Antibody composition and method for identifying chronic granulocyte immune typing by using flow cytometry

Abstract

The invention discloses a method for identifying chronic granulocyte immune typing by using a flow cytometer. The method comprises the following steps: incubating and purifying a to-be-detected sample and a fluorescein-labeled antibody composition; the method comprises the following steps: carrying out flow cytometry detection on resuspended cells: firstly, circling a liquid flow stable interval by utilizing Time / CD45, FSC INT / PEAK and FSC INT / SSC INT scatter diagrams, and removing sticky bodies and cell debris; all leukocyte groups are circled through combination of CD45 and SSC, and mononuclear cells are circled in all leukocyte gates through CD45 / SSC gate setting; excluding CD 19 + B lymphocytes; cD7 + T and NK cells are excluded; cD2 + T and NK cells are eliminated; immature and mature granulocytes of CD 16 and CD24 < + > are excluded through CD 16 / CD24 gate setting; the method comprises the following steps of: screening CD14-CD16, removing basophilic granulocytes of CD14-CD16-and residual NK (Natural Killer) cells, dividing a cross gate to determine the proportion of CD 14 + CD16-cMo cells, CD 14 + CD16 + iMo cells and CD 16 + CD 14 ncMo cells, and determining the proportion of the CD14 + CD16-cMo cells, the CD14 + CD16 + iMo cells and the CD16 + CD14 ncMo cells; cD34 / SSC gate setting is carried out, and CD34 + original cells are circled to calculate the proportion.

Description

Technical Field

[0001] The present invention relates to a method for identifying the immunophenotyping of chronic myelomonocytic leukemia, specifically a method for identifying the immunophenotyping of chronic myelomonocytic leukemia using a flow cytometer and a corresponding antibody composition, belonging to the technical field of immunophenotyping detection. Background Art

[0002] Chronic myelomonocytic leukemia (abbreviated as CMML) is a common myelodysplastic syndrome / myeloproliferative neoplasm (MDS / MPN), characterized by a persistent increase in the number of monocytes in peripheral blood and multiple somatic mutations involving epigenetic regulation, spliceosome, and signal transduction genes. Peripheral blood monocytes can be divided into three subsets according to the expression levels of CD14 and CD16: classical monocytes (cMo) highly express CD14 and do not express CD16; intermediate monocytes (iMo) express both CD14 and CD16; non-classical monocytes (ncMo) express CD16 at a level similar to iMo, but have low or no expression of CD14. In healthy individuals, cMo accounts for approximately 85%, iMo for approximately 5%, and ncMo for approximately 9%. The 2022 WHO fifth edition diagnostic guidelines use the abnormal distribution of peripheral blood monocyte subsets as a diagnostic support criterion for CMML, especially a significant increase in the proportion of cMo (threshold 94%).

[0003] Currently, there is no standardized detection protocol for CMML internationally. Clinically, monocytes are usually detected by gating with side scatter (SSC) and CD45 (human leukocyte common antigen), and CD45high / SSCint is labeled as the monocyte population. However, due to the small size difference between granulocytes and monocytes and the small difference in CD45 expression intensity between lymphocytes and monocytes, this method cannot accurately distinguish the monocyte population, resulting in an abnormal increase in the proportion of the cMo subset. In addition, the expression of ncMo in many classical monocyte markers is also weakened, such as CD33, CD64, and CD36, which underestimates the proportion of ncMo when using these markers.

[0004] Multicolor flow cytometry (abbreviated as MFC) has become the preferred method for detecting cell subsets due to its advantages such as rapidity, economy, reliable results, and precise quantification. To obtain accurate results and avoid difficulties in instrument compensation adjustment and result judgment caused by using too many antibodies, the antibody combination should be sufficiently streamlined to obtain the necessary information without redundancy. In practice, there is an urgent need to develop an antibody composition and a method for accurately obtaining the monocyte population that can be applied and popularized on common flow cytometers to solve the above technical problems, reduce inter-laboratory and intra-laboratory differences, and achieve accurate detection of monocyte subsets. Summary of the Invention

[0005] The primary technical problem to be solved by the present invention is to provide a method for accurately and rapidly identifying monocyte subsets on a common flow cytometer and using the identified monocyte subsets for immunophenotyping of chronic myelomonocytic leukemia.

[0006] Another technical problem to be solved by the present invention is to provide an antibody composition and a kit for the above method.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] According to the first aspect of the embodiments of the present invention, there is provided a method for identifying the immunophenotyping of chronic myelomonocytic leukemia using a flow cytometer for non-diagnosis of diseases, including the following steps:

[0009] (1) Incubating and purifying a sample to be tested with a fluorescein-labeled antibody composition;

[0010] (2) Performing flow cytometry on the resuspended cells in step (1): Using Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots, first circle the stable liquid flow interval, remove agglutinates and cell debris; circle all white blood cell populations through CD45 combined with SSC, and circle the monocyte population through CD45 / SSC gating in all white blood cell gates; exclude CD19+ B lymphocytes; exclude CD7+ T and NK cells; exclude CD2+ T and NK cells; exclude CD16, CD24+ immature and mature granulocytes through CD16 / CD24 gating; exclude basophils and residual NK cells with CD14-CD16-. The remaining cells are the monocyte population. Divide the cross gate to determine the proportions of CD14+CD16- cMo cells, CD14+CD16+ iMo cells, and CD16+CD14ncMo cells; circle CD34+ cells through CD34 / SSC gating;

[0011] The antibody composition consists of the following antibodies: anti-CD2 antibody, anti-CD7 antibody, anti-CD14 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD24 antibody, anti-CD34 antibody, and anti-CD45 antibody.

[0012] Preferably, the antibody composition is labeled with the following fluoresceins: fluorescein BV421-labeled anti-CD2 antibody, fluorescein APC-A700-labeled anti-CD7 antibody, fluorescein FITC and ECD-labeled anti-CD14 antibody, fluorescein APC-A750-labeled anti-CD16 antibody, fluorescein PE-Cy7-labeled anti-CD19 antibody, fluorescein PE-labeled anti-CD24 antibody, fluorescein APC-labeled anti-CD34 antibody, and fluorescein KO-labeled anti-CD45 antibody.

[0013] Preferably, in the step (1), the cell concentration of the sample to be tested is 5-10×10 6 / mL.

[0014] Preferably, the purification method is to add hemolysin to the mixture of the sample to be tested and the antibody composition labeled with fluorescein, mix and let stand, then centrifuge and wash, discard the supernatant, and resuspend the cells with phosphate buffered saline.

[0015] According to the second aspect of the embodiments of the present invention, a method for identifying the immunophenotyping of chronic myelomonocytic leukemia by flow cytometry is provided. Based on the above-mentioned subpopulation identification method, the calculation method is to divide the number of CD34+ cells circled by the above method by the total number of cells in the FSC INT / SSC INT scatter plot.

[0016] According to the third aspect of the embodiments of the present invention, an antibody composition for identifying the immunophenotyping of chronic myelomonocytic leukemia by flow cytometry is provided, which consists of the following antibodies: anti-CD2 antibody, anti-CD7 antibody, anti-CD14 antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD24 antibody, anti-CD34 antibody and anti-CD45 antibody.

[0017] Use of the above antibody composition in the preparation of a product for identifying the immunophenotyping of chronic myelomonocytic leukemia.

[0018] According to the fourth aspect of the embodiments of the present invention, a kit for identifying the immunophenotyping of chronic myelomonocytic leukemia by flow cytometry is provided, and the kit includes the above antibody composition.

[0019] According to the fifth aspect of the embodiments of the present invention, a device for identifying the immunophenotyping of chronic myelomonocytic leukemia by flow cytometry is provided, including: a detection unit and an analysis unit; the detection unit includes reagent materials for detecting the sample to be tested by flow cytometry to obtain the detection result of the sample; the reagent materials include the above antibody composition; the analysis unit is used to analyze the detection result of the detection unit.

[0020] Among them, the antibodies described in the present invention are all monoclonal antibodies.

[0021] Compared with the prior art, the present invention proposes an innovative exclusion gating strategy for excluding granulocytes, T cells, B cells, and NK cells from CD45+ cells to obtain a more precise monocyte population and calculate the percentages of each subset. This method uses a set of single-tube antibody combinations to achieve immunophenotyping detection of chronic myelomonocytic leukemia. Different from traditional methods, the present invention can accurately detect the percentage and subsets of monocytes only using one tube of a combination containing 8 antibodies. It is recommended to use peripheral blood as the specimen and adopt a "wash-free" method, eliminating the traditional washing steps. This method not only reduces the requirements for the specimen volume and the number of operations, lowers the requirements for specimen collection, reduces the labor intensity, but also saves operation time. Compared with the traditional forward gating strategy, the exclusion gating strategy adopted by the present invention can more accurately circle the monocyte population, improving the accuracy, specificity, and sensitivity of the results of detecting monocyte subsets, and effectively reducing the intra- and inter-laboratory differences caused by the previous method of gating monocytes using side scatter (SSC) / CD45. In addition, the present invention also uses SSC / CD34 gating to circle CD34+ primitive cells and calculate the percentage, providing effective assistance for the clinical diagnosis of chronic myelomonocytic leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A to 1G It is an analysis flowchart of detecting monocyte subsets using a six-color scheme (CD14 / CD16 / HLA-DR / CD33 / CD36 / CD45) in the initial stage of the research and development of the present invention;

[0023] Figures 2A to 2J It is an analysis flowchart of detecting monocyte subsets of the same specimen using the six-color scheme (CD24 / CD7 / CD14 / CD56 / CD16 / CD45) released by the Flow Cytometry Working Group of the European LeukemiaNet in 2024;

[0024] Figures 3A to 3K It is an analysis flowchart of detecting monocyte subsets of the same specimen using an eight-color scheme (CD2 / CD7 / CD14 / CD16 / CD19 / CD24 / CD34 / CD45 antibodies) in the present invention;

[0025] Figures 4A to 4K It is an analysis flowchart of detecting monocyte subsets of a healthy person sample using the present invention;

[0026] Figure 5 It is an analysis graph of the sensitivity and specificity of diagnosing CMML by establishing a receiver operating characteristic (ROC) curve for the percentage of cMo cells in peripheral blood monocytes for different groups of patients (CMML, Co, Non-CMML, Reactive patient groups);

[0027] Figures 6A to 6DThe figure shows the result differences with and without the washing step for the comparative example;

[0028] Figures 7A to 7D The figure shows the results of detecting monocyte subsets in different groups of patients (CMML patient group, healthy person group, other hematological tumor patient group without CMML, reactive monocytosis patient group) using MFC for the verification example. Detailed implementation manners

[0029] To enable those skilled in the art of this technology to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] It should be noted that the terms "including" and "having" and any variations thereof in the description and claims of the present invention and the above-mentioned accompanying drawings are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device including a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0031] The following embodiments are used to illustrate the present invention, but do not limit the protection scope of the present invention. Unless otherwise specified, the technical means used in the description of the embodiments are conventional means well-known to those skilled in the art. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. All materials, reagents, etc. in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] The samples used in the embodiments of the present invention are peripheral blood specimens of CMML patients (with continuous absolute (≥0.5×10 9 / L) and relative (≥10%) increase in peripheral blood monocytes).

[0033] Screening of the antibody combination scheme in Example 1

[0034] (1) In the initial stage of research and development of the present invention, a six-color scheme was applied, namely

[0035] CD 14 / CD16 / HLA-DR / CD33 / CD36 / CD45, as Figures 1A to 1G shown. Among them, Figures 1A to 1CBy using the Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots respectively, the stable flow interval was circled, and agglutinates and cell debris were removed; Figure 1D To circle all white blood cell populations by combining human leukemia common antigen CD45 with SS, and to roughly circle monocytes by gating with CD45 / SSC in all white blood cell gates; Figure 1E To circle the HLA-DR+ / CD33+ cell population by the HLA-DR / CD33 dual-parameter plot; Figure 1F After excluding the CD36-negative cell population, the remaining cells were the monocyte population; Figure 1G To divide the cross gate to determine that the proportion of CD14+CD16-cMo cells was 96.87%, the proportion of CD14+CD16+iMo cells was 3.26%, and the proportion of CD16+CD14(- / dim)ncMo cells was 0.05%. This was a specimen from a healthy individual.

[0036] Subsequently, in clinical applications, it was found that this six-color protocol had deficiencies: due to the downregulation of CD33, CD36, and HLA-DR expression in monocytes of patients with hematological malignancies or reactive diseases, the gating of monocytes was inaccurate, the proportion of cMo cells was too high, the proportion of iMo cells was too low, and the ncMo cell population was hardly detectable, thus affecting the clinical diagnosis of CMML.

[0037] (2) Subsequently, the inventor analyzed the same specimen using the six-color protocol (CD24 / CD7 / CD14 / CD56 / CD16 / CD45) published by the Flow Cytometry Working Group of the European LeukemiaNet (ELN) in 2024. This was a specimen from a healthy individual. Figures 2A to 2C By using the Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots respectively, the stable flow interval was circled, and agglutinates and cell debris were removed; Figure 2D To circle all white blood cell populations by combining human leukemia common antigen CD45 with SS, and to roughly circle monocytes by gating with CD45 / SSC in all white blood cell gates; Figure 2E To exclude CD7+ T lymphocytes; Figure 2F To exclude CD56+ NK cells; Figure 2G To exclude CD24+ B lymphocytes; Figure 2H To exclude CD16(hi)CD24+ immature and mature granulocytes by gating with CD16 / CD24; Figure 2I To exclude basophils and residual NK cells that were CD14-CD16-. The remaining cells were the monocyte population, with a proportion of 6.75%; Figure 2JTo determine the cross gate, the proportion of CD14+CD16-cMo cells was 94.67%, the proportion of CD14+CD16+iMo cells was 2.62%, and the proportion of CD16+CD14(- / dim)ncMo cells was 2.68%.

[0038] The inventors found that the above-mentioned solutions and combinations still have defects: on the one hand, due to the pathological up-regulation of CD56 expression in monocytes of CMML or reactive monocytosis patients, some monocytes are excluded by the F gate; on the other hand, due to the possible down-regulation of CD24 in B lymphocytes of patients with hematological malignancies or reactive diseases, the G gate cannot completely exclude B lymphocytes, resulting in an abnormal increase in the proportion of cMo cells and a decrease in the proportion of iMo / ncMo cells.

[0039] (3) In a further improved solution, the inventors replaced CD7 as a preliminary T and NK cell marker for exclusion, and then used CD2 to exclude the possibly missed CD7 - terminal T and NK cells. CD2 and CD7 are both expressed on NK cells and T cells, and can corroborate each other under special pathological conditions to reduce errors; at the same time, CD19 was replaced with CD24. CD19 is stably expressed on B lymphocytes, thus avoiding interference caused by the down-regulation of CD24 under pathological or physiological conditions. Adding the CD34 marker to detect the proportion of primitive cells helps in the auxiliary diagnosis of CMML and other hematological malignancies. As Figures 3A to 3K shown, Figures 3A to 3C Using the Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots respectively, the stable flow interval was circled, and agglutinates and cell debris were removed; Figure 3D To circle all white blood cell populations by the human leukemia common antigen CD45 combined with SS, and roughly circle monocytes through gating with CD45 / SSC in all white blood cell gates; Figure 3E To exclude CD19+ B lymphocytes; Figure 3F To first exclude CD7+ T and NK cells; Figure 3G To further exclude CD2+ T and NK cells; Figure 3H To exclude CD16(hi)CD24+ immature and mature granulocytes through gating with CD16 / CD24; Figure 3I To exclude CD14 - CD16 - basophils and residual NK cells, and the remaining cells are the monocyte population, with a proportion of 7.11%; Figure 3J To determine the cross gate, the proportion of CD14+CD16-cMo cells was 86.85%, the proportion of CD14+CD16+iMo cells was 4.77%, and the proportion of CD16+CD14(- / dim)ncMo cells was 8.20%; Figure 3KBy gating through CD34 / SSC, the proportion of CD34+ primitive cells was circled to be 0.02%. Since the end stage of T cell development is characterized by CD2+CD7-, the inventor can more accurately complete the exclusion gating by first excluding CD7+ T / NK cells and then excluding CD2+ T / NK cells. Therefore, Figure 3F and Figure 3G The order cannot be reversed. The analysis results show that the proportions of each subset of monocytes are normal, conforming to the differentiation pattern characteristics of healthy people. This improved scheme is an improvement on the ELN scheme, which can more accurately circle the subsets of monocytes for detection, greatly improving the specificity and sensitivity.

[0040] Compared with the prior art, the eight-color scheme (CD2 / CD7 / CD14 / CD16 / CD19 / CD24 / CD34 / CD45) finally provided by the present invention can accurately distinguish the subsets of monocytes that conform to the characteristics of CMML, providing an accurate basis for the immunophenotyping detection and diagnosis of CMML. In Example 2, the development and verification of the flow cytometry detection scheme using the eight-color scheme provided in Example 1 Research and development and scheme verification

[0041] 1. Preparation of reagents

[0042] Antibody combination for CMML immunophenotyping: Configure the antibody combination according to the combination in Table 1. Mix the above antibodies separately in 1 container according to the ratio for determining the immunophenotypic markers of monocytes in CMML. The above antibodies can be directly purchased commercially, and all the antibodies are monoclonal antibodies. The antibodies in the examples of the present invention are purchased from BD company (Becton, Dickinson and Company), Biol egend company, and Beckman company. The types of antibodies, fluorophores, and volume compatibility of the 8 antibody compositions are shown in Table 1.

[0043] Table 1

[0044]

[0045] Prepare CMML immunophenotyping detection kits with the above antibody combinations respectively. The kit also includes necessary red blood cell lysing solution, which can be prepared by oneself or purchased commercially (such as from BD company).

[0046] 2. Detection of CMML immunophenotype by flow cytometry with 8 antibody combinations

[0047] 2.1 Main materials and instruments for the experiment

[0048] 2.1.1 Materials: 10×PBS (Phosphate-Buffered Saline) buffer solution (prepared in the laboratory), flow cytometry-specific lysing solution (from BD company); 2.1.2 Instruments:

[0049] Navi os model 10-color flow cytometer, equipped with three lasers of 405nm, 488nm, and 635nm, and 10 fluorescence detectors. Desktop low-speed centrifuge, vortex mixer.

[0050] 2.2 Method:

[0051] 2.2.1 Sample collection:

[0052] Immediately place 1 - 2 mL of human peripheral venous blood obtained from the sample into an EDTA anticoagulation tube and quickly invert it several times to prevent specimen coagulation. After collection, it should be sent to the laboratory as soon as possible, stored at room temperature, and the flow cytometry (FCM) test must be completed within 48 hours. Operate according to the instructions.

[0053] 2.2.2 Sample preparation process:

[0054] (1) Cell counting: Count the number of white blood cells per microliter. According to the test results, adjust the cell concentration to 5 - 10x10 6 / 100 mL, and add 200 - 400 μL of cell suspension into the flow tube.

[0055] (2) Antigen staining:

[0056] a) Add the corresponding pre-mixed solution of fluorescein-labeled monoclonal antibody in Table 1 to each tube and mix well with the specimen, incubate at room temperature in the dark for 15 min;

[0057] b) Hemolysis: Add 2 mL of 1×FACS lysin, mix well at low speed by vortex, and let it stand at room temperature in the dark for 8 - 10 min. Centrifuge at 300 g for 5 min, discard the supernatant, add 200 μL of PBS to resuspend the cells, and wait for detection on the machine. If it cannot be detected on the machine in time, then add 0.5 mL of 1% paraformaldehyde, mix well and store in a refrigerator at 4℃, and complete the detection within 24 hours.

[0058] (3) Detection on the machine and data analysis:

[0059] a) Determine the optimal voltage and compensation: Set the voltage and compensation according to the conventional operation method of the flow cytometer.

[0060] b) Detection on the machine and data acquisition.

[0061] According to the set instrument conditions, obtain at least 200,000 CD45-positive white blood cells per tube, and use Kluza software to analyze the data.

[0062] The process of immunophenotyping of chronic myelomonocytic leukemia includes the following steps:

[0063] (1) Add the sample to be tested into the flow tube, and adjust the cell concentration to 5 - 10x106 / mL;

[0064] (2) Add the antibody composition labeled with the corresponding fluorophore in Table 1 to the flow tube, mix well, and incubate in the dark at room temperature for 15 min;

[0065] (3) Add 2 mL of 1×FACS lysing solution to the flow tube after incubation in step (2), mix well, and let it stand in the dark at room temperature for 8 - 10 min; centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells with 200 μL of PBS;

[0066] (4) Perform flow cytometry on the resuspended cells in step (3). Using the Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots, circle the stable flow interval, remove adherent bodies and cell debris; circle all white blood cell populations by combining CD45 (human leukocyte common antigen) with SSC, and roughly circle the monocyte population by gating with CD45 / SSC in all white blood cell gates; exclude CD19+ B lymphocytes; exclude CD7+ T and NK cells; exclude CD2+ T and NK cells; exclude CD16(hi)CD24+ immature and mature granulocytes by gating with CD16 / CD24; exclude basophils and residual NK cells that are CD14 - CD16-. The remaining cells are the monocyte population. Divide the cross gate to determine the proportions of CD14+CD16 - cMo cells, CD14+CD16+ iMo cells, and CD16+CD14(- / dim) ncMo cells; gate with CD34 / SSC to circle CD34+ primitive cells and calculate the proportion. The calculation method is the number of CD34+ cells divided by the total number of cells in the FSC INT / SSC INT scatter plot. Taking Figure 3J as an example, the number of CD34+ primitive cells in it is divided by the total number of cells in Figure C, that is, 79 / 461893 = 0.017%. If the peripheral blood CD34+ primitive cells < 20%, it meets the characteristics of CMML.

[0067] 2.3 Experimental results:

[0068] Figures 4A to 4K Show the flow chart of monocyte subset analysis using MFC for a healthy person sample from the health management center of a certain hospital. Among them, Figures 4A to 4C respectively use the Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots to circle the stable flow interval, remove adherent bodies and cell debris; Figure 4D : Circle all white blood cell populations by combining the human leukemia common antigen CD45 with SSC, and roughly circle the monocyte population by gating with CD45 / SSC in all white blood cell gates; Figure 4E : Exclude CD19+ B lymphocytes;Figure 4F : Exclude CD7+ T / NK cells; Figure 4G : Exclude CD2+ T / NK cells; Figure 4H : Exclude CD16(hi)CD24+ immature and mature granulocytes by gating on CD16 / CD24; Figure 4I : Exclude basophils that are CD14-CD16- and residual NK cells. The remaining cells are the monocyte population; Figure 4J : Divide the cross gate to determine that the proportion of CD14+CD16- cMo cells is 84.62%, the proportion of CD14+CD16+ iMo cells is 7.07%, and the proportion of CD16+CD14(- / dim) ncMo cells is 8.17%; Figure 4K : Gate on CD34 / SSC to circle CD34+ primitive cells. The calculation method is Figure 4J the number of CD34+ cells in Figure 4C divided by the total number of cells in

[0069] As Figure 5 shown, a receiver operating characteristic (ROC) curve was established for patients clinically suspected of having CMML (40 patients with a final diagnosis of CMML and 40 non-CMML patients; the CMML diagnostic criteria are: persistent absolute (≥0.5×109 / L) and relative (≥10%) increase in peripheral blood monocytes, consistent bone marrow morphology, peripheral blood or bone marrow blasts <20%, and clonal cytogenetic or molecular evidence) to analyze the sensitivity and specificity of diagnosing CMML based on the percentage of cMo cells in peripheral blood monocytes. The specificity of supporting the diagnosis of CMML with a cMo cell percentage >94% in the samples analyzed using the present invention is 95.0%, and the specificity is 97.5% (80 samples). The AUC of the ROC curve is 0.992 (95% confidence interval: 0.979 - 0.999).

[0070] Comparative example

[0071] Figures 6A to 6D Show the difference diagram of the results of a healthy person sample from the health management center of a certain hospital with and without the washing step. Figure 6A and Figure 6B are both the result diagrams after directly loading the sample onto the machine without the washing step during the hemolysis process in the antigen staining part using the operation steps provided by the present invention. Among them, Figure 6A the CD16 expression intensity at the arrow is clearly grouped; Figure 6BThe detected results are as follows: the proportion of CD14+CD16-cMo cells is 90.29%, the proportion of CD14+CD16+iMo cells is 3.36%, and the proportion of CD16+CD14(- / dim)ncMo cells is 6.01%; Figure 6C and Figure 6D is a comparative example, that is, the result diagram obtained by adding the traditional washing step during the hemolysis process in the antigen staining part and then loading the sample machine, with the remaining steps being the same as those of the present invention. Figure 6C At the position pointed by the arrow, the CD16 expression is not grouped; Figure 6D The detected results are as follows: the proportion of CD14+CD16-cMo cells is 90.56%, the proportion of CD14+CD16+iMo cells is 3.50%, and the proportion of CD16+CD14(- / dim)ncMo cells is 5.72%, which is basically consistent with Figure 6B the results without washing in. Therefore, omitting the washing step not only makes the results more accurate but also saves operation time.

[0072] Verification example

[0073] Verification was carried out using the eight-color scheme provided by the present invention. By Figures 7A to 7D showing the proportions of monocyte subsets in different groups, including the CMML patient group, the healthy control group (Co), the other hematological malignancies (Non-CMML) patient group, and the reactive monocytosis (Reactive) patient group, specifically, the proportions of cMo, iMo, and ncMo subsets. Figure 7A It shows that the proportion of cMo cells in the CMML patient group exceeds 94%, while Figure 7B (healthy control group), Figure 7C (other hematological malignancies patient group) and Figure 7D (reactive monocytosis patient group) all have proportions of cMo cells lower than 94%. This result is consistent with the significant increase in the proportion of cMo cells in CMML patients (the threshold is 94%). Therefore, the eight-color scheme provided by the present invention can effectively distinguish CMML patients, other hematological disease patients, and healthy people, providing an effective tool for clinical differential diagnosis.

[0074] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying chronic myeloid monocyte immunophenotyping using flow cytometry for non-disease diagnosis, characterized in that The steps include: (1) incubating and purifying the sample to be tested with the fluorescein-labeled antibody composition; (2) Perform flow cytometry on the resuspended cells in step (1): using the Time / CD45, FSC INT / PEAK and FSC INT / SSC INT scatter plots, first circle the stable flow interval and remove adhesions and cell debris; circle all white blood cell populations by combining CD45 with SSC, and circle monocytes in all white blood cell gates by setting a gate with CD45 / SSC; exclude CD19+B lymphocytes; exclude CD7+T and NK cells; exclude CD2+T and NK cells; exclude CD16, CD24+ immature and mature granulocytes by setting a gate with CD 16 / CD24; exclude CD14-CD16- basophils and residual NK cells. The remaining cells are monocytes, and a cross gate is used to determine the proportion of CD14+CD 16-cMo cells, CD14+CD16+iMo cells and CD16+CD 14ncMo cells; set a gate with CD34 / SSC to circle CD34+ cells; The antibody composition is composed of the following antibodies: anti-CD2 antibody, anti-CD7 antibody, anti-CD14 antibody, anti-CD16 antibody, anti-CD 19 antibody, anti-CD24 antibody, anti-CD34 antibody and anti-CD45 antibody.

2. The method for identifying chronic myeloid monocyte immunophenotyping using flow cytometry according to claim 1, characterized in that: The antibody composition is labeled with the following fluoresceins: anti-CD2 antibody labeled with fluorescein BV421, anti-CD7 antibody labeled with fluorescein APC-A700, anti-CD 14 antibody labeled with fluorescein FITC and ECD, anti-CD16 antibody labeled with fluorescein APC-A750, anti-CD 19 antibody labeled with fluorescein PE-Cy7, anti-CD24 antibody labeled with fluorescein PE, anti-CD34 antibody labeled with fluorescein APC, and anti-CD45 antibody labeled with fluorescein KO.

3. The method for identifying chronic myeloid monocyte immunophenotyping using flow cytometry according to claim 1, characterized in that: In step (1), the cell concentration of the sample to be tested is 5 to 10×10 6 / mL.

4. The method for identifying chronic myeloid monocyte immunophenotyping using flow cytometry according to claim 1, characterized in that: The purification method comprises adding hemolysin to the test sample and the antibody composition labeled with fluorescein, mixing and standing, then centrifuging to remove the supernatant, and resuspending the cells with phosphate buffered saline.

5. A method for identifying chronic myeloid monocyte immunophenotyping using flow cytometry, characterized in that The calculation is performed based on the method according to any one of claims 1 to 4, wherein the calculation method is to divide the number of circled CD34+ cells by the total number of cells in the FSCINT / SSCINT scatter plot.

6. An antibody composition for identifying chronic myeloid monocyte immunophenotyping using flow cytometry, characterized in that Composed of the following antibodies: Anti-CD2 antibody, anti-CD7 antibody, anti-CD 14 antibody, anti-CD 16 antibody, anti-CD19 antibody, anti-CD24 antibody, anti-CD34 antibody and anti-CD45 antibody.

7. Use of the antibody composition according to claim 6 in preparing a product for identifying chronic myeloid monocyte immunophenotyping.

8. A kit for identifying chronic myeloid monocyte immunophenotyping using flow cytometry, characterized in that Comprising the antibody composition of claim 6.

9. A device for identifying chronic myeloid monocyte immunophenotyping using flow cytometry, characterized in that comprising a detection unit and an analysis unit; The detection unit includes a reagent material for detecting the sample to be tested by flow cytometry, so as to obtain the detection result of the sample; wherein the reagent material includes the antibody composition according to claim 6; The analyzing unit is used to analyze the detection result of the detecting unit.

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