A reagent composition for detecting acute promyelocytic leukemia and its application

Abstract

The present invention provides a reagent composition for detecting acute promyelocytic leukemia and its application. The reagent composition comprises seven groups of antibodies and is a flow cytometric assay composition that can be used for screening and / or diagnosing acute promyelocytic leukemia. When used, a five-tube parallel assay is employed, wherein the first, second, and third groups of antibodies are used in different tubes of samples, the fourth and sixth groups of antibodies are used in the same tube of samples, and the fifth and seventh groups of antibodies are used in the same tube of samples. The reagent composition of the present invention can be used for flow cytometric screening of acute promyelocytic leukemia.

Description

Technical Field

[0001] The present invention relates to a reagent composition for rapid and efficient screening of acute promyelocytic leukemia and application thereof, belonging to the technical field of blood disease detection. Background Art

[0002] Acute promyelocytic leukemia (APL) is a specialized acute leukemia. The pathogenesis of typical cases is a balanced reciprocal translocation between chromosomes 15 and 17, termed t(15;17). This site fuses the promyelocytic (PML) gene on chromosome 15 to the retinoic acid receptor α (RARa) gene on chromosome 17, leading to the development of the disease, characterized by unique morphological and immunophenotypic characteristics. APL is a rare subtype of acute myeloid leukemia (AML), accounting for approximately 10% of all AML cases. It is often accompanied by disseminated intravascular coagulation (DIC), a consumptive coagulopathy. If promptly treated within 24 hours, the complete remission rate can reach over 94%. Therefore, APL is a rare emergency requiring urgent treatment in hematology departments, which also poses significant challenges for blood testing.

[0003] Typical cases of APL have a distinct immature cellular morphology, described as abnormal promyelocytes. Nuclei are characteristically reniform, lobed, or butterfly-shaped, with abundant cytoplasm consisting of inner and outer cytoplasms, the inner cytoplasm being filled with azure-philic granules. In some cells, the cytoplasm is filled with fine, dust-like particles. Numerous Oer bodies are present, giving the cells a faggot-like appearance. Cytochemical staining is strongly positive for peroxidase. The immunophenotype also exhibits characteristic changes, such as expression of myeloid markers such as CD117, CD13, CD33, MPO, and CD64, while expression of early markers CD34, HLA-DR, and myeloid differentiation markers CD11b and CD14 is absent. Side-angle light scatter (SSC) is also elevated. Therefore, typical cases are relatively easy to identify. However, approximately 20% of cases may show atypical changes: such as morphological hypogranularity (fine granular type) (APLv), with flow cytometry immunophenotyping showing decreased SSC, positive CD34, and decreased CD117; or APL with increased basophils or basophil changes (basophilic APL), with flow cytometry immunophenotyping showing weak expression of CD117 / weak expression of CD11b similar to basophils in areas with high CD45 / low SSC (basophil areas with CD45 / SSC gates). For a population of granulocytes, if this type of cells is the main one, the morphology may lead to misdiagnosis because the cell body becomes smaller, the inner and outer plasma is not obvious, and the coarse basophils cover the cell nucleus. However, the difference from basophils is that the immunophenotype of this group of cells is MPO positive, the background is high, and CD22 is negative. If CD25 is further tested, CD123 is weakly to moderately expressed. Typical basophils are MPO negative, with a low background and weak CD22 expression. If CD25 is further tested, CD123 is strongly expressed. More importantly, approximately 10% to 20% of non-APL AML (especially those characterized by genetic NPM1 mutations or KMT2A rearrangements) can also exhibit morphological and immunophenotypic changes similar to APL (APL-like). APL-like refers to a group of AMLs that exhibit morphological and / or flow cytometric immunophenotyping similar to typical APL or APLv, leading to a high risk of clinical misdiagnosis. These cases are most commonly seen in AML with NPM1 mutations and KMT2A rearrangements. For example, flow cytometric immunophenotyping often reveals an APL-like pattern of CD34- / HLA-DR- / CD117+ / MPO+ / CD33+ / CD11b-, with a 30% to 40% probability of CD9 positive. Furthermore, morphologically, these cases often exhibit granularity, strong MPO positivity, and nuclear notching, resulting in a "butterfly nucleus" resembling that of APL. Consequently, these cases are highly susceptible to misdiagnosis. However, the emergency treatment and prognosis of these diseases are completely different. If APL-like treatment is ineffective, chemotherapy or even transplantation should be performed according to typical AML.Therefore, the clinical differentiation of APL and APL-like poses a great challenge to clinical testing.

[0004] To quickly and accurately screen for APL, researchers have conducted research and exploration. Literature has reported that CD34 is negative, HLA-DR is negative, CD117 is positive, CD9 is positive, CD33 is strongly expressed, CD64 is positive, MPO is positive, CD11b is negative, CD11c is negative, and some cases are CD2 positive. These indicators are used to differentiate between APL and non-APL cases, and there is a statistical difference. However, existing APL screening technologies generally focus on screening for typical APL, and rarely study APLv, the subtype that is most in need of differentiation from other types of acute leukemia. Furthermore, they focus on comparing APL with common non-APL, while avoiding approximately 10% to 20% of special non-APL types, namely APL-like AML.

[0005] In view of the above reasons, to date, there is an urgent need for a method that can quickly and accurately screen APL in clinical practice, especially for newly diagnosed patients, who urgently need a universal, rapid and efficient solution for detecting APL. Summary of the Invention

[0006] One object of the present invention is to provide a reagent composition that can quickly and efficiently screen APL, especially including all variants, and distinguish APL like and other AML based on morphology and immunophenotyping to prevent misdiagnosis.

[0007] Another object of the present invention is to provide the use of the reagent composition in rapid and efficient screening of acute promyelocytic leukemia.

[0008] In one aspect, the present invention provides a reagent composition comprising a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, a sixth group of antibodies, and a seventh group of antibodies, wherein:

[0009] The first group of antibodies includes CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody, which are added to flow cytometry tube 1 in which the sample to be tested is in a single cell suspension state;

[0010] The second group of antibodies includes CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, HLA-DR antibody, CD11b antibody and CD45 antibody, which are added to the flow cytometry tube 2 in which the sample to be tested is in a single cell suspension state;

[0011] The third group of antibodies includes CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody and CD45 antibody, which are added to the flow cytometry tube 3 in which the sample to be tested is in a single cell suspension state and incubated with phosphate buffer and the supernatant is removed;

[0012] The fourth group of antibodies includes CD22 antibody, CD34 antibody, CD117 antibody, CD38 antibody and CD45 antibody, which are added to the flow cytometry tube 4 in which the sample to be tested is in a single cell suspension state;

[0013] The fifth group of antibodies includes CD64 antibody, CD34 antibody, CD117 antibody, CD42a antibody, CD36 antibody, CD14 antibody and CD45 antibody, which are added to flow cytometry tube 5 in which the sample to be tested is in a single cell suspension state and incubated with phosphate buffer and the supernatant is removed;

[0014] The sixth group of antibodies includes nuclear TdT antibody and cytoplasmic CD3 antibody, which are added to the flow cytometry tube 4 after the fourth group of antibodies have been added and the cells have been lysed;

[0015] The seventh group of antibodies includes cytoplasmic MPO antibodies, which are added to flow cytometry tube 5 after the fifth group of antibodies have been added and the cells have been lysed.

[0016] The reagent composition of the present invention can be applied to rapid and efficient flow cytometry screening for acute promyelocytic leukemia. In specific applications, a 5-tube parallel scheme is used, and each tube uses CD45 and / or myeloid primitive and immature cell antibodies CD117 in combination with SSC to set myeloid cell and primitive immature cell gates, and at the same time, CD45 / SSC is used to set control cell populations such as mature lymphocytes, mature granulocytes, and monocyte gates. If the ratio of mature granulocytes and monocytes is low, or the gate is unclear because the CD45 / SSC area overlaps with tumor cells, relevant markers such as CD4, CD16, CD11b, HLA-DR, CD371, CD15, CD11c, CD38, CD64, CD36, CD14, etc. are used to assist in setting gates, thereby accurately evaluating, especially using quantitative evaluation, improving detection accuracy, and being more objective, forming standardization, reducing misdiagnosis rate, and providing convenience for future automation and artificial intelligence.

[0017] According to a specific embodiment of the present invention, in the reagent composition of the present invention, each antibody is a monoclonal antibody.

[0018] According to a specific embodiment of the present invention, in the antibody composition of the present invention, each antibody is a fluorescein-labeled antibody. Preferably, in the first group of antibodies, the order of fluorescein labeling of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605. In the second group of antibodies, the order of fluorescein labeling of CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, anti-HLA-DR antibody, CD11b antibody and CD45 antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421 and V500. In the third group of antibodies, the order of fluorescein labeling for CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody and CD45 antibody was FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, BV421 and V500; in the fourth group of antibodies, the order of fluorescein labeling for CD22 antibody, CD34 antibody, CD117 antibody, CD38 antibody and CD45 antibody was PE, PerCP-Cy5.5, PE-Cy7, APC, BV421 and V500. 21 and V500; in the fifth group of antibodies, the order of fluorescein labeling of CD64 antibody, CD34 antibody, CD117 antibody, CD42a antibody, CD36 antibody, CD14 antibody and CD45 antibody is PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421 and V500; in the sixth group of antibodies, the order of fluorescein labeling of nuclear TdT antibody and cytoplasmic CD3 antibody is FITC and APC; in the seventh group of antibodies, the fluorescein labeling of cytoplasmic MPO antibody is FITC.

[0019] In the present invention, by combining different antibodies with specific fluoresceins, the reagent composition of the present invention can be used for rapid and efficient screening of acute promyelocytic leukemia, and all fluoresceins in each channel can achieve excellent staining effects.

[0020] According to a specific embodiment of the present invention, each antibody component in the reagent composition of the present invention can be commercially obtained and each antibody should comply with relevant industry standards.

[0021] According to a specific embodiment of the present invention, in the reagent composition of the present invention, the first group of antibodies is a mixture of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3. The second group of antibodies is a mixture of CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, anti-HLA-DR antibody, CD11b antibody and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3:3. The third group of antibodies is a mixture of CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3. The fourth group of antibodies is a mixture of CD22 antibodies, CD34 antibodies, CD117 antibodies, CD38 antibodies, and CD45 antibodies in a volume ratio of 5:5:3:3:3. The fifth group of antibodies is a mixture of CD64 antibodies, CD34 antibodies, CD117 antibodies, CD42a antibodies, CD36 antibodies, CD14 antibodies, and CD45 antibodies in a volume ratio of 5:5:3:2:3:3:3. The sixth group of antibodies is a mixture of nuclear TdT antibodies and cytoplasmic CD3 antibodies in a volume ratio of 2:2. The mixing ratios of the above antibodies refer to the mixing ratios when the titers of the antibodies are basically equivalent.

[0022] Another aspect of the present invention provides a kit comprising a first container, a second container, a third container, a fourth container, a fifth container, a sixth container, and a seventh container, each container respectively containing a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, a sixth group of antibodies, and a seventh group of antibodies of the reagent composition of the present invention.

[0023] According to specific embodiments of the present invention, the kit of the present invention may further include: one or more of: a hemolysin, a membrane permeabilizing agent, a buffer, and a flow cytometer for use with a flow cytometer. These reagents and consumables are commercially available. The membrane permeabilizing agent is preferably a membrane permeabilizing agent comprising Solution A and Solution B. Each reagent material may be housed in a separate container.

[0024] The kit of the present invention can be used for rapid and efficient screening of acute promyelocytic leukemia.

[0025] Another aspect of the present invention also provides the use of the reagent composition in preparing a flow cytometer sample for a reagent composition for rapid and efficient screening of acute promyelocytic leukemia.

[0026] According to a specific embodiment of the present invention, the process of preparing a flow cytometer sample for rapid and efficient screening of acute promyelocytic leukemia comprises the following steps:

[0027] (1) Add the sample to be tested into flow cytometry tubes 1 to 5 (also called tubes A, B, C, D, and E) respectively, to form a single cell suspension and ensure that the cell volume is 1×10 6 / tube-1×10 7 / Tube;

[0028] (2) Add phosphate buffer to tubes 3 and 5, mix well, incubate at 37°C, and centrifuge to remove the supernatant;

[0029] (3) adding the first group of antibodies in the reagent composition of the present invention to tube 1 obtained by the treatment in step (1), adding the second group of antibodies in the reagent composition of the present invention to tube 2 obtained by the treatment in step (1), adding the third group of antibodies in the reagent composition of the present invention to tube 3 obtained by the treatment in step (2), adding the fourth group of antibodies in the reagent composition of the present invention to tube 4 obtained by the treatment in step (1), and adding the fifth group of antibodies in the reagent composition of the present invention to tube 5 obtained by the treatment in step (2), and incubating each flow tube at room temperature in the dark;

[0030] (4) Add membrane permeabilization agent solution A to tubes 4 and 5 after incubation in step (3) and continue incubation at room temperature in the dark;

[0031] (5) Add 1× hemolysin to tubes 1, 2, and 3 after incubation in step (3) and tubes 4 and 5 after incubation in step (4), and continue incubation at room temperature in the dark;

[0032] (6) Centrifuge each flow cytometry tube after incubation in step (5) and remove the supernatant;

[0033] (7) Add membrane permeabilization agent B solution and the sixth group of antibodies in the reagent composition of the present invention to tube 4 after removing the supernatant in step (6), and add membrane permeabilization agent B solution and the seventh group of antibodies in the reagent composition of the present invention to tube 5 after removing the supernatant in step (6), and incubate at room temperature in the dark;

[0034] (8) PBS buffer was added to tubes 1, 2, and 3 after the supernatant was removed in step (6), and tubes 4 and 5 after the incubation in step (7), respectively, for washing. After centrifugation, the supernatant was removed and the cells were resuspended in PBS buffer to obtain flow cytometry samples.

[0035] In the present invention, the description of the operating steps does not limit the actual operation order of these steps unless otherwise specified or the existence of a sequence relationship can be clearly determined from the description in the context.

[0036] According to a specific embodiment of the present invention, in the present invention, the sample to be tested can be bone marrow or peripheral blood, and can also be used for tissue specimens, body fluid specimens, and all other samples that can be prepared into single living cells and are suitable for flow cytometry detection.

[0037] According to a specific embodiment of the present invention, in step (1), the volume of sample added to each tube does not exceed 160 μl (if the patient has a small amount of peripheral blood cells, a volume exceeding 160 μl may be added as needed, and the supernatant may be centrifuged and concentrated).

[0038] According to a specific embodiment of the present invention, the dosage of each reagent can refer to the conventional dosage in the prior art or follow the recommended dosage of the merchant.

[0039] According to a specific embodiment of the present invention, the reagent composition of the present invention, the added amount of the first group of antibodies is 16-64 μl / tube, the added amount of the second group of antibodies is 15-58 μl / tube, the added amount of the third group of antibodies is 13-52 μl / tube, the added amount of the fourth group of antibodies is 10-38 μl / tube, the added amount of the fifth group of antibodies is 12-48 μl / tube, the added amount of the sixth group of antibodies is 2-8 μl / tube, and the added amount of the seventh group of antibodies is 3-10 μl / tube.

[0040] According to a specific embodiment of the present invention, the operation of step (2) above can be selectively repeated one or more times as needed. According to a specific embodiment of the present invention, the amount of PBS added each time is 2-3 ml / tube, and the centrifugation conditions can be 1000-2000 rpm (or 300-450g) for 5 minutes.

[0041] According to a specific embodiment of the present invention, the incubation time in step (2) may be 5-30 minutes.

[0042] According to a specific embodiment of the present invention, the incubation time in step (3) may be 10-30 minutes.

[0043] According to a specific embodiment of the present invention, in step (4), the incubation time can be 5-20 minutes. The amount of membrane permeabilization agent A solution added can be based on the manufacturer's recommended dosage, usually 100 μl / tube.

[0044] According to a specific embodiment of the present invention, in step (5), the incubation time may be 5-30 minutes. The amount of 1× hemolysin added is 2-3 ml / tube.

[0045] According to a specific embodiment of the present invention, in step (6), the incubation period is generally about 10-30 minutes. The centrifugation condition may be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0046] According to a specific embodiment of the present invention, in step (7), the amount of membrane-breaking agent B solution added can be based on the recommended dosage of the merchant, usually 50 μl / tube.

[0047] According to a specific embodiment of the present invention, in step (8), the amount of PBS buffer added for washing is 2-3 ml / tube. The centrifugation conditions may be 1000-2000 rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspension is 0.5-1 ml / tube.

[0048] According to a specific embodiment of the present invention, when resuspending cells for flow cytometry, a deadhesive cell gate P1 and a live cell gate P2 are sequentially set for each tube to obtain single live cells; within the P2 gate, CD45 / SSC is used to set gates for each blood cell; within the P2 gate, either CD45 weak expression / medium to high SSC or CD117 positive / CD45 weak expression is used to set gates for myeloid immature cells (depending on the specific case, the gate method that indicates obvious tumor cells can be selected between the two methods); and a mature lymphocyte gate is set using CD45 strong expression / low SSC; and:

[0049] Tube 1 was gated as follows: within gate P2, CD45 weak / SSC high or SSC high / CD117 negative was used to gate differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD45 strong expression / CD4 weak expression was used to gate monocytes;

[0050] For tube 2, gates were set as follows: within gate P2, CD45 weak / SSC large or CD16 positive / CD11b positive were used to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD11b positive / HLA-DR positive were used to set the gate for monocytes;

[0051] For tube 3, gates were set as follows: within gate P2, use weak CD45 / high SSC or strong CD15 expression / CD371 positivity to set the gate for differentiation-stage granulocytes, and strong CD45 expression / intermediate SSC or positive CD371 / weak CD15 expression / CD11c positivity to set the gate for monocytes;

[0052] For tube 4, set gates as follows: within gate P2, use CD45 weak / SSC high or SSC high / CD117 negative to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD45 strong expression / CD38 positive to set the gate for monocytes;

[0053] For tube 5, set gates as follows: within gate P2, use CD45 weak / SSC high or MPO positive / CD64 positive to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD64 strong expression / CD36 positive to set the gate for monocytes.

[0054] In the present invention, the expression of CD45 refers to the strong expression of CD45 on mature lymphocytes, strong expression of CD45 on monocytes, weak expression of CD45 on granulocytes, and negative CD45 on nucleated red blood cells, based on normal bone marrow. In the present invention, the moderate expression of CD117 refers to the concentration of positive cells in the middle of the two-dimensional dot plot of CD117 (horizontal axis) / SSC (vertical axis). Figure 2 Strong CD117 expression is shown in the two-dimensional dot plot of CD117 (horizontal axis) / SSC (vertical axis). CD117 positive cells are concentrated in the area with strong expression (e.g., spindle-shaped) Figure 7 Unless otherwise specified, the expression levels of each antigen are based on normal bone marrow cells.

[0055] In the present invention, the SSC size refers to the normal bone marrow as the standard, the SSC of lymphocytes and nucleated red blood cells is small (or "smaller"), the SSC of primitive cells and monocytes is medium, and the SSC of granulocytes is large (or "larger").

[0056] In the detection technology of the present invention, CD371 is used as a marker for detecting APL. CD371 has an expression profile similar to CD33, with relatively high expression rates in both normal myeloid cells and AML cells. The present invention's research has found that, despite being positive for CD371, APL and other AML cells express CD371 in different ways. APL may express CD371 more strongly and consistently due to its unique promyelocyte stage. While almost all other AML cells express CD371, the vast majority express it only in a persistent or partial manner. Therefore, the CD371 expression pattern helps distinguish APL from non-APL.

[0057] In the detection technology of the present invention, CD38 is used as a marker for detecting APL. CD38 is a non-lineage-specific marker and a relatively early marker. Normally, CD38 is primarily expressed in activated mature lymphocytes, plasma cells, and most blasts of various lineages, including myeloid blasts, T blasts, and B blasts. In acute leukemia, because CD38 expression is diminished in cancer stem cells and the intensity of CD38 expression is altered in some tumor cells, CD38 is primarily used for the detection of minimal residual disease and the differentiation of myeloid subtypes. In this application, CD38 is used to detect APL for two main purposes: First, although the vast majority of AML cases express CD38, the present invention has found that almost all non-APL AMLs can find tumor stem cells with strong CD34 expression (bri) / HLA-DR+ / weak CD38 expression (dim). However, APL, whether typical APL or APLv, almost never has tumor stem cells. Therefore, the main function of CD38 is to find tumor stem cells in combination with CD34 and HLA-DR. Second, it can be used to differentiate APL from mast cell leukemia. Mast cell leukemia also expresses CD117, CD33, and CD9, but does not express CD34, HLA-DR, or CD11b, and has a relatively high SSC. If only these markers are used, it is difficult to distinguish. However, APL cases have a probability of expressing CD38 of over 80%, while mast cell leukemia does not express CD38.

[0058] In the detection technology of the present invention, cytoplasmic CD3 (cCD3) is used as a marker for distinguishing APL, particularly APLv. Determining the lineage of acute leukemia is crucial for the diagnosis of acute leukemia and is directly related to the treatment of leukemia patients. The most recognized diagnostic standard in the industry is the WHO diagnostic criteria, which use a single cCD3 antibody to determine T lineage composition. Even with increased expression intensity requirements, cCD3 expression in acute T lymphoblastic leukemia and mixed leukemias often fails to meet the WHO requirements. Commonly used flow cytometry for cCD3 positivity requires an expression rate exceeding 10% of the tumor cell population, making APL easily misdiagnosed as acute mixed cell leukemia (a biphenotypic T / myeloid phenotype). The present invention discovered that cCD3 expression in acute mixed cell leukemia partially exceeds 50% of the mature T level (even with partial expression, at least there is a clear demarcation between negative and positive cells), while cCD3 expression in APL is persistent, weak, and smearing (no demarcation between negative and positive cells is visible).

[0059] In the detection technology of the present invention, CD36 in tube 5 serves as a specific exclusion marker. Under normal circumstances, CD36 is primarily found in erythroid cells, megakaryocytes, and monocytes. In tumors, in addition to the aforementioned tumors such as erythroleukemia, megakaryocytic leukemia, and monocytic leukemia, it can also be found in rare primitive plasmacytoid dendritic cell tumors. CD36 has never been reported in APL, but other non-APL AMLs, particularly monocytic leukemia and megakaryocytic leukemia, may show positive CD36 expression.

[0060] On the other hand, the present invention also provides a device for rapid and efficient screening of acute promyelocytic leukemia, the device comprising a detection unit and an analysis unit, wherein:

[0061] The detection unit includes a reagent material for detecting a sample from a test individual by flow cytometry to obtain a test result of the sample; the reagent material includes the reagent composition of the present invention;

[0062] The analyzing unit is used to analyze the detection result of the detecting unit.

[0063] According to a specific embodiment of the present invention, in a rapid and efficient screening method for acute promyelocytic leukemia, the detection unit comprises: treating a sample to be tested with the reagent composition of the present invention to prepare a flow cytometer sample; and performing flow cytometric testing. The analysis unit comprises: analyzing the test results to quickly and accurately diagnose acute promyelocytic leukemia and exclude other diseases that may cause misdiagnosis, particularly AML with NPM1 gene mutations or KMT2A rearrangements, acute mixed cell leukemia, acute basophilic leukemia, etc.

[0064] According to a specific embodiment of the present invention, when the device of the present invention is used for rapid and efficient screening of acute promyelocytic leukemia, the gating analysis of each flow tube can be performed according to the above-mentioned operation.

[0065] According to a specific embodiment of the present invention, in a device for rapid and efficient screening of acute promyelocytic leukemia of the present invention, the analysis unit may further include a module for further determining the disease based on the gated analysis test results (the determination described in the present invention includes screening, prompting, auxiliary diagnosis, and diagnosis). The present invention compares each displayed cell group with the corresponding normal cells within a multi-marker combination gate to identify tumor cells, perform rapid and accurate diagnosis, and differentiate from other diseases that may be misdiagnosed or missed, especially APL with MPO+ / CD34- / HLA-DR- / CD117+ / CD33+ that are very similar in morphology and immunophenotype. Like, exemplified by AML with NPM1 gene mutations; APL, especially APLv, is characterized by smear expression of cCD3 in some cases, making it easy to misdiagnose as acute mixed cell leukemia (T / myeloid biphenotype) according to WHO diagnostic criteria; APL with basophils can be misdiagnosed as acute basophilic leukemia or other AML with basophilia; in particular, it was discovered that, regardless of the tumor cell phenotype, almost all APLlikes can be found with a very low proportion of tumor stem cells with the CD34bri / HLA-DR+ / CD38dim phenotype, while APL and APLv almost never have these. This discovery not only facilitates rapid and accurate diagnosis but also has the potential to lead to significant breakthroughs in understanding pathogenicity and prognosis, and particularly in treatment. In short, the above-mentioned difficulties can be avoided using this invention.

[0066] According to a specific embodiment of the present invention, when analyzing five tubes of samples, the CD45 / SSC ratio, common to each tube, is used to initially screen for normal cell populations (mature lymphocytes, monocytes, differentiated granulocytes, and nucleated red blood cells) and the presence of a high proportion of tumor cells. This is further combined with other markers in each tube to determine the presence of various diseases. Specifically, the determination of various diseases can be performed as follows:

[0067] (1) Judgment of acute promyelocytic leukemia: In 80% of typical types, the SSC of leukemia cells is relatively large on the CD45 / SSC images, and they consistently express CD64, MPO, CD33, CD371, and CD9, express CD117 with moderate intensity, express CD13 and CD38, and do not express CD34, HLA-DR, CD11b, CD14, and CD36. In about 20% of atypical types (APLv), some (all in a few cases) of the leukemia cells on the CD45 / SSC images have a small SSC. These cells with small SSC express CD34, while the cells with large SSC do not express CD34. That is, one case includes two parts of tumor cells: CD34+ / small SSC and CD34- / large SSC. They consistently express CD64, MPO, CD33, CD371, and CD9, express CD117, CD13, and CD38, and do not express HLA-DR, CD11b, CD14, and CD36.

[0068] (2) Other morphologically and immunophenotypically similar acute myeloid leukemias (APLlike) represented by NPM1 gene mutations: The primitive cells have a smaller SSC on the CD45 / SSC plot, express CD117 (generally consistent expression) and CD33, and differentially express MPO, CD64, CD371, CD13, and CD38 in intensity or proportion. Most cases do not express CD34 and HLA-DR, and most do not express CD9, CD11b, CD14, and CD36.

[0069] (3) cCD3-positive APLv cases are differentiated from acute mixed cell leukemia (T and myeloid biphenotype). The cCD3 intensity of acute mixed cell leukemia partially exceeds 50% of the mature T level (even if it is partially expressed, at least there is a clear boundary between negative and positive cells), while the cCD3 expression of APL is continuous weak expression and smear expression (the boundary between negative and positive cells cannot be seen), and the SSC is large. It often consistently expresses CD64, MPO, CD9, expresses CD117, does not express or partially expresses CD34, and does not express HLA-DR. CD7, CD5, and CD2 have a good auxiliary role. Almost 100% of T lineage components express CD7, 90% express CD5, and 60% express CD2, while APL almost does not express CD7 and CD5, and a few APLv express CD2.

[0070] (4) Rare APL may be CD117 negative. To prevent misdiagnosis of such cases: if the SSC is large and other characteristics are consistent (such as strong and consistent expression of CD9, MPO, CD33, CD64, and CD371, no expression of CD34, HLA-DR, CD11b, and CD36, and no tumor stem cells with the CD34bri / HLA-DR+ / CD38dim phenotype), it should also be suggested that it may be APL.

[0071] (5) Differential diagnosis between APL with basophilic granules and acute basophilic leukemia: APL cells with basophilic granules have basophilic granules that are easily lost during operation, resulting in a low SSC, but a high background, and the presence of other characteristics. The difference between APL cells with basophilic granules and acute basophilic leukemia is that APL cells with basophilic granules are MPO-positive and do not express CD22, while basophils do the opposite, not expressing MPO but weakly expressing CD22. If CD123 and CD25 are added, further differentiation can be achieved. APL cells express CD123 weakly to moderately and do not express CD25; basophils express CD123 strongly and express CD25.

[0072] (6) AML is differentiated. Almost all AML except APL have more or less the earliest myeloid primitive cells (tumor stem cells), with the phenotype of CD34bri / HLA-DR+ / CD38dim. APL does not have tumor stem cells, which may be one of the reasons why APL has a good prognosis.

[0073] (7) Other characteristic manifestations of APL that need to be observed include: APL tumor cells have a large SSC, resulting in the FSC / SSC image being the same as that of differentiated granulocytes (i.e., the characteristic FSC / SSC image of tumor cells and granulocytes has the same center); frustrated expression of CD117 (CD117 expression in the main part of the tumor cells is not at the strongest position, but is moderately to weakly positive, and there are negative parts, resulting in CD117 expression not being a clean center). Non-APL (including APL-like) often shows differences in FSC / SSC images between tumor cells and differentiated granulocytes (two centers); although the expression of CD117 in non-APL (including APL-like) tumor cells may vary from case to case, it is often relatively strong and consistent.

[0074] (8) Based on the immunophenotypes of APL and its subtypes, a scoring system was proposed:

[0075] Table 1: Diagnosis and differential diagnosis of AML in APL and non-APL

[0076]

[0077] The scoring system for the immunophenotype of APL and its subtypes of the present invention can roughly divide each indicator into level 0, level 1, level 2, and level 3 according to weight, and the corresponding coefficients can be 0, 1, 2, and 3, respectively. Specifically, the present invention comprehensively identifies the following characteristics of APL: MPO positivity, HLA-DR negativity, and crossover of CD34 and SSC (80% of typical APL or typical portions of APLv have high CD34- / SSC ratios; some tumor cells in APLv have low CD34+ / SSC ratios). These three criteria are key for diagnosing APL, with 3 points added for each finding and 3 points subtracted for each finding. Absence of tumor stem cells and characteristic FSC / SSC positional expression, both of which have sensitivity and specificity exceeding 90%, are both associated with a 2-point increase for each finding and a 2-point reduction for each finding. Other characteristics, such as moderate CD117 expression, strong CD33 expression, CD9 positivity, CD64 consistency, and CD371 consistency, are subjective or subject to variability, with 1 point added for each finding and 1 point subtracted for each finding. Because cCD3 is limited to specific cases, a positive finding adds 1 point and a negative finding does not subtract points. CD22, involved in differential diagnosis with basophils, is not scored (a score of 0). See Table 1. Using this scoring scale, a score of 12 or higher indicates APL, with the vast majority of APL and APLv cases scoring as high as 18 to 19. Only two cases (0.64%) of rare hereditary types and one case (0.32%) of HLA-DR-positive APL scored 15 and 12, respectively. APL-like scores ranged from -2 to 6, while non-APL scores ranged from -18 to -4. Using this method, sensitivity, specificity, positive predictive value, and negative predictive value all exceeded 99%. This demonstrates that this invention represents an important detection and analysis solution for improving efficiency, reducing costs, and reducing misdiagnosis rates across the entire clinical diagnostic field.

[0078] In summary, when the analysis unit of the present invention is used to analyze the detection result of the detection unit, the detection result can be output according to one of the following judgment methods:

[0079] Method 1:

[0080] Typical type of acute promyelocytic leukemia: Leukemic cells have a large SSC in the CD45 / SSC gate, and consistently express CD64, MPO, CD33, CD371, and CD9, moderately express CD117, express CD13 and CD38, and do not express CD34, HLA-DR, CD11b, CD14, and CD36;

[0081] APLv: CD45 / SSC includes both CD34+ / small SSC and CD34- / large SSC tumor cells, with strong and consistent expression of CD64, MPO, CD33, CD371, and CD9, moderate expression of CD117, expression of CD13 and CD38, and no expression of HLA-DR, CD11b, CD14, and CD36;

[0082] CD117-negative acute promyelocytic leukemia: SSC is larger, with consistent expression of CD9, MPO, CD33, CD64, and CD371, as well as expression of CD13 and CD38, but no expression of CD117, CD34, HLA-DR, CD11b, CD14, or CD36;

[0083] Rare HLA-DR-positive acute promyelocytic leukemia: large SSC, strong and consistent expression of CD9, MPO, CD33, CD64, CD371, moderate expression of CD117, expression of CD13 and CD38, and no expression of CD34, CD11b, CD14, and CD36;

[0084] Method 2:

[0085] MPO positive, HLA-DR negative, CD34 and SSC showed crossover; if each of these three items is met, 3 points will be added, and if not met, 3 points will be subtracted;

[0086] Lack of characteristic expression of tumor stem cells or FSC / SSC positions; 2 points will be added if each of these two items is met, and 2 points will be subtracted if each item is not met;

[0087] CD117 moderate frustration, CD33 strong expression, CD9 positivity, CD64 consistency, CD371 consistency, if each of these five items is met, 1 point will be added, and 1 point will be subtracted if each of these five items is not met;

[0088] cCD3 positive adds 1 point, negative does not reduce points;

[0089] A score of 12 or above is APL.

[0090] In summary, the present invention provides a reagent composition for rapid and efficient screening of acute promyelocytic leukemia and its application. The present invention has the following advantages: ① Based on the understanding of the diagnosis and differential diagnosis of the immune phenotypes of a large number of tumor cases, a comprehensive diagnosis and differential diagnosis scheme is designed to minimize the missed diagnosis and misdiagnosis rates. ② In terms of application, an accurate diagnosis can be made quickly. ③ The present invention adopts a rapid and efficient method for screening acute promyelocytic leukemia, which can pre-mix antibodies, greatly reducing the workload, improving work efficiency, and speeding up the reporting time. Clinical patients can obtain an accurate diagnosis early to take appropriate treatment, thereby improving the remission rate and survival rate. ④ A very important reason that has long restricted the development of flow cytometry is the individualization of the scheme and excessive manual operations, which makes it difficult to achieve automation, standardization, and normalization. The present invention can create conditions for subsequent sample pre-processing machines, automatic loading of flow cytometers, and immobilization of data analysis, especially for the future development of artificial intelligence. ⑤ The present invention further developed a scoring system based on the contribution, specificity, and coverage of marker combinations for diagnosis and differential diagnosis. Combined with computer software, this system enables accurate and rapid assessments with high sensitivity and specificity. In particular, it can distinguish rare APLv and APL with basophilic granules, as well as APL-like. The detection and analysis methods of the present invention meet the current clinical needs of flow cytometry diagnosis and are suitable for widespread application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 A scoring system was proposed based on the immunophenotypic characteristics of APL, its subtypes, and non-APL (including APL-like).

[0092] Figure 2-Figure 6 Shown are the results of flow cytometry gating analysis of bone marrow specimens from typical APL cases according to one embodiment of the present invention.

[0093] Figure 7-11 The results of flow cytometry gating analysis of bone marrow specimens from NPM1 gene mutation acute myeloid leukemia (APL like) cases with similar phenotypes that are easily misdiagnosed are shown in one embodiment of the present invention.

[0094] Figure 12-16 The results of flow cytometry gating analysis of bone marrow specimens of cCD3-positive, CD34+ APLv, which are easily missed in one embodiment of the present invention, are shown. DETAILED DESCRIPTION

[0095] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the following detailed description is now given in conjunction with specific embodiments and the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the embodiments, all raw materials and reagents are commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0096] Example 1, preparation of reagents

[0097] The antibody combination used in this example is:

[0098] The first group consists of: CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody. The order of fluorescein labeling of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605. The above nine monoclonal antibody reagents are mixed in a first container at a volume ratio of 5:5:5:3:2:3:3:3:3;

[0099] The second group consists of: CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, anti-HLA-DR antibody, CD11b antibody, and CD45 antibody. The order of fluorescein labeling of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500. The above eight monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3 and placed in a second container;

[0100] The third group consists of: CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody, and CD45 antibody. The fluorescein labels of each antibody are FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, BV421, and V500 in the following order. The above seven monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3 and placed in a third container.

[0101] The fourth group consists of: CD22 antibody, CD34 antibody, CD117 antibody, CD38 antibody, and CD45 antibody, and the fluorescein labeling order of each antibody is PE, PerCP-Cy5.5, PE-Cy7, BV421, and V500; a mixture of the above five monoclonal antibody reagents is mixed in a volume ratio of 5:5:3:3:3 and placed in a fourth container;

[0102] The fifth group includes: CD64 antibody, CD34 antibody, CD117 antibody, CD42a antibody, CD36 antibody, CD14 antibody and CD45 antibody, and the fluorescein labeling order of each antibody is PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421 and V500; the above seven monoclonal antibody reagents are mixed in a volume ratio of 5:5:3:2:3:3:3 and placed in the fifth container;

[0103] The sixth group comprises: nuclear TdT antibody and cCD3 antibody, wherein the fluorescent labeling order of each antibody is FITC and APC; the above two monoclonal antibody reagents are mixed in a volume ratio of 2:2 and placed in the sixth container;

[0104] The seventh component is a cytoplasmic MPO monoclonal antibody, which is labeled with FITC and is placed in a seventh container.

[0105] All antibodies in this example are commercially available. Among them, TdT-FITC and cCD3-APC are products of Beckman Coulter, USA, and the other fluorescein-directly labeled antibodies are products of Becton Dickinson, USA.

[0106] Optionally, prepare a cell lysis buffer (hemolysin) and place it in the eighth container, permeabilization agent A in the ninth container, permeabilization agent B in the tenth container, and PBS buffer in the eleventh container. Hemolysin, permeabilization agent, and PBS buffer are commercially available. The cell lysis buffer and permeabilization agent are both products of Becton Dickinson, and the PBS buffer is a product of Beckman Coulter.

[0107] Example 2: Sample processing

[0108] According to the cell count results, add heparin or EDTA anticoagulated bone marrow or peripheral blood samples into flow cytometry tube A, ensuring that the amount of cells added is approximately 2×10 6 , in a state of single cell suspension; then, add 32 μl of nine kinds of cell membrane monoclonal antibody reagents labeled with different fluorescein into the flow tube according to Table 2, mix thoroughly with the cell suspension and incubate at room temperature in the dark for 15 minutes, add 3 ml of 1× hemolysin, incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml of PBS, mix thoroughly, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and add 0.5 ml of PBS buffer to resuspend the cells. The processed specimen is ready for detection on the machine.

[0109] According to the cell count results, add heparin or EDTA anticoagulated bone marrow or peripheral blood samples to flow cytometry tube B, ensuring that the amount of cells added is about 2×106 , in a single cell suspension state; then, add 29 μl of eight cell membrane monoclonal antibody reagents labeled with different fluorescein into the flow tube according to Table 2, mix thoroughly with the cell suspension, incubate at room temperature in the dark for 15 minutes, add 3 ml of 1× hemolysin, incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml of PBS, mix thoroughly, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and add 0.5 ml of PBS buffer to resuspend the cells. This is the processed specimen ready for detection on the machine.

[0110] According to the cell count results, add heparin or EDTA anticoagulated bone marrow or peripheral blood samples into flow cytometry tube C, ensuring that the amount of cells added is about 2×10 6 , in a single cell suspension state; add 3 ml PBS, mix well, incubate in a 37 degree water bath for 5 minutes, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml PBS again, mix well, incubate in a 37 degree water bath for 5 minutes, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml PBS again, mix well, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, then add 26 μl of nine kinds of cell membrane monoclonal antibody reagents labeled with different fluorescein in the third container according to Table 2 to the flow tube, mix well with the cell suspension, incubate at room temperature in the dark for 15 minutes, add 3 ml 1× hemolysin, mix well, incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500 rpm for 5 minutes to remove the supernatant, add 3 ml PBS buffer, mix well, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 0.5 ml PBS buffer. The processed specimen is ready for machine detection.

[0111] According to the cell count results, heparin or EDTA anticoagulated bone marrow or peripheral blood samples were added to the flow cytometry tube to ensure that the amount of cells added was about 2×10 6 , in a single cell suspension state; then, according to Table 2, add 19 μl of the five membrane monoclonal antibody reagents in the fourth container labeled with different fluorescein to the flow tube, mix thoroughly with the cell suspension, and incubate at room temperature in the dark for 15 minutes. Then, add 100 μl of membrane permeabilization agent A solution, incubate at room temperature in the dark for 5 minutes, add 3 ml of 1× hemolysin, mix well, incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and add 50 μl of membrane permeabilization agent B solution and 4 μl of the components in the sixth container (2 μl of nuclear monoclonal antibody TdT-FITC and 2 μl of cytoplasmic antibody CD3-APC), incubate at room temperature in the dark for 15 minutes, and finally add 3 ml of PBS buffer, mix well, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 0.5 ml of PBS buffer. This is the processed specimen ready for detection on the flow cytometer.

[0112] According to the cell count results, heparin or EDTA anticoagulated bone marrow or peripheral blood samples were added to the flow cytometry tube to ensure that the amount of cells added was about 2×10 6 Add 3 ml PBS, mix well, place in a 37-degree water bath and incubate for 5 minutes, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml PBS again, mix well, place in a 37-degree water bath and incubate for 5 minutes, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and add 3 ml Mix with PBS, centrifuge at 1500rpm for 5 minutes, remove the supernatant, and then add 24μl of seven kinds of cell membrane monoclonal antibody reagents in the fifth container with different fluorescein labels to the flow tube according to Table 2. Mix thoroughly with the cell suspension and incubate at room temperature in the dark for 15 minutes. Add 100μl of membrane permeabilization agent A solution, incubate at room temperature in the dark for 5 minutes, add 3ml of 1× hemolysin and mix, incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500rpm for 5 minutes, remove the supernatant, add 50μl of membrane permeabilization agent B solution and 5μl of the cytoplasmic monoclonal antibody reagent MPO-FITC in the seventh container, incubate at room temperature in the dark for 15 minutes, finally add 3ml of PBS buffer and mix. Centrifuge at 1500rpm for 5 minutes, remove the supernatant, and resuspend the cells in 0.5ml of PBS buffer. The processed specimen is ready for detection on the machine.

[0113] Table 2: Antibody composition for one-step screening / diagnosis of APL and CD34- / DR- AML

[0114]

[0115] Example 3: Detection of specimens

[0116] The specimens processed according to the method of Example 2 were detected on a Becton Dickinson 3-laser 10-color FACS Canto plus flow cytometer. Preferably, 1 million cells per tube (at least 300,000 cells are recommended) were obtained, and the data were analyzed using Diva 2.8 software or other software such as Kaluza.

[0117] Among them, when testing on a flow cytometer, gates are set in the following ways: ① Fixed gate setting: remove the adhesion cell gate, live cell gate, and blood cell gate in sequence; ② Multi-marker combination gate setting: start with a single live cell. To prevent missing tumor cells, the gate setting and definition of all cells need to be performed in parallel with the blood cell gate; ③ Within the multi-marker combination gate setting, the common expression patterns and development patterns of various marker combinations are displayed, and tumor cells are identified based on their differences from normal cells. Specifically:

[0118] 1. Fixed gate: Consists of the de-adhesive cell gate, live cell gate, and blood cell gate, which are connected in series.

[0119] De-adhesion cell gate: First, use the forward scatter (FSC) area (A) and height (H) to set the de-adhesion cell gate (denoted by P1). Adhesion cells can be removed by FSC-area (Area, A) / height (Height, H). The principle is that cells are spherical and A is positively correlated with H. Figure 2-Figure 16 The first gate picture in the top row from left to right.

[0120] Live cell gate: For cells within P1, use FSC / side scatter (SSC) to set the live cell gate (denoted by P2) to obtain single live cells. The FSC / SSC principle is that live cells have a near-normal distribution in size and granularity, clustering around a central point and clearly separated from dead cells, apoptotic cells, debris, and background noise. Figure 2-Figure 16 The second gate picture from left to right in the top row.

[0121] Blood cell gate: Within the single live cell gate (P2 gate), use CD45 / SSC to set each blood cell gate to roughly observe lymphocytes, monocytes, granulocytes, and nucleated red blood cells, as well as whether there are obvious tumor cells or abnormal cells. CD45 / SSC is used to roughly distinguish each blood cell group based on the different fluorescence intensities of CD45 expression on hematopoietic cells (mature lymphocytes > monocytes > granulocytes > nucleated red blood cells) and the differences in SSC size (eosinophils > granulocytes > monocytes > mature lymphocytes > nucleated red blood cells). See Figure 2-Figure 16 CD45 / SSC gate image in.

[0122] 2. Multi-marker combination gating: Performed simultaneously with CD45 / SSC gating, starting from single live cells (P2).

[0123] Each tube has different observation purposes and gating methods:

[0124] Tube A: In gate P2, use weak CD45 expression / medium to large SSC or CD117 positive / weak CD45 expression to set the gate for myeloid immature cells (choose the gate method that easily locks on tumor cells between the two methods). Use strong CD45 expression / small SSC to set the gate for mature lymphocytes. Use weak CD45 / large SSC or large SSC / CD117 negative to set the gate for differentiated granulocytes. Use strong CD45 expression / medium SSC or strong CD45 expression / weak CD4 expression to set the gate for monocytes. The main purpose of A tube is to observe the expression of myeloid immature cells with lymphocytes, and to exclude abnormalities of T and NK cells (CD2, CD3, CD4, CD5, CD7, CD8, and CD56 are T and NK markers) and CD45dim or - / CD56+ tumor cells (metastatic cancer, some plasma cell tumors, BPDCN, some acute megakaryocytic leukemia, etc.). Among them, CD7, CD2, CD4, and CD56 are T and NK markers, but they are lymphocyte markers that are easily expressed in non-APL AML. CD2 is an APLv Common accompanying expression markers include CD7, CD5, CD2, CD3, and CD8, which are used to differentiate cCD3-positive APLv from acute T-lineage myeloid mixed leukemia. APLv only weakly expresses cCD3 and does not express other T-lineage markers. Almost 100% of the T-lineage components of mixed leukemia express CD7, 90% express CD5, and 60% express CD2. Due to the contribution of A-ducts, APL may be accompanied by abnormal expression of CD56 and CD2. The expression of CD56 may be associated with poor prognosis. CD2 is seen in some APLv. The main contribution of CD7 is its use in differentiating APL from acute mixed cell leukemia (ACS) when cCD3 is positive. This is because the expression rate of CD7 and cCD3 in primitive T-lineage cells is nearly 100%, with few CD7-negative, cCD3-positive T-lineage blasts. Furthermore, while CD7 is expressed in approximately 30% of AML, it is rarely seen in APL. Furthermore, the fluorescence intensity of CD7 expressed on companion lineages is generally diminished, rarely reaching that of normal T and NK cells in the specimen. Therefore, gates are set using CD45 / SSC and CD117 / SSC to observe the intensity of CD117 expression on myeloid blasts and the presence of lymphoid markers. In AML other than APL, CD7 and CD56 are both relatively common markers of abnormal expression of companion lineages.

[0125] Comprehensively identifying the key features of APL: MPO positivity, HLA-DR negativity, and crossover of CD34 and SSC (80% of typical APL or typical portions of APLv have high CD34- / SSC; some tumor cells in APLv have low CD34+ / SSC). These key features are diagnostic of APL, resulting in an additional 3 points if the diagnosis is positive and a subtraction of 3 points if it is negative. Absence of tumor stem cells and characteristic FSC / SSC expression, with sensitivity and specificity exceeding 90%, result in an additional 2 points if the diagnosis is positive and a subtraction of 2 points if it is negative. Other features, such as moderate CD117 expression, strong CD33 expression, CD9 positivity, consistent CD64 expression, and consistent CD371 expression, are subjective or subject to variability, resulting in an additional 1 point if the diagnosis is positive and a subtraction of 1 point if it is negative. Since cCD3 is limited to specific cases, points are added if positive and not subtracted if negative. CD22 is not assigned a point due to differential diagnosis with basophils. (See Table 1.) Using this scoring scale, the present invention found that scores above 12 indicate APL, with the vast majority of APL and APLv cases scoring as high as 18 to 19. Only two cases (0.64%) of rare hereditary types and one case (0.32%) of HLA-DR-positive APL scored 15 and 12, respectively. APL-like scores ranged from -2 to 6, while non-APL scores ranged from -18 to -4. Using this method, sensitivity, specificity, positive predictive value, and negative predictive value all exceeded 99%. This demonstrates that the present invention represents an important testing and analysis solution for improving efficiency, reducing costs, and reducing misdiagnosis rates in the entire field of clinical diagnosis.

[0126] Tube B mainly observes the presence of myeloid primitive immature cells and abnormalities in the development of granulocytes and monocytes. Use CD45 / SSC, CD117 / SSC, and CD34 / SSC to set gates, or use CD16 positive / CD11b positive to set the gate for differentiation-stage granulocytes, and use CD11b positive / HLA-DR positive to set the gate for monocytes. In particular, observe the differential expression of CD34, CD117, CD33, CD13, HLA-DR, and CD11b between APL and non-APL. In addition, the presence of CD34bri / HLA-DR+ tumor stem cells is key to distinguishing APL (without tumor stem cells) from non-APL AML (with tumor stem cells).

[0127] Tube C is a relatively critical tube for determining APL. It uses CD45 / SSC to set the blood cell gate and the myeloid immature cell gate. It mainly observes the expression of CD9, CD371, CD11c, and CD15 on myeloid tumor cells. APL characteristically expresses CD9 and CD371 consistently, some express CD11c, and a few express CD15. Non-APL AML (including APL-like) differentially expresses CD371 and CD11c, with a probability of weakly expressing CD9 in about 30%, and a few expressing CD15.

[0128] Dingguan mainly observes the lineage of tumor cells, using CD45 / SSC, CD117 / SSC, CD34 / SSC, TdT / SSC, CD22 / SSC, and CD45 / cCD3 to set gates, respectively observing whether there are TdT+ / cCD3+ T-lineage and TdT+ / CD22+ B-lineage primitive cells; after excluding lymphoid and mixed leukemias, APL cases express CD117 moderately strongly, express CD38, and have almost no CD34bri / CD38dim tumor stem cells; non-APL AML (including APL-like) does not express CD22 and cCD3, a few cases express TdT, and differentially express CD38, and CD34bri / CD38dim tumor stem cells can be found in almost 95% of cases.

[0129] The pentoses tube is primarily used to determine whether myeloid tumor cells are granulocyte, monocytic, erythroid, or megakaryocytic. Using CD45 / SSC, CD34 / SSC, and CD45 / CD42a gates, the expression of myeloid tumor cells is observed, excluding CD14+ / CD36+ monocytes, CD42a+ / CD36+ and megakaryocytes, and CD45- / CD36+ erythroid cells. APL cases characteristically and consistently express MPO and CD64, but lack CD36, CD14, and CD42a. Non-APL AML (including APL-like) differentially expresses MPO and CD64. Monocytic components will differentially express CD36 and CD14. Megakaryocytes express CD42a and CD36, and nucleated erythrocytes are CD45- / CD36+.

[0130] 3. Accurately target target cells based on observed marker combinations

[0131] Preliminary criteria for immunophenotyping of acute leukemia include: ① Malignant blasts occupy more than 20% of nucleated cells and express early markers (CD34, HLA-DR, TdT, CD38) or no mature markers (granulocytes lack CD11b and CD16, monocytes lack or weakly express CD14, B cells lack a normal distribution of cκ and cλ, and T cells lack a normal distribution and intensity of CD4 and CD8). ② Single-lineage leukemia cells typically express two or more antigens specific to that lineage: myeloid lineage: MPO, CD13, CD33, CD117, CD11b, CD14, CD64; B lineage: CD19, CD22, CD10, CD20; T lineage: CD2, cCD3, CD5, CD7, CD4, CD8, CD3. ③ Acute mixed cell leukemia occurs in a 3%-5% chance, with tumor cells expressing two or more of the aforementioned lineage antigens.

[0132] TdT is expressed in over 95% of acute lymphoblastic leukemias, is almost absent in APL, and has a positive rate of 20% in non-APL AML. Therefore, TdT and CD22 are selected in the present invention to exclude B-lineage components, while TdT, cCD3, CD7, CD2, CD5, CD3, CD4, CD8, and CD56 are used to exclude T and NK lineages. Furthermore, CD2 and cCD3 are commonly expressed abnormally in APLv. APLv is distinguished from mixed leukemias with T-lineage components by the high expression of CD7, CD5, and TdT in T-lineage cells, while APLv barely expresses these three markers.

[0133] Granulocyte differentiation is generally divided into blasts, promyelocytes, myelocytes, metamyelocytes, band nuclei, and lobed granulocytes. The expression intensity changes with cell development; CD34 is only expressed on blasts, while CD117 and HLA-DR expression begins in blasts but stops in the early stages of promyelocytes. CD15 begins to appear in the promyelocyte stage and is highly expressed in normal cells once it appears. However, CD15 is generally weakly expressed on monocytes and is therefore often used as a marker to distinguish granulocytes from monocytes. CD64, CD11b, and CD11c are expressed on neutrophils, basophils, eosinophils, and monocytes at all stages after myelocytes. CD16 is mainly expressed on mature granulocytes such as band nuclei and lobed nuclei.

[0134] MPO, CD13, CD33, and CD371 are pan-lineage markers of the myeloid lineage. However, because AML is a group of diseases with extremely different differentiation stages and expression markers, MPO is mainly found in relatively differentiated granulocyte components. Therefore, the expression rate of MPO in overly primitive AML tumor cells is not high. Among AML, APL is AML that has differentiated to the promyelocytic stage. Therefore, the expression rates of MPO, CD33, and CD13 in APL are all above 95%. However, because non-APL AML also has an expression rate of 50% to 90%, these markers can only be used as one of them and cannot be used alone to distinguish APL from non-APL AML.

[0135] CD64, CD14, CD36, and CD11c are monocyte markers. APL consistently expresses CD64, but rarely expresses CD14 and CD36, and differentially expresses CD11c. CD42a is a megakaryocytic marker, and CD36 is found on monocytes, megakaryocytes, and erythroid cells, but is almost absent in APL. Non-APL AML differentially expresses these myeloid markers.

[0136] Generally, only eosinophils, basophils, platelets, megakaryocytes, and proliferating B progenitor cells express CD9. However, APL shows moderate CD9 expression with over 95% consistency. However, CD9 alone cannot be used to distinguish APL from non-APL, as non-APL also has a 30% probability of expressing CD9. While this statistically significant number was used when writing the article, a 30% misdiagnosis rate is unacceptable in clinical practice. Therefore, a combination of more markers is needed to make rapid and accurate judgments.

[0137] Except for APL, almost all AML, regardless of how CD34 and HLA-DR are expressed, even CD34- / HLA-DR- APL-like, has a probability of more than 95% to contain early myeloid primitive cells (cancer stem cells) with the phenotype of CD34bri / HLA-DR+ / CD38dim.

[0138] Initially, this study enrolled 130 AML patients, divided into three groups: typical APL (44 cases), APLv (24 cases), and APL-like (62 cases, including 51 cases of NPM1-mutated AML and 11 cases of AML with other genetic abnormalities) requiring differential morphology and / or MFC analysis. The results, shown in Table 3, show statistically significant differences in CD64, MPO, CD34, and HLA-DR due to the heterogeneity of APL-like. The APL group showed relatively good consistency, with typical cases expressing CD64 and MPO but lacking HLA-DR and CD34. However, APLv had the highest CD34 expression rate, exceeding that of APL-like. HLA-DR, which is highly suggestive of APL and has an extremely low positive rate, was also observed in a significant number of cases in the APL-like group, displaying immunophenotypes similar to APL and similar to those found in APL. This is a challenge not addressed in the literature and a most pressing issue in clinical practice. Clinical work requirements differ from those of research articles, requiring sensitivity and specificity exceeding 95%. Therefore, the present invention further quantified the fluorescence expression intensity of markers with high positive rates and found that the median expression intensity (fluorescence intensity) of CD9, MPO, and CD64 was statistically significant for differentiating APL from APL-like. To eliminate the influence of instrument stability, the present invention used lymphocytes as a reference and found that the ratio of the median expression intensity (fluorescence intensity) of SSC and MPO on tumor cells to lymphocytes was statistically significant for differentiating APL from APL-like (see Table 4).

[0139] Table 3: Comparison of flow cytometric immunophenotypes of classic APL, APLv, and APL-like

[0140]

[0141] Table 4: Diagnostic value of flow cytometry immunophenotyping parameter expression intensity in distinguishing APL cases from APL-like

[0142]

[0143] Note: MDFI is the median expression intensity (fluorescence intensity) of this parameter, T / L MDFIR is the ratio of the median expression intensity (fluorescence intensity) of this parameter on tumor cells to that on lymphocytes, AUC is the area under the curve, and CI is the confidence interval.

[0144] On this basis, the present invention makes a fast, simple and efficient scheme and score table which is very suitable for clinical work, see Table 1 and Figure 1 The sensitivity, specificity, positive predictive value, and negative predictive value were all above 99%.

[0145] In this study, tubes B, C, and E primarily focus on myeloid markers and are key to disease diagnosis, while tubes A and D primarily serve as differential diagnosis or APLv subtype expression markers. However, each marker, and their combination, contributes to diagnosis and differential diagnosis. In newly diagnosed APL and non-APL (including APL-like conditions requiring differential diagnosis), the proportion of tumor cells is generally high, and CD45 / SSC can be used to delineate tumor cells. However, because APL generally has a higher SSC, it can be difficult to distinguish them from relatively mature granulocytes and monocytes. Therefore, CD117 / SSC is typically used for gating. If the boundaries are unclear or there is overlap, the expression of important markers such as CD16, CD11b, HLA-DR, CD13, CD33, CD9, CD371, CD11c, CD15, CD38, CD64, MPO, CD36, and CD14 can be used for auxiliary gating.

[0146] Regarding the contribution of APL, APL may be accompanied by abnormal expression of CD56 and CD2. CD56 expression may be associated with a poor prognosis, while CD2 is observed in some APLv. The primary contribution of CD7 is that it can be used to differentiate APLv from acute mixed cell leukemia when cCD3 is positive. This is because CD7 and cCD3 expression rates are nearly 100% in primitive T-lineage cells, with few CD7-negative, cCD3-positive T-lineage blasts. Furthermore, while CD7 is expressed in approximately 30% of AML, it is rarely seen in APL. Furthermore, the fluorescence intensity of CD7 expressed by myeloid blasts is generally diminished, rarely reaching that of normal T and NK cells in the specimen. Therefore, gates are set based on CD45 / SSC and CD117 / SSC to assess the intensity of CD117 expression on myeloid blasts and the presence of concomitant lymphoid marker expression. In non-APL AML, CD7 and CD56 are both common markers of abnormal expression of concomitant lineages.

[0147] Tube B is the key. Although AML can be CD34-negative and HLA-DR-negative, there are certain patterns for diagnosis and differential diagnosis. ① The correspondence between CD34 and SSC, as well as the presence of small clones of tumor blasts in CD34-negative AML, is key to differentiating APL from APLv and APL-like. Typical APL is CD34-negative with a moderately high SSC, while APLv is CD34+ with a low SSC. In other words, the expression of these two parameters, SSC and CD34, is typical (high SSC and CD34-negative), while atypical (low SSC and CD34-positive), is atypical. If there is overlap, there is a >95% probability that it is APL-like rather than APL. ② Another important parameter is that in CD34-negative AML, if it is APL, there will be almost no CD34+ / HLA-DR+ early tumor blasts, while in non-APL, CD34bri / HLA-DR+ tumor stem cells will be found in >95% of cases. ③In addition, if it is APL with basophils, myeloid immature cells can be seen in the basophil area (medium CD45 / small SSc). Although they look like basophils and may be CD117 negative, they are different from basophils in that this group of tumor cells has a high background, is MPO positive, and is CD22 negative, while the background of basophils is not high, is MPO negative, and is weakly CD22 positive; ④The expression intensity of CD117 is also helpful for diagnosis. The expression of CD117 in APL is not strong, but generally of medium intensity and with heterogeneity. In other types of AML, the expression rate of CD117 is 95%, and the expression intensity is basically consistent and relatively strong; ⑤The expression intensity of CD33 and CD13 also plays a role in disease diagnosis and differential diagnosis. The expression intensity of CD33 in APL is very strong, almost close to that of monocytes, and CD13 is weaker than that of typical normal promyelocytes. ⑥ HLA-DR negativity is a typical characteristic of APL. Although there have been case reports of HLA-DR-positive APL, both literature reports and the statistics presented here indicate that over 96% of APL is HLA-DR-negative. Therefore, in clinical practice, despite the diverse phenotypes and morphologies of APL, which can easily lead to missed and misdiagnosed cases, it is recommended to remain vigilant against APL and APLv in cases with HLA-DR negativity and a CD34 / SSC high or CD34+ / SSC low pattern.

[0148] Tube C is a relatively critical tube for determining APL. It mainly observes the expression of CD9, CD371, CD11c, and CD15 on myeloid primitive cells. Typical APL and APLv consistently express CD9 and CD371, and rarely express CD11c and CD15. In non-APL cases, regardless of whether they are APL-like or not, except for rare megakaryocytic leukemia, the expression rate of CD9 is generally less than 20%. The expression of CD371 in cells of all stages of normal myeloid lineage is very consistent with CD33. In AML, the intensity of CD371 is generally inconsistent with that of CD33, but CD33 and CD371 in APL are generally consistently strongly expressed. CD11c and CD15 are differentially expressed in other types of AML and rarely expressed in APL. Therefore, the literature uses these two parameters as the basis for differential diagnosis. However, it can be seen that these parameters, which are expressed in 40% to 50% of one type of disease and rarely in another type of disease, can be statistically differentiated when writing articles. However, if used as a clinical diagnosis, it is completely imprecise because the positive proportion is only half, and clinical work requires almost 100% accuracy. Therefore, in the invention patent of this invention, these parameters that do not contribute much to clinical diagnosis are not included in the points.

[0149] Dingguan mainly observes the lineage of primitive cells and the expression of CD38. It has a high probability of expression in both AML and APL. Therefore, even if there is a statistical difference, it is of little use in clinical work. Almost no articles mention APL.

[0150] CD38, but the present invention found that tumor stem cells are very valuable for distinguishing APL from non-APL, and the characteristic expression of tumor stem cells is CD34bri / CD38dim. If it is APLv, there may be weak expression of cCD3, and the differentiation from acute mixed cell leukemia is CD7 and TdT negative. Other characteristics are consistent with APLv; 100% of the T-lineage components of acute mixed cell leukemia express CD7 and 95% express TdT. On the one hand, the help of CD22 is to exclude the B lineage. On the other hand, some APLs will have promyelocytes with basophilic granules. Rare cases can even be easily misdiagnosed as acute basophilic leukemia. APL expresses MPO and does not express CD22, while basophils express the opposite.

[0151] The pentameric tube is primarily used to determine whether myeloid tumor cells are granulocyte, monocytic, erythroid, or megakaryocytic. APL is almost always consistently positive for MPO and CD64, but lacks expression of CD14, CD36, or CD42a. Other types of AML, while expressing MPO, rarely show consistent expression of CD64. Myeloid leukemias may show smeared expression of CD64, while monocytic leukemias show strong CD64 expression accompanied by some or partial expression of CD36 and CD14.

[0152] In short, the present invention uses the marker combination CD34bri / HLA-DR+ / CD38dim of early myeloid primitive cells (cancer stem cells) and common APL markers within the multi-marker combination gate. MPO, CD64, CD33, CD9, CD13, and CD371 are expressed with strong consistency, CD117 is expressed with moderate intensity, and HLA-DR, CD11b, CD15, CD36, CD14, and CD42a are not expressed. APLv easily expresses CD2, CD56, and cCD3, and therefore, T-lineage markers are included for identification. At the same time, these markers are easily expressed in companion lineages of non-APL AML. MPO and CD22 are helpful in distinguishing basophils from APL with basophilic granules.

[0153] In this example, 20 APL and APL-like bone marrow specimens were first selected for testing. The timing of antigen appearance, expression intensity, and pairwise combinations all formed regular differentiation patterns, as well as the status of tumor stem cells. Based on this, the protocol was designed and further expanded to 130 specimens to obtain further experimental data.

[0154] This example provides one case each of typical APL, NPM1 gene mutation APL like, and microparticle variant APLv as examples. Figure 2-Figure 6 For the immunophenotyping of typical APL specimens from A to E, the gates were set and observed strictly according to the above description. Figure 7-11 In this specific implementation scheme, the alpha to epinephrine immunophenotyping of patients with APL-like NPM1 gene mutations with similar phenotypes that can easily lead to misdiagnosis is performed. Figure 12-16 Immunophenotyping of A to E tubes in patients with microparticulate variant APLv.

[0155] Specifically, Figure 2-Figure 6 : Five bone marrow specimens from the same typical APL case were analyzed together.

[0156] Specifically, Figure 2 A typical APL bone marrow specimen A analysis is shown. The following settings are made in sequence: ① FSC-A / H sets P1 as the gate for de-adhesive cells, and P1 contains single cells; ② FSC / SSC is displayed in P1, and P2 is set as the gate for living cells, and P2 contains single living cells; ③ CD45 / SSC is used to set the blood cell gate in the P2 gate, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) are obtained respectively; ④ CD45 weak expression / SSC medium to large is used to set the gate to detect myeloid immature cells (blast) in the P2 gate; ⑤ Observe the expression of blast. In this case, the tumor cells (blast) express CD117 with moderate intensity and heterogeneity. Figure 2As shown in the picture marked I, CD117 expression is strong and continuous, and most cells express it with medium intensity. CD2, CD3, CD4, CD5, CD7, CD8, and CD56 are not expressed. FSC / SSC of blast ( Figure 2 II), and FSC / SSC of granulocytes ( Figure 2 The images marked III in the figure are almost identical and are all within gates A' and A''.

[0157] Specifically, Figure 3 A typical APL bone marrow specimen is shown, and tube B is analyzed. The following settings are made in sequence: ① Set P1 as the gate for de-adhesive cells using FSC-A / H, and obtain single cells within P1; ② Set P2 as the gate for live cells using FSC / SSC within P1, and obtain single live cells within P2; ③ Use CD45 / SSC to set the blood cell gate within P2, and obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Use CD45 / SSC to set the gate for myeloid blasts within P2; ⑤ Use CD16 positive / CD11b positivity assisted in setting the differentiation stage granulocyte gate (Gra') to further determine the FSC / SSC relationship between blast and Gra'; ⑥ In the P2 gate, CD34bri / SSC small was used to set the early myeloid primitive cell gate. This case had almost no CD34 expression, and even if there was a small amount of weak expression of CD34, it was HLA-DR negative; ⑦ Observe the tumor cell gate set with CD45 medium / SSC large. This case expressed strong expression of CD33, CD13 and CD117, and did not express CD34, HLA-DR, CD16, or CD11b. Figure 2 Similarly, FSC / SSC of blast ( Figure 3 The FSC / SSC of Gra' ( Figure 3 The images marked II in the figure are almost identical and are all within gates A' and A''.

[0158] Specifically, Figure 4A typical APL bone marrow specimen, analyzed in tube C, is shown. The following settings were made: ① Using FSC-A / H ratios, set P1 as the gate for deadhesive cells, identifying single cells within P1; ② Using FSC / SSC ratios within P1, set P2 as the gate for live cells, identifying single live cells within P2; ③ Using CD45 / SSC ratios within the P2 gate to gate blood cells, gates were set for lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Using CD45 / SSC ratios within the P2 gate to gate myeloid blasts; ⑤ Using CD371 positivity / strong CD15 positivity to gate differentiated granulocytes (Gra'); ⑥ Using CD45 medium / SSC high to gate tumor cells, this case consistently expressed strong expression of CD9 and CD371, but not CD15 or CD11c. Figure 4 As shown in the picture marked I, CD9 is strongly expressed and CD371 is evenly expressed.

[0159] Specifically, Figure 5 Figure 3 shows a typical APL bone marrow specimen analysis. The following settings were used: ① Using FSC-A / H to gate P1 for deadhesive cells, P1 contained single cells; ② Using FSC / SSC to gate P2 for live cells, P2 contained single live cells; ③ Using CD45 / SSC to gate blood cells within P2, lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) were gated; ④ Using CD45 / SSC to gate myeloid blasts within P2; ⑤ Using CD34 / SSC to gate early myeloid blasts, which were almost absent in this case; ⑥ Observing tumor cell expression: In this case, tumor cells expressed CD38 and CD117, but not CD22, cCD3, TdT, or CD34.

[0160] Specifically, Figure 6 The penta-tube analysis of a typical APL bone marrow specimen was shown. The following settings were performed: ① FSC-A / H was used to set P1 as the gate for de-adhesive cells, resulting in single cells within P1; ② FSC / SSC was used to set P2 as the gate for live cells within P1, resulting in single live cells within P2; ③ CD45 / SSC was used to set the blood cell gate within the P2 gate, resulting in lymphocyte (lym), granulocyte (Gra), monocyte (mono), nucleated erythrocyte (NEC) cell gates, and eosinophil (eo) gates; ④ CD45 / SSC was used to set the myeloid blast gate within the P2 gate; ⑤ The expression of tumor cells was observed. In this case, the tumor cells consistently expressed MPO and CD64, differentially expressed CD117 with moderate intensity, and did not express CD14, CD36, CD42a, or CD34. Blast ( Figure 6 The expression intensities of MPO and CD64 in the images marked with I were significantly different from those in Gra ( Figure 6 The results are almost the same as those in the picture marked II).

[0161] Specifically, Figure 7-11 Immunophenotyping of APL-like bone marrow specimens from patients with the same NPM1 mutation.

[0162] Specifically, Figure 7 The analysis of bone marrow specimens from AML patients is shown in Figure 1. The following settings are made in sequence: ① FSC-A / H is used to set P1 as the gate for de-adhesive cells, and single cells are obtained in P1; ② FSC / SSC is displayed in P1, and P2 is set as the gate for living cells, and single living cells are obtained in P2; ③ CD45 / SSC is used to set the blood cell gate in the P2 gate, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophil gates (eo) are obtained respectively; ④ CD117 / CD45dim is used to set the myeloid blast gate in the P2 gate; ⑤ Observe the expression of blast, and the proportion of malignant myeloid blasts is increased, expressing CD117, but not expressing CD2, CD3, CD4, CD5, CD7, CD8, and CD56. In this case, the tumor cells (blast) and APL ( Figure 2 Unlike the image marked with I in the figure, CD117 expression is not very continuous, and most cells express it strongly ( Figure 7 The FSC / SSC of blast ( Figure 7 II), and FSC / SSC of granulocytes ( Figure 7 The difference between the images marked III is very large. Figure 7 In the picture marked II, the FSC / SSC of blasts is significantly reduced, most cells are outside the A' gate, and the FSC / SSC of granulocytes are within the A'' gate.

[0163] Specifically, Figure 8APL-like bone marrow specimen showing NPM1 mutation, analyzed in tube B. The following settings were performed: ① Using FSC-A / H to set P1 as the gate for dissociated cells, P1 contained single cells; ② Using FSC / SSC to set P2 as the gate for live cells, P2 contained single live cells; ③ Using CD45 / SSC to set the blood cell gate within P2, lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) were gated; ④ Using CD45 / SSC to set the gate for myeloid blasts within P2; ⑤ Using CD16 Positive / CD11b positive assists in setting the differentiation stage granulocyte gate (Gra') to further determine the FSC / SSC relationship between blasts and Gra'; ⑥ Using CD34bri / SSC small within the P2 gate to set the early myeloid primitive cell gate, a small number of CD34bri / HLA-DR+ tumor stem cells can be seen in this case; ⑦ Blast cell expression: In this case, tumor cells mainly and consistently express CD117, CD33, and CD13, but do not express CD34, HLA-DR, CD11b, or CD16. However, a small number of tumor stem cells can be seen, as shown by the darkest thick dots in the figure. Figure 3 Different, blast FSC / SSC ( Figure 8 The FSC / SSC of Gra' ( Figure 8 Unlike the picture marked II in the figure, the FSC / SSC of blast is mostly outside the A' gate, while Gra' is within the A'' gate.

[0164] Specifically, Figure 9 Showing APL-like bone marrow specimens showing NPM1 mutations, tube C analysis. Sequential settings: ① FSC-A / H set P1 as the de-adhesion cell gate, and obtained single cells in P1; ② Display FSC / SSC in P1 and set P2 as the live cell gate, and obtained single live cells in P2; ③ Use CD45 / SSC to set the blood cell gate in the P2 gate, and obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) respectively; ④ Use CD45 / SSC or CD117 / SSC to set the myeloid immature cell gate (blast) in the P2 gate; ⑤ Use CD371 positive / CD15 strong positive to assist in setting the differentiation stage granulocyte gate; ⑥ Observe blast in the P2 gate, which heterogeneously expresses CD371, and does not express CD9, CD15, and CD11c. Figure 4 The pictures marked with I are different, the blast of APL like ( Figure 9 (Image marked with I) CD9 is negative, and CD371 is concentrated, not evenly distributed like APL.

[0165] Specifically, Figure 10 Figure 3 shows APL-like bone marrow specimens displaying NPM1 mutations. The following settings were performed in order: ① Using FSC-A / H, set P1 as the de-adhesive cell gate to obtain single cells within P1; ② Using FSC / SSC within P1, set P2 as the live cell gate to obtain single live cells within P2; ③ Using CD45 / SSC within the P2 gate to set the blood cell gate to obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo), respectively; ④ Using CD45 / SSC or CD117 / SSC within the P2 gate to set the myeloid blast gate; ⑤ Using CD34bri / SSC within the P2 gate to set the early myeloid primitive cell gate. In this case, a small number of CD34bri / CD38dim cancer stem cells were seen, as shown by the darkest dots in the figure; ⑥ Observing blast expression, the tumor cells in this case expressed CD38 and CD117, but did not express CD22, cCD3, TdT, or CD34 (except for a small number of cancer stem cells).

[0166] Specifically, Figure 11 Penetrant tube analysis of APL-like bone marrow specimens from patients with NPM1 mutations. The following settings were performed: ① FSC-A / H was used to set P1 as the gate for de-adhesive cells, and single cells were obtained within P1; ② FSC / SSC was used to set P2 as the gate for live cells within P1, and single live cells were obtained within P2; ③ CD45 / SSC was used to set the blood cell gate within the P2 gate, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) were obtained respectively; ④ CD45 / SSC was used to set the myeloid immature cell gate (blast) within the P2 gate; ⑤ Observe blast expression. The tumor cells in this case expressed CD117, a small amount of MPO and CD64, and no CD36, CD42a, or CD34. Figure 6 Completely different, the tumor cells in this case had MPO and CD64 ( Figure 11 The expression of MPO / CD64 in the middle image is weak, which is different from that of MPO / CD64 in granulocytes ( Figure 11 The differences are quite large (see the image marked II).

[0167] Specifically, Figure 12-16 : Five normal bone marrow specimens from the same APLv case were analyzed together.

[0168] Specifically, Figure 12Analysis of APLv bone marrow specimens in tube A is shown. The following settings are made in sequence: ① FSC-A / H is used to set P1 as the gate for dissociated cells, and single cells are obtained in P1; ② FSC / SSC is displayed in P1, and P2 is set as the gate for living cells, and single living cells are obtained in P2; ③ CD45 / SSC is used to set the blood cell gate in the P2 gate, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophil gates (eo) are obtained respectively; ④ CD45 weak expression / SSC medium size is used to set the gate for myeloid blasts in the P2 gate; ⑤ SSC small / CD45 weak expression is used to set the gate for myeloid tumor cells (APL2) with basophilic granules in the P2 gate, and ⑥ Observe the expression of blasts. The tumor cells in this case heterogeneously express CD117 with moderate intensity. Figure 12 As shown in the picture marked I, CD117 expression is strong and continuous, and most cells express it with medium intensity. CD2, CD3, CD4, CD5, CD7, CD8, and CD56 are not expressed. FSC / SSC of blast ( Figure 12 II in the middle), although smaller than typical APL ( Figure 2 -I) is smaller, but similar to the FSC / SSC of granulocytes ( Figure 12 The images (marked in the image III) are also nearly identical, falling within gates A' and A''. ⑦ Observing the expression of APL2 cells, the background is high (reflected by the thick lines at a 45-degree angle in the CD3 / CD5 and CD4 / CD8 combined images), and SSC and CD117 expression is weaker than that of blasts.

[0169] Specifically, Figure 13Display APLv bone marrow specimen, analysis of tube B. Set in sequence: ① FSC-A / H set P1 as the de-adhesion cell gate, and obtain single cells in P1; ② Display FSC / SSC in P1 and set P2 as the live cell gate, and obtain single live cells in P2; ③ Use CD45 / SSC to set the blood cell gate in the P2 gate, and obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophils (eo); ④ Use CD45 weak expression / SSC medium size to set the myeloid immature cell gate (blast) in the P2 gate; ⑤ Use CD16 positive / CD11b positive to assist in setting the differentiation stage granulocyte gate (Gra'), the purpose is to further Step 1: Determine the FSC / SSC relationship between blast and Gra'; ⑥ Use CD34bri / SSCsmall to set the gate for early myeloid primitive cells in the P2 gate. Although some of the cases express CD34, they all have SSClarge / CD34weak expression, and there are almost no tumor stem cells with CD34bri / SSCsmall; ⑦ Use SSCsmall / CD45dim to set the gate for myeloid tumor cells (APL2) with basophilic granules in the P2 gate; ⑧ Observe the tumor cell gate set with CD45medium / SSClarge. This case expresses strong CD33, CD13 and CD117, partially expresses CD34, and does not express HLA-DR, CD16, or CD11b. Figure 12 Similarly, FSC / SSC of blast ( Figure 13 The FSC / SSC of Gra' ( Figure 13 The expression of APL2 cells was almost the same as that of blast cells (the picture marked II in the figure), and they were all within the A' and A'' gates; ⑨ The background was high (reflected in the thick line at a 45-degree angle in the CD34 / HLA-DR combination graph), the expression of SSC and CD117 was weaker than that of blast cells, CD13 and CD33 were strongly expressed, CD11b was partially weakly expressed, and CD34, HLA-DR, and CD16 were not expressed.

[0170] Specifically, Figure 14Display APLv bone marrow specimen, tube C analysis. Sequential settings: ① FSC-A / H set P1 as the de-adhesive cell gate, and obtained single cells in P1; ② Display FSC / SSC in P1 and set P2 as the live cell gate, and obtained single live cells in P2; ③ Use CD45 / SSC to set the blood cell gate in P2 gate, and obtain lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophils (eo); ④ Use CD45 weak expression / SSC medium size to set the myeloid immature cell gate (blast) in P2 gate; ⑤ Use CD371 positive / CD15 strong positive auxiliary cells to set the blood cell gate. ⑥ Use CD34bri / SSC small to set the gate for early myeloid primitive cells in the P2 gate. Although some tumor cells in this case weakly express CD34, the SSC is medium and there are almost no tumor stem cells with CD34bri / SSC small. ⑦ Use CD45dim / SSC small to set the gate for myeloid tumor cells with basophilic granules (APL2) in the P2 gate. ⑧ Observe the tumor cell gate set with CD45 medium / SSC large. This case consistently expresses CD9 and CD371 strongly, some weakly express CD34, and do not express CD15 and CD11c. Figure 4 The image marked with I in the figure is similar to the blast of APL like ( Figure 9 Different from the picture marked with I in the figure), APLv expresses CD371 ( Figure 14 ⑨Observe the expression of APL2 cells, the background is high (reflected in the high background of CD34 and CD11c), the expression of SSC and CD117 is weaker than that of blast, CD9 and CD371 are strongly expressed, and CD34, CD11c, and CD15 are not expressed.

[0171] Specifically, Figure 15The analysis of APLv bone marrow specimens is shown. The following settings are made in sequence: ① FSC-A / H is used to set P1 as the gate for de-adhesive cells, and single cells are obtained in P1; ② FSC / SSC is displayed in P1, and P2 is set as the gate for living cells, and single living cells are obtained in P2; ③ CD45 / SSC is used to set the blood cell gate in the P2 gate, and lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated red blood cells (NEC), and eosinophil gates (eo) are obtained respectively; ④ CD45 weak expression / SSC medium size is used to set the myeloid immature cell gate (blast) in the P2 gate; ⑤ CD34bri / SSC small is used to set the early myeloid primitive cell gate in the P2 gate. In this case, although some tumor cells weakly express CD34, the SSC is medium, and there are almost no tumor stem cells with small CD34bri / SSC. ⑥ Use CD45dim / SSC to set a gate for myeloid neoplasm cells with basophilic granules (APL2) within the P2 gate; ⑦ Observe the expression of tumor cells. In this case, the tumor cells expressed CD38 and CD117, and some expressed CD34 and cCD3, but did not express CD22 or TdT. APLv can partially express cCD3, but it is distinguished from T-ALL in that T-ALL almost 100% expresses CD7, more than 85% expresses TdT, and the expression intensity of cCD3 is also relatively strong (disconnected from the negative part), while APLv almost does not express CD7 and TdT, and the expression intensity of cCD3 is weak (showing continuity with the negative part); ⑧ Observe the expression of APL2 cells, which express CD38 but do not express CD34, TdT, CD22, or cCD3.

[0172] Specifically, Figure 16The penta-tube analysis of bone marrow specimens from patients with APLv is shown. The following settings were performed: ① Using FSC-A / H to set P1 as the gate for de-adhesive cells, P1 contained single cells; ② Using FSC / SSC to set P2 as the gate for live cells within P1, P2 contained single live cells; ③ Using CD45 / SSC to set the blood cell gate within P2, lymphocytes (lym), granulocytes (Gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo) were obtained, respectively; ④ Using weak CD45 expression / medium SSC to set the myeloid blast gate within P2; ⑤ Using P2 gate ⑥ Use CD34bri / SSC small to set the gate for early myeloid primitive cells. In this case, although some tumor cells weakly express CD34, the SSC is medium-sized and there are almost no tumor stem cells with CD34bri / SSC small. ⑥ Use CD45dim / SSC small to set the gate for myeloid tumor cells with basophilic granules (APL2) in the P2 gate. ⑦ Observe the expression of tumor cells. In this case, tumor cells consistently express MPO and CD64, differentially express CD117 with moderate intensity, partially express CD34, and do not express CD14, CD36, or CD42a. Compared with typical APL ( Figure 6 ) is similar to APLlike ( Figure 11 The tumor cells in this case have MPO and CD64 levels that are not as high as those in typical APL, but they are very similar, with consistent expression of MPO and CD64 ( Figure 16 The intensity of MPO / CD64 on granulocytes ( Figure 16 (Image labeled II) is weaker. ⑧ Observe the expression of APL2 cells: they express MPO and CD64, but not CD14, CD36, or CD42a. APL2 cells are APL cells with basophilic granules. They are distinguished from basophils by their high background expression of MPO and lack of CD22, while basophils have a low background expression, lack of MPO, and weak expression of CD22.

[0173] The method of this example was clinically validated using 6,319 AML patients admitted to Hebei Yanda Lu Daopei Hospital from January 1, 2016, to December 20, 2024. Of these, 313 were APL patients (165 males, 148 females), including 236 with classic APL (124 males, 112 females) and 77 with APLv (41 males, 36 females). A total of 426 APL-like patients (311 with NPM1 mutations and 115 with other genetic abnormalities) were identified, including 219 males and 207 females. Other non-APL and non-APL-like AMLs were identified in 5,580 cases, including 2,902 males and 2,678 females. Simultaneous morphological, genetic, and clinical validation confirmed the correctness of the diagnosis in all cases. Using the method in Table 1, MPO positivity, HLA-DR negativity, and crossover of CD34 and SSC (80% of typical APL or typical parts of APLv have large CD34- / SSC; some tumor cells of APLv have small CD34+ / SSC) are the main features for diagnosing APL, so 3 points are added if the condition is met, and 3 points are subtracted if the condition is not met; the absence of tumor stem cells and characteristic expression of FSC / SSC positions, with sensitivity and specificity both above 90%, adds 2 points if the condition is met, and subtracts 2 points if the condition is not met; other features, such as moderate CD117 expression, strong CD33 expression, CD9 positivity, CD64 consistency, and CD371 consistency, are relatively subjective or have differences, so 1 point is added if the condition is met, and 1 point is subtracted if the condition is not met; cCD3 is limited to special cases, so points are added if the condition is positive, and no points are subtracted if the condition is negative; CD22 is involved in the differential diagnosis with basophils, and no points are given. Figure 1 All APL cases scored above 12, with 99% of APL and APLv cases scoring 18-19. Two cases (0.64%) with rare genotypes scored 15, and one case (0.32%) with HLA-DR-positive APL scored 12. APL-like scores ranged from -2 to 6, while non-APL scores ranged from -18 to -4. Using this method, sensitivity, specificity, positive predictive value, and negative predictive value all exceeded 99%. This demonstrates that this invention improves efficiency, saves costs, and reduces misdiagnosis rates.

Claims

1. A reagent composition for detecting acute promyelocytic leukemia by flow cytometry, characterized in that: The reagent composition includes seven groups of antibodies, wherein: The first group of antibodies consists of fluorescein-labeled CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody. The order of fluorescein labeling of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500 and BV605, and is used to add to flow cytometry tube 1 in which the sample to be tested is in the state of a single cell suspension; The second group of antibodies consists of fluorescein-labeled CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, HLA-DR antibody, CD11b antibody and CD45 antibody. The order of fluorescein labeling of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421 and V500, and is added to the second flow cytometry tube in which the sample to be tested is in the state of a single cell suspension; The third group of antibodies consists of fluorescein-labeled CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody, and CD45 antibody. The order of fluorescein labeling of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, BV421, and V500. They are added to flow cytometry tube 3 in which the sample to be tested is in a single cell suspension state and incubated in phosphate buffer, after which the supernatant is removed; The fourth group of antibodies consists of fluorescein-labeled CD22 antibody, CD34 antibody, CD117 antibody, CD38 antibody, and CD45 antibody. The order of fluorescein labeling of each antibody is PE, PerCP-Cy5.5, PE-Cy7, BV421, and V500. They are added to flow cytometry tube 4 in which the sample to be tested is in the form of a single cell suspension. The fifth group of antibodies consists of fluorescein-labeled CD64 antibody, CD34 antibody, CD117 antibody, CD42a antibody, CD36 antibody, CD14 antibody and CD45 antibody. The order of fluorescein labeling of each antibody is PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421 and V500. It is added to flow cytometry tube 5 in which the sample to be tested is in a single cell suspension state and incubated with phosphate buffer, and the supernatant is removed; The sixth group of antibodies consists of FITC-labeled nuclear TdT antibody and APC-labeled cytoplasmic CD3 antibody, and is added to flow cytometry tube 4 after the fourth group of antibodies has been added and the cells have been lysed; The seventh group of antibodies is FITC-labeled cytoplasmic MPO antibody, which is added to flow cytometry tube 5 after the fifth group of antibodies has been added and the cells have been lysed; Among them, each antibody is a monoclonal antibody.

2. The reagent composition according to claim 1, wherein: The first group of antibodies is a mixture of CD7 antibody, CD117 antibody, CD3 antibody, CD4 antibody, CD5 antibody, CD8 antibody, CD56 antibody, CD45 antibody and CD2 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3; The second group of antibodies is a mixture of CD16 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD33 antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3:3; The third group of antibodies is a mixture of CD9 antibody, CD371 antibody, CD34 antibody, CD117 antibody, CD11c antibody, CD15 antibody, and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3; The fourth group of antibodies is a mixture of CD22 antibody, CD34 antibody, CD117 antibody, CD38 antibody, and CD45 antibody in a volume ratio of 5:5:3:3:3; The fifth group of antibodies is a mixture of CD64 antibody, CD34 antibody, CD117 antibody, CD42a antibody, CD36 antibody, CD14 antibody, and CD45 antibody in a volume ratio of 5:5:3:2:3:3:3; The sixth group of antibodies is a mixture of nuclear TdT antibody and cytoplasmic CD3 antibody at a volume ratio of 2:

2.

3. A kit for detecting acute promyelocytic leukemia by flow cytometry, the kit comprising a first container, a second container, a third container, a fourth container, a fifth container, a sixth container, and a seventh container, each container containing a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, a sixth group of antibodies, and a seventh group of antibodies of the reagent composition according to any one of claims 1 to 2, respectively.

4. The kit according to claim 3, wherein The kit also includes: one or more of a cell lysis solution, a membrane permeabilizing agent, a buffer solution, and a flow tube used in conjunction with a flow cytometer.

5. Use of the reagent composition according to any one of claims 1 to 2 in preparing flow cytometric samples for detecting acute promyelocytic leukemia.

6. The use according to claim 5, characterized in that The process of preparing a flow cytometer sample for detecting acute promyelocytic leukemia comprises the following steps: (1) Add the sample to be tested into flow tubes 1 to 5 respectively to form a single cell suspension and ensure that the cell volume is 1×10 6 / tube-1×10 7 / Tube; (2) Add phosphate buffer to tubes 3 and 5, mix well, incubate at 37°C, and centrifuge to remove the supernatant; (3) Add the first group of antibodies in the reagent composition of any one of claims 1 to 2 to the tube 1 obtained by the treatment in step (1), add the second group of antibodies in the reagent composition of any one of claims 1 to 2 to the tube 2 obtained by the treatment in step (1), add the third group of antibodies in the reagent composition of any one of claims 1 to 2 to the tube 3 obtained by the treatment in step (2), add the fourth group of antibodies in the reagent composition of any one of claims 1 to 2 to the tube 4 obtained by the treatment in step (1), and add the fifth group of antibodies in the reagent composition of any one of claims 1 to 2 to the tube 5 obtained by the treatment in step (2), and incubate each flow tube at room temperature in the dark; (4) Add membrane permeabilization agent solution A to tubes 4 and 5 after incubation in step (3) and continue incubation at room temperature in the dark; (5) Add 1× hemolysin to tubes 1, 2, and 3 after incubation in step (3) and tubes 4 and 5 after incubation in step (4), and continue incubation at room temperature in the dark; (6) Centrifuge each flow cytometry tube after incubation in step (5) and remove the supernatant; (7) Add the membrane permeabilization agent B solution and the sixth group of antibodies in the reagent composition of any one of claims 1 to 2 to tube 4 after removing the supernatant in step (6), and add the membrane permeabilization agent B solution and the seventh group of antibodies in the reagent composition of any one of claims 1 to 2 to tube 5 after removing the supernatant in step (6), and incubate at room temperature in the dark; (8) PBS buffer was added to tubes 1, 2, and 3 after the supernatant was removed in step (6), and tubes 4 and 5 after the incubation in step (7), respectively, for washing. After centrifugation, the supernatant was removed and the cells were resuspended in PBS buffer to obtain flow cytometry samples.

7. A device for detecting acute promyelocytic leukemia, characterized in that: The device comprises a detection unit and an analysis unit, wherein: The detection unit includes a reagent material for detecting a sample from a test individual by flow cytometry to obtain a test result of the sample; the reagent material includes the reagent composition according to any one of claims 1 to 2; The analyzing unit is used to analyze the detection result of the detecting unit.

8. The device according to claim 7, characterized in that The device is used to detect acute promyelocytic leukemia, wherein: The process of testing a sample from an individual by flow cytometry involves: Treating a sample to be tested with the reagent composition according to any one of claims 1 to 2 to prepare a flow cytometry sample; Conduct flow cytometry testing; When flow cytometry is performed, the de-adhesive cell gate P1 and the live cell gate P2 are set for each tube in sequence to obtain single live cells; within the P2 gate, the CD45 / SSC ratio is used to set the gates for each blood cell; within the P2 gate, the CD45 weak expression / medium to large SSC ratio or CD117 positive / CD45 weak expression ratio is used to set the gates for myeloid immature cells, and the CD45 strong expression / small SSC ratio is used to set the gates for mature lymphocytes; and: Tube 1 was gated as follows: within gate P2, CD45 weak / SSC high or SSC high / CD117 negative was used to gate differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD45 strong expression / CD4 weak expression was used to gate monocytes; For tube 2, gates were set as follows: within gate P2, CD45 weak / SSC large or CD16 positive / CD11b positive were used to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD11b positive / HLA-DR positive were used to set the gate for monocytes; For tube 3, gates were set as follows: within gate P2, use weak CD45 / high SSC or strong CD15 expression / CD371 positivity to set the gate for differentiation-stage granulocytes, and strong CD45 expression / intermediate SSC or positive CD371 / weak CD15 expression / CD11c positivity to set the gate for monocytes; For tube 4, set gates as follows: within gate P2, use CD45 weak / SSC high or SSC high / CD117 negative to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD45 strong expression / CD38 positive to set the gate for monocytes; For tube 5, set gates as follows: within gate P2, use CD45 weak / SSC high or MPO positive / CD64 positive to set the gate for differentiation-stage granulocytes, and CD45 strong expression / SSC medium or CD64 strong expression / CD36 positive to set the gate for monocytes.

9. The device according to claim 7 or 8, characterized in that When the analysis unit is used to analyze the detection result of the detection unit, the detection result is output according to one of the following judgment methods: Method 1: Typical type of acute promyelocytic leukemia: Leukemic cells have a large SSC in the CD45 / SSC gate, and consistently express CD64, MPO, CD33, CD371, and CD9, moderately express CD117, express CD13 and CD38, and do not express CD34, HLA-DR, CD11b, CD14, and CD36; APLv: CD45 / SSC includes both CD34+ / small SSC and CD34- / large SSC tumor cells, with strong and consistent expression of CD64, MPO, CD33, CD371, and CD9, moderate expression of CD117, expression of CD13 and CD38, and no expression of HLA-DR, CD11b, CD14, and CD36; CD117-negative acute promyelocytic leukemia: SSC is larger, with consistent expression of CD9, MPO, CD33, CD64, and CD371, as well as expression of CD13 and CD38, but no expression of CD117, CD34, HLA-DR, CD11b, CD14, or CD36; Rare HLA-DR-positive acute promyelocytic leukemia: large SSC, strong and consistent expression of CD9, MPO, CD33, CD64, CD371, moderate expression of CD117, expression of CD13 and CD38, and no expression of CD34, CD11b, CD14, and CD36; Method 2: MPO positive, HLA-DR negative, CD34 and SSC cross; if each of these three items is met, 3 points will be added; if not met, 3 points will be subtracted; Lack of characteristic expression of tumor stem cells or FSC / SSC positions; 2 points will be added if each of these two items is met, and 2 points will be subtracted if each item is not met; CD117 moderate frustration, CD33 strong expression, CD9 positivity, CD64 consistency, CD371 consistency, if each of these five items is met, 1 point will be added, and 1 point will be subtracted if each of these five items is not met; cCD3 positive adds 1 point, negative does not reduce points; A score of 12 or above is APL.

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