Antibody composition, kit and system for rapidly screening acute promyelocytic leukemia

Abstract

The invention relates to an antibody composition, a kit and a system for rapidly screening 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 a CD71 antibody, a CD33 antibody, a CD117 antibody, a CD34 antibody, an HLA-DR antibody, a CD19 antibody, a CD10 antibody, a CD7 antibody and a CD45 antibody; the second group of antibodies comprises a CD38 antibody, a CD56 antibody, a CD16 antibody, a CD34 antibody, a CD13 antibody, a CD11b antibody, a CD64 antibody, a CD15 antibody and a CD45 antibody; the third group of antibodies comprises a CD9 antibody, an MPO antibody, a CD117 antibody and a CD45 antibody; and each antibody is a monoclonal antibody marked with a detection marker. The antibody composition comprises a CD117 + CD33 + region as a main target cell population, and covers three groups of antibodies for identifying acute promyelocytic leukemia, such as CD33, CD34, CD64, CD13, CD9 and MPO, the acute promyelocytic leukemia can be rapidly, simply and conveniently detected by adopting the antibody composition, the detection sensitivity is high, the specificity is high, the accuracy rate is high, and the antibody composition can be widely applied to detection of acute promyelocytic leukemia. The acute promyelocytic leukemia patient can be accurately and quickly identified, and early diagnosis and early treatment are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and particularly relates to an antibody composition, a kit and a system for rapidly screening acute promyelocytic leukemia. Background Art

[0002] Acute promyelocytic leukemia (APL) is a special type of acute myeloid 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 product leads to cell differentiation arrest and insufficient apoptosis, which is the main molecular mechanism of APL occurrence. APL is common in young and middle-aged people, with an average onset age of 44 years. APL accounts for 10% - 15% of AML during the same period, and the incidence rate is about 0.23 / 100,000. The clinical manifestations of APL are severe, and bleeding and embolism are likely to occur during the onset and induction treatment processes, leading to death. Once APL is suspected, clinical treatment needs to be carried out as an emergency, and tretinoin should be used promptly for preemptive treatment. Therefore, accurate and rapid identification of this disease is particularly crucial for the treatment prognosis of patients.

[0003] 98% of APL patients have the PML-RARa fusion gene, and less than 2% of APL patients have other types of fusion genes. Currently, clinically, real-time quantitative PCR technology or RNA-seq technology is used to detect the PML-RARa fusion gene or other types of fusion genes for the diagnosis, efficacy evaluation, prognosis analysis and recurrence prediction of APL. However, because the reporting cycle of using PCR technology to detect the PML-RARa fusion gene is relatively long, generally about 2 - 3 days, it is very unfavorable for the rapid detection of APL and cannot meet the clinical need for rapid diagnosis of this disease.

[0004] In recent years, it has been found that immunophenotype detection plays a good auxiliary role in the diagnosis of APL. Its typical tumor cell phenotype is: strongly expressing CD13, CD33, CD117 and MPO, not expressing CD34, HLA-DR, CD11b, CD56. Moreover, the flow cytometry immunophenotype detection technology is easy to operate, and the result can be obtained in 2 - 3 hours, greatly shortening the waiting time of patients, enabling rapid reporting and guiding clinical preemptive treatment, thus gaining a treatment advantage for patients. However, currently, different hospitals may have different combination configurations, operation and data analysis processes on the flow cytometry detection platform, and due to large differences, the repeatability of the results is relatively poor, which is not conducive to the accuracy of the results of acute promyelocytic leukemia immunophenotype detection.

[0005] Therefore, there is a need in the market for an antibody composition and a detection kit for the rapid screening of acute promyelocytic leukemia, which include a standard detection protocol, operation process, and analysis idea to ensure the accuracy of the results and assist in the early identification and diagnosis of acute promyelocytic leukemia (APL) in clinical practice. Summary of the Invention

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

[0007] In the first aspect of the present invention, an antibody composition for detecting acute promyelocytic leukemia is provided, which includes 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 is a monoclonal antibody labeled with a detection marker.

[0012] In some embodiments, the detection marker is a fluorophore; preferably, the fluorophore is selected from FITC, PE, ECD, 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 fluorophore;

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

[0015] The CD117 antibody and the 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 fluorophore;

[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 fluorophore;

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

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

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

[0023] The CD33, CD56, and MPO antibodies are labeled with the fluorophore PE;

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

[0025] 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 fluorophore PE-Cy TM 5.5;

[0026] The HLA-DR and CD13 antibodies are labeled with the fluorophore PE-Cy7;

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

[0028] The CD10 and CD64 antibodies are labeled with the fluorophore APC-750;

[0029] The CD7 and CD15 antibodies are labeled with the fluorophore PB;

[0030] The CD45 antibodies in the first group of antibodies, the second group of antibodies, and the third group of antibodies are labeled with the fluorophore KO.

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

[0032] The catalog number of the CD71 antibody labeled with fluorescein FITC is 665339, the catalog number of the CD33 antibody labeled with fluorescein PE is 663527, and the catalog number of the CD11b antibody labeled with fluorescein APC is 982604, and the manufacturer is BD;

[0033] The catalog number of the CD10 antibody labeled with fluorescein APC750 is 982208, the catalog number of the CD45 antibody labeled with fluorescein KO is 982320, the catalog number of the CD38 antibody labeled with fluorescein FITC is 980304, the catalog number of the CD13 antibody labeled with fluorescein PECY7 is 982806, and the catalog number of the CD15 antibody labeled with fluorescein PB is 3230409, and the manufacturer is biolegend.

[0034] In a second aspect of the present invention, there is provided a kit for detecting acute promyelocytic leukemia, and the kit contains the antibody composition as described above.

[0035] In a third aspect of the present invention, there is provided the use of the antibody composition or the kit as described above in the auxiliary detection of acute promyelocytic leukemia.

[0036] In a fourth aspect of the present invention, there is provided a system for detecting acute promyelocytic leukemia, including:

[0037] A detection module, which uses the antibody composition as described above or the kit as described above to perform flow cytometry detection on the cells to be tested;

[0038] A data acquisition module, which acquires the data of the flow cytometry detection results;

[0039] Data analysis module, which analyzes the acquired data: Use CD45-SSC gating, and divide the cell population into granulocyte population, monocyte population, lymphocyte population, nucleated red blood cell or platelet region, and blast cell population according to the expression of CD45. Further analyze the target cells in the granulocyte population, and analyze the fluorescence expression intensity of the 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 acquired 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 test cell falls into the antibody expression pattern template of abnormal promyelocytes, the test cell is determined to be a neoplastic promyelocyte;

[0042] If the antibody expression pattern of the test cell does not fall into the antibody expression pattern template of the abnormal promyelocytes, the test cell is determined to be a non-neoplastic promyelocyte.

[0043] In some embodiments, the antibody expression pattern of the test cell and the antibody expression pattern of abnormal promyelocytes are established through the following steps: Use CD45-SSC gating for the data of flow cytometry detection results, and divide the cell population into granulocyte population, monocyte population, lymphocyte population, nucleated red blood cell or platelet region, and blast cell population according to the expression of CD45. Further analyze the target cells in the granulocyte population, and analyze the fluorescence expression intensity of the 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.

[0044] The inventors of the present invention have obtained an antibody composition for rapid screening of acute promyelocytic leukemia based on their own many years of experience and a large amount of research and screening. The antibody composition comprises three groups of antibodies with CD117+CD33+ region as the main target cell population, covering CD33, CD34, CD64, CD13, CD9 and MPO, etc., which can identify acute promyelocytic leukemia. The antibody composition can be used to quickly and easily detect acute promyelocytic leukemia, and has high detection sensitivity, high specificity and high accuracy. It can accurately and quickly identify patients with acute promyelocytic leukemia, realize early diagnosis and treatment, and gain golden treatment time for patients. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0047] Figure 3 The first group of antibodies used in Example 3 of the present invention is used to detect the expression of the immunophenotype CD117-FS of the APL sample to be tested.

[0048] Figure 4 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD117-CD34 of the APL sample to be tested.

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

[0050] Figure 6 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD71-CD45 of the APL sample to be tested.

[0051] Figure 7 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD34-CD19 of the APL sample to be tested.

[0052] Figure 8 The first group of antibodies is used in Example 3 of the present invention to detect the expression of the immunophenotype CD19-CD10 of the APL sample to be tested.

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

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

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

[0056] Figure 12 This is the situation of the immunophenotype CD13 - CD16 expression detected using the second group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0057] Figure 13 This is the situation of the immunophenotype CD38 - CD56 expression detected using the second group of antibodies for the normal APL sample to be tested in Example 3 of the present invention.

[0058] Figure 14 This is the situation of the immunophenotype CD34 - CD38 expression detected using the second group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0059] Figure 15 This is the situation of the immunophenotype CD34 - CD13 expression detected using the second group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0060] Figure 16 This is the situation of the immunophenotype CD34 - CD56 expression detected using the second group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0061] Figure 17 This is the situation of the immunophenotype CD34 - CD11b expression detected using the second group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0062] Figure 18 This is the situation of the immunophenotype CD117 - CD9 expression detected using the third group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0063] Figure 19 This is the situation of the immunophenotype CD117 - MPO expression detected using the third group of antibodies for the APL sample to be tested in Example 3 of the present invention.

[0064] Figure 20 This is the situation of the immunophenotype CD9 - CD45 expression detected using the third group of antibodies for the APL sample to be tested in Example 3 of the present invention.

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

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

[0067] Figure 23 This is the expression of CD117-FS, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0068] Figure 24 This is the expression of CD117-CD34, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0069] Figure 25 This is the expression of CD117-HLA-DR, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0070] Figure 26 This is the expression of CD71-CD45, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0071] Figure 27 This is the expression of CD34-CD19, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0072] Figure 28 This is the expression of CD19-CD10, an immunophenotype, detected using the first set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

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

[0074] Figure 30 This is a scatter plot of the expression of CD13-CD64, an immunophenotype, detected using the second set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0075] Figure 31 This is a scatter plot of the expression of CD15-CD11b, an immunophenotype, detected using the second set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0076] Figure 32 This is the expression of CD13-CD16, an immunophenotype, detected using the second set of antibodies in non-APL samples to be tested in Example 3 of the present invention.

[0077] Figure 33This is to detect the expression of immunophenotype CD38-CD56 in the normal non-APL sample to be tested using the second group of antibodies in Example 3 of the present invention.

[0078] Figure 34 This is to detect the expression of immunophenotype CD34-CD38 in the non-APL sample to be tested using the second group of antibodies in Example 3 of the present invention.

[0079] Figure 35 This is to detect the expression of immunophenotype CD34-CD13 in the non-APL sample to be tested using the second group of antibodies in Example 3 of the present invention.

[0080] Figure 36 This is to detect the expression of immunophenotype CD34-CD56 in the non-APL sample to be tested using the second group of antibodies in Example 3 of the present invention.

[0081] Figure 37 This is to detect the expression of immunophenotype CD34-CD11b in the non-APL sample to be tested using the second group of antibodies in Example 3 of the present invention.

[0082] Figure 38 This is to detect the expression of immunophenotype CD117-CD9 in the non-APL sample to be tested using the third group of antibodies in Example 3 of the present invention.

[0083] Figure 39 This is to detect the expression of immunophenotype CD117-MPO in the non-APL sample to be tested using the third group of antibodies in Example 3 of the present invention.

[0084] Figure 40 This is to detect the expression of immunophenotype CD9-CD45 in the non-APL sample to be tested using the third group of antibodies in Example 3 of the present invention. Detailed implementation manners

[0085] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0086] For the experimental methods without specific conditions noted in the following examples, they are usually in accordance with conventional conditions or the conditions recommended by the manufacturers. All kinds of common chemical reagents used in the examples are commercially available products.

[0087] Unless otherwise defined, all technical and scientific terms used in the present invention are the same as those belonging to the technology of the present invention

[0088] The meanings commonly understood by those skilled in the art are the same. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0089] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0090] In some embodiments of the present invention, there is provided 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 is a monoclonal antibody labeled with a detection marker.

[0095] The antibody composition of the present invention includes three groups of antibodies. Using the CD117+CD33+ region as the main target cell population, it covers antibody compositions that recognize abnormal promyelocytic cell populations (i.e., neoplastic promyelocytic cell populations), such as CD9, CD13, CD64, HLA-DR, MPO, etc. Through comprehensive logical analysis strategies, it can better identify neoplastic abnormal promyelocytic cells and achieve the detection of APL. The present invention reasonably applies various antibody indicators with a wide coverage. Using this antibody combination through multi-parameter flow cytometry detection technology, it can quickly and simply (only 1-2 hours from sample receipt to test results) and highly sensitively detect abnormal promyelocytic cells. Moreover, the present invention optimizes the result analysis template. Before optimization, only a single gate (CD117+) was used to check the expression of HLA-DR and CD34. However, since tumor cells of other types of AML patients can all express CD117, the inaccurate gate led to deviations in the analysis results. After optimization, the present invention first uses CD117-CD33 to circle suspicious cells and check the expression of HLA-DR, CD34, CD13, CD64, etc. In addition, the analysis template is fixed, and the data can be directly analyzed by dragging it in, which is simple to operate, reduces the requirements for the professional knowledge level of analysts, and makes up for the limitations of existing detection technologies.

[0096] The antibody composition of the present invention includes 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; except for using CD45 as a leukocyte gating antibody, the first group of antibodies can identify suspicious abnormal promyelocytic cells through the backbone antibodies CD117-CD33, and then check whether CD34 and HLA-DR are expressed. If neither is expressed, it can be initially considered as abnormal promyelocytic cells, and then further confirmed in combination with the second / third group of antibodies. The first group of antibodies also adds the normal erythroblast marker CD71 to check the proportion of erythroblasts, and also adds lymphoid markers such as CD19, CD10, and CD7 to initially screen whether they are B-ALL or T-ALL cells. If there is expression, it is necessary to consider the possibility of lymphoid leukemia or mixed leukemia.

[0098] 2. The second group of antibodies, including: CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15 and CD45 antibodies. The second group of antibodies needs to be used in combination with the first group of antibodies. Using CD38-CD45 can identify a suspicious abnormal promyelocyte population, and then check whether CD34, CD11b, CD15, CD13, CD64, CD16 are expressed. Negative CD11b / CD16 indicates that immature monocytes are not considered. Negative CD34, high expression of CD13 and expression of CD64 are consistent with the immunophenotype of abnormal promyelocytes; if CD38 is strongly expressed and CD56 is positive, plasma cells also need to be excluded for differential diagnosis.

[0099] 3. The third group of antibodies, including: CD9, MPO, CD117 and CD45 antibodies. The third group of antibodies needs to be used in combination with the first group and the second group of antibodies. Using CD117-CD45 can identify a suspicious abnormal promyelocyte population, and then check whether CD9 and MPO are expressed. If both CD9 and MPO are highly expressed, it is determined that its phenotype is 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 fluorescein; the CD33, CD56 and MPO antibodies are labeled with the same fluorescein; the CD117 antibody and the CD16 antibody in the first group of antibodies are labeled with the same fluorescein; 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 fluorescein: the CD19 and CD11b antibodies are labeled with the same fluorescein; the CD10 and CD64 antibodies are labeled with the same fluorescein; the CD7 and CD15 antibodies are labeled with the same fluorescein; the CD45 antibodies in the first group of antibodies, the second group of antibodies and the third group of antibodies are labeled with the same fluorescein.

[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 antibody and the CD16 antibody in the first group of antibodies are labeled with fluorescein ECD; 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 fluorescein PE-Cy TM5.5; the HLA-DR and CD13 antibodies are labeled with the fluorophore PE-Cy7; the CD19 and CD11b antibodies are labeled with the fluorophore APC; the CD10 and CD64 antibodies are labeled with the fluorophore APC-750; the CD7 and CD15 antibodies are labeled with the fluorophore PB; the CD45 antibody in the first group of antibodies, the second group of antibodies, and the third group of antibodies is labeled with the fluorophore KO.

[0103] In some preferred embodiments, the catalog number of the CD117 antibody labeled with the fluorophore ECD is B38307, the catalog number of the CD34 antibody labeled with the fluorophore PECY5.5 is 343522, the catalog number of the HLA-DR antibody labeled with the fluorophore PE-Cy7 is B49180, the catalog number of the CD19 antibody labeled with the fluorophore APC is IM2470, the catalog number of the CD7 antibody labeled with the fluorophore PB is B06499, the catalog number of the CD56 antibody labeled with the fluorophore PE is A07788, the catalog number of the CD16 antibody labeled with the fluorophore ECD is B49216, the catalog number of the CD34 antibody labeled with the fluorophore PECY5.5 is 343522, the catalog number of the CD64 antibody labeled with the fluorophore APC750 is B96769, the catalog number of the CD9 antibody labeled with the fluorophore FITC is IM1755U, the catalog number of the MPO antibody labeled with the fluorophore PE is B36288, the catalog number of the CD117 antibody labeled with the fluorophore PECY5.5 is B96754, and the manufacturer is Beckman Coulter;

[0104] The catalog number of the CD71 antibody labeled with the fluorophore FITC is 665339, the catalog number of the CD33 antibody labeled with the fluorophore PE is 663527, and the catalog number of the CD11b antibody labeled with the fluorophore APC is 982604, and the manufacturer is BD;

[0105] The catalog number of the CD10 antibody labeled with the fluorophore APC750 is 982208, the catalog number of the CD45 antibody labeled with the fluorophore KO is 982320, the catalog number of the CD38 antibody labeled with the fluorophore FITC is 980304, the catalog number of the CD13 antibody labeled with the fluorophore PECY7 is 982806, and the catalog number of the CD15 antibody labeled with the fluorophore PB is 3230409, and the manufacturer is biolegend.

[0106] In some embodiments of the present invention, a kit for detecting acute promyelocytic leukemia is involved, and the kit contains the antibody composition as described above.

[0107] In some embodiments of the present invention, a system for detecting acute promyelocytic leukemia is involved, including:

[0108] A detection module, which uses the antibody composition as described above or the kit as described above to perform flow cytometry detection on the cells to be tested;

[0109] A data acquisition module that acquires data from flow cytometry detection results;

[0110] A data analysis module that analyzes the acquired data: gates using CD45-SSC, and divides cell populations into granulocyte population, monocyte population, lymphocyte population, nucleated red blood cell or platelet region, and blast cell population according to the expression of CD45, further analyzes target cells in the granulocyte population, and analyzes 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; determines whether the acquired 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 performing antigen-antibody reactions by incubating the single cell suspension and the reagent composition separately in the dark; a resuspension module for adding hemolysin to the incubated cells, centrifuging, washing, and then resuspending the cells to prepare a suspension; and a measurement module for performing flow cytometry measurement 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 antibody expression pattern template of abnormal promyelocytes, the cell to be tested is determined to be a neoplastic promyelocyte;

[0114] If the antibody expression pattern of the cell to be tested does not fall into the antibody expression pattern template of the abnormal promyelocytes, the cell to be tested is determined to be a non-neoplastic promyelocyte.

[0115] In some embodiments, the antibody expression patterns of the cells to be tested and the antibody expression patterns of abnormal promyelocytes are established through the following steps: The data of the flow cytometry detection results are gated using CD45-SSC, and the cell population is divided into 5 regions according to the expression of CD45, namely: granulocyte population (Gran region in the upper middle part of the figure), monocyte population (mono region in the upper right part of the figure), lymphocyte population (lym region in the lower right part of the figure), nucleated red blood cell or platelet region (CD45neg region in the lower left part of the figure), and blast cell population (CD45dim region in the lower middle part of the figure), which facilitates further analysis of the target cells in the granulocyte population; then analyze the fluorescence expression intensity of the 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.

[0116] For the system for detecting acute promyelocytic leukemia of the present invention, the technical process can refer to the conventional techniques of flow cytometry, and the equipment and consumables used are selected from the equipment and consumables for flow cytometry analysis, such as special flow 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 antibodies used in the following examples are as follows: CD117ECD (product number B38307), CD34PECY5.5 (product number 343522), HLA-DR PECY7 (product number B49180), CD19APC (product number IM2470), CD7 PB (product number B06499), CD56 PE (product number A07788), CD16 ECD (product number B49216), CD34 PECY5.5 (product number 343522), CD64APC750 (product number B96769), CD9 FITC (product number IM1755U), MPO PE (product number B36288), CD117 PECY5.5 (product number B96754), manufacturer Beckman Coulter; CD71FITC (product number 665339), CD33 PE (product number 663527), CD11b APC (product number 982604), manufacturer BD; CD10 APC750 (product number 982208), CD45 KO (product number 982320), CD38FITC (product number 980304), CD13 PECY7 (product number 982806), CD15 PB (product number 3230409), manufacturer bio1egend.

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

[0120] This example provides an antibody composition for rapid screening of acute promyelocytic leukemia (APL), including 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 fluorescence labeling and dosage of the relevant monoclonal antibodies in the above antibodies are shown in Table 1.

[0125] Table 1

[0126]

[0127] Note: The above-mentioned commercially available antibodies were verified with concentration gradients to determine the optimal dosage, and then the monoclonal antibodies at the above dosages were respectively filled into flow cytometry special tubes numbered 1, 2, and 3.

[0128] Example 2 A System and Method for Rapid Screening of Acute Promyelocytic Leukemia (APL)

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

[0130] The specific workflow for detecting acute promyelocytic leukemia (APL) using the above system of this example 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] Adjust the concentration of the sample to be tested (bone marrow fluid) to 1×10 6 cells / ml according to the number of cells to make a single-cell suspension.

[0134] 3. Sample detection.

[0135] (1) Take flow cytometry tubes, label them 1 and 2, and add the first group of antibodies, the second group of antibodies, and the third group of antibodies in Example 1 respectively, with the addition amounts of 25 μl, 15 μl, and 16 μl respectively. Then add 100 μl of the suspension in step 2 respectively, vortex and mix well, and incubate in the dark at room temperature for 15 min.

[0136] (2) Add 400 μl of Bc lysin to each of the incubated flow cytometry tubes 1, 2, and 3, vortex, and let it stand until hemolysis is clear. After hemolysis is clear, centrifuge the flow cytometry tubes 1, 2, and 3 at 1500 r / min for 5 min, discard the supernatant, add 2 ml of calf serum, vortex, centrifuge at 1500 r / min for 5 min, and discard the supernatant. Resuspend the flow cytometry tubes 1 and 2 with 400 μl of 1% paraformaldehyde.

[0137] (3) Intracellular antibody experiment operation: After staining the cell surface markers in the above-mentioned tube 3 according to the detection methods in (1) and (2) above, add 450 μL of 1X FACS permeabilization reagent, mix well, incubate in the dark for 5 minutes, then add PBS to wash once and discard the supernatant. Add the anti-intracellular fluorescent antibodies (Granzyme B, Perforin, Ki67) according to the above antibody amounts, incubate at room temperature in the dark for 30 minutes, add 2 ml of calf serum, vortex, centrifuge at 1500 r / min for 5 minutes, discard the supernatant, and resuspend with 400 μl of 1% paraformaldehyde.

[0138] (4) Use a Beckman Coulter Navios ten-color flow cytometer to detect the flow cytometry tubes 1, 2, and 3 and analyze their immunophenotypes.

[0139] 4. Data analysis.

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

[0141] Perform the above-mentioned experimental detections on 40 patients with acute promyelocytic leukemia (APL) who are positive for the detection of the fusion gene PML-RARA at the first diagnosis to obtain detection data. Gate the cell populations by setting gates, preferably using CD45-SSC (side scatter) to set the gate, circle the target cell population, and then analyze the expression of each fluorescent antibody in this cell population. Select the following antibody pairs: 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, CD34-56, 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 detection result data of the cells to be tested. Gate the cell populations according to the method in the antibody expression pattern of acute promyelocytic leukemia (APL) above, circle the target cell population, and then analyze the expression of each fluorescent antibody in the target cell population according to the method 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 within the expression pattern template of the antibody of abnormal promyelocytes, the cell to be tested is determined to be neoplastic promyelocytes;

[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 promyelocytes, the cell to be tested is determined to be non-neoplastic promyelocytes.

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

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

[0149] Table 2

[0150]

[0151] The following uses two cases as examples to detect the tumor cells of two cells to be tested (1 APL sample + 1 AML control sample of non-APL), and perform antibody expression pattern analysis to display the analysis strategy of the detection scheme of the present invention.

[0152] I. APL sample

[0153] This sample is an APL bone marrow fluid sample positive for PML-RARA fusion gene detection, and the antibody expression pattern analysis is performed according to the following steps:

[0154] Obtain the flow cytometry detection result data of the sample to be tested. Use CD45-SSC (side scatter) to set the gate. According to the expression of CD45-SSC, the cell population is divided into 5 regions, namely granulocytes ( Figure 1 middle upper Gran region), monocytes ( Figure 1 upper right mono region), lymphocytes ( Figure 1 lower right 1ym region), nucleated red / platelet region (Figure 1 in the lower left CD45neg region) and the primitive cell population ( Figure 1 in the middle lower CD45dim region) in 5 regions (such as Figure 1 shown), the granulocyte region is the region where the target cell population is located. The immunophenotype analysis of this group of cells will be carried out below.

[0155] Figure 2 is the scatter plot of CD117-CD33 immunophenotype for all nucleated cells. It can be seen from the figure that the proportion of CD117+CD33+ abnormal cells is 83.90% (shown in red in the figure), and the proportion is significantly increased, suggesting a high possibility of AML.

[0156] Figure 3 is the scatter plot of CD117-FS expression for all nucleated cells. It can be seen from the figure that the FS of CD117+ abnormal cells is very large, indicating that the abnormal cells are relatively large in volume.

[0157] Figure 4 is the scatter plot of CD117-CD34 expression for all nucleated cells. It can be seen from the figure that CD34 of CD117+ abnormal cells is negative (not expressed).

[0158] Figure 5 is the scatter plot of CD117-HLA-DR expression for all nucleated cells. It can be seen from the figure that HLA-DR of CD117+ abnormal cells is negative (not expressed).

[0159] Figure 6 is the scatter plot of CD71-CD45 expression for all nucleated cells. It can be seen from the figure that CD71 of CD117+ abnormal cells is negative (not expressed), and the proportion of nucleated red blood cells with CD71+CD45- is 2.78%.

[0160] Figure 7 is the scatter plot of CD34-CD19 expression for all nucleated cells. It can be seen from the figure that CD19 of CD117+ abnormal cells is negative (not expressed), and CD19+ normal B lymphocytes can be seen (the blue population in the upper left corner of the figure).

[0161] Figure 8 is the scatter plot of CD19-CD10 expression for all nucleated cells. It can be seen from the figure that CD10 of CD117+ abnormal cells is negative (not expressed).

[0162] Figure 9 is the scatter plot of CD117-CD7 expression for all nucleated cells. It can be seen from the figure that CD7 of CD117+ abnormal cells is negative (not expressed), and CD7+ normal T lymphocytes can be seen (the blue population in the upper left corner of the figure).

[0163] Figure 10Scatter plot of CD13 - CD64 expression in all nucleated cells. It can be seen from the figure that both CD13 and CD64 are positive (expressed) in CD117+ abnormal cells.

[0164] Figure 11 Scatter plot of CD15 - CD11b expression in all nucleated cells. It can be seen from the figure that both CD15 and CD11b are negative (not expressed) in CD117+ abnormal cells.

[0165] Figure 12 Scatter plot of CD13 - CD16 expression in all nucleated cells. It can be seen from the figure that CD16 is negative (not expressed) in CD117+ abnormal cells.

[0166] Figure 13 Scatter plot of CD38 - CD56 expression in all nucleated cells. It can be seen from the figure that CD38 is positive (expressed) and CD56 is negative (not expressed) in CD117+ abnormal cells.

[0167] Figure 14 Scatter plot of CD34 - CD38 expression in all nucleated cells. It can be seen from the figure that CD34 is negative (not expressed) in CD117+ abnormal cells, and no obvious CD38++ plasma cells are seen.

[0168] Figure 15 Scatter plot of CD34 - CD13 expression in all nucleated cells. It can be seen from the figure that CD13 is positive (expressed) and CD34 is negative (not expressed) in CD117+ abnormal cells.

[0169] Figure 16 Scatter plot of CD34 - CD56 expression in all nucleated cells. It can be seen from the figure that both CD34 and CD56 are negative (not expressed) in CD117+ abnormal cells, and CD56+ normal NK lymphocytes can be seen (blue population in the upper left corner of the figure).

[0170] Figure 17 Scatter plot of CD34 - CD11b expression in all nucleated cells. It can be seen from the figure that both CD34 and CD11b are negative (not expressed) in CD117+ abnormal cells, and CD11b is positive in normal monocytes (purple population in the figure).

[0171] Figure 18 Scatter plot of CD117 - CD9 expression in all nucleated cells. It can be seen from the figure that CD9 is positive (expressed) in CD117+ abnormal cells.

[0172] Figure 19 Scatter plot of CD117 - MPO expression in all nucleated cells. It can be seen from the figure that MPO is positive (expressed) in CD117+ abnormal cells.

[0173] Figure 20 It is a scatter plot of CD9-CD45 expression in all nucleated cells. It can be seen from the figure that the expression intensity of CD9 in CD117+ abnormal cells is relatively strong.

[0174] The abnormal cells in this test sample have relatively large cell volume (high FS), express CD117, CD9, CD33 (strong expression), MPO (strong expression), CD13, CD38, CD64, do not express early primitive cell markers (CD34, HLA-DR), do not express lymphoid markers (CD19, CD7, CD56), and do not express mature granulocyte-monocyte markers (CD11b, CD16, CD10), which is in line with the immunophenotype of abnormal promyelocytes, and is considered to be acute promyelocytic leukemia (APL), consistent with the positive result of the fusion gene PML-RARA fusion gene detection.

[0175] II. AML control sample of non-APL

[0176] This sample is a bone marrow fluid specimen of AML of non-APL with negative detection of PML-RARA fusion gene. The antibody expression pattern analysis is carried out according to the following steps:

[0177] Obtain the flow cytometry test result data of this test sample. Use CD45-SSC (side scatter light) to set the gate. According to the expression of CD45-SSC, the cell population is divided into 5 regions, namely granulocytes ( Figure 21 the middle upper Gran region), monocytes ( Figure 21 the upper right mono region), lymphocytes ( Figure 21 the lower right 1ym region), nucleated red / platelet region ( Figure 21 the lower left CD45neg region) and primitive cell population ( Figure 21 the middle lower CD45dim region) 5 regions (as Figure 21 shown). The granulocyte region is the region where the target cell population is located. The immunophenotype analysis of this group of cells will be carried out below.

[0178] Figure 22 It is a scatter plot of CD117-CD33 immunophenotype of all nucleated cells. It can be seen from the figure that the CD117+CD33+ abnormal cells are 78.34% (red in the figure), and the proportion is significantly increased, suggesting a high possibility of AML.

[0179] Figure 23 It is a scatter plot of CD117-FS expression of all nucleated cells. It can be seen from the figure that the FS of CD117+ abnormal cells is very large, suggesting that the abnormal cells have a relatively large volume.

[0180] Figure 24Scatter plot of CD117-CD34 expression in all nucleated cells. It can be seen from the figure that CD34 in CD117+ abnormal cells is partially positive (partially expressed).

[0181] Figure 25 Scatter plot of CD117-HLA-DR expression in all nucleated cells. It can be seen from the figure that HLA-DR in CD117+ abnormal cells is partially positive (partially expressed).

[0182] Figure 26 Scatter plot of CD71-CD45 expression in all nucleated cells. It can be seen from the figure that CD71 in CD117+ abnormal cells is negative (not expressed), and the proportion of nucleated red blood cells with CD71+CD45- is 1.05%.

[0183] Figure 27 Scatter plot of CD34-CD19 expression in all nucleated cells. It can be seen from the figure that CD19 in CD117+ abnormal cells is negative (not expressed), and normal CD19+ B lymphocytes can be seen (blue population in the upper left corner of the figure).

[0184] Figure 28 Scatter plot of CD19-CD10 expression in all nucleated cells. It can be seen from the figure that CD10 in CD117+ abnormal cells is negative (not expressed).

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

[0186] Figure 30 Scatter plot of CD13-CD64 expression in all nucleated cells. It can be seen from the figure that CD13 in CD117+ abnormal cells is positive (expressed), and CD64 is partially positive (partially expressed).

[0187] Figure 31 Scatter plot of CD15-CD11b expression in all nucleated cells. It can be seen from the figure that both CD15 and CD11b in CD117+ abnormal cells are negative (not expressed).

[0188] Figure 32 Scatter plot of CD13-CD16 expression in all nucleated cells. It can be seen from the figure that CD16 in CD117+ abnormal cells is negative (not expressed).

[0189] Figure 33Scatter plot of CD38-CD56 expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are positive (expressing) for CD38 and negative (not expressing) for CD56.

[0190] Figure 34 Scatter plot of CD34-CD38 expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are negative (not expressing) for CD34, and no obvious CD38++ plasma cells are seen.

[0191] Figure 35 Scatter plot of CD34-CD13 expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are positive (expressing) for CD13 and partially positive (partially expressing) for CD34.

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

[0193] Figure 37 Scatter plot of CD34-CD11b expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are partially positive (partially expressing) for CD34 and all negative (not expressing) for CD11b.

[0194] Figure 38 Scatter plot of CD117-CD9 expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are all negative (not expressing) for CD9.

[0195] Figure 39 Scatter plot of CD117-MPO expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are positive (expressing) for MPO.

[0196] Figure 40 Scatter plot of CD9-CD45 expression in all nucleated cells. It can be seen from the figure that the CD117+ abnormal cells are negative (not expressing) for CD9.

[0197] The abnormal cells in the sample to be tested were also large in size (high FS), expressed early primitive cell markers (CD34, HLA-DR), expressed CD117, CD33 (strong expression), MPO (strong expression), CD13, CD38, partially expressed CD64, did not or partially expressed T lymphocyte marker CD7, did not express B and NK markers (CD19, CD56), did not express mature granule single markers (CD11b, CD16, CD10), and did not express CD9, which was consistent with the immunophenotype of abnormal primitive myeloid cells rather than abnormal promyelocytes. It 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 uses CD117+CD33+ region as the main target cell population, covers CD9, CD13, CD64, HLA-DR, MPO and other antibody combinations for identifying abnormal promyelocytic cell populations, and a comprehensive logical analysis strategy can quickly identify tumorous abnormal promyelocytic cells, help clinicians quickly identify acute promyelocytic leukemia (APL), and gain golden treatment time for patients.

[0199] Example 4 Clinical Compliance Verification of the Detection Method of the Present Invention

[0200] Bone marrow fluid was collected from 40 patients clinically diagnosed with APL and non-APL, and the antibody composition and detection system of the present invention were used for detection and analysis to verify the clinical compliance of the detection of the present invention.

[0201] As shown in Table 3 below, all 40 APL patients were positive for PML-RARA fusion gene detection, and all of them could be detected using the antibody combination of the present invention, with a 100% coincidence rate with the clinical diagnosis.

[0202] Table 3

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

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

[0205] Table 4

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

[0207] Example 5

[0208] Collect the bone marrow fluid of 40 patients clinically diagnosed with APL and 40 patients with non-APL respectively, and use the antibody composition and detection system of the present invention for comparison with other antibody compositions.

[0209] Through a large number of screenings and studies, the present invention has obtained an antibody composition for rapid screening of APL with high detection sensitivity, good specificity and high accuracy (as shown in Table 1). Taking the antibody composition in Table 5 as an example for comparison:

[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] For each specimen, parallel detection of the antibody combination of the present invention and the antibody composition in Table 5 was carried out according to the detection steps in Example 2 above, and data was obtained for analysis.

[0213] As shown in Table 6 below, for 40 APL patients, the detection of the PML-RARA fusion gene was all positive. Using the antibody composition of the present invention, all could be detected, and the detection accuracy rate was 100%; when using the antibody composition in Table 5 for detection, 3 APL samples had detection results of AML-M5 (non-APL), and the detection accuracy rate was only 75%.

[0214] Table 6

[0215]

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

[0217] Table 7

[0218]

[0219] The above results show that the antibody composition of the present invention can effectively distinguish APL and non-APL AML patients, and has good sensitivity and specificity when used for APL detection, and the accuracy rate is as high as 100%.

[0220] Example 6

[0221] This example is the screening and research of the antibody composition of the present invention.

[0222] In order to accurately and rapidly identify the antibody composition for patients with acute promyelocytic leukemia, we have conducted a large number of studies and screenings, and finally obtained the antibody composition of the present invention with high detection sensitivity, high specificity and high accuracy. Taking the following 4 detection schemes as examples for illustration, the antibody combinations, antibody uses and analysis strategies in the specific screening scheme are shown in Table 8 below.

[0223] Table 8

[0224]

[0225] Using the bone marrow fluid of 20 patients clinically diagnosed with APL and 20 patients with non-APL respectively, the above antibody combination scheme was tested, and the test results are shown in Table 9 below.

[0226] Table 9

[0227]

[0228] The above results show that the antibody composition (the fourth round) of the present invention has high accuracy and specificity, and the detection results are completely consistent with the clinical diagnosis results.

[0229] Compared with the other 3 antibody combination schemes (the 1st to 3rd rounds), the antibody selection and fluorochrome matching of the antibody composition of the present invention have at least the following advantages:

[0230] 1. Spectral optimization of the multicolor fluorescence system: Using long-wavelength fluorochromes (APC750, PB) to label low-expression antigens (such as CD10, CD7), avoiding the spillover interference of high-expression antigens (CD33, CD117), and CD71-FITC (high expression) and CD33-PE (high expression) belong to different channels, avoiding signal overlap.

[0231] 2. Interference signal exclusion system: Combining the addition of lymphoid markers such as CD19, CD7, CD10, etc. can exclude lymphoid diseases, and through the triple-tube linkage detection, it can 100% exclude mixed phenotype leukemia (MPAL) and lymphoid tumor infiltration.

[0232] 3. Introducing MPO and CD9 double labeling to lock tumor cells: The strong expression of MPO and the high expression of CD9 are effective combinations for differentiating APL from AML with NPM1, greatly improving the detection accuracy and specificity.

[0233] The above-described embodiments only express several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An antibody composition for detecting acute promyelocytic leukemia, characterized in that: including group I antibodies, group II antibodies and group III antibodies; The first group of antibodies includes CD71, CD33, CD117, CD34, HLA-DR, CD19, CD10, CD7 and CD45 antibodies; The second group of antibodies includes CD38, CD56, CD16, CD34, CD13, CD11b, CD64, CD15 and CD45 antibodies; The third group of antibodies includes CD9, MPO, CD117 and CD45 antibodies; Each of the antibodies is a monoclonal antibody labeled with a detection marker.

2. The antibody composition according to claim 1, characterized in that The detection marker is fluorescein, which is selected from FITC, PE, ECD, 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 the CD16 antibody in the first group of antibodies are labeled with the same fluorescein; 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 fluorescent substance; The HLA-DR and CD13 antibodies are labeled with the same fluorescein; The CD19 and CD11b antibodies are labeled with the same fluorescein; The CD10 and CD64 antibodies are labeled with the same fluorescein; The CD7 and CD15 antibodies are labeled with the same fluorescein; The CD45 antibodies in the first group of antibodies, the second group of antibodies and the third group of antibodies are labeled with the same fluorescent substance.

4. The antibody composition according to claim 3, characterized in that The CD71, CD38 and CD9 antibodies are labeled with fluorescein FITC; The CD33, CD56 and MPO antibodies are labeled with fluorescein PE; The CD117 antibody and CD16 antibody in the first group of antibodies are labeled with fluorescein ECD; 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 fluorescein PE-Cy TM 5.5; 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 antibodies in the first group of antibodies, the second group of antibodies and the third group of antibodies are labeled with fluorescein KO.

5. The antibody composition according to claim 4, characterized in that The catalog number of CD117 antibody labeled with fluorescein ECD is B38307, the catalog number of CD34 antibody labeled with fluorescein PECY5.5 is 343522, the catalog number of HLA-DR antibody labeled with fluorescein PE-Cy7 is B49180, the catalog number of CD19 antibody labeled with fluorescein APC is IM2470, the catalog number of CD7 antibody labeled with fluorescein PB is B06499, the catalog number of CD56 antibody labeled with fluorescein PE is A07788, and the catalog number of CD16 antibody labeled with fluorescein The catalog number of ECD is B49216, the catalog number of CD34 antibody labeled with fluorescein PECY5.5 is 343522, the catalog number of CD64 antibody labeled with fluorescein APC750 is B96769, the catalog number of CD9 antibody labeled with fluorescein FITC is IM1755U, the catalog number of MPO antibody labeled with fluorescein PE is B36288, and the catalog number of CD117 antibody labeled with fluorescein PECY5.5 is B96754. The manufacturer is BeckmanCoulter; The product number of CD71 antibody labeled with fluorescein FITC is 665339, the product number of CD33 antibody labeled with fluorescein PE is 663527, and the product number of CD11b antibody labeled with fluorescein APC is 982604. The manufacturer is BD; The catalog number of CD10 antibody labeled with fluorescein APC750 is 982208, the catalog number of CD45 antibody labeled with fluorescein KO is 982320, the catalog number of CD38 antibody labeled with fluorescein FITC is 980304, the catalog number of CD13 antibody labeled with fluorescein PECY7 is 982806, and the catalog number of CD15 antibody labeled with fluorescein PB is 3230409. The manufacturer is biolegend.

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

7. Use of the antibody composition according to any one of claims 1 to 5 or the kit according to claim 6 in assisting the detection of acute promyelocytic leukemia.

8. A system for detecting acute promyelocytic leukemia, characterized in that: include: A detection module, wherein the detection module uses the antibody composition according to any one of claims 1 to 5 or the kit according to claim 6 to perform flow cytometry detection on the cells to be detected; A data acquisition module, which acquires data of flow cytometry detection results; The data analysis module analyzes the acquired data: uses CD45-SSC to set a gate, and divides the cell population into a granulocyte population, a monocyte population, a lymphocyte population, a nucleated red or platelet region, and a primitive cell population according to the expression of CD45, further analyzes the target cells in the granulocyte population, and analyzes the fluorescence expression intensity of the antibody pair in the target cells; the antibody pair includes: 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.

9. The system for detecting acute promyelocytic leukemia according to claim 8, 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 is determined to be a tumorous promyelocyte; If the antibody expression pattern of the cell to be tested does not fall into the expression pattern template of the antibody of the abnormal promyelocyte, the cell to be tested is determined to be a non-neoplastic promyelocyte.

10. The system for detecting acute promyelocytic leukemia according to claim 9, characterized in that: The antibody expression pattern of the cells to be tested and the antibody expression pattern of abnormal promyelocytes are established by the following steps: the data of the flow cytometry test results are gated using CD45-SSC, and the cell population is divided into a granulocyte population, a monocyte population, a lymphocyte population, a nucleated red or platelet region, and a primitive cell population according to the expression of CD45, and the target cells in the granulocyte population are further analyzed, and the fluorescence expression intensity of the antibody pair in the target cells is analyzed; the antibody pair includes: CD45-SSC, CD117-CD33, 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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