization in ethanol to obtain purplish-red crystals, product A. The molar ratio of the quinoline ring intermediate to N,N-dimethylformamide can be 1:1, 1:3, 1:5, 1:8, 1:10, etc., the oil bath temperature can be 70℃, 100℃, 200℃, 250℃, 300℃, etc., and the melting reaction time can be 10 min, 50 min, 100 min, 125 min, 150 min. Understandably, the process parameters for reaction III can be selected according to actual needs and are not specifically limited.

[0044] S4. React the thiophene ring intermediate generated in reaction I with product A generated in reaction III in reaction IV to obtain the nucleic acid dye. The molar ratio of the thiophene ring intermediate to product A is 1:(1-10). Add the reaction reagents, react in the dark, heat in an oil bath to 50-200℃, and stir continuously for 1-15 hours. Then recrystallize, filter and dry the reaction solution, and then perform column chromatography separation, elution, rotary evaporation and drying.

[0045] Specifically, the thiophene ring intermediate and product A are weighed separately and placed in a reaction vessel. Reaction reagents are added, and the reaction is carried out in the dark. The mixture is heated in an oil bath to 50-200°C and stirred continuously for 1-15 hours. Subsequently, the reaction solution is recrystallized to precipitate dark purple particles, which are filtered and vacuum dried. The dark purple particles are separated by silica gel column chromatography, followed by gradient elution with eluents including dichloromethane and methanol. The blue fraction is collected, rotary evaporated, and vacuum dried to obtain the nucleic acid dye. In some embodiments, a round-bottom flask can be used as the reaction vessel, and the flask is wrapped with aluminum foil to protect it from light.

[0046] The molar ratio of the thiophene ring intermediate to product A can be 1:1, 1:3, 1:5, 1:8, 1:10, etc., the heating temperature can be 50℃, 80℃, 100℃, 150℃, 200℃, etc., and the stirring reaction time can be 1h, 5h, 8h, 10h, 15h. Understandably, the process parameters for reaction IV can be selected according to actual needs and are not specifically limited.

[0047] The reaction reagents in steps S1 to S4 are all polar solvents, such as dichloromethane, ethanol, acetonitrile, ethyl acetate, toluene, or xylene.

[0048] The antibody treatment agents, with a total volume of 1L, include the following components for CD13 antibody treatment: 1-50mg of fluorescently labeled CD13 antibody, with the remainder being antibody diluent. The CD34 antibody treatment agents, with a total volume of 1L, include the following components for CD34 antibody treatment: 1-100mg of fluorescently labeled CD34 antibody, with the remainder being antibody diluent. The antibody diluent, with a total volume of 1L, includes the following components for CD34 antibody treatment: 0.5-15g of buffer, 0.01-8g of preservative, with the remainder being water. The content of fluorescently labeled CD13 antibody can be 1mg, 10mg, 20mg, 25mg, 50mg, etc.; the content of fluorescently labeled CD34 antibody can be 1mg, 25mg, 50mg, 80mg, 100mg, etc.; the content of buffer can be 0.5g, 1g, 5g, 10g, 15g, etc.; and the content of preservative can be 0.01g, 0.5g, 2g, 5g, 7g, 8g, etc.

[0049] Both the fluorescently labeled CD13 and CD34 antibodies contain fluorescently labeled dyes. These dyes are fluorescein isothiocyanate (FITC), cyanine fluorescent dyes, or Alexa Fluor series dyes. Cyanine fluorescent dyes include Cy3, Cy5, or Cy7 dyes; Alexa Fluor series dyes include Alexa Fluor 405, Alexa Fluor 488, or Alexa Fluor 594, etc. All fluorescently labeled dyes are commercially available. The selection of the fluorescently labeled dye and the nucleic acid dye in the staining agent must ensure that the fluorescence emission of the fluorescently labeled dye and the nucleic acid dye does not overlap. The buffer composition in the antibody diluent is the same as that in the hemolysing agent, and the preservative composition in the antibody diluent is the same as that in the hemolysing agent. Understandably, fluorescently labeled CD13 antibodies can be FITC-labeled CD13 monoclonal antibodies, Cy3-labeled CD13 monoclonal antibodies, etc., and fluorescently labeled CD34 antibodies can be FITC-labeled CD34 monoclonal antibodies, Cy5-labeled CD34 monoclonal antibodies, etc.

[0050] This invention also proposes a method for detecting abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia. The method uses the aforementioned detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia, and employs flow cytometry technology with a hematology analyzer to analyze blast cells and abnormal lymphocytes. The detection method includes the following steps:

[0051] S1. Obtaining the blood sample to be tested: Blood is drawn from the subject and processed to obtain the blood sample to be tested. Understandably, blood collection and processing are existing technologies and will not be described in detail here.

[0052] S2. Preparation of the first sample: Mix the blood sample to be tested with the hemolytic agent and staining agent in the first reagent, and incubate at 35-39℃ for 40-60s to obtain the first sample. The incubation temperature can be 35℃, 36℃, 37℃, 38℃, or 39℃, and the incubation time can be 40s, 45s, 50s, 55s, or 60s, etc. The mixing ratio of the blood sample to be tested and the first reagent is 1:(10-1000), and can be 1:10, 1:100, 1:500, 1:700, or 1:1000, etc.; the mixing ratio of the hemolytic agent and staining agent is (10-1000):1, and can be 10:1, 200:1, 500:1, 800:1, or 1000:1, etc.

[0053] S3. Detect the first sample: Perform forward scattering and fluorescence detection on the first sample to obtain the forward scattering intensity (FSC) and lateral fluorescence intensity (SFL), and count the abnormal lymphocytes in the first sample. Specifically, count the proportion of abnormal lymphocytes among normal mature white blood cells based on the two-dimensional scatter plot of forward scattering intensity-lateral fluorescence intensity.

[0054] S4. Preparation of the second sample: Mix the blood sample to be tested with the hemolysin, staining agent, and antibody treatment agent in the second reagent, and incubate at 35-39℃ for 40-60s to obtain the second sample. The incubation temperature can be 35℃, 36℃, 37℃, 38℃, or 39℃, and the incubation time can be 40s, 45s, 50s, 55s, or 60s, etc. The mixing ratio of the blood sample to be tested and the second reagent is 1:(10-1000), and can be 1:10, 1:100, 1:500, 1:700, 1:1000, etc. The mixing ratio of hemolytic agent, staining agent, CD13 antibody treatment agent and CD34 antibody treatment agent is (10-1000):1:1:1, and can be 10:1:1:1, 200:1:1:1, 500:1:1:1, 800:1:1:1, 1000:1:1:1, etc.

[0055] S5. Detection of the second sample: The second sample is subjected to scattered light and fluorescence detection to obtain the forward scattered light intensity (FSC), side scattered light intensity (SSC), side fluorescence intensity (SFL), and antibody fluorescence labeling intensity. Myeloid blast cells in the second sample are counted to determine whether the subject has acute promyelocytic leukemia. The antibody fluorescence labeling intensity includes the fluorescence intensity of the CD13-labeled antibody and the fluorescence intensity of the CD34-labeled antibody.

[0056] Specifically, the proportion of myeloid blast cells among normal mature leukocytes was counted based on two-dimensional scatter plots of forward-scattered light intensity versus lateral fluorescence intensity and two-dimensional scatter plots of lateral-scattered light intensity versus labeled CD13 antibody fluorescence intensity. Based on the two-dimensional scatter plots of labeled CD13 antibody fluorescence intensity versus labeled CD34 antibody fluorescence intensity, a higher fluorescence intensity of labeled CD13 antibody (or labeled CD34 antibody) indicated positive expression of CD13 antibody (or CD34 antibody); otherwise, it was negative. If the second sample showed positive expression of CD13 antibody and negative expression of CD34 antibody, the subject could be diagnosed with APL.

[0057] In some embodiments, if the fluorescence emission wavelengths of the staining agent, CD13 antibody treatment agent, and CD34 antibody treatment agent are different, these three reagents can be used simultaneously. That is, the second reagent includes a hemolysin, staining agent, CD13 antibody treatment agent, and CD34 antibody treatment agent, which can be used to detect abnormal lymphocytes, myeloid blast cells, and acute promyelocytic cells.

[0058] In other embodiments, if the fluorescence emission of the nucleic acid dye in the staining agent and the fluorescently labeled dye in the CD34 antibody treatment agent overlaps, the test kit includes two second reagents, denoted as Second Reagent A and Second Reagent B. Second Reagent A includes a hemolysin, a staining agent, and a CD13 antibody treatment agent, while Second Reagent B includes a hemolysin, a CD13 antibody treatment agent, and a CD34 antibody treatment agent. Second Reagent A is used to detect myeloid blast cells, and Second Reagent B is used to detect acute promyelocytic cells to determine whether the subject has acute promyelocytic leukemia.

[0059] This invention utilizes nucleic acid fluorescence staining and semiconductor laser flow cytometry to detect abnormal lymphocytes in peripheral blood, and employs immunofluorescence technology to detect myeloid blast cells using fluorescently labeled CD13 monoclonal antibodies. Based on this, if a certain amount of myeloid blast cells are detected, fluorescently labeled CD13 and CD34 monoclonal antibodies are further used to diagnose whether acute promyelocytic leukemia (APL) is present. If CD13 expression is positive but CD34 expression is negative, the subject can be diagnosed with APL. Therefore, the detection kit and method of this invention can detect abnormal lymphocytes and myeloid blast cells, and can also differentiate acute promyelocytic leukemia.

[0060] In this invention, the hemolytic agent has a weak effect on the cell membrane of primitive cells, which is beneficial for antibody binding to cell surface antigens. However, it can perforate the membranes of mature leukocytes and abnormal lymphocytes, facilitating the staining of nucleic acid substances in these cells by nucleic acid dyes. The nucleic acid dye solution has difficulty penetrating primitive cells to stain their nucleic acid substances, resulting in relatively low lateral fluorescence intensity in primitive cells. In contrast, abnormal lymphocytes differ from normal mature leukocytes in their nucleic acid content, leading to relatively high lateral fluorescence intensity in abnormal lymphocytes, thus distinguishing them from other cells. Furthermore, since myeloid primitive cells express CD13 antigen strongly, fluorescently labeled CD13 monoclonal antibodies can differentially detect myeloid primitive cells. Since the surface CD34 antigen of primitive cells in APL patients is negative, simultaneous treatment with fluorescently labeled CD13 monoclonal antibodies and different fluorescently labeled CD34 monoclonal antibodies, if CD13 is positive but CD34 is negative, a preliminary diagnosis of APL can be made. The detection kit and detection method based on the present invention can detect abnormal lymphocytes and primitive cells, distinguish whether the primitive cells are myeloid or lymphoid, and determine whether the subtype of acute myeloid leukemia patients is M3.

[0061] The following is an illustration through specific examples:

[0062] The detection kits of Examples 1-1 to 1-3 were prepared according to the detection kits of abnormal lymphocytes, myeloid blast cells and acute promyelocytic leukemia of the present invention. The detection kits include a first reagent and a second reagent. The first reagent includes a hemolytic agent and a staining agent. The second reagent includes a hemolytic agent, a staining agent and an antibody treatment agent. The antibody treatment agent includes a CD13 antibody treatment agent and a CD34 antibody treatment agent. The components and contents of the hemolytic agent, the staining agent and the antibody treatment agent are shown in Table 1.

[0063] Table 1. Components and contents of hemolytic agents, staining agents, and antibody treatment agents in Examples 1-1 to 1-3.

[0064]

[0065] In the table, " / " indicates that it does not contain.

[0066] Example 2

[0067] A method for detecting abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia (APIL) is disclosed. The method utilizes the detection kit for these cells and employs flow cytometry technology with a hematology analyzer to analyze blast cells and abnormal lymphocytes. The detection method includes the following steps:

[0068] S1. Obtaining the blood sample to be tested: Blood is collected from the subject and processed to obtain the blood sample to be tested.

[0069] S2. Preparation of the first sample: Mix the blood sample to be tested with the hemolytic agent and staining agent in the first reagent, and incubate at 37°C for 50 seconds to obtain the first sample. The amount of hemolytic agent used is 1000 μL, the amount of staining agent is 50 μL, and the amount of blood sample to be tested is 15 μL.

[0070] S3. Detect the first sample: Perform forward scattering and fluorescence detection on the first sample to obtain the forward scattering intensity (FSC) and lateral fluorescence intensity (SFL), and count the abnormal lymphocytes in the first sample. Specifically, count the proportion of abnormal lymphocytes among normal mature white blood cells based on the two-dimensional scatter plot of forward scattering intensity-lateral fluorescence intensity.

[0071] S4. Preparation of the second sample: Mix the blood sample to be tested with the hemolytic agent, staining agent, and antibody treatment agent in the second reagent, and incubate at 37°C for 50 seconds to obtain the second sample. The amount of hemolytic agent used is 1000 μL, the amount of staining agent is 50 μL, the amount of CD13 antibody treatment agent is 10 μL, the amount of CD34 antibody treatment agent is 10 μL, and the amount of blood sample to be tested is 15 μL.

[0072] In some embodiments, the second reagent includes a second reagent A and a second reagent B. 15 μL of the blood sample to be tested is mixed with 1000 μL of hemolysin, 50 μL of staining agent and 10 μL of CD13 antibody treatment agent of the second reagent A to obtain the second sample A; 15 μL of the blood sample to be tested is mixed with 1000 μL of hemolysin, 10 μL of CD13 antibody treatment agent and 10 μL of CD34 antibody treatment agent of the second reagent B to obtain the second sample B.

[0073] S5. Detection of the Second Sample: The second sample is subjected to scattered light and fluorescence detection to obtain the forward scattered light intensity (FSC), side scattered light intensity (SSC), side fluorescence intensity (SFL), and antibody fluorescence labeling intensity. Myeloid blast cells in the second sample are counted, and the presence of acute promyelocytic leukemia (APLE) in the subject is determined. The antibody fluorescence labeling intensity includes the fluorescence intensity of CD13-labeled antibodies and CD34-labeled antibodies. Based on the two-dimensional scatter plots of forward scattered light intensity versus side fluorescence intensity and side scattered light intensity versus CD13-labeled antibody fluorescence intensity, the proportion of myeloid blast cells among normal mature white blood cells is counted. Based on the two-dimensional scatter plot of CD13-labeled antibody fluorescence intensity versus CD34-labeled antibody fluorescence intensity, the presence of APLE in the subject is determined.

[0074] In steps S3 and S5, the WPC channel of the DH800CS hematology analyzer (Shenzhen Dimai Biotechnology Co., Ltd.) was used to detect the first and second samples.

[0075] Verification experiment:

[0076] According to the detection method for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia in Example 2, the detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia of the present invention was used to process and detect normal blood samples, blood samples containing abnormal lymphocytes, blood samples without abnormal lymphocytes but containing myeloid blast cells, blood samples from APL patients, and blood samples from patients with other subtypes of AML (except M3 AML), respectively, to verify whether the detection kit and detection method of the present invention can correctly detect abnormal lymphocytes, myeloid blast cells, and determine acute promyelocytic leukemia.

[0077] (1) Detection of abnormal lymphocytes

[0078] Normal blood samples and blood samples containing abnormal lymphocytes were tested. The abnormal lymphocyte-containing blood samples were then counted under a microscope. The counting results of this invention were compared with the microscopic counting results to verify whether the detection kit and method of this invention can accurately detect abnormal lymphocytes. The microscopic examination method involves preparing a smear using bone marrow fluid, highlighting the shape of the cell nucleus, chromatin distribution, and cytoplasmic contents through Wright staining, and then observing the proportion of abnormal lymphocytes among nucleated white blood cells under a microscope, counting at least 200 white blood cells.

[0079] Test results as follows Figure 1 As shown in the figures, a) is a schematic diagram of the distribution areas of various cell types in the FSC-SFL two-dimensional scatter plot; b) is a two-dimensional scatter plot of FSC-SFL for a normal blood sample, showing no abnormal lymphocytes; c) is a two-dimensional scatter plot of FSC-SFL for a blood sample containing abnormal lymphocytes, showing that their lateral fluorescence intensity is significantly higher than that of normal cells. Counting revealed that the number of abnormal lymphocytes accounted for 2.5% of the total number of normal mature white blood cells, with a microscopic count of 2.9%; d) is a two-dimensional scatter plot of FSC-SFL for another blood sample containing abnormal lymphocytes, showing that the number of abnormal lymphocytes accounted for 6.9% of the total number of normal mature white blood cells, with a microscopic count of 8.1%. It is evident that the counting results and microscopic results of this invention are close, indicating that the detection kit and detection method of this invention can accurately detect abnormal lymphocytes.

[0080] (2) Detection of myeloid blast cells

[0081] Normal blood samples and blood samples containing myeloid blast cells but without abnormal lymphocytes were tested. Blood samples containing myeloid blast cells but without abnormal lymphocytes were also examined and counted under a microscope. The counting results of this invention were compared with the microscopic counting results to verify whether the detection kit and method of this invention can correctly detect myeloid blast cells. The microscopic examination method is the same as described above and will not be repeated here.

[0082] Test results as follows Figure 2 As shown in the figures, CD13 refers to the fluorescence intensity of the CD13-labeled antibody, and CD34 refers to the fluorescence intensity of the CD34-labeled antibody. Figure a) shows the distribution areas of various cell types in the FSC-SFL two-dimensional scatter plot (left) and SSC-CD13 two-dimensional scatter plot (right). Figure b) shows the FSC-SFL two-dimensional scatter plot (left) and SSC-CD13 two-dimensional scatter plot (right) of a normal blood sample, showing neither abnormal lymphocytes nor myeloid blast cells. Figure c) shows the FSC-SFL two-dimensional scatter plot (left) and SSC-CD13 two-dimensional scatter plot (right) of a blood sample without abnormal lymphocytes but containing myeloid blast cells. The figure shows that this blood sample has no abnormal lymphocytes, and the CD13 fluorescence intensity is significantly higher than that of normal cells, indicating that the blood sample contains myeloid blast cells. Counting shows that their number accounts for 7.6% of the total number of normal mature white blood cells, and the microscopic count result is 8.4%. d) The figure shows the FSC-SFL two-dimensional scatter plot (left) and SSC-CD13 two-dimensional scatter plot (right) of another blood sample without abnormal lymphocytes but containing myeloid blasts. As can be seen from the figure, this blood sample does not contain abnormal lymphocytes but contains myeloid blasts. Counting shows that the number of myeloid blasts accounts for 33.7% of the total number of normal mature white blood cells, while the microscopic count result is 32.9%. It is evident that the counting results and microscopic results of this invention are close, indicating that the detection kit and detection method of this invention can accurately detect myeloid blasts.

[0083] (3) Diagnosis of acute promyelocytic leukemia

[0084] The test kits were used to test normal blood samples, blood samples from APL patients, and blood samples from other subtypes of AML patients to determine whether CD13 and CD34 expression was positive or negative. This allowed the cells in the blood samples to be identified as normal mature white blood cells, acute promyelocytic cells, or myeloid blast cells. By comparing the test results of different blood samples, the test kits and test methods of the present invention can be verified to correctly identify acute promyelocytic leukemia.

[0085] Test results as follows Figure 3As shown in the figure, CD13 refers to the fluorescence intensity of the CD13-labeled antibody, and CD34 refers to the fluorescence intensity of the CD34-labeled antibody. A high fluorescence intensity of the CD34-labeled antibody indicates that the cell binds a large amount of CD34 antibody, further indicating that the cell highly expresses CD34. Similarly, a high fluorescence intensity of the CD13-labeled antibody indicates that the cell positively expresses CD13. a) The figure is a schematic diagram of the distribution areas of various cell types in a two-dimensional scatter plot of CD13-CD34; b) The figure is a two-dimensional scatter plot of CD13-CD34 in a normal blood sample, showing normal mature white blood cells without acute promyelocytes or myeloid blast cells. c) The figure is a two-dimensional scatter plot of CD13-CD34 in a blood sample from an APL patient. As can be seen from the figure, the particles in the acute promyelocyte region have a high CD13 fluorescence intensity, indicating that the particle is positive for CD13, but the CD34 fluorescence intensity is low, indicating that the particle is negative for CD34. Based on this, the particle can be identified as an acute promyelocyte, indicating acute promyeloid leukemia, i.e., M3 type AML. d) The figure shows a two-dimensional scatter plot of CD13-CD34 in blood samples from other subtypes of AML. It can be seen that the fluorescence intensity of CD13 and CD34 in the myeloid blast cell region is high, indicating that these cells are positive for both CD13 and CD34, proving that these cells are myeloid blast cells, and thus not M3 type AML. Therefore, the detection kit and method of this invention can accurately determine whether a patient with acute myeloid leukemia has the M3 subtype, i.e., whether they have acute promyelocytic leukemia.

[0086] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0087] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia, characterized in that, It includes a first reagent and a second reagent; the first reagent includes a hemolytic agent and a staining agent; the second reagent includes a hemolytic agent, a staining agent, and an antibody treatment agent, wherein the antibody treatment agent includes a CD13 antibody treatment agent and a CD34 antibody treatment agent; The hemolytic agent, based on a total volume of 1 L, comprises the following components: 0.1-10 g of nonionic surfactant, 0.01-10 g of cationic surfactant, 0.5-15 g of buffer, 0.01-8 g of preservative, with the remainder being water. The pH value of the hemolytic agent is 5-9. The nonionic surfactant is a castor oil polyoxyethylene ether nonionic surfactant with the structural formula: Ricinoleic Acid-O-(CH2CH2O). n In the formula, n = 10 to 90; the staining agent, based on a total volume of 1L, includes the following components: 0.001-0.2g nucleic acid dye, the remainder being solvent; The CD13 antibody treatment agent, with a total volume of 1L, comprises the following components: 1-50mg of fluorescently labeled CD13 antibody, with the remainder being antibody diluent; the CD34 antibody treatment agent, with a total volume of 1L, comprises the following components: 1-100mg of fluorescently labeled CD34 antibody, with the remainder being antibody diluent; the antibody diluent, with a total volume of 1L, comprises the following components: 0.5-15g of buffer, 0.01-8g of preservative, with the remainder being water.

2. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The cationic surfactant includes at least one alkyl quaternary ammonium salt, wherein the alkyl quaternary ammonium salt is decaalkyltrimethylammonium bromide, dodecyltrimethylammonium bromide, or hexadecyltrimethylammonium chloride.

3. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The nucleic acid dye is a compound with the following structure: In the formula, R1 and R2 are C1-C6 alkyl, C1-C6 alkoxy, acyl or hydrogen atoms, and X is a halogen atom, boron halide or phosphorus compound.

4. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 3, characterized in that, The nucleic acid dye is prepared from raw material A, compound R1X, raw material B, compound R2X, and N,N-dimethylformamide. Raw material A is a compound with the following structure: The raw material B is a compound with the following structure: R1 and R2 are both C1-C6 alkyl groups, C1-C6 alkoxy groups, acyl groups, or hydrogen atoms, and X is a halogen atom, boron halide, or phosphorus compound.

5. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, Both the fluorescently labeled CD13 antibody and the fluorescently labeled CD34 antibody contain a fluorescent labeling dye, which is fluorescein isothiocyanate, a cyanine fluorescent dye, or an Alexa Fluor series dye, and the cyanine fluorescent dye is Cy3 dye, Cy5 dye, or Cy7 dye.

6. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The buffer is HEPES buffer, MOPS buffer, citrate buffer, or phosphate buffer.

7. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The preservative is benzoic acid and its salts, isothiazolinone, sodium hydroxymethylglycinate, phenoxyethanol or benzyl alcohol.

8. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The hemolytic agent also includes 0-20g of an osmotic pressure regulator, which is an inorganic salt, sugar, or amino acid; the sugar is at least one of glucose, fructose, and mannitol; the inorganic salt includes at least one of sodium sulfate, sodium chloride, and potassium chloride; and the amino acid includes at least one of glycine, valine, and alanine.

9. The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to claim 1, characterized in that, The hemolytic agent also includes 0.01-50g of a chelating agent, wherein the chelating agent is sodium ethylenediaminetetraacetate or potassium ethylenediaminetetraacetate.

10. A method for detecting abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia, characterized in that, The detection kit for abnormal lymphocytes, myeloid blast cells, and acute promyelocytic leukemia according to any one of claims 1 to 9, wherein the detection method includes the following steps: S1. Obtaining the blood sample to be tested: Blood is collected from the subject and processed to obtain the blood sample to be tested; S2. Preparation of the first sample: The blood sample to be tested is mixed with the hemolytic agent and the staining agent in the first reagent, and incubated at 35-39℃ for 40-60s to obtain the first sample; the mixing ratio of the blood sample to be tested to the first reagent is 1:(10-1000), and the mixing ratio of the hemolytic agent to the staining agent is (10-1000):

1. S3. Detect the first sample: Perform scattered light detection and fluorescence detection on the first sample to obtain the forward scattered light intensity and the lateral fluorescence intensity, and count the abnormal lymphocytes in the first sample. S4. Preparation of the second sample: The blood sample to be tested is mixed with the hemolytic agent, staining agent and antibody treatment agent in the second reagent, and incubated at 35-39℃ for 40-60s to obtain the second sample; the mixing ratio of the blood sample to be tested and the second reagent is 1:(10-1000), and the mixing ratio of the hemolytic agent, the staining agent and the antibody treatment agent is (10-1000):1:1; S5. Detect the second sample: Perform scattered light detection and fluorescence detection on the second sample to obtain the forward scattered light intensity, side scattered light intensity, side fluorescence intensity and antibody fluorescence labeling intensity, count the myeloid blast cells in the second sample, and determine whether the subject has acute promyelocytic leukemia.

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