Acute megakaryocytic leukemia minimal residual disease detection test reagent composition and use thereof

Abstract

The present application provides a kind of acute megakaryocyte leukemia micro residual disease detection reagent composition and its application.The reagent composition includes two groups of antibodies, and is used for the composition of flow cytometry detection for following-up micro residual disease after the treatment of acute megakaryocyte leukemia, and when used, 2 tubes are used in parallel, wherein the first group of antibodies and the second group of antibodies are used for different tube samples respectively.The reagent composition of the present application can be applied to flow cytometry detection of micro residual disease after the treatment of acute megakaryocyte leukemia.

Description

Technical Field

[0001] This invention relates to a reagent composition for detecting minimal residual disease in acute megakaryocytic leukemia and its application, belonging to the field of hematological disease detection technology. Background Technology

[0002] Acute megakaryoblastic leukemia (AMKL), also known as acute myeloid leukemia (AML) M7 according to the FAB classification, is a subtype of AML defined by cell differentiation. This type of leukemia is defined as having ≥20% primitive cells, with half of the tumor cells exhibiting megakaryoblastic differentiation characteristics. AMKL occurs more frequently in children than adults, accounting for 4%-15% of all AML cases in children and less than 1% in adults. Diagnosis of AMKL relies on a comprehensive diagnostic approach combining bone marrow cell morphology, immunophenotype, cytogenetics, and molecular biology. Immunophenotypic analysis plays a crucial role in the diagnosis and treatment of AMKL, particularly in monitoring minimal residual diseases (MRD) after treatment.

[0003] Although AMKL has a low incidence rate, it is a type of AML with a very poor prognosis, especially affecting the health and survival of children. Therefore, MRD testing is very important. Since most AMKL cases lack specific fusion genes, flow cytometry has become an important tool for clinical prognostic assessment and relapse prediction.

[0004] However, due to the small number of AMKL cases and the complexity of its immunophenotype, only a few teams have conducted flow cytometry immunophenotyping studies on AMKL. One important study is an AMKL immunophenotyping study published in 2022 by a member of the European Flow Cytometry Consortium (euroflow). This study selected 72 AMKL patients and 114 non-AMKL AML patients for immunophenotyping analysis using the EuroFlow AML protocol, employing a large number of biomarkers and a highly complex protocol (see Table 1).

[0005]

[0006] The above protocol's characteristic immunophenotype results for AMKL showed that, compared to other AMLs, AMKL expressed megakaryocyte-related markers but lacked CD13 and HLA-DR. Since megakaryocyte markers are a prerequisite, this study focused on the contributions of tubes 6 and 7 of the EuroFlow protocol. CD42a and CD61 (both antibodies combined) showed the highest expression rates (59 / 65, 91%), followed by CD42b (52 / 65, 80%) and CD41 (46 / 65, 71%). The incidence of expression of all three megakaryocyte markers was 39 / 65 (60%). However, this study was limited to treatment-naïve cases and only evaluated the positive expression intensity of a single parameter, without addressing how to detect MRD post-treatment. Furthermore, this extensive combination protocol lacked common aberrantly expressed markers in the two tubes containing megakaryocyte markers, potentially leading to missed diagnoses.

[0007] In practice, detecting MRD in AMKL using flow cytometry is extremely challenging and prone to missed or misdiagnosis. This is because AMKL exhibits diverse phenotypes, with approximately half of the cases not expressing primitive cell markers such as CD34 and CD117. Some cases even show no abnormal markers other than megakaryocyte markers. Furthermore, megakaryocyte markers are easily lost after AMKL treatment, which is a major reason why AMKL MRD is easily missed. Additionally, megakaryocyte markers such as CD61, CD42a, CD42b, and CD110 are expressed in both normal megakaryocytes and platelets. Megakaryocytes that do not express primitive markers need to be differentiated from normal megakaryocytes. Platelet aggregation and platelet adhesion to monocytes and granulocytes can also easily lead to false positives, further contributing to the misdiagnosis of AMKL MRD.

[0008] Currently, there are a few reports on immunophenotyping of AMKL, but few studies on MRD of AMKL detected by flow cytometry. Clinically, there is an urgent need for a method that can rapidly and accurately detect MRD of AMKL using flow cytometry. Summary of the Invention

[0009] One object of the present invention is to provide a reagent composition that can rapidly and efficiently detect MRD of AMKL, especially in cases of phenotypic changes after treatment, to prevent missed diagnoses.

[0010] Another object of the present invention is to provide the application of the reagent composition described herein in the rapid and efficient flow cytometry detection of minimal residual disease in acute megakaryocytic leukemia, so as to prevent missed diagnoses and misdiagnoses.

[0011] On one hand, the present invention provides a reagent composition comprising two groups of antibodies, wherein:

[0012] The first group of antibodies includes fluorescently labeled CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500. These antibodies are added to the first flow cytometer tube containing the test sample in a single-cell suspension.

[0013] The second group of antibodies includes fluorescently labeled CD61, CD42b, CD34, CD117, CD110, CD14, CD7, CD45, and CD56 antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are added to the second flow cytometer tube containing the test sample in a single-cell suspension.

[0014] The reagent composition of this invention can be applied to the rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia using flow cytometry. In specific applications, a two-tube parallel approach is used, wherein: (1) each tube uses CD45 weak expression and megakaryocytic markers to set up a megakaryocytic gate, and false positives caused by platelet aggregation, platelet contamination of granulocytes and / or monocytes need to be excluded; (2) simultaneously, myeloid primitive and immature cell antibodies CD34 and CD117 are combined with SSC to set up myeloid cell and primitive / immature cell gates respectively, to prevent missing minimal residual disease that does not express megakaryocytic markers; (3) simultaneously, CD45 weak expression / CD56 positive and CD45 weak expression / CD7 positive are used to set up abnormal cell gate 1 and abnormal cell gate 2, and one or more common markers need to be expressed simultaneously. By setting up gates for positive detection and adding exclusion conditions, missed diagnoses and misdiagnoses can be reduced, detection accuracy can be improved, and convenience can be provided for future automation and artificial intelligence.

[0015] According to a specific embodiment of the present invention, each antibody in the reagent composition of the present invention is a monoclonal antibody.

[0016] According to a specific embodiment of the present invention, in the antibody composition of the present invention, each antibody is a fluorescently labeled antibody. Specifically, in the first group of antibodies, the fluorescent labeling order of CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500. In the second group of antibodies, the fluorescent labeling order of CD61 antibody, CD42b antibody, CD34 antibody, CD117 antibody, CD110 antibody, CD14 antibody, CD7 antibody, CD45 antibody, and CD56 antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. In this invention, by combining different antibodies with specific fluorescein, the reagent composition of this invention can achieve excellent staining effects for all fluorescein in each channel when used for rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia.

[0017] According to a specific embodiment of the present invention, all antibody components in the reagent composition of the present invention are commercially available. Each antibody should meet the requirements of relevant industry standards.

[0018] According to a specific embodiment of the present invention, in the reagent composition of the present invention, the first group of antibodies is a mixture of CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3:3. The second group of antibodies is a mixture of CD61 antibody, CD42b antibody, CD34 antibody, CD117 antibody, CD110 antibody, CD14 antibody, CD7 antibody, CD45 antibody, and CD56 antibody in a volume ratio of 5:5:5:3:2:3:3:3:3. The mixing ratios of the antibodies mentioned above refer to the mixing ratios when the titers of each antibody are substantially equivalent.

[0019] Another aspect of the present invention provides a reagent kit comprising a first container and a second container, each container containing a first group of antibodies and a second group of antibodies of the reagent composition described in the present invention.

[0020] According to a specific embodiment of the present invention, the kit may further include one or more of the following: hemolysin, buffer solution, and flow cytometry tubes for use with a flow cytometer. These reagents and consumables are commercially available. Each reagent material may be contained in a separate container.

[0021] The kit of this invention can be used for rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia by flow cytometry.

[0022] Another aspect of the present invention provides the use of the reagent composition in the preparation of flow cytometry samples for the detection of minimal residual disease in acute megakaryocytic leukemia.

[0023] According to a specific embodiment of the present invention, the process of preparing flow cytometry samples for detecting minimal residual disease in acute megakaryocytic leukemia includes the following steps:

[0024] (1) Add the sample to be tested into flow cytometry tube one and flow cytometry tube two (or tube A and tube B) respectively, so that it is in a single cell suspension state, and ensure that the cell quantity is 1×10 6 / tube-1×10 7 / Tube;

[0025] (2) Add phosphate-buffered saline (PBS) to tube 1 and tube 2, mix well, incubate at 37°C, and centrifuge to remove the supernatant;

[0026] (3) Add the first group of antibodies in the reagent composition of the present invention to tube one obtained in step (2), and add the second group of antibodies in the reagent composition of the present invention to tube two obtained in step (2). Incubate each flow cytometer at room temperature in the dark.

[0027] (4) Add 1×hemolysin to tube 1 and tube 2 after incubation in step (3) respectively, and continue incubation at room temperature in the dark;

[0028] (5) After centrifuging each flow cytometer tube incubated in step (4), remove the supernatant;

[0029] (6) Add PBS buffer to tube 1 and tube 2 after removing the supernatant in step (5) to wash, centrifuge and remove the supernatant, resuspend the cells with PBS buffer to obtain the flow cytometry sample.

[0030] In this invention, unless otherwise specified or clearly determined from the context, the order of the described operation steps is not intended to limit the actual order of these steps.

[0031] According to a specific embodiment of the present invention, the sample to be tested can be bone marrow or peripheral blood, or it can be any sample that can be prepared into a single live cell and is suitable for flow cytometry detection, such as tissue samples or body fluid samples.

[0032] According to a specific embodiment of the present invention, in step (1), the volume of each sample added does not exceed 160 μl (if the patient has a low number of peripheral blood cells, the volume can exceed 160 μl if necessary, and the sample is centrifuged to remove the supernatant and concentrated).

[0033] According to a specific embodiment of the present invention, the dosage of each reagent can be based on the conventional dosage in the art or the manufacturer's recommended dosage.

[0034] According to a specific embodiment of the present invention, the reagent composition of the present invention has the following addition amounts: the first group of antibodies is 15-58 μl / tube, and the second group of antibodies is 16-64 μl / tube.

[0035] According to a specific embodiment of the present invention, the operation of step (2) above can be selectively repeated once or multiple times as needed to remove platelet adhesion, avoid the aggregation of platelets expressing megakaryocyte markers or their adhesion to other cells, which would affect the MRD detection of AMKL, thereby eliminating false positives. According to a specific embodiment of the present invention, the amount of PBS added each time is 2-3 ml / tube, and the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0036] According to a specific embodiment of the present invention, the incubation time in step (2) can be 5-30 minutes.

[0037] According to a specific embodiment of the present invention, the incubation time in step (3) can be 10-30 minutes.

[0038] According to a specific embodiment of the present invention, in step (4), the incubation time can be 5-30 minutes. The amount of 1×hemolysin added is 2-3 ml / tube.

[0039] According to a specific embodiment of the present invention, in step (5), the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0040] According to a specific embodiment of the present invention, in step (6), the amount of PBS buffer added for washing is 2-3 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspension is 0.5-1 ml / tube.

[0041] According to a specific embodiment of the present invention, when resuspending cells for flow cytometry analysis, each tube is sequentially equipped with a de-adhesion cell gate P1 and a viable cell gate P2 to obtain single viable cells; within gate P2, CD45 / SSC is used to set each blood cell gate; and:

[0042] In tube one, gating is set up as follows: within the P2 gate, a weakly positive CD45 / CD42a positive (generally moderate to weakly positive) gate is set up for immature megakaryocytes; a weakly positive CD45 / CD42a strongly positive gate is set up for platelets and mature megakaryocytes; and a medium-small SSC / CD34 positive and a medium-small SSC / CD117 positive gate are set up for myeloid primitive immature cells, respectively. In this tube one, the expression of other markers in cells within the immature megakaryocyte gate and the myeloid primitive immature cell gate can also be observed, and further gating can be set up as needed.

[0043] Gate 2 is set up as follows: Within gate P2, immature megakaryocyte gates are set up using CD45 weak expression / CD61 positive (generally moderate to weak positive), CD45 weak expression / CD42b positive (generally moderate to weak positive), and CD45 weak expression / CD110 positive, respectively; platelet and mature megakaryocyte gates are set up using CD45 weak expression / CD110 strong positive, CD45 weak expression / CD61 strong positive, and CD45 weak expression / CD42b strong positive, respectively; myeloid blast cell gates are set up using SSC medium / CD34 positive and SSC medium / CD117 positive, respectively; abnormal cell gate 1 is set up using CD45 weak expression / CD56 positive; and abnormal cell gate 2 is set up using CD45 weak expression / CD7 positive.

[0044] In this invention, CD45 expression strength refers to the following: using normal bone marrow as the standard, mature lymphocytes show strong CD45 expression (strong positive), monocytes show strong CD45 expression, granulocytes show weak CD45 expression (weak positive), and nucleated erythrocytes are CD45 negative (no expression).

[0045] In this invention, the expression strength of CD61, CD42a, CD42b, and CD110 refers to the expression strength of mature megakaryocytes and platelets as a standard, with normal bone marrow as the standard (the lowest point in the two-dimensional dot plot is the lower left, and strong expression means that the expression intensity basically reaches the rightmost side of the coordinate axis); the expression intensity of primitive immature megakaryocytes cannot reach this level, and they are all moderate or weakly expressed.

[0046] In this invention, the size of SSC refers to the following based on normal bone marrow: granulocytes are SSC-large, eosinophils are SSC-extra-large, monocytes and myeloid blasts are SSC-medium, and lymphocytes, nucleated erythrocytes and the earliest stem cells in bone marrow are SSC-small.

[0047] In this invention, the size of the FSC refers to the size of cells compared to normal bone marrow, with lymphocytes, nucleated erythrocytes, and some primitive cells being considered small, and granulocytes and some myeloid immature cells being considered medium. Cells similar to or larger than granulocytes, but not significantly larger than the SSC, are classified as large FSC / medium-small SSC.

[0048] In this invention, the expression strength of CD33, CD13, CD34, CD117, and HLA-DR refers to the expression intensity exceeding that of normal myeloid primitive immature cells in the bone marrow as a standard. The expression intensity is considered strong when it exceeds that of cells at this stage, and weak when it is lower than that of cells at this stage. Nucleated erythrocytes are negative.

[0049] According to a specific embodiment of the present invention, CD42a, CD61, CD42b, and CD110 are used as phylogenetic markers for megakaryocytes. This is mainly based on years of clinical work (see Table 2, which shows the detection results of 230 cases of AMKL at Hebei Yanda Lu Daopei Hospital from June 2008 to June 2025). The findings indicate that in newly diagnosed AMKL, the positive rate of CD110 is as high as 98.33%, the positive rate of CD42a is 97.8%, the positive rate of CD42b is 95.61%, and the positive rate of CD61 is 93.01%. CD41 is a traditional megakaryocyte marker; however, the study of this invention found that while normal platelets and megakaryocytes show strong positive expression of CD41, the positive rate of CD41 in AMKL is only 79.33%. Normal bone marrow does not contain CD34 and / or CD117-positive primitive immature megakaryocytes, and the proportion of mature megakaryocytes is less than 0.05%. However, various causes, such as myelodysplastic syndromes, idiopathic thrombocytosis, idiopathic thrombocytopenic purpura, autoimmune diseases, and certain infections, can lead to an increase in megakaryocytes. Reactive megakaryocytes only express megakaryocyte markers and do not express myeloid markers CD33, CD13, CD14, and CD11b, nor early markers HLA-DR, CD34, and CD117, or lymphoid markers CD7 and CD56. These markers can be used as indicators to distinguish between normal and malignant megakaryocytes. This invention selects CD42a, CD61, CD42b, and CD110 as the main megakaryocyte markers, which are used in the first and second tubes, respectively. Combined with the expression of other specific granulocytic and monocytic markers on tumor cells, as well as the expression of megakaryocyte markers in normal granulocytes and monocytic cells remaining in the sample, this invention provides a comprehensive assessment of minimal residual disease in acute megakaryocyte leukemia.

[0050] CD42a, CD42b, CD61, and CD110 are expressed in both normal megakaryocytes and platelets, but the expression intensity differs. Mature megakaryocytes strongly express CD42a, CD42b, CD61, and CD110, while primitive immature megakaryocytes express these markers at a moderate or even weak intensity, thus excluding mature megakaryocytes. Platelets are significantly smaller than the smallest white blood cells in bone marrow—lymphocytes and nucleated red blood cells. In the two-dimensional dot plot of FSC / SSC, the lower left is the lowest point, and platelets are located at the lower left. Using FSC / SSC with no centroid, the fact that FSC is lower than that of lymphocytes and nucleated red blood cells excludes platelets. In addition, platelets can easily adhere to monocytes and granulocytes, causing false positives. In this invention, the strong expression of CD45 / CD33 / CD11b / HLA-DR in the first tube and the strong expression of CD45 / CD14 in the second tube can rule out mature monocyte adhesion. Granulocyte adhesion can be ruled out by the large SSC and the granulocyte development pattern of CD13 / CD11b.

[0051]

[0052] Further research in this invention revealed that some cases experienced loss of megakaryocyte markers during follow-up, particularly the statistically significant loss rates of CD61, CD42a, and CD42b. After treatment, the positivity rate of CD42a decreased from 97.8% to 89.23%, CD42b from 95.61% to 78.22%, and CD61 from 93.01% to 72.48%. In this invention, the inclusion of CD110 reduced the MRD (Misdiagnosis Related Groups) missed diagnosis rate; after treatment, the positivity rate of CD110 decreased from 98.33% to 94.74%.

[0053] According to a specific embodiment of the present invention, in addition to megakaryocyte markers, the identification of myeloid blast cells is also emphasized. CD34 and CD117 are used for gating myeloid blast cells in the first and second tubes. CD11b in the first tube and CD7 and CD56 in the second tube are markers not expressed by normal myeloid blast cells. In AMKL, the expression rates of CD11b, CD7, and CD56 are 36.77%. Therefore, myeloid blast cells positive for CD34 and / or CD117, with any positive result for CD11b, CD7, or CD56, can be considered malignant blast cells. CD34, CD117, CD33, CD13, HLA-DR, and CD45 are all markers expressed by normal myeloid blast cells. Combinations of these markers can be used to observe the presence of abnormal myeloid blast cells, thus allowing the identification of CD34 and / or CD117-positive malignant tumor cells in a different manner than normal, even in the absence of megakaryocyte markers.

[0054] According to the research in this case, 27.63% to 47.06% of cases showed weak CD45 positivity and CD7 and / or CD56 positivity. In order to prevent missed diagnosis of cases where megakaryocyte markers were lost and CD34 and CD117 were not expressed, this invention further sets abnormal cell gates 1 and 2 using weak CD45 positivity / CD7 positivity and weak CD45 positivity / CD56 positivity after gating with megakaryocyte markers and early myeloid markers. If any one of CD13, CD33, HLA-DR, or CD11b is expressed (any one of CD61, CD42a, CD42b, CD110, CD34, or CD117 positivity has been identified above), it is judged as MRD of AMKL.

[0055] On the other hand, the present invention also provides a device for rapid and efficient examination of minimal residual disease in acute megakaryocytic leukemia, the device comprising a detection unit and an analysis unit, wherein:

[0056] The detection unit includes reagent materials for detecting samples from the individual to be tested by flow cytometry, and for obtaining the detection results of the samples; the reagent materials include the reagent composition described in this invention;

[0057] The analysis unit is used to analyze the detection results of the detection unit.

[0058] According to a specific embodiment of the present invention, when the device for rapid and efficient detection of minimal residual disease in acute megakaryoblastic leukemia is used to detect minimal residual disease in acute megakaryoblastic leukemia, the process of detecting samples from the test individual by flow cytometry includes: processing the test sample using the reagent composition described in the present invention to prepare a flow cytometry sample; and performing flow cytometry detection.

[0059] According to a specific embodiment of the present invention, in the device for rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia, the analysis process of the analysis unit includes: analyzing the detection results to rapidly and accurately detect minimal residual disease in acute megakaryocytic leukemia, excluding misdiagnosis and missed diagnosis caused by other possible factors, especially misdiagnosis caused by normal mature megakaryocytes, platelets, platelet-stained granulocytes and monocytes, and missed diagnosis caused by loss of megakaryocyte markers after treatment.

[0060] According to a specific embodiment of the present invention, the device of the present invention is used for rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia. When performing flow cytometry analysis, the gating analysis of each flow cytometer tube can be performed according to the aforementioned operation.

[0061] According to a specific embodiment of the present invention, in the device for rapid and efficient detection of minimal residual disease in acute megakaryocytic leukemia, the analysis unit may further include a module for further determining the disease based on the gating analysis results (the determination described in this invention includes detection, confirmation, and differential diagnosis by excluding false positives and false negatives). The present invention, through multi-marker gating, compares the displayed cell populations with corresponding normal cells to identify tumor cells, enabling rapid and accurate diagnosis and eliminating misdiagnosis and missed diagnosis caused by other factors, especially misdiagnosis and missed diagnosis caused by normal megakaryocytes, platelets, platelet adhesion to monocytes and granulocytes, and malignant immature megakaryocytes that have lost megakaryocyte markers after treatment.

[0062] According to a specific embodiment of the present invention, when analyzing two tubes of samples, the CD45 / SSC used in each tube is used for preliminary screening to determine whether the proportions of common cell populations (mature lymphocytes, monocytes, differentiated granulocytes, and nucleated erythrocytes) in normal samples are normal, and whether a high proportion of tumor cells are present. In particular, the use of these markers, along with the location of FSC and SSC, serves as a reference for differential diagnosis, further combined with other markers from each tube for further judgment. Specifically, the test results can be output according to one of the following judgment methods (the judgment of various diseases can be performed according to one or more of the following methods):

[0063] Method 1:

[0064] Minimal residual disease in typical acute megakaryocytic leukemia: CD45 weak expression / CD42a positive (generally moderate to weak positive) and / or CD45 weak expression / CD61 positive (generally moderate to weak positive) and / or CD45 weak expression / CD42b positive (generally moderate to weak positive) cell population and / or CD45 weak expression / CD110 positive (generally moderate to weak positive), and meeting the following six conditions: (1) there is a centroid on the FSC / SSC plot, (2) FSC is larger than lymphocytes and SSC is not larger than lymphocytes, (3) CD14 is not positive, (4) no CD11b positive / CD45 high expression / HLA-DR positive / CD33 high expression, (5) no high SSC, and the CD13 / CD11b two-dimensional dot plot shows a normal granulocyte development pattern, (6) at least one of CD61, CD42a, CD42b and CD110 is not strongly expressed;

[0065] Method 2:

[0066] Malignant myeloid blast cells lacking megakaryocyte markers: CD34 positive and / or CD117 positive cells, and phenotypes exhibiting at least one of the following conditions: (1) abnormal expression of CD7, (2) abnormal expression of CD56, (3) abnormal expression of CD11b, (4) loss or weakened or enhanced expression of any of the markers CD13, CD33, HLA-DR, CD34, CD117, or CD45, (5) CD13 / CD33 two-dimensional dot plot, CD33 / HLA-DR two-dimensional dot plot, CD13 / HLA-DR two-dimensional dot plot, CD34 / HLA-DR two-dimensional dot plot, and CD34 / CD117 two-dimensional dot plot show expression patterns different from those of normal myeloid blast cells;

[0067] Method 3:

[0068] Malignant myeloid blast cells that have lost megakaryocyte markers and do not express primitive blast cell markers: CD45 weakly expressed / CD56 positive and / or CD45 weakly expressed / CD7 positive cells, and expressing any one of CD13, CD33, HLA-DR, or CD11b.

[0069] According to a specific embodiment of the present invention, in the detection and assessment of minimal residual disease in typical acute megakaryocytic leukemia, 72.48%–94.74% of the typical types retain megakaryocytic markers such as CD42a and / or CD42b and / or CD61 and / or CD110, and all six conditions must be met simultaneously. By using these six conditions, the present invention can eliminate false positives caused by normal mature megakaryocytic cells, platelets, platelet adhesion to monocytes and granulocytes, etc. The present invention can also examine and assess malignant myeloid immature cells that have lost megakaryocytic markers, and can also detect and assess malignant myeloid immature cells that have lost megakaryocytic markers and do not express primitive immature markers, thereby minimizing the misdiagnosis and missed diagnosis rates.

[0070] In summary, this invention provides a rapid and efficient reagent composition for detecting minimal residual disease (MRD) in acute megakaryocytic leukemia (AML) and its applications. This invention offers the following advantages: ① Based on immunophenotypic analysis of numerous newly diagnosed and follow-up cases, continuous optimization of the protocol, and new research findings, particularly new insights into tumor cells expressing and lacking megakaryocytic markers, a comprehensive MRD detection protocol has been designed, minimizing missed and misdiagnosed rates. ② In terms of application, standardized procedures and gates ensure good reproducibility in clinical practice, reducing variability caused by personnel experience. ③ This invention employs a rapid and efficient method for detecting MRD in AML, allowing for premixed antibodies, significantly reducing workload, improving efficiency, and accelerating reporting time. Clinical patients can obtain accurate clinical reports quickly to implement appropriate treatment, thereby improving remission and survival rates. ④ This invention can create conditions for subsequent sample preprocessing machines, automated sample loading in flow cytometry, immobilization of data analysis, and especially for the future development of artificial intelligence. ⑤ This invention, based on the combined application of biomarkers, employs a method that simultaneously detects positive results and excludes negative ones, thereby eliminating various false positives and false negatives and improving detection specificity and coverage. These analytical models can be further programmed into computer language as artificial intelligence software, enabling accurate and rapid judgments with high sensitivity and specificity. The detection and analysis methods of this invention meet the current needs of clinical flow cytometry diagnosis and are suitable for widespread application and promotion. Attached Figure Description

[0071] Figures 1-2 This invention shows the results of flow cytometry analysis of bone marrow samples from non-tumor patients according to a specific embodiment of the present invention.

[0072] Figures 3-4 This invention shows the results of flow cytometry gating analysis of a typical acute megakaryocytic leukemia minimal residual disease bone marrow sample according to a specific embodiment of the present invention.

[0073] Figures 5-6 This invention presents flow cytometry gating analysis results of bone marrow samples from minimal residual disease of acute megakaryocyte leukemia, which is prone to being missed due to the loss of megakaryocyte markers, according to a specific embodiment of the present invention. Detailed Implementation

[0074] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the following detailed description, in conjunction with specific embodiments and the technical solutions of this invention, is provided. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this invention. In the embodiments, all original reagent materials are commercially available. Experimental methods not specifically specified are conventional methods and conditions well-known in the art, or are performed according to the conditions recommended by the instrument manufacturer.

[0075] Example 1: Preparation of Reagents

[0076] The antibody combination used in this embodiment is,

[0077] The first group consists of: CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500. The above eight monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3 and placed in the first container.

[0078] The second group consists of: CD61 antibody, CD42b antibody, CD34 antibody, CD117 antibody, CD110 antibody, CD14 antibody, CD7 antibody, CD45 antibody, and CD56 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. The above nine monoclonal antibody reagents are mixed in a volume ratio of 5:5:5:3:2:3:3:3:3 and placed in the second container.

[0079] All antibodies in this embodiment are commercially available. CD61 and CD42a are products of Henan Kaipuri Company, and CD110 and CD42b are products of BD Company in the United States.

[0080] Optionally, prepare cell lysis buffer (hemolysin) and place it in the third container, and PBS buffer in the fourth container. Hemolysin and PBS buffer are commercially available; the cell lysis buffer is from Becton Dickinson, USA, and the PBS buffer is from Beckman Coulter, USA.

[0081] Example 2, Sample Processing

[0082] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples into flow cytometry tube A, ensuring that the added cell quantity is approximately 2 × 10⁶ cells / mL. 6The cells were in a single-cell suspension state. 3 ml of PBS was added, mixed, and incubated in a 37°C water bath for 5 minutes. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added again, mixed, and incubated in a 37°C water bath for 5 minutes. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added again, mixed, and centrifuged at 1500 rpm for 5 minutes and removal of the supernatant. Then, 29 μl of eight different fluorescently labeled monoclonal antibodies were added to the flow cytometer according to Table 3. After thorough mixing with the cell suspension, the cells were incubated at room temperature in the dark for 15 minutes. 3 ml of 1×hemolysin was added, and the cells were incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added, mixed, and centrifuged at 1500 rpm for 5 minutes and removal of the supernatant. Finally, 0.5 ml of PBS buffer was added to resuspend the cells. This is the processed sample, ready for flow cytometry analysis.

[0083] Based on the cell count results, add heparinized or EDTA-anticoagulated bone marrow or peripheral blood samples to tube B of the flow cytometry tube, ensuring that the added cell quantity is approximately 2 × 10⁻⁶. 6 The cells were in a single-cell suspension state. 3 ml of PBS was added, mixed, and incubated in a 37°C water bath for 5 minutes. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added again, mixed, and incubated in a 37°C water bath for 5 minutes. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added again, mixed, and centrifuged at 1500 rpm for 5 minutes and removal of the supernatant. Then, 32 μl of nine different fluorescently labeled monoclonal antibodies were added to the flow cytometer according to Table 3. After thorough mixing with the cell suspension, the cells were incubated at room temperature in the dark for 15 minutes. 3 ml of 1×hemolysin was added, and the cells were incubated in the dark for 10 minutes to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes and removal of the supernatant, 3 ml of PBS was added, mixed, and centrifuged at 1500 rpm for 5 minutes and removal of the supernatant. Finally, 0.5 ml of PBS buffer was added to resuspend the cells. This is the processed sample, ready for flow cytometry analysis.

[0084]

[0085] Example 3: Sample Detection

[0086] The samples processed according to the method in Example 2 were analyzed on a Becton Dickinson 3-laser 10-color FACS Canto Plus or Lyric flow cytometer. It is preferred to obtain 1 million cells per tube (at least 300,000 is recommended). The data were then analyzed using Diva 2.8 software or other software such as Kaluza.

[0087] The flow cytometry analysis was performed using the following gating methods: ① Fixed gating: This involved sequentially removing adherent cells, live cells, and blood cells; ② Multi-marker gating: Starting with a single live cell, gating and defining all cells were performed concurrently with the blood cell gating to prevent missing tumor cells; ③ Within the multi-marker gating, common expression and developmental patterns of various marker combinations were displayed, allowing for the identification of tumor cells based on their differences from normal cells. Specifically:

[0088] 1. Fixed gate: It consists of deadhesion cell gate, live cell gate, and blood cell gate, which are connected in series.

[0089] De-adhesion cell gate: First, the de-adhesion cell gate (denoted by P1) is set using the area (A) and height (H) of the forward scatter (FSC). Adhesive cells can be removed via FSC-A / FSC-H, based on the principle that cells are spherical and A and H are positively correlated. See... Figures 1-6 The first image from left to right in the top row features a door.

[0090] Viable cell phylogenetics: Cells in P1 are phylogenetically gorged using FSC / side scatter (SSC) (denoted as P2) to obtain single viable cells. The principle of FSC / SSC is that viable cells exhibit a near-normal distribution in size and granularity, clustering around a central point and clearly demarcated from dead cells, apoptotic cells, debris, and background noise. See [link to FSC / SSC]. Figures 1-6 The second picture from left to right in the top row shows a door.

[0091] Blood cell phylogenetic analysis: Within a single live cell phylogenetic stage (P2 phylogenetic stage), CD45 / SSCs are used to establish various blood cell phylogenetic groups, allowing for the general observation of lymphocytes (lym), monocytes (mono), granulocytes (gra), nucleated erythrocytes (NEC), eosinophils (eo), and the presence of obvious tumor cells or abnormal cells. CD45 / SSCs are used to roughly differentiate between blood cell groups based on differences in CD45 expression fluorescence intensity among hematopoietic cells (mature lymphocytes > monocytes > granulocytes > nucleated erythrocytes) and differences in SSC size (eosinophils > granulocytes > monocytes > mature lymphocytes > nucleated erythrocytes). See [link to documentation]. Figures 1-6 Image of CD45 / SSC door in the image.

[0092] 2. Multi-marker gating: Performed simultaneously with CD45 / SSC gating, starting from a single live cell (P2).

[0093] Each tube has a different observation purpose and a different gate setting:

[0094] Tube A: Within the P2 gate, a primitive immature megakaryocyte gate is established using CD45 weak expression / CD42a positive (generally moderate to weakly positive) to detect malignant immature megakaryocytes. A platelet and mature megakaryocyte gate is established using CD45 weak expression / CD42a strong expression, where large FSCs indicate megakaryocytes. Primitive immature myeloid cells are established using small / medium SSCs / CD34 positive and small / medium SSCs / CD117 positive, respectively. For easier visualization, a CD34 or CD117 gate can also be established to collectively display CD34-positive or CD117-positive cells. Tube A is mainly used to observe the presence of malignant immature megakaryocytes expressing or not expressing megakaryocyte markers, and to exclude false positives caused by normal megakaryocytes, platelets, platelets contaminated with granulocytes and monocytes, as well as false negatives that may result from the loss of megakaryocyte markers.

[0095] Tube B: Within gate P2, primitive immature megakaryocyte gates are established using CD45 weak expression / CD42b positive (generally moderate to weakly positive), CD45 weak expression / CD61 positive (generally moderate to weakly positive), and CD45 weak expression / CD110 positive (generally moderate to weakly positive). Malignant immature megakaryocytes are detected using gates for platelets and mature megakaryocytes, respectively, using CD45 weak expression / CD42b strong expression, CD45 weak expression / CD61 strong expression, and CD45 weak expression / CD110 strong expression. Larger FSC values ​​indicate megakaryocytes. For easier visualization, the most prominent primitive immature megakaryocyte gate can be selected to display tumor cells; other gates are not displayed. Primitive immature myeloid cell gates are established using small / medium SSC / CD34 positive and small / medium SSC / CD117 positive. For easier visualization, a CD34 or CD117 gate can also be created to collectively display CD34-positive or CD117-positive cells. Abnormal cell gates 1 and 2 were established using CD45 weak expression / CD56 positive and CD45 weak expression / CD7 positive. Tube B was primarily used to observe the presence of malignant immature megakaryocytes expressing or not expressing megakaryocyte markers, as well as cells abnormally expressing CD7 and CD56. False positives were excluded due to normal megakaryocytes, platelets, platelet-contaminated granulocytes, and monocytes, as well as false negatives that might result from the loss of megakaryocyte and immature cell markers.

[0096] 3. Precisely locate target cells based on observed marker combinations.

[0097] The criteria for identifying malignant immature megakaryocytes include the presence of any of the following conditions: (1) Minimal residual disease of typical acute megakaryocyte leukemia: a population of cells with weak CD45 expression / CD42a positivity (generally moderate to weak positivity) and / or weak CD45 expression / CD61 positivity (generally moderate to weak positivity) and / or weak CD45 expression / CD42b positivity (generally moderate to weak positivity) and / or weak CD45 expression / CD110 positivity (generally moderate to weak positivity), and must simultaneously meet the criteria for malignant immature megakaryocytes. The following six conditions must be met: ① There is a centroid on the FSC / SSC plot; ② FSC is larger than lymphocytes and SSC is not large; ③ CD14 positivity is not present; ④ CD11b positivity / CD45 high expression / HLA-DR positivity / CD33 high expression is not present; ⑤ SSC is not high and the CD13 / CD11b two-dimensional dot plot shows a normal granulocyte development pattern; ⑥ CD61, CD42a, CD42b, and CD110 are not strongly expressed; (2) Malignant myeloid naïve cells that have lost megakaryocyte markers Cells: CD34-positive and / or CD117-positive cells, with a phenotype different from normal myeloid immature cells, exhibiting one of the following: ① abnormal expression of CD7, ② abnormal expression of CD56, ③ abnormal expression of CD11b, ④ loss or weakened or enhanced expression of any of the markers CD13, CD33, HLA-DR, CD34, CD117, or CD45, ⑤ CD13 / CD33, CD33 / HLA-DR, CD13 / HLA-DR, CD34 / HLA-DR, and C The two-dimensional dot plot of the D34 / CD117 combination shows an expression pattern different from that of normal myeloid blast cells; (3) Malignant myeloid blast cells that have lost megakaryocyte markers and do not express blast cell markers: CD45 weak expression / CD56 positive and / or CD45 weak expression / CD7 positive cells, any of which express CD13, CD33, HLA-DR, CD11b (CD61, CD42a, CD42b, CD110, CD34, CD117 any positive see the first two methods).

[0098] In this invention, immunophenotypic analysis was performed on 230 newly treated cases (see Table 2), and the feasibility of the proposed method was verified by testing bone marrow samples from 108 follow-up cases with minimal residual disease after treatment. Furthermore, using the proposed method, minimal residual disease of AMKL was detected in 952 cases from January 2020 to June 2025, with 149 positive cases and 803 negative cases, resulting in only one missed diagnosis. The concordance rate with clinical findings and prognosis was 99.89%. Therefore, the proposed method is highly suitable for rapid, simple, and efficient detection and analysis in clinical practice, with sensitivity, specificity, positive predictive value, and negative predictive value all exceeding 99%.

[0099] In this invention, in addition to the megakaryocyte marker CD42a, the main markers selected for tube A are CD13, CD33, CD34, CD117, and HLA-DR, which are markers of normal myeloid primitive immature cells. This can efficiently identify malignant immature megakaryocytes. Normal megakaryocytes can be excluded by CD42a expression intensity, platelets can be excluded by the FSC / SSC pattern, and platelet-infected monocytes can be excluded by strong CD45 expression / strong CD33 expression / HLA-DR positivity / CD11b positivity. Platelet-infected granulocytes can be excluded by the CD13 / CD11b granulocyte development pattern and SSC. The main markers for megakaryocytes are CD42b, CD61, and CD110, along with myeloid immaturity markers CD34 and CD117, plus common companion lineage markers CD7 and CD56. This allows for efficient identification of malignant immature megakaryocytes. Normal megakaryocytes can be excluded based on the expression intensity of CD42b, CD61, and CD110. Platelets can be excluded based on the FSC / SSC pattern. Strong CD45 expression / CD14 positivity can exclude platelet-infected monocytes. Large SSC can exclude platelet-infected granulocytes. CD7 and CD56 can exclude T and NK cells.

[0100] In summary, this invention, within a multi-marker gating system, firstly uses a megakaryocyte marker gating system per tube to select megakaryocytes. Simultaneously, based on the characteristics of high megakaryocyte expression intensity, small FSC / SSC ratio, and lack of a center of gravity, normal mature megakaryocytes and platelets are excluded. The first tube, based on strong CD45 / CD33 expression / HLA-DR positivity / CD11b positivity, and the second tube, based on CD14 positivity, excludes platelet-infected monocytes. Platelet-infected granulocytes are excluded based on the CD13 / CD11b granulocyte development pattern and large SSC ratio. Secondly, using the myeloid primordial cell markers CD34 and CD117, a primordial cell gating system is established. The first tube identifies the benign or malignant nature of myeloid primordial cells by checking for changes in the intensity and combination of expression patterns of these markers. The second tube identifies the benign or malignant nature of myeloid primordial cells by checking for abnormal expression of CD7 and CD56. Third, a small number of malignant immature cells do not express megakaryocyte markers and early markers, but some cases show abnormal expression of CD7 and / or CD56. We used CD45 weak expression / CD7 positive and / or CD56 positive cells and excluded NK cells with strong CD45 expression / CD7 strong expression / CD56 positive cells. In addition, AMKL cells almost always show expression of at least one of CD13, CD33, HLA-DR, and CD11b. Further detection of malignant immature megakaryocytes was performed.

[0101] This embodiment provides normal bone marrow samples, bone marrow samples from patients with typical acute megakaryocytic leukemia and minimal residual disease, and bone marrow samples from tubes A and B of acute megakaryocytic leukemia and minimal residual disease cases that have lost megakaryocytic markers for immunophenotyping. The gating and observation were strictly carried out according to the above description.

[0102] Specifically, Figures 1-2 Bone marrow samples from the same non-tumor patient, tubes A and B were analyzed together.

[0103] Specifically, Figure 1 Analysis of bone marrow samples from non-tumor patients. The following settings were executed sequentially: ① FSC-A / H was set to P1 as the de-adhesion cell gate, resulting in single cells within P1; ② FSC / SSC was displayed within P1, and P2 was set to the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, yielding lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, a CD42a+ gate (red cell population) was set using weakly positive CD45 / positive CD42a. This cell population had large SSCs, and CD13 / CD11b showed a normal granulocyte development pattern; therefore, a false positive was considered to be caused by platelet contamination of granulocytes. A strong CD42a gate (straw-yellow) is set up for CD45 weakly positive / CD42a strongly positive cells. This cell population is extremely low in proportion, mostly consisting of normal platelets with small FSC / SSC ratios and no centroid. A few are normal mature megakaryocytes with large FSC ratios. The strong CD42a cell population does not express CD34, CD117, CD13, CD33, HLA-DR, or CD11b. Myeloid primitive immature cells are gated using small / medium SSC / CD34 positive and small / medium SSC / CD117 positive cells, respectively. For clearer display, either a CD34 or CD117 gate is set up, showing either CD34 positive (dark green) or CD117 positive (sapphire blue) cell populations. The CD13 / CD33, CD13 / CD34, CD33 / CD34, CD34 / CD117, CD34 / HLA-DR, CD13 / HLA-DR, and CD33 / HLA-DR combinations that showed CD34 or CD117 positive cell populations all had normal expression patterns, therefore no malignant immature megakaryocytes were observed.

[0104] Specifically, Figure 2This section displays bone marrow samples from the same non-tumor patient, analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was set to P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was set to P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, primitive immature megakaryocyte gates were set using CD45 weak expression / CD42b positive, CD45 weak expression / CD61 positive, and CD45 weak expression / CD110 positive cells. For clearer display, CD42b+ cells with clear boundaries were highlighted (red). CD61 and CD110 positive cells were not highlighted. The large SSC value of this cell group indicates a false positive caused by platelet contamination of granulocytes. CD45 weakly positive / CD42b strongly positive, CD45 weakly expressed / CD61 strongly positive, and CD45 weakly expressed / CD110 strongly positive are respectively gated with strong CD42b (straw yellow), strong CD61, and strong CD110. For clarity, only the clearly defined strong CD42b cells are displayed in color; strong CD61 and strong CD110 cells are not displayed. The proportion of cells with strong CD42b is extremely low; most are normal platelets with small FSC / SSC ratios and no centroid, and a few are normal mature megakaryocytes with large FSCs. The cell population with strong CD42a does not express CD34, CD117, CD14, CD7, or CD56. Myeloid blast cells were selected using small / CD34-positive and small / CD117-positive SSCs, respectively. For clear visualization, CD34 or CD117 gates were set up to display CD34-positive (dark green) or CD117-positive (sapphire blue) cell populations. No abnormal expression of CD7, CD56, or CD14 was observed in this patient. Abnormal cell gate 1 was set up using weak CD45 expression / CD56-positive, and abnormal cell gate 2 was set up using weak CD45 expression / CD7-positive. No abnormal cells with weak CD45 expression / CD56-positive or weak CD45 expression / CD7-positive were observed in this sample.

[0105] Specifically, Figures 3-4 Immunophenotypic analysis of tubes A and B of a typical acute megakaryocytic leukemia minimal residual disease-positive bone marrow sample.

[0106] Specifically, Figure 3Bone marrow samples showing typical acute megakaryocytic leukemia with minimal residual disease were analyzed in tube A. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, a CD42a+ gate (red) was set using weakly positive CD45 / CD42a positive cells. This cell group expressed weakly positive CD42a, CD117, and CD33, but did not express CD34, CD13, HLA-DR, or CD11b, indicating malignant immature megakaryocytic cells. The tube shows a weakly positive CD45 / strongly positive CD42a result with a strong CD42a cell gate (grass green), a small FSC / SSC ratio, and no center of gravity, indicating normal platelets. Myeloid primitive cells were phylogenized using small / CD34-positive and small / CD117-positive SSCs, respectively. For clearer visualization, CD34 or CD117 phylogenetic cells were also phylogenized to display the expression patterns of CD13 / CD33, CD13 / CD34, CD33 / CD34, CD34 / CD117, CD34 / HLA-DR, CD13 / HLA-DR, and CD33 / HLA-DR in CD34-positive (dark green) or CD117-positive (sapphire blue and red) cell populations. Dark green and sapphire blue cells represent normal myeloid primitive cells, as tumor cells express CD117. The red cell population within the CD34 or CD117 phylogenetic cells represents CD42a+ malignant immature megakaryocytes, which express weakly positive CD33 and CD117, but do not express CD34, CD13, HLA-DR, or CD11b.

[0107] Specifically, Figure 4Bone marrow samples showing typical acute megakaryocytic leukemia with minimal residual disease (MRDL) were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FSC / SSC was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, primitive immature megakaryocytic cell gates were set using CD45 weak expression / CD42b positive, CD45 weak expression / CD61 positive, and CD45 weak expression / CD110 positive gates, respectively. For clarity, the CD42b+ gate (red) with the clearest boundaries was selected; CD110 and CD61 positive cell populations were not displayed. This cell population shows expression of CD42b and CD110, with some weak expression of CD61, abnormally strong expression of CD117 and CD56, and no expression of CD14 and CD7. The tube was further divided into CD45 weakly positive / CD42b strongly positive, CD45 weakly positive / CD61 strongly positive, and CD45 weakly positive / CD110 strongly positive groups, with strong CD42b (grass green), strong CD61, and strong CD110 groups respectively. For clearer visualization, the clearly defined strong CD42b groups were displayed in color; strong CD61 and strong CD110 groups were not displayed. The strong CD42b cell population strongly expresses CD61 and CD110, but does not express CD56, CD34, CD117, CD7, or CD14. The FSC / SSC ratio is low and lacks a center of gravity, indicating normal platelet count. Myeloid blast cells were selected using small / CD34-positive and small / CD117-positive SSCs, respectively. For clarity, CD34 or CD117 gates were established, displaying either CD34-positive (dark green) or CD117-positive (bright blue and red) cell populations. The dark green and bright blue cells represent normal myeloid blast cells, without abnormal expression of CD7, CD56, or CD14. Since tumor cells express CD117, the red cell population within the CD34 or CD117 gates represents CD42b+ malignant immature megakaryocytes, strongly positive for CD56 and CD117, but without expression of CD7, CD14, or CD34. Abnormal cell gate 1 was established using weak CD45 expression / CD56 positivity, which overlapped with the red CD42b+ tumor cell gate. Abnormal cell gate 2 was established using weak CD45 expression / CD7 positivity; no abnormal cells with weak CD45 expression / CD7 positivity were observed in this sample.

[0108] Specifically, Figures 5-6 Immunophenotypic analysis of tube A and tube B of bone marrow samples from acute megakaryocyte leukemia with minimal residual disease who have lost megakaryocyte markers.

[0109] Specifically, Figure 5 Bone marrow samples from acute megakaryocytic leukemia with minimal residual disease (MRDL) showing loss of megakaryocyte markers were analyzed in tube A. The following settings were applied sequentially: ① FSC-A / H was set to P1 as the de-adhesion cell gate, resulting in single cells within P1; ② FSC / SSC was displayed within P1, and P2 was set to the viable cell gate, resulting in single viable cells within P2; ③ Within P2, CD45 / SSC was used to set the blood cell gate, resulting in gates for lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NEC), and eosinophils (eo); ④ Within P2, a CD42a+ gate was set using weakly positive CD45 / CD42a positive cells; no CD42a+ tumor cells were observed in this tube. A strong CD42a gate (grass green) was set for weakly positive CD45 / CD42a expression cells; this cell group had a small FSC / SSC ratio and lacked a centroid, indicating normal platelets. Myeloid primitive cell phyla were set up using small / CD34-positive and small / CD117-positive SSC cells respectively. For clearer display, CD34 or CD117 phyla were set up to jointly display CD34-positive (dark green) or CD117-positive cell populations with CD13 / CD33, CD13 / CD34, CD33 / CD34, CD34 / CD117, CD34 / HLA-DR, CD13 / HLA-DR, and CD33 / HLA-DR combinations. CD33-positive / HLA-DR-negative tumor cells were visible and set up as MRD1 phyla. This cell population expressed CD33 and CD117, but did not express CD34, HLA-DR, CD13, CD11b, or CD42a. These were malignant immature megakaryocytes that had lost CD42a. Dark green and bright blue cells are normal myeloid primitive naive cells, with normal expression patterns of CD13 / CD33, CD13 / CD34, CD33 / CD34, CD34 / CD117, CD34 / HLA-DR, CD13 / HLA-DR, and CD33 / HLA-DR.

[0110] Specifically, Figure 6Bone marrow samples showing minimal residual disease (MRD) in acute megakaryocytic leukemia with missing megakaryocyte markers were analyzed in tube B. The following settings were executed sequentially: ① FSC-A / H setting P1 as the de-adhesion cell gate, resulting in single cells within P1; ② FSC / SSC setting P2 as the live cell gate within P1, resulting in single live cells within P2; ③ Using CD45 / SSC to set the blood cell gate within P2, lymphocytes (lym), granulocytes (gra), and monocytes were obtained; ④ Primitive immature megakaryocytic cell gates were set within P2 using CD45 weak expression / CD42b positive, CD45 weak expression / CD61 positive, and CD45 weak expression / CD110 positive. Only CD61 showed partial weak positivity, but the intensity was insufficient to clearly establish the gate. Monocytes (brown cell cluster) and granulocytes (sky blue cell cluster) showed nonspecific expression, while CD42b and CD110 showed no positive cell clusters. The tube was designed with CD45 weakly positive / CD42b strongly positive, CD45 weakly positive / CD61 strongly positive, and CD45 weakly positive / CD110 strongly positive cell groups, respectively, with strong CD42b cells (grass green), strong CD61 cells, and strong CD110 cells displayed. For clarity, only the clearly defined strong CD42b cells were shown in color; strong CD61 and strong CD110 cells were not displayed. The strong CD42b cell population strongly expressed CD61 and CD110, but did not express CD56, CD34, CD117, CD7, or CD14. The FSC / SSC ratio was low and lacked a centroid, indicating normal platelets. An abnormal cell group AB1 (red cell population) was created using CD45 weakly expressed / CD56 positive. This cell group was strongly positive for CD56 and CD117, partially expressed weakly positive for CD61, and did not express CD7, CD34, CD14, CD42b, or CD110. These were malignant immature megakaryocytes lacking megakaryocyte markers. No cells showing weak CD45 expression or CD7 positivity were observed. Phylogenetic blast cells were established using small / CD34-positive and small / CD117-positive SSCs, respectively, revealing a red AB1 cell population. Dark green and royal blue cells represent normal myeloid blast cells, expressing CD117 and CD34, but not CD7, CD56, CD14, CD61, CD42b, or CD110.

[0111] Clinical validation was conducted using the method described in this embodiment: From January 1, 2020 to June 30, 2025, 952 patients with AMKL (Amyotrophic Lateral Sclerosis) who visited Hebei Yanda Lu Daopei Hospital were tested for minimal residual disease. Of these, 149 were positive and 803 were negative. Among the patients, 540 were male and 412 were female. Simultaneous validation using morphological, genetic, clinical, and follow-up methods confirmed the diagnostic accuracy of all cases. Using this method, the sensitivity, specificity, positive predictive value, and negative predictive value were all above 99%. Therefore, this invention can improve efficiency, save costs, and reduce the misdiagnosis rate.

Claims

1. A reagent composition for flow cytometry detection of minimal residual disease in acute megakaryocytic leukemia, characterized in that, The reagent composition comprises two groups of antibodies, wherein: The first group of antibodies consists of fluorescently labeled CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody, and CD45 antibody. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500. These antibodies are used to add to the first flow cytometer tube in which the sample to be tested is in a single-cell suspension state. The second group of antibodies consists of fluorescently labeled CD61, CD42b, CD34, CD117, CD110, CD14, CD7, CD45, and CD56 antibodies. The fluorescent labeling order of each antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, V500, and BV605. These antibodies are added to the second flow cytometer tube containing the test sample in a single-cell suspension.

2. The reagent composition according to claim 1, characterized in that, All antibodies are monoclonal antibodies.

3. The reagent composition according to claim 1 or 2, characterized in that: The first group of antibodies is a mixture of CD33 antibody, CD117 antibody, CD34 antibody, CD13 antibody, CD42a antibody, HLA-DR antibody, CD11b antibody and CD45 antibody in a volume ratio of 5:5:5:3:2:3:3:3; The second group of antibodies is a mixture of CD61 antibody, CD42b antibody, CD34 antibody, CD117 antibody, CD110 antibody, CD14 antibody, CD7 antibody, CD45 antibody and CD56 antibody in a volume ratio of 5:5:5:3:2:3:3:3:

3.

4. A kit for flow cytometry detection of minimal residual disease in acute megakaryocytic leukemia, characterized in that, The kit includes a first container and a second container, each container containing a first group of antibodies and a second group of antibodies of the reagent composition according to any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes one or more of the following: cell lysis buffer, buffer solution, and flow cytometry tubes for use with a flow cytometer.

6. The use of the reagent composition according to any one of claims 1-3 in the preparation of flow cytometry samples for detecting minimal residual disease in acute megakaryocytic leukemia.

7. The application according to claim 6, characterized in that, The process for preparing flow cytometry samples for detecting minimal residual disease in acute megakaryocytic leukemia includes the following steps: (1) Add the sample to be tested into flow cytometry tube one and flow cytometry tube two respectively to make it into a single cell suspension, and ensure that the cell quantity is 1×10 6 / tube-1×10 7 / Tube; (2) Add phosphate buffer to tube 1 and tube 2, mix well, incubate at 37°C, and centrifuge to remove the supernatant; (3) Add the first group of antibodies in the reagent composition of any one of claims 1-3 to tube one obtained in step (2), and add the second group of antibodies in the reagent composition of any one of claims 1-3 to tube two obtained in step (2). Incubate each flow cytometer at room temperature in the dark. (4) Add 1×hemolysin to tube 1 and tube 2 after incubation in step (3) respectively, and continue incubation at room temperature in the dark; (5) After centrifuging each flow cytometer tube incubated in step (4), remove the supernatant; (6) Add PBS buffer to tube 1 and tube 2 after removing the supernatant in step (5) to wash, centrifuge and remove the supernatant, resuspend the cells with PBS buffer to obtain the flow cytometry sample.

8. A device for detecting minimal residual disease in acute megakaryocytic leukemia, characterized in that, The device includes a detection unit and an analysis unit, wherein: The detection unit includes reagent materials for detecting samples from the individual to be tested by flow cytometry, and obtaining the test results of the samples; the reagent materials include the reagent composition according to any one of claims 1-3; The analysis unit is used to analyze the detection results of the detection unit.

9. The apparatus according to claim 8, characterized in that, This device is used to detect minimal residual disease in acute megakaryocytic leukemia, in which... The process of detecting samples from the individual being tested using flow cytometry includes: The sample to be tested is treated with the reagent composition according to any one of claims 1-3 to prepare a flow cytometry sample; Perform flow cytometry analysis; During flow cytometry analysis, each tube is sequentially configured with an anti-adhesion cell gate (P1) and a viable cell gate (P2) to obtain single viable cells. Within gate P2, CD45 / SSC is used to configure each blood cell gate, and: The gates in tube 1 are set up as follows: In the P2 gate, the immature megakaryocyte gate is set up using CD45 weak positive / CD42a positive, the platelet and mature megakaryocyte gates are set up using CD45 weak positive / CD42a strong positive, and the myeloid primitive cell gates are set up using SSC medium-small / CD34 positive and SSC medium-small / CD117 positive, respectively. Gate 2 is set up as follows: Within gate P2, immature megakaryocyte gates are set up using CD45 weak expression / CD61 positive, CD45 weak expression / CD42b positive, and CD45 weak expression / CD110 positive, respectively; platelet and mature megakaryocyte gates are set up using CD45 weak expression / CD110 strong positive, CD45 weak expression / CD61 strong positive, and CD45 weak expression / CD42b strong positive, respectively; myeloid blast cell gates are set up using SSC small / CD34 positive and SSC small / CD117 positive, respectively; abnormal cell gate 1 is set up using CD45 weak expression / CD56 positive; and abnormal cell gate 2 is set up using CD45 weak expression / CD7 positive.

10. The apparatus according to claim 8 or 9, characterized in that, When the analysis unit analyzes the detection results of the detection unit, it outputs the detection results according to one of the following judgment methods: Method 1: Minimal residual disease in typical acute megakaryocytic leukemia: a population of cells with weak CD45 expression / CD42a positivity and / or weak CD45 expression / CD61 positivity and / or weak CD45 expression / CD42b positivity and / or weak CD45 expression / CD110 positivity, and meeting the following six conditions: (1) centroid on the FSC / SSC plot, (2) FSC larger than lymphocytes and SSC not larger than lymphocytes, (3) CD14 not positive, (4) no CD11b positivity / CD45 high expression / HLA-DR positivity / CD33 high expression, (5) no high SSC, and the CD13 / CD11b two-dimensional dot plot combination expression pattern presents a normal granulocyte development pattern, (6) at least one of CD61, CD42a, CD42b and CD110 is not strongly expressed; Method 2: Malignant myeloid blast cells lacking megakaryocyte markers: CD34 positive and / or CD117 positive cells, and phenotypes exhibiting at least one of the following conditions: (1) abnormal expression of CD7, (2) abnormal expression of CD56, (3) abnormal expression of CD11b, (4) loss or weakened or enhanced expression of any of the markers CD13, CD33, HLA-DR, CD34, CD117, or CD45, (5) CD13 / CD33 two-dimensional dot plot, CD33 / HLA-DR two-dimensional dot plot, CD13 / HLA-DR two-dimensional dot plot, CD34 / HLA-DR two-dimensional dot plot, and CD34 / CD117 two-dimensional dot plot show expression patterns different from those of normal myeloid blast cells; Method 3: Malignant myeloid blast cells that have lost megakaryocyte markers and do not express primitive blast cell markers: CD45 weakly expressed / CD56 positive and / or CD45 weakly expressed / CD7 positive cells, and expressing any one of CD13, CD33, HLA-DR, or CD11b.

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