An antibody composition, kit, and system for rapid screening of acute promyelocytic leukemia.

Abstract

This invention relates to an antibody composition, kit, and system for rapid screening of acute promyelocytic leukemia. The antibody composition comprises a first group of antibodies, a second group of antibodies, and a third group of antibodies. The first group of antibodies comprises antibodies against CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7, and CD45. The second group of antibodies comprises antibodies against CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15, and CD45. The third group of antibodies comprises antibodies against CD9, MPO, CD117, and CD45. Each of the antibodies is a monoclonal antibody labeled with a detection marker. The antibody composition contains a cell population targeting the CD117+CD33+ region, and includes three groups of antibodies that recognize acute promyelocytic leukemia, including CD33, CD34, CD64, CD13, CD9, and MPO. Using this antibody composition, acute promyelocytic leukemia can be detected quickly and easily with high sensitivity, specificity, and accuracy. It can accurately and rapidly identify patients with acute promyelocytic leukemia, enabling early diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to an antibody composition, kit, and system for rapid screening of acute promyelocytic leukemia. Background Technology

[0002] Acute promyelocytic leukemia (APL) is a specific type of acute myelogenous leukemia (AML). The vast majority of patients have a specific chromosomal translocation t(15;17)(q22;q12), forming the PML-RARa fusion gene. Its protein products lead to cell differentiation arrest and insufficient apoptosis, which is the main molecular mechanism of APL development. APL is more common in young and middle-aged adults, with an average age of onset of 44 years. APL accounts for 10%–15% of AML cases during the same period, with an incidence rate of approximately 0.23 / 100,000. APL presents with a severe clinical manifestation, and bleeding and embolism are common during onset and induction therapy, leading to death. Once APL is suspected, clinical treatment should be emergency, with timely administration of retinoic acid as a preemptive measure. Therefore, accurate and rapid identification of this disease is crucial for the patient's treatment prognosis.

[0003] 98% of APL patients have the PML-RARa fusion gene, while less than 2% have other types of fusion genes. Currently, clinical practice uses real-time quantitative PCR or RNA-seq technology to detect the PML-RARa fusion gene or other types of fusion genes for the diagnosis, efficacy evaluation, prognostic analysis, and recurrence prediction of APL. However, the current PCR technology for detecting the PML-RARa fusion gene has a long reporting cycle, generally around 2-3 days, which is not conducive to the rapid detection of APL and cannot meet the clinical need for rapid diagnosis of this disease.

[0004] In recent years, immunophenotyping has been found to play a significant auxiliary role in the diagnosis of APL. Typical tumor cell phenotypes in APL include strong expression of CD13, CD33, CD117, and MPO, while lacking expression of CD34, HLA-DR, CD11b, and CD56. Furthermore, flow cytometry immunophenotyping is simple to operate, providing results in 2-3 hours, greatly reducing patient waiting time and enabling rapid reporting to guide early clinical treatment and give patients a competitive edge. However, different hospitals may use different combinations of flow cytometry platforms, and the varying operational and data analysis procedures can lead to poor reproducibility of results, negatively impacting the accuracy of immunophenotyping findings in acute promyelocytic leukemia.

[0005] Therefore, there is a need in the market for a systematic rapid screening antibody composition and test kit for acute promyelocytic leukemia (APL), which includes standard testing protocols, operating procedures and analytical approaches to ensure the accuracy of results and help clinicians identify and diagnose APL at an early stage. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide an antibody composition, kit, and system for acute promyelocytic leukemia. The antibody composition can rapidly screen for acute promyelocytic leukemia and has the advantages of high accuracy, high sensitivity, and strong specificity, and is simple and convenient to use.

[0007] In a first aspect, the present invention provides an antibody composition for detecting acute promyelocytic leukemia, comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies;

[0008] The first group of antibodies includes CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7, and CD45 antibodies;

[0009] The second group of antibodies includes CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15, and CD45 antibodies;

[0010] The third group of antibodies includes CD9, MPO, CD117, and CD45 antibodies;

[0011] Each of the antibodies described is a monoclonal antibody labeled with a detection marker.

[0012] In some embodiments, the detection marker is a fluorescein; preferably, the fluorescein is selected from FITC, PE, ECD, and PE-Cy. TM 5.5, PE-Cy7, APC, APC-750, PB, KO.

[0013] In some embodiments, the CD71, CD38, and CD9 antibodies are labeled with the same fluorescein;

[0014] The CD33, CD56, and MPO antibodies are labeled with the same fluorescein;

[0015] The CD117 antibody and CD16 antibody in the first group of antibodies are labeled with the same fluorophore;

[0016] The CD34 antibody in the first group of antibodies, the CD34 antibody in the second group of antibodies, and the CD117 antibody in the third group of antibodies are labeled with the same fluorescein;

[0017] The HLA-DR and CD13 antibodies are labeled with the same fluorophore;

[0018] The CD19 and CD11b antibodies are labeled with the same fluorophore;

[0019] The CD10 and CD64 antibodies are labeled with the same fluorescein;

[0020] The CD7 and CD15 antibodies are labeled with the same fluorescein;

[0021] The CD45 antibody in the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with the same fluorophore.

[0022] In some embodiments, the CD71, CD38, and CD9 antibodies are labeled with FITC;

[0023] The CD33, CD56, and MPO antibodies were used to label fluorescein PE.

[0024] The CD117 and CD16 antibodies in the first group of antibodies are labeled with fluorescein ECD;

[0025] The CD34 antibody in the first group, the CD34 antibody in the second group, and the CD117 antibody in the third group are labeled with fluorescein PE-Cy. TM 5.5;

[0026] The HLA-DR and CD13 antibodies were labeled with fluorescein PE-Cy7;

[0027] The CD19 and CD11b antibodies are labeled with fluorescein APC;

[0028] The CD10 and CD64 antibodies were labeled with fluorescein APC-750;

[0029] The CD7 and CD15 antibodies were labeled with fluorescein PB;

[0030] The CD45 antibody in the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with fluorescein KO.

[0031] In some embodiments, the catalog number of CD117 antibody-labeled fluorescein ECD is B38307, the catalog number of CD34 antibody-labeled fluorescein PECY5.5 is 343522, the catalog number of HLA-DR antibody-labeled fluorescein PE-Cy7 is B49180, the catalog number of CD19 antibody-labeled fluorescein APC is IM2470, the catalog number of CD7 antibody-labeled fluorescein PB is B06499, the catalog number of CD56 antibody-labeled fluorescein PE is A07788, and the catalog number of CD16 antibody-labeled... The catalog numbers for the following products are listed: ECD (fluorescein ECD) B49216, PECY5.5 (CD34 antibody-labeled fluorescein) 343522, APC750 (CD64 antibody-labeled fluorescein) B96769, FITC (CD9 antibody-labeled fluorescein FITC) IM1755U, PE (MPO antibody-labeled fluorescein PE) B36288, and PECY5.5 (CD117 antibody-labeled fluorescein PECY5.5) B96754. The manufacturer is Beckman Coulter.

[0032] The catalog numbers for CD71 antibody-labeled fluorescein FITC are 665339, CD33 antibody-labeled fluorescein PE are 663527, and CD11b antibody-labeled fluorescein APC are 982604. The manufacturer is BD.

[0033] The catalog numbers for CD10 antibody-labeled fluorescein APC750 are 982208, CD45 antibody-labeled fluorescein KO are 982320, CD38 antibody-labeled fluorescein FITC are 980304, CD13 antibody-labeled fluorescein PECY7 are 982806, and CD15 antibody-labeled fluorescein PB are 3230409. The manufacturer is Biolegend.

[0034] In a second aspect, the present invention provides a kit for detecting acute promyelocytic leukemia, the kit comprising the antibody composition described above.

[0035] A third aspect of the invention provides the use of the antibody composition or kit described above in the adjunctive detection of acute promyelocytic leukemia.

[0036] A fourth aspect of the present invention provides a system for detecting acute promyelocytic leukemia, comprising:

[0037] The detection module utilizes the antibody composition described above or the kit described above to perform flow cytometry detection on the cells to be tested.

[0038] The data acquisition module acquires data from flow cytometry analysis results.

[0039] The data analysis module analyzes the acquired data: using CD45-SSC gating, and dividing the cell population into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and primitive cells based on CD45 expression; further analyzing the target cells within the granulocyte population, and analyzing the fluorescence expression intensity of antibody pairs in the target cells; the antibody pairs include: CD45-SSC, CD117-CD33, CD117-FS, CD117-CD34, CD117-HLA- DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO, and CD9-CD45; determine whether the obtained promyelocytes are neoplastic promyelocytes according to the judgment criteria.

[0040] In some embodiments, the judgment criteria include:

[0041] If the antibody expression pattern of the cell to be tested falls into the expression pattern template of the antibody of abnormal promyelocytes, the cell to be tested will be identified as a neoplastic promyelocyte.

[0042] If the antibody expression pattern of the cell to be tested does not fall within the expression pattern template of the antibody of the abnormal promyelocyte, the cell to be tested will be identified as a non-tumor promyelocyte.

[0043] In some embodiments, the antibody expression patterns of the test cells and abnormal promyelocytes are established through the following steps: flow cytometry data are gated using CD45-SSC, and the cell population is divided into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and primitive cells based on CD45 expression. Further analysis is performed on the target cells within the granulocyte population to analyze the fluorescence expression intensity of antibody pairs in the target cells. The antibody pairs include: CD45-SSC and CD117-CD117. 33. CD117-FS, CD117-CD34, CD117-HLA-DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD 11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO and CD9-CD45.

[0044] The inventors of this invention, combining years of experience and extensive research, have developed an antibody composition for rapid screening of acute promyelocytic leukemia (APLE). This antibody composition contains a cell population primarily targeting the CD117+CD33+ region, and includes three groups of antibodies that recognize APL, such as CD33, CD34, CD64, CD13, CD9, and MPO. Using this antibody composition, APL can be detected quickly and easily with high sensitivity, specificity, and accuracy. It can accurately and rapidly identify APL patients, enabling early diagnosis and treatment and providing patients with valuable time for treatment. Attached Figure Description

[0045] Figure 1 This is a scatter plot of the CD45-SSC expression immunophenotype of the APL sample to be tested, detected using the first group of antibodies in Example 3 of the present invention.

[0046] Figure 2 This is a scatter plot of the CD117-CD33 expression immunophenotype of the APL sample to be tested, which was detected using the first group of antibodies in Example 3 of the present invention.

[0047] Figure 3 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the immunophenotype CD117-FS in the APL sample to be tested.

[0048] Figure 4 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the immunophenotype CD117-CD34 in the APL sample to be tested.

[0049] Figure 5 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the immunophenotype CD117-HLA-DR in the APL sample to be tested.

[0050] Figure 6 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the immunophenotype CD71-CD45 in the APL sample to be tested.

[0051] Figure 7 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD34-CD19 immunophenotype in the APL sample to be tested.

[0052] Figure 8 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD19-CD10 immunophenotype in the APL sample to be tested.

[0053] Figure 9 This is a scatter plot of the CD117-CD7 expression of the APL sample to be tested, detected using the first group of antibodies in Example 3 of the present invention.

[0054] Figure 10 This is a scatter plot of the CD13-CD64 expression immunophenotype of the APL sample to be tested, detected using the second group of antibodies in Example 3 of the present invention.

[0055] Figure 11 This is a scatter plot of the CD15-CD11b expression immunophenotype of the APL sample to be tested, detected using the second group of antibodies in Example 3 of the present invention.

[0056] Figure 12 In Example 3 of this invention, the expression of the CD13-CD16 immunophenotype in the APL sample to be tested was detected using the second group of antibodies.

[0057] Figure 13 In Example 3 of this invention, the expression of the CD38-CD56 immunophenotype in the normal APL sample to be tested was detected using the second group of antibodies.

[0058] Figure 14 In Example 3 of this invention, the expression of the CD34-CD38 immunophenotype in the APL sample to be tested was detected using the second group of antibodies.

[0059] Figure 15 In Example 3 of this invention, the expression of CD34-CD13 in the immunophenotype of the APL sample to be tested was detected using the second group of antibodies.

[0060] Figure 16 In Example 3 of this invention, the expression of the CD34-CD56 immunophenotype in the APL sample to be tested was detected using the second group of antibodies.

[0061] Figure 17 In Example 3 of this invention, the expression of the CD34-CD11b immunophenotype in the APL sample to be tested was detected using the second group of antibodies.

[0062] Figure 18 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD117-CD9 immunophenotype in the APL sample to be tested.

[0063] Figure 19 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD117-MPO immunophenotype in the APL sample to be tested.

[0064] Figure 20 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD9-CD45 immunophenotype in the APL sample to be tested.

[0065] Figure 21 This is a scatter plot of the CD45-SSC expression immunophenotype of the non-APL sample being tested using the first group of antibodies in Example 3 of the present invention.

[0066] Figure 22 This is a scatter plot of the CD117-CD33 expression immunophenotype of the non-APL sample to be tested, using the first group of antibodies in Example 3 of the present invention.

[0067] Figure 23 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD117-FS immunophenotype in the non-APL sample to be tested.

[0068] Figure 24 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD117-CD34 immunophenotype in the non-APL sample to be tested.

[0069] Figure 25 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD117-HLA-DR immunophenotype in the non-APL sample to be tested.

[0070] Figure 26 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD71-CD45 immunophenotype in the non-APL sample to be tested.

[0071] Figure 27 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD34-CD19 immunophenotype in the non-APL sample to be tested.

[0072] Figure 28 In Example 3 of this invention, the first group of antibodies was used to detect the expression of the CD19-CD10 immunophenotype in the non-APL sample to be tested.

[0073] Figure 29 This is a scatter plot of the CD117-CD7 expression immunophenotype of the non-APL sample to be tested, using the first group of antibodies in Example 3 of the present invention.

[0074] Figure 30 This is a scatter plot of the CD13-CD64 expression immunophenotype of the non-APL sample being tested using the second group of antibodies in Example 3 of the present invention.

[0075] Figure 31 This is a scatter plot of the CD15-CD11b expression immunophenotype of the non-APL sample being tested, using the second group of antibodies in Example 3 of the present invention.

[0076] Figure 32 In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD13-CD16 immunophenotype in the non-APL sample to be tested.

[0077] Figure 33In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD38-CD56 immunophenotype in normal non-APL samples.

[0078] Figure 34 In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD34-CD38 immunophenotype in the non-APL sample to be tested.

[0079] Figure 35 In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD34-CD13 immunophenotype in the non-APL sample to be tested.

[0080] Figure 36 In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD34-CD56 immunophenotype in the non-APL sample to be tested.

[0081] Figure 37 In Example 3 of this invention, the second group of antibodies was used to detect the expression of the CD34-CD11b immunophenotype in the non-APL sample to be tested.

[0082] Figure 38 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD117-CD9 immunophenotype in the non-APL sample to be tested.

[0083] Figure 39 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD117-MPO immunophenotype in the non-APL sample to be tested.

[0084] Figure 40 In Example 3 of this invention, the third group of antibodies was used to detect the expression of the CD9-CD45 immunophenotype in the non-APL sample to be tested. Detailed Implementation

[0085] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0086] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0087] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with those belonging to this invention.

[0088] The meanings are generally understood to be the same by those skilled in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0089] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0090] Some embodiments of the present invention relate to an antibody composition for detecting acute promyelocytic leukemia, comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies;

[0091] The first group of antibodies includes CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7, and CD45 antibodies;

[0092] The second group of antibodies includes CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15, and CD45 antibodies;

[0093] The third group of antibodies includes CD9, MPO, CD117, and CD45 antibodies;

[0094] Each of the antibodies described is a monoclonal antibody labeled with a detection marker.

[0095] The antibody composition of this invention comprises three groups of antibodies, targeting the CD117+CD33+ region as the primary cell population, and including antibodies that recognize abnormal promyelocyte populations (i.e., neoplastic promyelocyte populations) such as CD9, CD13, CD64, HLA-DR, and MPO. Through a comprehensive logical analysis strategy, this composition can effectively identify neoplastic abnormal promyelocytes, thus enabling the detection of APL. This invention rationally applies various antibody indicators, covering a wide range. Using this antibody combination with multi-parameter flow cytometry detection technology, it can quickly, easily (from sample reception to test results, only 1-2 hours), and with high sensitivity, detect abnormal promyelocytes. Furthermore, this invention optimizes the result analysis template. Previously, only a single gate (CD117+) was used to check for HLA-DR and CD34 expression. However, because tumor cells from other types of AML patients can express CD117, the inaccurate gate led to biased analysis results. After optimization, this invention first uses CD117-CD33 to circle suspicious cells and observes the expression of HLA-DR, CD34, CD13, and CD64. In addition, the analysis template is fixed, and data can be directly analyzed by dragging and dropping in. The operation is simple, reduces the professional knowledge required of the analysts, and makes up for the limitations of existing detection technologies.

[0096] The antibody composition of the present invention comprises a first group of antibodies, a second group of antibodies, and a third group of antibodies:

[0097] 1. The first group of antibodies includes: CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7, and CD45 antibodies. Besides CD45 as a leukocyte gating antibody, the first group of antibodies uses the backbone antibody CD117-CD33 to identify suspected abnormal promyelocytes. Then, check the expression of CD34 and HLA-DR. If neither is expressed, abnormal promyelocytes can be initially considered. Further confirmation is then made using the second / third group of antibodies. The first group of antibodies also includes the normal erythroblast marker CD71 to check the proportion of erythroblasts. It also includes lymphoid markers such as CD19, CD10, and CD7 for preliminary screening of B-ALL and T-ALL cells. If these are expressed, the possibility of lymphoid leukemia or mixed leukemia needs to be considered.

[0098] 2. The second group of antibodies includes: CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15, and CD45 antibodies. The second group of antibodies must be used in conjunction with the first group of antibodies. Using CD38-CD45 can identify suspected abnormal promyelocyte populations. Then, check the expression of CD34, CD11b, CD15, CD13, CD64, and CD16. Negative CD11b / CD16 suggests that immature monocytes are not a possibility. Negative CD34, high CD13 expression, and CD64 expression are consistent with the immunophenotype of abnormal promyelocytes. If CD38 is strongly expressed and CD56 is positive, plasma cells need to be excluded for further differential diagnosis.

[0099] 3. The third group of antibodies includes CD9, MPO, CD117 and CD45 antibodies. The third group of antibodies must be used in combination with the first and second groups of antibodies. Using CD117-CD45 can identify suspicious abnormal promyelocyte populations. Then check whether CD9 and MPO are expressed. If both CD9 and MPO are highly expressed, the phenotype is confirmed to be consistent with abnormal promyelocytes.

[0100] In some embodiments, the detection marker is a fluorescein; preferably, the fluorescein is selected from FITC, PE, ECD, PE-CyT. M 5.5, PE-Cy7, APC, APC-750, PB, KO.

[0101] In some embodiments, the CD71, CD38, and CD9 antibodies are labeled with the same fluorophore; the CD33, CD56, and MPO antibodies are labeled with the same fluorophore; the CD117 and CD16 antibodies in the first group of antibodies are labeled with the same fluorophore; the CD34 antibody in the first group of antibodies, the CD34 antibody in the second group of antibodies, and the CD117 antibody in the third group of antibodies are labeled with the same fluorophore; the HLA-DR and CD13 antibodies are labeled with the same fluorophore; the CD19 and CD11b antibodies are labeled with the same fluorophore; the CD10 and CD64 antibodies are labeled with the same fluorophore; the CD7 and CD15 antibodies are labeled with the same fluorophore; and the CD45 antibody in the first, second, and third groups of antibodies is labeled with the same fluorophore.

[0102] In some embodiments, the CD71, CD38, and CD9 antibodies are labeled with fluorescein FITC; the CD33, CD56, and MPO antibodies are labeled with fluorescein PE; the CD117 and CD16 antibodies from the first group of antibodies are labeled with fluorescein ECD; and the CD34 antibody from the first group of antibodies, the CD34 antibody from the second group of antibodies, and the CD117 antibody from the third group of antibodies are labeled with fluorescein PE-Cy. TM5.5; the HLA-DR and CD13 antibodies are labeled with fluorescein PE-Cy7; the CD19 and CD11b antibodies are labeled with fluorescein APC; the CD10 and CD64 antibodies are labeled with fluorescein APC-750; the CD7 and CD15 antibodies are labeled with fluorescein PB; the CD45 antibody from the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with fluorescein KO.

[0103] In some preferred embodiments, the catalog number of CD117 antibody-labeled fluorescein ECD is B38307, the catalog number of CD34 antibody-labeled fluorescein PECY5.5 is 343522, the catalog number of HLA-DR antibody-labeled fluorescein PE-Cy7 is B49180, the catalog number of CD19 antibody-labeled fluorescein APC is IM2470, the catalog number of CD7 antibody-labeled fluorescein PB is B06499, the catalog number of CD56 antibody-labeled fluorescein PE is A07788, and the catalog number of CD16 antibody-labeled fluorescein ECD is B06499. The catalog numbers for the following products are as follows: ECD (labeled with antibody), PECY5.5 (labeled with CD34 antibody), APC750 (labeled with CD64 antibody), FITC (labeled with CD9 antibody), PECY5.5 (labeled with MPO antibody), and PECY5.5 (labeled with CD117 antibody). The manufacturer is Beckman Coulter.

[0104] The catalog numbers for CD71 antibody-labeled fluorescein FITC are 665339, CD33 antibody-labeled fluorescein PE are 663527, and CD11b antibody-labeled fluorescein APC are 982604. The manufacturer is BD.

[0105] The catalog numbers for CD10 antibody-labeled fluorescein APC750 are 982208, CD45 antibody-labeled fluorescein KO are 982320, CD38 antibody-labeled fluorescein FITC are 980304, CD13 antibody-labeled fluorescein PECY7 are 982806, and CD15 antibody-labeled fluorescein PB are 3230409. The manufacturer is Biolegend.

[0106] Some embodiments of the present invention relate to a kit for detecting acute promyelocytic leukemia, the kit comprising the antibody composition described above.

[0107] Some embodiments of the present invention relate to a system for detecting acute promyelocytic leukemia, comprising:

[0108] The detection module utilizes the antibody composition described above or the kit described above to perform flow cytometry detection on the cells to be tested.

[0109] The data acquisition module acquires data from flow cytometry analysis results.

[0110] The data analysis module analyzes the acquired data: using CD45-SSC gating, and dividing the cell population into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and primitive cells based on CD45 expression; further analyzing the target cells within the granulocyte population, and analyzing the fluorescence expression intensity of antibody pairs in the target cells; the antibody pairs include: CD45-SSC, CD117-CD33, CD117-FS, CD117-CD34, and CD117-HLA-D. R, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO, and CD9-CD45; determine whether the obtained promyelocytes are neoplastic abnormal promyelocytes according to the judgment criteria.

[0111] In some embodiments, the detection module includes: a single-cell suspension module for preparing a single-cell suspension; an incubation module for incubating the single-cell suspension and the reagent composition in the dark to perform an antigen-antibody reaction; a resuspension module for adding hemolysin to the incubated cells, centrifuging, washing, and resuspending the cells to prepare a suspension; and an assay module for performing flow cytometry assays on the resuspended suspension.

[0112] In some embodiments, the judgment criteria include:

[0113] If the antibody expression pattern of the cell to be tested falls into the expression pattern template of the antibody of abnormal promyelocytes, the cell to be tested will be identified as a neoplastic promyelocyte.

[0114] If the antibody expression pattern of the cell to be tested does not fall within the expression pattern template of the antibody of the abnormal promyelocyte, the cell to be tested will be identified as a non-tumor promyelocyte.

[0115] In some embodiments, the antibody expression patterns of the test cells and abnormal promyelocytes are established through the following steps: flow cytometry data are gated using CD45-SSC, and the cell population is divided into five regions based on CD45 expression: granulocyte population (upper middle Gran region in the figure), monocyte population (upper right mono region in the figure), lymphocyte population (lower right lym region in the figure), nucleated erythrocyte or platelet population (lower left CD45neg region in the figure), and primitive cell population (lower middle CD45dim region in the figure), facilitating further analysis of the target cells within the granulocyte population; further subdivision... The fluorescence expression intensity of the antibody pairs in the target cells was analyzed; the antibody pairs included: CD45-SSC, CD117-CD33, CD117-FS, CD117-CD34, CD117-HLA-DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO, and CD9-CD45.

[0116] The system for detecting acute promyelocytic leukemia of the present invention can refer to the conventional techniques of flow cytometry. The equipment and consumables used are those for flow cytometry analysis, such as flow cytometry tubes, shakers, pipettes, and other consumables.

[0117] The present invention will be further described in detail below with reference to specific embodiments.

[0118] The specific sources of some of the antibodies used in the following examples are: CD117ECD (catalog number B38307), CD34PECY5.5 (catalog number 343522), HLA-DR PECY7 (catalog number B49180), CD19APC (catalog number IM2470), CD7 PB (catalog number B06499), CD56 PE (catalog number A07788), CD16 ECD (catalog number B49216), CD34 PECY5.5 (catalog number 343522), CD64APC750 (catalog number B96769), CD9 FITC (catalog number IM1755U), MPO PE (catalog number B36288), CD117 PECY5.5 (catalog number B96754), manufactured by Beckman Coulter; CD71FITC (catalog number 665339), CD33 PE (part number 663527), CD11b APC (part number 982604), manufacturer BD; CD10 APC750 (part number 982208), CD45 KO (part number 982320), CD38FITC (part number 980304), CD13 PECY7 (part number 982806), CD15 PB (part number 3230409), manufacturer bio1egend.

[0119] Example 1: An antibody composition for rapid screening of acute promyelocytic leukemia (APL)

[0120] This embodiment provides an antibody composition for rapid screening of acute promyelocytic leukemia (APL), comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies, wherein...

[0121] The first group of antibodies includes: CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7 and CD45 antibodies;

[0122] The second group of antibodies includes: CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15, and CD45 antibodies;

[0123] The third group of antibodies includes: CD9, MPO, CD117 and CD45 antibodies.

[0124] The fluorescent labeling and dosage of the relevant monoclonal antibodies mentioned above are shown in Table 1.

[0125] Table 1

[0126]

[0127] Note: The above-mentioned commercially available antibodies were subjected to concentration gradient verification to determine the optimal dosage. The above-mentioned dosage of monoclonal antibody was then loaded into flow cytometry tubes numbered 1, 2, and 3 respectively.

[0128] Example 2: A system and method for rapid screening of acute promyelocytic leukemia (APL).

[0129] This embodiment provides a system for rapid screening of acute promyelocytic leukemia (APL), comprising: a detection module, a data acquisition module, and a data analysis module. The detection module performs flow cytometry analysis on the cells to be tested; the data acquisition module acquires the flow cytometry results of the cells to be tested stained with the detection reagent composition described in Example 1; the data analysis module analyzes the acquired data and determines whether the cells to be tested are neoplastic promyelocytic cells according to predetermined judgment criteria.

[0130] The specific workflow for detecting acute promyelocytic leukemia (APL) using the system described in this embodiment is as follows:

[0131] 1. Prepare each antibody in the antibody combination of Example 1, as shown in Table 1.

[0132] 2. Sample processing.

[0133] The concentration of the sample to be tested (bone marrow fluid) was adjusted to 1×10⁻⁶ cells based on the cell count. 6 Cells / ml were prepared into a single-cell suspension.

[0134] 3. Sample testing.

[0135] (1) Take a flow cytometer, label it 1 and 2, and add the first group antibody, the second group antibody and the third group antibody in Example 1 respectively. The amount added is 25 μl, 15 μl and 16 μl respectively. Then add 100 μl of the suspension in step 2, vortex to mix, and incubate at room temperature in the dark for 15 min.

[0136] (2) Add 400 μl of Bc hemolysin to each of the incubated flow cytometry tubes 1, 2, and 3, vortex, and let stand until hemolysis and clarity are achieved. After hemolysis and clarity are achieved, centrifuge flow cytometry tubes 1, 2, and 3 at 1500 r / min for 5 min, discard the supernatant, add 2 ml of fetal bovine serum, vortex, centrifuge at 1500 r / min for 5 min, and discard the supernatant. Resuspend flow cytometry tubes 1 and 2 in 400 μl of 1% paraformaldehyde.

[0137] (3) Intracellular antibody experiment procedure: After staining the cell surface according to the above (1, 2) steps, add 450 μL of 1X FACS membrane permeabilizing agent, mix well and incubate in the dark for 5 min. After washing once with PBS, discard the supernatant. Add anti-intracellular fluorescent antibody (Granzyme B, Perforin, Ki67) according to the above antibody amount and incubate in the dark at room temperature for 30 min. Add 2 ml of fetal bovine serum, vortex, centrifuge at 1500 r / min for 5 min, discard the supernatant, and resuspend in 400 μl of 1% paraformaldehyde.

[0138] (4) The flow cytometer tubes 1, 2 and 3 were tested and their immunophenotypes were analyzed using a Beckman Coulter Navios ten-color flow cytometer.

[0139] 4. Data analysis.

[0140] (1) Establish an expression pattern template for patients with acute promyelocytic leukemia (APL).

[0141] The above experimental data were obtained from 40 newly diagnosed acute promyelocytic leukemia (APL) patients who tested positive for the PML-RARA fusion gene. Cells were grouped by gating, with CD45-SSC (side-scattered light) gating being the preferred method to identify the target cell population. The expression of various fluorescent antibodies in this cell population was then analyzed, and the following antibody pairs were selected: CD45-SS... C. CD117-CD33, CD117-FS, CD117-CD34, CD117-HLA-DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-56, CD34-11b, CD117-CD9, CD117-MPO, CD9-CD45.

[0142] (2) Establish the antibody expression pattern of the cells to be tested

[0143] Obtain the flow cytometry results of the cells to be tested, and then, according to the method described in the antibody expression pattern of acute promyelocytic leukemia (APL), set the gates to divide the cells into groups, circle the target cell group, and then analyze the expression of each fluorescent antibody in the target cell group according to the method described in the antibody expression pattern of acute promyelocytic leukemia (APL) to obtain the antibody expression pattern of the cells to be tested.

[0144] (3) Analyze the antibody expression pattern of the cells to be tested.

[0145] If the antibody expression pattern of the cell to be tested falls into the expression pattern template of the antibody of abnormal promyelocytes, the cell to be tested will be identified as a neoplastic promyelocyte.

[0146] If the antibody expression pattern of the cell to be tested does not fall within the expression pattern template of the antibody of the abnormal promyelocyte, the cell to be tested will be identified as a non-tumor promyelocyte.

[0147] Example 3: Validation of the detection sensitivity and specificity of the detection method of the present invention

[0148] Statistical analysis of 757 AML cases reported using this invention between January 1, 2021 and December 31, 2023 at our center included 110 APL cases and 647 AML (non-M3) cases. The genetic results and final clinical diagnoses were tracked, and the results are shown in Table 2 below. Among the 110 patients with APL reported by flow cytometry, 104 were positive for the PMR-RARA fusion gene (quantitative PCR), and 5 were positive for other variant RARA fusion genes (next-generation sequencing). Among the 647 patients with AML (non-M3) reported by flow cytometry, 3 were positive for the PMR-RARA fusion gene (quantitative PCR), and 5 were positive for other variant RARA fusion genes (next-generation sequencing). The detection sensitivity of the combined antibody of this invention can reach 99.09%, and the detection specificity can reach 98.76%. Furthermore, the detection time is 1.5-2.5 hours, which can well meet the clinical requirements for rapid screening of acute promyelocytic leukemia.

[0149] Table 2

[0150]

[0151] The following two case studies illustrate the analytical strategy of the detection scheme of this invention by detecting tumor cells in two test cells (1 APL sample + 1 non-APL AML control sample) and performing antibody expression pattern analysis.

[0152] I. APL Sample

[0153] This sample is an APL bone marrow fluid sample that tested positive for the PML-RARA fusion gene. Antibody expression pattern analysis was performed according to the following steps:

[0154] Obtain the flow cytometry results of the sample. Using CD45-SSC (side-scattered light) gating, the cell population was divided into five regions based on CD45-SSC expression, namely granulocytes (…). Figure 1 Middle upper Gran region), monocytes ( Figure 1 (middle right upper mono region), lymphocytes ( Figure 1 The lower right 1-ym region), the nucleated red blood / platelet region ( Figure 1 The lower left CD45neg region and the primitive cell population ( Figure 1 The middle and lower part of the CD45dim area) are divided into 5 areas (e.g. Figure 1 As shown in the figure, the granulocyte region is the area where the target cell population is located. The following will perform immunophenotypic analysis on this cell population.

[0155] Figure 2 The scatter plot of CD117-CD33 immunophenotypes for all nucleated cells shows that 83.90% of the cells are CD117+CD33+ abnormal (red in the figure), which is significantly higher and suggests a high probability of AML.

[0156] Figure 3 A scatter plot of CD117-FS expression in all nucleated cells shows that CD117+ abnormal cells have a large FS, indicating that the abnormal cells are relatively large.

[0157] Figure 4 A scatter plot showing the expression of CD117-CD34 in all nucleated cells. The plot shows that CD117+ abnormal cells are CD34 negative (not expressed).

[0158] Figure 5 A scatter plot showing the expression of CD117-HLA-DR in all nucleated cells. The plot shows that CD117+ abnormal cells are HLA-DR negative (not expressed).

[0159] Figure 6 A scatter plot of CD71-CD45 expression in all nucleated cells shows that CD117+ abnormal cells are CD71 negative (not expressed), and the proportion of CD71+CD45- nucleated erythrocytes is 2.78%.

[0160] Figure 7 A scatter plot showing the expression of CD34-CD19 in all nucleated cells. The plot shows that CD117+ abnormal cells are CD19 negative (not expressed), and CD19+ normal B lymphocytes are visible (blue population in the upper left corner of the plot).

[0161] Figure 8 A scatter plot showing the expression of CD19-CD10 in all nucleated cells shows that CD117+ abnormal cells are CD10 negative (not expressed).

[0162] Figure 9 A scatter plot showing the expression of CD117-CD7 in all nucleated cells. The plot shows that CD117+ abnormal cells are CD7-negative (not expressed), and CD7+ normal T lymphocytes are visible (blue population in the upper left corner of the plot).

[0163] Figure 10A scatter plot showing the expression of CD13-CD64 in all nucleated cells shows that CD117+ abnormal cells are positive for both CD13 and CD64.

[0164] Figure 11 A scatter plot showing the expression of CD15-CD11b in all nucleated cells. The plot shows that CD117+ abnormal cells are negative for both CD15 and CD11b (not expressed).

[0165] Figure 12 A scatter plot showing the expression of CD13-CD16 in all nucleated cells shows that CD117+ abnormal cells are CD16 negative (not expressed).

[0166] Figure 13 A scatter plot showing the expression of CD38-CD56 in all nucleated cells. The plot shows that CD117+ abnormal cells are positive for CD38 (expressed) and negative for CD56 (not expressed).

[0167] Figure 14 A scatter plot of CD34-CD38 expression in all nucleated cells shows that CD117+ abnormal cells are CD34 negative (not expressed), and no plasma cells with obvious CD38++ are observed.

[0168] Figure 15 A scatter plot showing the expression of CD34-CD13 in all nucleated cells shows that CD117+ abnormal cells are positive for CD13 (expressed) and negative for CD34 (not expressed).

[0169] Figure 16 A scatter plot showing the expression of CD34-CD56 in all nucleated cells. The plot shows that CD117+ abnormal cells are negative for both CD34 and CD56 (not expressed), while CD56+ normal NK lymphocytes are visible (blue population in the upper left corner of the plot).

[0170] Figure 17 A scatter plot showing the expression of CD34-CD11b in all nucleated cells. The plot shows that CD117+ abnormal cells are negative for both CD34 and CD11b (not expressed), while normal monocytes are positive for CD11b (the purple population in the figure).

[0171] Figure 18 A scatter plot showing the expression of CD117-CD9 in all nucleated cells shows that CD117+ abnormal cells are positive for CD9 (expression).

[0172] Figure 19 A scatter plot showing the expression of CD117-MPO in all nucleated cells. The plot shows that CD117+ abnormal cells are positive for MPO (expression).

[0173] Figure 20 A scatter plot of CD9-CD45 expression in all nucleated cells shows that CD117+ abnormal cells have a stronger expression intensity of CD9.

[0174] The abnormal cells in this sample were large (high FS), expressed CD117, CD9, CD33 (strong expression), MPO (strong expression), CD13, CD38, and CD64, but did not express early primitive cell markers (CD34, HLA-DR), lymphoid markers (CD19, CD7, CD56), or mature granulocyte markers (CD11b, CD16, CD10). This immunophenotype was consistent with abnormal promyelocytes, and the case was considered to be acute promyeloid leukemia (APL), which was consistent with the positive result of the PML-RARA fusion gene detection.

[0175] II. Non-APL AML Control Samples

[0176] This sample is a non-APL AML bone marrow fluid specimen that tested negative for the PML-RARA fusion gene. Antibody expression pattern analysis was performed following these steps:

[0177] Obtain the flow cytometry results of the sample. Using CD45-SSC (side-scattered light) gating, the cell population was divided into five regions based on CD45-SSC expression, namely granulocytes (…). Figure 21 Middle upper Gran region), monocytes ( Figure 21 (middle right upper mono region), lymphocytes ( Figure 21 The lower right 1-ym region), the nucleated red blood / platelet region ( Figure 21 The lower left CD45neg region and the primitive cell population ( Figure 21 The middle and lower part of the CD45dim area) are divided into 5 areas (e.g. Figure 21 As shown in the figure, the granulocyte region is the area where the target cell population is located. The following will perform immunophenotypic analysis on this cell population.

[0178] Figure 22 The scatter plot of CD117-CD33 immunophenotypes for all nucleated cells shows that 78.34% of the cells are CD117+CD33+ abnormal (red in the figure), a significantly increased proportion, suggesting a high probability of AML.

[0179] Figure 23 A scatter plot of CD117-FS expression in all nucleated cells shows that CD117+ abnormal cells have a large FS, indicating that the abnormal cells are relatively large.

[0180] Figure 24A scatter plot showing the expression of CD117-CD34 in all nucleated cells shows that CD117+ abnormal cells are partially positive for CD34 (partial expression).

[0181] Figure 25 A scatter plot showing the expression of CD117-HLA-DR in all nucleated cells. The plot shows that CD117+ abnormal cells are partially positive for HLA-DR (partially expressed).

[0182] Figure 26 A scatter plot of CD71-CD45 expression in all nucleated cells shows that CD117+ abnormal cells are CD71 negative (not expressed), and the proportion of CD71+CD45- nucleated erythrocytes is 1.05%.

[0183] Figure 27 A scatter plot showing the expression of CD34-CD19 in all nucleated cells. The plot shows that CD117+ abnormal cells are CD19 negative (not expressed), and CD19+ normal B lymphocytes are visible (blue population in the upper left corner of the plot).

[0184] Figure 28 A scatter plot showing the expression of CD19-CD10 in all nucleated cells shows that CD117+ abnormal cells are CD10 negative (not expressed).

[0185] Figure 29 The scatter plot shows the CD117-CD7 expression of all nucleated cells. It can be seen from the figure that CD117+ abnormal cells are partially positive for CD7 (partial expression), and CD7+ normal T lymphocytes can be seen (blue population in the upper left corner of the figure).

[0186] Figure 30 A scatter plot showing the expression of CD13-CD64 in all nucleated cells. The plot shows that CD117+ abnormal cells are positive for CD13 (expression) and partially positive for CD64 (partial expression).

[0187] Figure 31 A scatter plot showing the expression of CD15-CD11b in all nucleated cells. The plot shows that CD117+ abnormal cells are negative for both CD15 and CD11b (not expressed).

[0188] Figure 32 A scatter plot showing the expression of CD13-CD16 in all nucleated cells shows that CD117+ abnormal cells are CD16 negative (not expressed).

[0189] Figure 33A scatter plot showing the expression of CD38-CD56 in all nucleated cells. The plot shows that CD117+ abnormal cells are positive for CD38 (expressed) and negative for CD56 (not expressed).

[0190] Figure 34 A scatter plot of CD34-CD38 expression in all nucleated cells shows that CD117+ abnormal cells are CD34 negative (not expressed), and no plasma cells with obvious CD38++ are observed.

[0191] Figure 35 A scatter plot showing the expression of CD34-CD13 in all nucleated cells shows that CD117+ abnormal cells are positive for CD13 (expression) and partially positive for CD34 (partial expression).

[0192] Figure 36 The scatter plot shows the expression of CD34-CD56 in all nucleated cells. It can be seen from the figure that CD117+ abnormal cells are partially positive for CD34 (partially expressed) and all are negative for CD56 (not expressed). CD56+ normal NK lymphocytes can be seen (blue population in the upper left corner of the figure).

[0193] Figure 37 A scatter plot of CD34-CD11b expression in all nucleated cells shows that CD117+ abnormal cells are partially positive for CD34 (partially expressed) and completely negative for CD11b (not expressed).

[0194] Figure 38 A scatter plot showing the CD117-CD9 expression of all nucleated cells shows that CD117+ abnormal cells are all CD9 negative (not expressed).

[0195] Figure 39 A scatter plot of CD117-MPO expression in all nucleated cells shows that CD117+ abnormal cells are positive for MPO (expression).

[0196] Figure 40 A scatter plot showing the expression of CD9-CD45 in all nucleated cells shows that CD117+ abnormal cells are CD9 negative (not expressed).

[0197] The abnormal cells in this sample were also large in size (high FS), expressed early primitive cell markers (CD34, HLA-DR), expressed CD117, CD33 (strongly expressed), MPO (strongly expressed), CD13, CD38, partially expressed CD64, did not or partially expressed the T lymphoid marker CD7, did not express B and NK markers (CD19, CD56), did not express mature granulocyte markers (CD11b, CD16, CD10), and did not express CD9. This immunophenotype was consistent with abnormal primitive myeloid cells rather than abnormal promyeloid cells, and was considered to be acute myeloid leukemia (AML, not APL), which was consistent with the negative result of the PML-RARA fusion gene test.

[0198] The results of this embodiment show that the antibody composition of the present invention targets the CD117+CD33+ region as the main target cell population, and includes antibody combinations that recognize abnormal promyelocyte populations such as CD9, CD13, CD64, HLA-DR, and MPO. With a comprehensive logical analysis strategy, it can quickly identify tumor-related abnormal promyelocytes, helping clinicians to quickly identify acute promyeloid leukemia (APL) and buy patients the golden time for treatment.

[0199] Example 4: Clinical conformity verification of the detection method of the present invention

[0200] Bone marrow fluid was collected from 40 patients clinically diagnosed with APL and 40 patients without APL. The antibody composition and detection system of the present invention were used for detection and analysis to verify the clinical conformity of the detection method of the present invention.

[0201] As shown in Table 3 below, all 40 APL patients tested positive for the PML-RARA fusion gene. The antibody combination described in this invention could detect it, and the concordance rate with the clinical diagnosis was 100%.

[0202] Table 3

[0203] Sample number Clinical diagnosis PML-RARAR fusion gene The detection conclusion of this method Does it meet the requirements? 1 APL Positive APL conform to 2 APL Positive APL conform to 3 APL Positive APL conform to 4 APL Positive APL conform to 5 APL Positive APL conform to 6 APL Positive APL conform to 7 APL Positive APL conform to 8 APL Positive APL conform to 9 APL Positive APL conform to 10 APL Positive APL conform to 11 APL Positive APL conform to 12 APL Positive APL conform to 13 APL Positive APL conform to 14 APL Positive APL conform to 15 APL Positive APL conform to 16 APL Positive APL conform to 17 APL Positive APL conform to 18 APL Positive APL conform to 19 APL Positive APL conform to 20 APL Positive APL conform to 21 APL Positive APL conform to 22 APL Positive APL conform to 23 APL Positive APL conform to 24 APL Positive APL conform to 25 APL Positive APL conform to 26 APL Positive APL conform to 27 APL Positive APL conform to 28 APL Positive APL conform to 29 APL Positive APL conform to 30 APL Positive APL conform to 31 APL Positive APL conform to 32 APL Positive APL conform to 33 APL Positive APL conform to 34 APL Positive APL conform to 35 APL Positive APL conform to 36 APL Positive APL conform to 37 APL Positive APL conform to 38 APL Positive APL conform to 39 APL Positive APL conform to 40 APL Positive APL conform to

[0204] As shown in Table 4 below, 40 patients clinically diagnosed as non-APL, including various types of AML, all tested negative for the PML-RARA fusion gene. Using the antibody composition described in this invention, all were reported as AML (non-M3), with a concordance rate of 100% with the clinical diagnosis.

[0205] Table 4

[0206] Sample number Clinical diagnosis PML-RARAR fusion gene The detection conclusion of this method Does it meet the requirements? 41 AML with NPM1 gene mutation Negative AML (non-M3) conform to 42 AML-MR Negative AML (non-M3) conform to 43 AML-M1 Negative AML (non-M3) conform to 44 AML-M2 Negative AML (non-M3) conform to 45 AML-M5 Negative AML-M5 conform to 46 AML-M5 Negative AML (non-M3) conform to 47 AML with NPM1 gene mutation Negative AML (non-M3) conform to 48 AML with RUNX1::RUNX1T1 Negative AML (non-M3) conform to 49 AML-M4 Negative AML (non-M3) conform to 50 AML-M4 Negative AML (non-M3) conform to 51 AML-M5 Negative AML-M5 conform to 52 AML with NPM1 gene mutation Negative AML (non-M3) conform to 53 AML with NPM1 gene mutation Negative AML (non-M3) conform to 54 AML-M4 Negative AML (non-M3) conform to 55 AML-M2 Negative AML (non-M3) conform to 56 AML-M1 Negative AML (non-M3) conform to 57 AML-M2 Negative AML (non-M3) conform to 58 AML with NPM1 gene mutation Negative AML (non-M3) conform to 59 AML with CBFB::MYH11 Negative AML (non-M3) conform to 60 AML with RUNX1::RUNX1T1 Negative AML (non-M3) conform to 61 AML-M5 Negative AML (non-M3) conform to 62 AML with NPM1 gene mutation Negative AML-M5 conform to 63 AML-M4 Negative AML (non-M3) conform to 64 AML-M5 Negative AML (non-M3) conform to 65 AML with NPM1 gene mutation Negative AML-M5 conform to 66 AML-M2 Negative AML (non-M3) conform to 67 AML-MR Negative AML (non-M3) conform to 68 AML-M0 Negative AML (non-M3) conform to 69 AML with RUNX1::RUNX1T1 Negative AML (non-M3) conform to 70 AML-M4 Negative AML (non-M3) conform to 71 AML-M2 Negative AML (non-M3) conform to 72 AML with RUNX1::RUNX1T1 Negative AML (non-M3) conform to 73 AML with NPM1 gene mutation Negative AML (non-M3) conform to 74 AML with NPM1 gene mutation Negative AML (non-M3) conform to 75 AML-M1 Negative AML (non-M3) conform to 76 AML-M2 Negative AML (non-M3) conform to 77 AML with CBFB::MYH11 Negative AML (non-M3) conform to 78 AML with RUNX1::RUNX1T1 Negative AML (non-M3) conform to 79 AML-M2 Negative AML (non-M3) conform to 80 AML with NPM1 gene mutation Negative AML (non-M3) conform to

[0207] Example 5

[0208] Bone marrow fluid was collected from 40 patients clinically diagnosed with APL and 40 patients without APL, and the antibody composition and detection system of the present invention were compared with other antibody compositions.

[0209] Through extensive screening and research, this invention has obtained antibody compositions for rapid screening of APL with high sensitivity, specificity, and accuracy (as shown in Table 1). The antibody compositions in Table 5 are used as examples for comparative illustration:

[0210] Table 5

[0211] FITC PE ECD PECY5.5 PECY7 Group 1 <![CDATA[ HLA-DR ]]> CD33 CD34 CD117 CD45 Group 2 CD11b CD13 CD16 CD15 CD45 Group 3 CD36 CD64 CD14 CD117 CD45

[0212] Each specimen was tested according to the detection steps in Example 2 above, and the antibody combination of the present invention and the antibody composition in Table 5 were tested in parallel and the data obtained were analyzed.

[0213] As shown in Table 6 below, all 40 APL patients tested positive for the PML-RARA fusion gene. The antibody composition described in this invention could detect it in all cases with a detection accuracy of 100%. However, when using the antibody composition in Table 5, 3 APL samples tested positive for AML-M5 (non-APL), resulting in a detection accuracy of only 75%.

[0214] Table 6

[0215]

[0216] As shown in Table 7 below, 40 non-APL patients, including various types of AML, all tested negative for the PML-RARA fusion gene. Using the antibody composition described in this invention, all were reported as AML (non-M3), with a detection accuracy of 100%. When using the antibody composition in Table 5, 3 non-APL samples (2 AML-M5 and 1 AML with NPM1 gene mutation) tested positive for APL, with a detection accuracy of only 75% and a false positive rate of 25%.

[0217] Table 7

[0218]

[0219] The above results indicate that the antibody composition of the present invention can effectively distinguish between APL and non-APL AML patients, and has excellent sensitivity and specificity when used for APL detection, with an accuracy of up to 100%.

[0220] Example 6

[0221] This embodiment is a screening study of the antibody composition of the present invention.

[0222] To accurately and rapidly identify antibody compositions for patients with acute promyelocytic leukemia, we conducted extensive research and screening, ultimately obtaining the antibody compositions of this invention with high sensitivity, specificity, and accuracy. The following four detection schemes are used as examples for illustration; the specific antibody combinations, antibody uses, and analytical strategies in each screening scheme are shown in Table 8 below.

[0223] Table 8

[0224]

[0225] Bone marrow fluid from 20 patients clinically diagnosed with APL and 20 patients without APL was used to test the above antibody combination regimen. The test results are shown in Table 9 below.

[0226] Table 9

[0227]

[0228] The above results indicate that the antibody composition of the present invention (fourth round) has high accuracy and specificity, and the detection results are in complete agreement with the clinical diagnostic results.

[0229] Compared with the other three antibody combination schemes (rounds 1-3), the antibody selection and fluorescein combination of the antibody composition of the present invention have at least the following advantages:

[0230] 1. Spectral optimization of the multicolor fluorescence system: Long-wavelength fluorophores (APC750, PB) are used to label low-expression antigens (such as CD10, CD7) to avoid leakage interference from high-expression antigens (CD33, CD117). CD71-FITC (high expression) and CD33-PE (high expression) belong to different channels to avoid signal overlap.

[0231] 2. Interference signal elimination system: The addition of lymphatic markers such as CD19, CD7, and CD10 can exclude lymphatic diseases, and through three-tube linkage detection, mixed phenotype leukemia (MPAL) and lymphatic tumor infiltration can be 100% excluded.

[0232] 3. Introduction of MPO and CD9 dual labeling to target tumor cells: Strong MPO expression and high CD9 expression are an effective combination that distinguishes APL from AML with NPM1, greatly improving detection accuracy and specificity.

[0233] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An antibody composition for detecting acute promyelocytic leukemia, characterized in that, Including group 1 antibodies, group 2 antibodies, and group 3 antibodies; The first group of antibodies consists of CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7 and CD45 antibodies; The second group of antibodies consists of CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15 and CD45 antibodies; The third group of antibodies consists of CD9, MPO, CD117 and CD45 antibodies; Each of the antibodies described is a monoclonal antibody labeled with a detection marker; The method for detecting acute promyelocytic leukemia using the antibody composition includes the following steps: gating with CD45-SSC, and dividing the cell population into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and blast cells based on CD45 expression; further analyzing the target cells in the granulocyte population, and analyzing the fluorescence expression intensity of the antibody pairs in the target cells; the antibody pairs include: CD45-SSC, CD117-CD33, CD117-FS, CD45-SSC ... 117-CD34, CD117-HLA-DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD1 3-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO and CD9-CD45.

2. The antibody composition according to claim 1, characterized in that, The detection marker is a fluorescein, selected from FITC, PE, ECD, and PE-Cy. TM 5.5, PE-Cy7, APC, APC-750, PB, KO.

3. The antibody composition according to claim 2, characterized in that, The CD71, CD38, and CD9 antibodies are labeled with the same fluorescein; The CD33, CD56, and MPO antibodies are labeled with the same fluorescein; The CD117 antibody and CD16 antibody in the first group of antibodies are labeled with the same fluorophore; The CD34 antibody in the first group of antibodies, the CD34 antibody in the second group of antibodies, and the CD117 antibody in the third group of antibodies are labeled with the same fluorescein; The HLA-DR and CD13 antibodies are labeled with the same fluorophore; The CD19 and CD11b antibodies are labeled with the same fluorophore; The CD10 and CD64 antibodies are labeled with the same fluorescein; The CD7 and CD15 antibodies are labeled with the same fluorescein; The CD45 antibody in the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with the same fluorophore.

4. The antibody composition according to claim 3, characterized in that, The CD71, CD38, and CD9 antibodies were labeled with FITC; The CD33, CD56, and MPO antibodies were used to label the fluorescent pigment PE. The CD117 and CD16 antibodies in the first group of antibodies are labeled with fluorescein ECD; The CD34 antibody in the first group, the CD34 antibody in the second group, and the CD117 antibody in the third group are labeled with fluorescein PE-Cy. TM 5.5; The HLA-DR and CD13 antibodies were labeled with fluorescein PE-Cy7; The CD19 and CD11b antibodies are labeled with fluorescein APC; The CD10 and CD64 antibodies were labeled with fluorescein APC-750; The CD7 and CD15 antibodies were labeled with fluorescein PB; The CD45 antibody in the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with fluorescein KO.

5. The antibody composition according to claim 4, characterized in that, The catalog numbers for the following antibodies are as follows: CD117 antibody-labeled fluorescein ECD (Catalog No. B38307), CD34 antibody-labeled fluorescein PECY5.5 (Catalog No. 343522), HLA-DR antibody-labeled fluorescein PE-Cy7 (Catalog No. B49180), CD19 antibody-labeled fluorescein APC (Catalog No. IM2470), CD7 antibody-labeled fluorescein PB (Catalog No. B06499), CD56 antibody-labeled fluorescein PE (Catalog No. A07788), and CD16 antibody-labeled fluorescein... The catalog numbers for ECD (B49216), CD34 antibody-labeled fluorescein PECY5.5 (343522), CD64 antibody-labeled fluorescein APC750 (B96769), CD9 antibody-labeled fluorescein FITC (IM1755U), MPO antibody-labeled fluorescein PE (B36288), and CD117 antibody-labeled fluorescein PECY5.5 (B96754) are all manufactured by Beckman Coulter. The catalog numbers for CD71 antibody-labeled fluorescein FITC are 665339, CD33 antibody-labeled fluorescein PE are 663527, and CD11b antibody-labeled fluorescein APC are 982604. The manufacturer is BD. The catalog numbers for CD10 antibody-labeled fluorescein APC750 are 982208, CD45 antibody-labeled fluorescein KO are 982320, CD38 antibody-labeled fluorescein FITC are 980304, CD13 antibody-labeled fluorescein PECY7 are 982806, and CD15 antibody-labeled fluorescein PB are 3230409. The manufacturer is Biolegend.

6. A kit for detecting acute promyelocytic leukemia, characterized in that, The kit comprises the antibody composition as described in any one of claims 1 to 5.

7. A system for detecting acute promyelocytic leukemia, characterized in that, include: The detection module utilizes the antibody composition as described in any one of claims 1 to 5 or the kit as described in claim 6 to perform flow cytometry detection on the cells to be tested. The data acquisition module acquires data from flow cytometry analysis results. The data analysis module analyzes the acquired data: using CD45-SSC gating, and dividing the cell population into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and primitive cells based on CD45 expression; further analyzing the target cells within the granulocyte population, and analyzing the fluorescence expression intensity of antibody pairs in the target cells; the antibody pairs include: CD45-SSC, CD117-CD33, CD117-FS, CD117-CD34, CD117-HLA- DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11b, CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO, and CD9-CD45; determine whether the obtained promyelocytes are neoplastic promyelocytes according to the judgment criteria.

8. The system for detecting acute promyelocytic leukemia as described in claim 7, characterized in that, The judgment criteria include: If the antibody expression pattern of the cell to be tested falls into the expression pattern template of the antibody of abnormal promyelocytes, the cell to be tested will be identified as a neoplastic promyelocyte. If the antibody expression pattern of the cell to be tested does not fall within the expression pattern template of the antibody of the abnormal promyelocyte, the cell to be tested will be identified as a non-tumor promyelocyte.

9. The system for detecting acute promyelocytic leukemia as described in claim 8, characterized in that, The antibody expression patterns of the target cells and abnormal promyelocytes were established through the following steps: flow cytometry data were gated using CD45-SSC, and the cell population was divided into granulocytes, monocytes, lymphocytes, nucleated erythrocytes or platelets, and primitive cells based on CD45 expression. Further analysis was performed on the target cells within the granulocyte population to analyze the fluorescence expression intensity of antibody pairs in the target cells. The antibody pairs included: CD45-SSC, CD117-CD33, and C... D117-FS, CD117-CD34, CD117-HLA-DR, CD71-CD45, CD34-CD19, CD19-CD10, CD117-CD7, CD13-CD64, CD15-CD11 b. CD13-CD16, CD38-CD56, CD34-CD38, CD34-CD13, CD34-CD56, CD34-CD11b, CD117-CD9, CD117-MPO and CD9-CD45.

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