Antibody combination and system for detecting leukemia stem cells

Abstract

The invention discloses an antibody combination and a system for detecting leukemia stem cells. The antibody combination comprises a CD7 antibody, a CD371 antibody, a CD34 antibody, a CD45RA antibody, a CD123 antibody, a CD133 antibody, a CD38 antibody, a CD45 antibody, a CD90 antibody, a CD33 antibody and a CD19 antibody. The antibody combination provided by the invention can accurately identify target cells and distinguish normal HSCs and pluripotent progenitor cells in a CD34 + CD38-cell population, and the specific antibody has abnormal expression or expression intensity change, so that leukemia stem cells can be rapidly, simply and conveniently detected with high accuracy, high sensitivity and high specificity, the sensitivity of MRD evaluation is favorably improved, and the kit has a good application prospect. Therefore, signs of disease recurrence can be found earlier, more information about disease prognosis can be provided, and doctors and patients can be helped to better understand disease prognosis and possible results.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease detection, and more specifically, relates to an antibody combination and system for detecting leukemia stem cells. Background Art

[0002] Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the clonal proliferation of primitive myeloid cells in the bone marrow. These cells are blocked from maturing and differentiation, leading to the accumulation of immature cells in the bone marrow, peripheral blood, and extramedullary tissues. The clinical manifestations of AML vary, with common symptoms including fatigue, pallor, easy bruising and bleeding, fever, and infection. Diagnosis is typically confirmed through blood and bone marrow tests. Long-term survival in AML is poor, and treatment failure in AML may be due to the presence of leukemia stem cells (LSCs) at diagnosis and their persistence after treatment.

[0003] Leukemia stem cells are a type of leukemia cell that has the ability to self-renew and generate a heterogeneous population of leukemia cells. They are one of the most classic tumor stem cells (TSCs) and were first successfully isolated in the 1990s. Leukemia stem cells play a key role in the occurrence, development, treatment resistance, and relapse of leukemia. They have the ability to self-renew for a long time and can produce more LSCs or differentiate into various mature leukemia cells through asymmetric division. In addition, LSCs have the ability to migrate and home, and can survive and proliferate in specific microenvironments such as the bone marrow.

[0004] Currently, methods for detecting residual tumor cells include cell morphology, multiparameter flow cytometry, karyotyping, and molecular genetic testing. Cytomorphology analysis is not very sensitive, and the number of tumor cells may decrease after treatment, limiting its application. Karyotyping is time-consuming. While molecular methods are highly sensitive, they are not suitable for cases with normal karyotypes. In contrast, multiparameter flow cytometry is widely considered an effective means of detecting minimal residual disease due to its rapidity and simplicity. This method primarily detects leukemia-associated immunophenotype (LAIP) or "different from normal expression pattern" (DFN). However, the LAIP method carries the risk of missed diagnoses, while the DFN method requires a high level of analyst experience and expertise. The frequency of these minimal or measurable residual disease (MRD) cells is an important risk factor for post-remission AML. Measurable residual disease (MRD) measured by multiparameter flow cytometry (MFC) covers over 90% of AML cases and has become an important variable for predicting relapse and guiding treatment selection. However, a significant number (20-70%) of patients with low / negative MRD levels will relapse, providing a limitation of providing false-negative results.Despite extensive research, comprehensive characterization of leukemic stem cells (LSCs) and information on their immunophenotypic differences from normal hematopoietic stem cells (HSCs) is lacking.

[0005] Therefore, there is an urgent need in clinical practice for a sensitive, rapid, and simple detection technology to detect leukemia stem cells in a timely manner. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a method for sensitive, rapid and simple detection of leukemia stem cells.

[0007] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0008] In a first aspect of the present invention, an antibody combination for detecting leukemia stem cells is provided, comprising CD7 antibody, CD371 antibody, CD34 antibody, CD45RA antibody, CD123 antibody, CD133 antibody, CD38 antibody, CD45 antibody, CD90 antibody, CD33 antibody and CD19 antibody.

[0009] In a second aspect, the present invention provides a kit for detecting leukemia stem cells, wherein the kit comprises the above-mentioned antibody combination for detecting leukemia stem cells.

[0010] A third aspect of the present invention provides a system for detecting leukemia stem cells, comprising:

[0011] A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected;

[0012] A data acquisition module, which acquires data of flow cytometry detection results;

[0013] The data analysis module analyzes the acquired data and determines whether the acquired cells are leukemia stem cells based on predetermined analysis logic and judgment criteria.

[0014] In the present invention, after a large number of experiments, the inventors found that the antibody combination consisting of CD34 antibody + CD38 antibody + CD45 antibody (gating antibody), CD33 antibody + CD123 antibody + CD371 antibody + CD133 antibody (myeloid antibody), CD7 antibody + CD19 antibody (lymphoid antibody), and CD90 antibody + CD45RA can accurately identify target cells and distinguish normal HSCs and multipotent progenitor cells in the CD34+CD38- cell population. In addition, specific antibodies show abnormal expression or changes in expression intensity. Therefore, leukemia stem cells can be detected quickly, easily, accurately, sensitively and specifically, which helps to increase the sensitivity of MRD assessment, thereby detecting signs of disease recurrence earlier, providing more information about disease prognosis, helping doctors and patients better understand the disease process and possible outcomes, and promoting the development of anti-leukemia drugs targeting LSCs, providing the possibility of developing new treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The antibody combination used in Example 1 of the present invention was used to detect the expression of the immunophenotype CD38-SSC of HSCs cells.

[0016] Figure 2 The antibody combination used in Example 1 of the present invention was used to detect the expression of the immunophenotype CD34-CD38 of HSCs cells.

[0017] Figure 3 The antibody combination used in Example 1 of the present invention was used to detect the expression of the immunophenotype CD45RA-CD90, CD7-CD19, CD371-CD33, and CD133-CD123 of HSCs cells.

[0018] Figure 4 This is the antibody combination used in Example 2 of the present invention to detect the expression of the immunophenotype CD38-SSC of LSCs cells.

[0019] Figure 5 This is the antibody combination used in Example 2 of the present invention to detect the expression of the immunophenotype CD34-CD38 of LSCs cells.

[0020] Figure 6The antibody combination used in Example 2 of the present invention was used to detect the expression of the immunophenotype CD45RA-CD90, CD7-CD19, CD371-CD33, and CD133-CD123 of LSCs cells.

[0021] Figure 7 The antibody combination used in Example 2 of the present invention was used to detect the expression of the immunophenotype CD38-SSC of HSCs and LSCs.

[0022] Figure 8 The antibody combination used in Example 2 of the present invention was used to detect the expression of CD34-CD38, the immunophenotype of HSCs and LSCs.

[0023] Figure 9 The antibody combination used in Example 2 of the present invention was used to detect the expression of the immunophenotypes CD45RA-CD90, CD7-CD19, CD133-CD123, and CD371-CD33 of HSCs and LSCs.

[0024] Figure 10 The antibody combination of Comparative Example 1 was used in Comparative Example 1 to detect the expression of the immunophenotypes CD45-SSC, CD34-SSC, and CD34-CD38 of normal healthy human samples.

[0025] Figure 11 In Comparative Example 1, the antibody combination of Example 1 was used to detect the expression of the immunophenotypes CD34-SSC, CD34-CD38, CD33-CD133, CD7-CD19, CD371-CD123, and CD45RA-CD90 in normal healthy human samples.

[0026] Figure 12 The antibody combination of Comparative Example 2 was used to detect the expression of the immunophenotype CD34-SS INT, CD34-CD38, CD33-CD133, and CD371-CD123 of AML cells.

[0027] Figure 13 In Comparative Example 2, the antibody combination of Example 1 was used to detect the expression of the immunophenotype CD34-SS INT, CD34-CD38, CD33-CD133, CD19-CD7, CD45RA-CD90, and CD371-CD123 of AML cells.

[0028] Figure 14 The antibody combination of Comparative Example 3 was used to detect the expression of the immunophenotypes CD45-SSC, CD34-SSC, CD34-CD38, and CD7-CD19 of AML cells.

[0029] Figure 15 In Comparative Example 3, the antibody combination of Example 1 was used to detect the expression of the immunophenotypes CD45-SSC, CD34-SSC, CD34-CD38, CD7-CD19, CD133-CD123, CD45RA-CD90, and CD371-CD33 of AML cells.

[0030] Figure 16 The antibody combination of Comparative Example 4 was used to detect the expression of the immunophenotype CD45-SSC, CD33-CD133, CD19-CD7, CD371-CD123, CD45RA-CD90, and CD45-CD38 of AML cells.

[0031] Figure 17 In Comparative Example 4, the antibody combination of Example 1 was used to detect the expression of the immunophenotypes CD45-SSC, CD34-SSC, CD33-CD133, CD19-CD7, CD371-CD123, CD45RA-CD90, and CD34-CD38 of AML cells.

[0032] Figure 18 In Comparative Example 5, the antibody combination of Comparative Example 5 was used to detect the expression of the immunophenotype CD45-SSC, CD34-SSC, CD33-CD133, CD19-CD7, CD371-CD123, and CD45RA-CD90 of AML cells.

[0033] Figure 19 In Comparative Example 5, the antibody combination of Example 1 was used to detect the expression of the immunophenotype CD34-CD38 of AML cells.

[0034] Figure 20 In Comparative Example 6, the antibody combination of Comparative Example 6 was used to detect the expression of the immunophenotypes CD45-SSC, CD34-SSC, CD34-CD38, CD33-CD133, and CD19-CD7 of AML cells.

[0035] Figure 21 In Comparative Example 6, the antibody combination of Example 1 was used to detect the expression of the immunophenotype CD45-SSC, CD34-SSC, CD34-CD38, CD33-CD133, CD7-CD19, CD45RA-CD90, and CD371-CD123 of AML cells.

[0036] Figure 22 The antibody combination of Comparative Example 7 was used to detect the expression of the immunophenotype CD34-SSC, CD34-CD38, CD45RA-CD90, CD19-CD7, and CD371-CD123 of AML cells.

[0037] Figure 23 In Comparative Example 7, the antibody combination of Example 1 was used to detect the expression of the immunophenotypes CD34-SSC, CD34-CD38, CD45RA-CD90, CD19-CD7, CD371-CD123, and CD33-CD133 of AML cells.

[0038] Figure 24 The antibody combination of Comparative Example 8 was used to detect the expression of the immunophenotype CD34-SS INT, CD34-CD38, CD33-CD133, CD45RA-CD90, and CD371-CD123 of AML cells.

[0039] Figure 25 In Comparative Example 8, the antibody combination of Example 1 was used to detect the expression of the immunophenotype CD34-SS INT, CD34-CD38, CD33-CD133, CD19-CD7, CD45RA-CD90, and CD371-CD123 of AML cells.

[0040] Figure 26 In Comparative Example 9, the antibody combination of Comparative Example 9 was used to detect the expression of the immunophenotypes CD34-CD38, CD123-CD38, CD45RA-CD38, CD33-CD38, CD44-CD38, and COCKTAIL-CD38 in normal healthy human samples. DETAILED DESCRIPTION

[0041] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or the conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0044] In some embodiments of the present invention, an antibody combination for detecting leukemia stem cells is disclosed, comprising CD7 antibody, CD371 antibody, CD34 antibody, CD45RA antibody, CD123 antibody, CD133 antibody, CD38 antibody, CD45 antibody, CD90 antibody, CD33 antibody and CD19 antibody.

[0045] In some embodiments, the antibodies of the antibody combination are monoclonal antibodies.

[0046] In some embodiments, the monoclonal antibody is a fluorescein-labeled antibody, and the fluorescein is selected from the group consisting of FITC, PE, PC5.5, PC7, APC, BV421, efluor 450, BV510, BV605, BV750 and BV786.

[0047] In some embodiments, the CD7 antibody is labeled with fluorescein FITC, the CD371 antibody is labeled with fluorescein PE, the CD34 antibody is labeled with fluorescein PC5.5, the CD45RA antibody is labeled with fluorescein PC7, the CD123 antibody is labeled with fluorescein APC, the CD133 antibody is labeled with fluorescein BV421, the CD38 antibody is labeled with fluorescein efluor 450, the CD45 antibody is labeled with fluorescein BV510, the CD90 antibody is labeled with fluorescein BV605, the CD33 antibody is labeled with fluorescein BV750, and the CD19 antibody is labeled with fluorescein BV786.

[0048] In other embodiments of the present invention, a kit for detecting leukemia stem cells is disclosed, wherein the kit comprises the above-mentioned antibody combination for detecting leukemia stem cells.

[0049] In other embodiments of the present invention, the use of an antibody combination or a kit for detecting leukemia stem cells in assisting the detection of residual tumor cells is disclosed.

[0050] In other embodiments of the present invention, a system for detecting leukemia stem cells is disclosed, comprising:

[0051] A detection module, wherein the detection module performs flow cytometry detection on the cells to be detected;

[0052] A data acquisition module, which acquires data of flow cytometry detection results;

[0053] The data analysis module analyzes the acquired data and determines whether the acquired cells are leukemia stem cells based on predetermined analysis logic and judgment criteria.

[0054] In some embodiments, the judgment criteria include: if the antibody expression pattern of the test cell falls within the antibody expression pattern template of the normal control population, the test cell is judged to be a normal hematopoietic stem cell; if the antibody expression pattern of the test cell does not fall within the antibody expression pattern template of the normal control population, the test cell is judged to be a suspected leukemia stem cell.

[0055] In some embodiments, the antibody expression pattern of the cells to be tested and the antibody expression pattern of the normal control population are established by the following steps: the data of the flow cytometry test results are gated to circle out the mononuclear cells, CD34-CD38 is selected under the mononuclear cell gate, CD34+CD38- cells are circled, and then the expression of each fluorescent antibody pair is analyzed.

[0056] In some embodiments, the fluorescent antibody pairs include: CD90-CD45RA, CD19-CD7, CD33-CD371, CD123-CD133.

[0057] In the following examples of the present invention, the antibodies used include: CD7 FITC, catalog number A07755, purchased from Beckman Coulter; CD371 PE, catalog number 353604, purchased from biolegend; CD34 PC5.5, catalog number 343522, purchased from biolegend; CD45RA PC7, catalog number 25-0458-42, purchased from eBioscience; CD123 APC, catalog number 560087, purchased from BDP; CD133 BV421, catalog number 566595, purchased from BD; CD38 efluor 450, catalog number 48-0388-42, purchased from eBioscience; CD45BV510, catalog number 563204, purchased from BDP; CD90BV605, catalog number 328128, purchased from biolegend; CD33BV750, catalog number 746985, purchased from BDP; CD19BV786, catalog number 563325, purchased from BD.

[0058] In the present invention, the dosage of each monoclonal antibody in the antibody combination can be adjusted according to different manufacturers, different titers, different instruments, etc.

[0059] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] Example 1 Antibody Combination and Method for Detecting Leukemia Stem Cells

[0061] This example provides an antibody combination for detecting leukemia stem cells, comprising the following antibodies: CD7, CD371, CD34, CD45RA, CD123, CD133, CD38, CD45, CD90, CD33, and CD19. Each monoclonal antibody was fluorescently labeled. The type of fluorescent label and the volume used for each monoclonal antibody are shown in Table 1.

[0062] Table 1

[0063] Fluorescent labeling Monoclonal antibodies volume FITC CD7 5μL PE CD371 0.625μL PC5.5 CD34 0.625μL PC7 CD45RA 2.5 μL APC CD123 2.5 μL BV421 CD133 0.625μL efluor450 CD38 2.5 μL BV510 CD45 1 μL BV605 CD90 2.5 μL BV750 CD33 2.5 μL BV786 CD19 1.25 μL

[0064] This embodiment also provides a method for monitoring leukemia stem cells using the above antibody combination, comprising the following steps:

[0065] 1. Preparation of cell suspension

[0066] Will contain 10*10 6 Transfer the bone marrow sample (volume less than 2 mL) containing nucleated cells to a 50 mL centrifuge tube and add lysis buffer to the 50 mL mark. Dissolve the sample at room temperature for 10 minutes, centrifuge at 500 g for 5 minutes, discard the supernatant, wash the sample once with 50 mL of calf serum to remove the remaining lysis buffer, and adjust the concentration to (1-5) × 10 6 cells / mL.

[0067] 2. Sample processing

[0068] Remove the flow cytometry tube, add the antibody combination, then add 100 μL of the cell suspension, vortex to mix, and incubate at room temperature in the dark for 15 minutes. Add 2 mL of calf serum, vortex, and centrifuge at 500 g for 5 minutes. Discard the supernatant. Resuspend in 400 μL of 1% paraformaldehyde.

[0069] 3. Testing and analysis

[0070] Flow cytometry tubes were tested and data collected using an NL-CLC spectral flow cytometer. SpectroFlo software was used for data acquisition. After completing the standard startup checklist, the instrument was set up using the standard setup procedure, monitoring laser power, laser current, sheath pressure, and other parameters. An instrument setup report was generated. Samples could only be tested after the standard setup was passed. After instrument quality control, the prepared samples were placed on the sample tray for testing. The FSC vs. SSC dot plot was observed, and the threshold was adjusted to exclude debris. The voltage was adjusted to center the cell population (avoiding signal saturation or low signal levels). Compensation was set to eliminate fluorescence spillover. Single-color control tubes were used to adjust compensation and minimize interference between fluorescent channels. When setting compensation, it was important to ensure that the single-color control tube contained only one fluorescently labeled antibody, which allowed for more accurate adjustment of compensation parameters. ≥1,000,000 total events were acquired, the raw FCS files were saved, and the immunophenotypes were analyzed using Kaluza software.

[0071] Specifically, antibody expression pattern analysis was performed according to the following steps:

[0072] (1) Use CD38-SSC (side scattered light) to set the gate. According to the expression of CD38-SSC, mononuclear cells (MNCs) are circled to exclude granulocytes for better gate setting in the future.

[0073] (2) Select CD34-CD38 under the mononuclear cell (MNC) gate to divide the lineage cells into four areas. The CD34+CD38- area in the lower right corner of the cross gate is the target cell population.

[0074] (3) Under the CD34+CD38- gate, the expression of CD90-CD45RA, CD19-CD7, CD33-CD371, and CD123-CD133 were analyzed in turn.

[0075] (4) If the antibody expression pattern of the cells to be tested falls within the normal control population antibody expression pattern template, the cells to be tested are determined to be HSCs; if the antibody expression pattern of the cells to be tested does not fall within the normal control population antibody expression pattern template, the cells to be tested are determined to be suspected LSCs.

[0076] Among them, the results of the antibody expression pattern template of the normal control population (i.e., bone marrow samples of healthy people with only HSCs cell populations) are as follows Figures 1 to 3 As shown, mononuclear cells (MNCs) are located in Figure 1 In the lower area, the target cell population CD34+CD38- is the orange cell population ( Figure 2 ), the immunophenotype of normal hematopoietic stem cells (HSCs) is usually CD90+, CD45RA-, CD19-, CD7-, CD371-, CD33+, CD133+, CD123-( Figure 3 ).

[0077] Example 2 Practical application of the detection method of the present invention

[0078] In this example, the method of Example 1 was used to test two bone marrow samples (bone marrow containing only LSCs cell population and bone marrow containing both HSCs and LSCs cell populations) using the same method as in Example 1.

[0079] The results of bone marrow samples with only LSCs population are as follows Figures 4 to 6 As shown, mononuclear cells (MNCs) are located in Figure 4 In the lower area, the target cell population CD34+CD38- is the orange cell population ( Figure 5 ), the immunophenotype was CD90-, CD45RA+, CD371+, CD33+, CD123+, CD133+, CD7-, CD19-( Figure 6 Therefore, combined with the abnormal positive expression of CD45RA, CD371, and CD123 in the immunophenotype, and the enhanced fluorescence intensity of CD33 and CD133, it suggests that this group of cells in the sample to be tested may be LSCs tumor cells.

[0080] The results of bone marrow samples with both HSCs and LSCs are as follows Figures 7 to 9 As shown, mononuclear cells (MNCs) are located in Figure 7 In the lower area, the target cell population CD34+CD38- contains both normal HSCs cells (pink cell population) and abnormal LSCs cells (orange cell population) ( Figure 8 The immunophenotype of HSCs is CD90+, CD33+, CD45RA-, CD123-, CD133+, CD7-, CD19-, and CD371-. The immunophenotype of LSCs is CD90-, CD33++, CD45RA+, CD123+, CD371+, weak expression of CD133, CD7-, and CD19-( Figure 9 ).

[0081] Example 3 Precision and Sensitivity of the Method for Monitoring Leukemia Stem Cells of the Present Invention

[0082] 1. Intra-batch precision

[0083] Five clinically diagnosed positive bone marrow samples were selected and tested under stable conditions according to the method of Example 1. The determination was repeated three times, and the SD and CV of the results were analyzed. The results are shown in Table 2.

[0084] Table 2

[0085]

[0086] From the results in Table 2, we can see that the intra-batch precision %CV of the above 5 samples is less than 3%.

[0087] 2. Inter-batch precision

[0088] Two clinically positive bone marrow samples were selected and tested under stable conditions according to the method of Example 1. Three replicates were performed, with an interval of at least 3 hours between each batch. The SD and CV of each sample were analyzed. The results are shown in Table 3.

[0089] Table 3

[0090]

[0091]

[0092] From the results in Table 3, it can be seen that the inter-batch precision %CV of the above two samples is less than 5%.

[0093] 3. Sensitivity

[0094] One clinically diagnosed positive bone marrow sample and one clinically diagnosed negative bone marrow sample were diluted (1:1, 1:9, 1:99, 1:999, 1:9999, 1:99999) and tested under stable conditions according to the method of Example 1, with three repeated measurements. The negative sample was selected as a control. The results showed that R 2 The correlation was >0.995, indicating good correlation and acceptable results. The analytical sensitivity was 0.01%.

[0095] Comparative Example 1

[0096] The antibody combination provided in this comparative example includes: CD34 (fluorescently labeled PC5.5, 0.625 μL), CD38 (fluorescently labeled efluor 450, 2.5 μL) and CD45 (fluorescently labeled BV510, 1 μL). Normal healthy human samples were tested.

[0097] After removing debris and adhesions, circle the CD34+ cells and display the expression of CD38. The results are as follows Figure 10 As shown. Figure 10 As can be seen, the CD34+ cell count is 0.98%, a relatively low proportion. CD34+CD38+ cells account for 85.01% of the CD34+ cells, and CD34+CD38- cells account for 14.9% of the CD34+ cells. Therefore, when the antibody panel only contains CD34, CD38, and CD45, and the CD34+ count is low, it is impossible to determine which lineage these CD34+ cells are from or whether they are abnormal.

[0098] The same normal healthy human sample was tested using the antibody combination of Example 1 and the method of Example 1. The results are as follows: Figure 11 As shown. Figure 11 As can be seen, the myeloid markers CD33 and CD133, as well as the lymphoid markers CD7 and CD19, can separate CD34+ cells into pink and red cells. The pink cells have an immunophenotype of CD34+CD38+CD19+CD7-CD33-CD133-, suggesting a B progenitor origin. The red cells have an immunophenotype of CD34+CD38+dimCD19-CD7-CD33+CD133+, suggesting a myeloid progenitor origin. Because the target cells are myeloid progenitors, interference from B progenitors must be eliminated during analysis. After removing the interference from B progenitors and combining the immunophenotype of CD371-CD123-CD90+CD45RA-, these myeloid progenitors are considered normal, early CD34+ myeloid precursors.

[0099] Comparative Example 2

[0100] The antibody combination provided in this comparative example includes: CD34, CD38, CD45, CD33, CD123, CD133 and CD371, and the fluorescent labeling and volume are the same as those in Example 1.

[0101] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 12 As shown. Figure 12 The results showed that the proportion of CD34+ cells was 5.86%, which was slightly higher. They expressed myeloid markers CD133 and CD33, but no abnormal expression of CD371 and CD123 was found, and CD38 was not lost. The current immunophenotype cannot support the judgment of whether this group of myeloid primitive cells is the source of tumor cells.

[0102] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results are as follows: Figure 13 As shown. Figure 13 It can be seen that among the CD34+ cells, there is a group of CD19+CD7-CD33-CD133-CD38++ cells, which are derived from normal B progenitors (blue cell group). Excluding the interference of B progenitors, the remaining CD34+ cells express CD7 and a small amount of CD19. Since CD7 and CD19 are both lymphocyte markers, the presence of cross-lineage expression clearly excludes the possibility that the remaining CD34+ cells after interference with B progenitors are leukemic cells.

[0103] Comparative Example 3

[0104] The antibody combination provided in this comparative example includes: CD34, CD38, CD45, CD7 and CD19, and the fluorescent labeling and volume are the same as those in Example 1.

[0105] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 14 As shown. Figure 14 The results showed 48.64% of CD34+ cells, with an immunophenotype of CD34+CD38+dimCD19-CD7-, which can be considered as acute leukemia. However, the current immunophenotype is not sufficient to determine the tumor's origin, and subsequent MRD testing does not have a good screening indicator.

[0106] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results are as follows: Figure 15 As shown. Figure 15 It can be seen that the immunophenotype of this group of tumor cells is CD123+CD133+CD45RA+CD90-CD33+CD371+, which can be diagnosed as acute myeloid leukemia. Subsequent clinical treatment can select relevant target therapy and post-treatment testing, especially the detection of CD34+CD38- cells, which are considered to be the most resistant.

[0107] Comparative Example 4

[0108] The antibody combination provided in this comparative example includes: CD38, CD45, CD90, CD45RA, CD33, CD123, CD133, CD371, CD7 and CD19, and the fluorescent labeling and volume are the same as those in Example 1.

[0109] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 16 As shown. Figure 16 The results showed that when CD34 was lacking, CD45 was selected to group the cells. The immunophenotype analysis of CD45dim cells showed that CD7, CD19, and CD45RA were expressed in small amounts.

[0110] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results are as follows: Figure 17 As shown. Figure 17 It can be seen that CD7, CD19, and CD45RA are not expressed.

[0111] Therefore, when gating on CD45-ss, some granulocytes and lymphocytes cannot be separated from the target cells, resulting in impure target cells, which can easily lead to misinterpretation of the results and affect the reporting of the target cell ratio.

[0112] Comparative Example 5

[0113] The antibody combination provided in this comparative example includes: CD34, CD45, CD90, CD45RA, CD33, CD123, CD133, CD371, CD7 and CD19, and the fluorescent labeling and volume are the same as those in Example 1.

[0114] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 18 As shown. Figure 18 The results show that when CD38 is lacking, it is impossible to distinguish normal from abnormal cells based on the immunophenotype CD34+CD33+CD133+CD90-CD45RA-CD123-CD371-CD7-CD19-, because CD133+CD33+ is also expressed in normal myeloid primitive cells.

[0115] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The immunophenotype of the target cells was as follows: Figure 19 As shown, from Figure 19 It can be clearly seen that CD38 expression is lost. CD38 is highly expressed in normal hematopoietic stem cells, but is often lowly expressed in leukemia cells, especially AML transformed by myelodysplastic syndrome.

[0116] Therefore, CD38 loss of expression helps to identify abnormal leukemia cells and thus improve the accuracy of diagnosis.

[0117] Comparative Example 6

[0118] The antibody combination provided in this comparative example includes: CD34, CD38, CD45, CD33, CD133, CD7 and CD19, and the fluorescent labeling and volume are the same as those in Example 1.

[0119] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 20 As shown. Figure 20 The results showed that the immunophenotype of CD34+CD38- cells was CD33+CD133+CD7-CD19-, which confirmed that they were derived from myeloid primitive cells. The current combination of markers alone could not distinguish LSCs from HSCs.

[0120] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results are as follows: Figure 21 As shown. Figure 21As can be seen, when CD90, CD45RA, CD123, and CD371 are added to the antibody panel, LSCs (pink cell population) and HSCs (orange cell population) can be identified. The immunophenotype of the pink cell population is strongly CD33 positive, abnormally positive expression of CD371 and CD123, positive expression of CD45RA, and absent expression of CD90, suggesting that they are LSCs tumor cells. The immunophenotype of the orange cell population is positive for CD33, negative for CD371 and CD123, negative for CD45RA, and positive for CD90, suggesting that they are normal HSCs.

[0121] Comparative Example 7

[0122] The antibody combination provided in this comparative example includes: CD34, CD38, CD45, CD90, CD45RA, CD123, CD371, CD7 and CD19, and the fluorescent labeling and volume are the same as those in Example 1.

[0123] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 22 As shown. Figure 22 The results showed that 3.58% of CD34+ cells were accompanied by severe loss of CD38 expression. This group of cells were the target cells. No expression of CD19 and CD7 was found. Taking all factors into consideration, the CD34+CD38-CD19-CD7- cells may be myeloid tumor cells, but a conclusion cannot be drawn based on this phenotypic combination alone.

[0124] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results showed that when CD133 and CD33 were added to the antibody combination, the tumor cells expressed CD33 and CD133 ( Figure 23 ), so there is sufficient evidence to determine that this group of cells are myeloid tumor cells.

[0125] Comparative Example 8

[0126] The antibody combination provided in this comparative example includes: CD34, CD38, CD45, CD90, CD45RA, CD33, CD123, CD133, and CD371. The fluorescent labeling and volume are the same as those in Example 1.

[0127] The antibody combination of this comparative example was used to detect clinically diagnosed AML samples according to the method of Example 1. The results are as follows: Figure 24 As shown. Figure 24The results show that the immunophenotype is CD34+CD38+CD33+CD133+CD45RA-CD90-CD371-CD123-. The current immunophenotype is not sufficient to diagnose whether CD34+ cells are tumor cell origin. There may be doubts about the judgment of the results. At the same time, there may also be interference from normal B progenitor cells, affecting the proportion of the final results.

[0128] The same AML sample was tested using the antibody combination of Example 1 and the method of Example 1. The results showed that after adding CD7 and CD19, a group of CD19+CD7-CD34+CD38++CD33-CD133-CD90-CD45RA-CD90-CD123-CD371- cells (blue, Figure 25 ), considered to be derived from normal B progenitor cells, the remaining cell population (red) is CD34+CD38+CD33+CD133+CD7+CD19+a small amount of CD45RA-CD90-CD371-CD123-. Since CD7 is a T cell marker and CD19 is a B cell marker, myeloid cells have cross-lineage expression, and there is sufficient evidence to support that this group of cells is derived from tumor cells.

[0129] Comparative Example 9

[0130] The antibody combination provided in this comparative example (from existing literature) includes CD45RA, CLL 1, TIM 3, CD7, CD11b, CD22, CD123, CD33, CD38, CD44, CD34, CD45, and CD56, using a labeled mixture cocktail (CLL-1 / TIM-3 / CD7 / CD11b / CD22 / CD56).

[0131] The antibody combination of this comparative example was used to test a bone marrow sample from a healthy person, and the results were as follows: Figure 26 As shown. Figure 26The results revealed a population of CD34+CD38- cells with the following immunophenotypic expression: CD123-, CD45RA-, CD33+, CD44+, and Cocktail+. These abnormalities could lead inexperienced individuals to mistakenly identify these cells as suspected tumor cells. Clinically, this population of cells is considered to represent interference from mature granulocytes, as Cocktail includes CD11b, and CD11b expression gradually increases during neutrophil maturation. Normal B progenitor cells also express CD22, and CLL-1 and CD7 may also be expressed at trace levels on normal cells. If these cells are present, the positive signal from the cocktail can interfere with the interpretation of the results. Furthermore, in the case of a positive specimen, the positive signal from the cocktail makes it difficult to identify the specific CD molecule responsible, complicating subsequent target drug selection and testing.

[0132] Therefore, when using this comparative antibody combination to detect LSCs, it is impossible to determine which marker the mixture is positive for, making it difficult to screen for subsequent monitoring markers and select target therapies. Furthermore, when the mixture is positive for a marker, it is impossible to accurately determine whether it is a false positive due to tumor cell expression or interference from other cells.

[0133] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0134] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An antibody combination for detecting leukemia stem cells, characterized in that: Including CD7 antibody, CD371 antibody, CD34 antibody, CD45RA antibody, CD123 antibody, CD133 antibody, CD38 antibody, CD45 antibody, CD90 antibody, CD33 antibody and CD19 antibody.

2. The antibody combination for detecting leukemia stem cells according to claim 1, characterized in that: The antibodies of the antibody combination are monoclonal antibodies.

3. The antibody combination for detecting leukemia stem cells according to claim 2, characterized in that: The monoclonal antibody is a fluorescein-labeled antibody, and the fluorescein is selected from the group consisting of FITC, PE, PC5.5, PC7, APC, BV421, efluor 450, BV510, BV605, BV750 and BV786.

4. The antibody combination for detecting leukemia stem cells according to claim 3, characterized in that: The CD7 antibody is labeled with fluorescein FITC, the CD371 antibody is labeled with fluorescein PE, the CD34 antibody is labeled with fluorescein PC5.5, the CD45RA antibody is labeled with fluorescein PC7, the CD123 antibody is labeled with fluorescein APC, the CD133 antibody is labeled with fluorescein BV421, the CD38 antibody is labeled with fluorescein efluor 450, the CD45 antibody is labeled with fluorescein BV510, the CD90 antibody is labeled with fluorescein BV605, the CD33 antibody is labeled with fluorescein BV750, and the CD19 antibody is labeled with fluorescein BV786.

5. A kit for detecting leukemia stem cells, characterized in that: The kit comprises the antibody combination for detecting leukemia stem cells according to any one of claims 1 to 4.

6. Use of the antibody combination for detecting leukemia stem cells according to any one of claims 1 to 4 or the kit according to claim 5 in assisting the detection of residual tumor cells.

7. A system for detecting leukemia stem cells, characterized in that: include: A detection module, wherein the detection module performs 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 and determines whether the acquired cells are leukemia stem cells based on predetermined analysis logic and judgment criteria.

8. The system for detecting leukemia stem cells according to claim 7, characterized in that: The judgment criteria include: If the antibody expression pattern of the cells to be tested falls within the expression pattern template of antibodies of the normal control population, the cells to be tested are determined to be normal hematopoietic stem cells; If the antibody expression pattern of the cells to be tested does not fall within the antibody expression pattern template of the normal control population, the cells to be tested are determined to be suspected leukemia stem cells.

9. The system for detecting leukemia stem cells according to claim 8, characterized in that: The antibody expression pattern of the cells to be tested and the antibody expression pattern of the normal control population are established by the following steps: using the data of the flow cytometry test results, circling the mononuclear cells by setting a gate, selecting CD34-CD38 under the mononuclear cell gate, circling the CD34+CD38- cells, and then analyzing the expression of each fluorescent antibody pair.

10. The system for detecting leukemia stem cells according to claim 9, characterized in that: The fluorescent antibody pairs include: CD90-CD45RA, CD19-CD7, CD33-CD371, and CD123-CD133.

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