Antibody compositions and methods for identifying chronic myelomonocytic immunophenotypes using flow cytometry

Abstract

The application discloses a method for identifying chronic granulocyte mononuclear cell immune typing by using a flow cytometer, and comprises the following steps: incubating a sample to be detected with a fluorescein-labeled antibody composition and purifying; resuspending cells and performing flow cytometry detection; using Time / CD45, FSC INT / PEAK and FSC INT / SSC INT scatter diagrams, first, a stable liquid flow interval is circled, and clumps and cell fragments are removed; all white blood cell groups are circled by combining CD45 with SSC, and mononuclear cells are circled by setting a gate in all white blood cell gates; CD 19+ B lymphocytes are excluded; CD7+ T and NK cells are excluded; CD2+ T and NK cells are excluded; CD 16, CD24+ immature and mature granulocytes are excluded by setting a gate through CD 16 / CD24; CD14-CD 16- basophilic granulocytes and residual NK cells are excluded, and the remaining cells are mononuclear cell groups, a cross gate is divided to determine the proportions of CD 14+CD16-cMo cells, CD 14+CD16+iMo cells and CD16+CD 14ncMo cells; and CD34+ original cells are circled by setting a gate through CD34 / SSC, and the proportion is calculated.

Description

Technical Field

[0001] This invention relates to a method for identifying chronic myelomonocyte immunophenotypic (CMP) immunophenotypic cells, specifically a method for identifying CMP immunophenotypic cells using flow cytometry and a corresponding antibody composition, belonging to the field of immunophenotypic detection technology. Background Technology

[0002] Chronic myelomonocytic leukemia (CMML) is a common myelodysplastic syndrome / myeloproliferative neoplasm (MDS / MPN) characterized by a persistently high number of monocytes in peripheral blood and various somatic mutations involving epigenetic regulation, spliceosomes, and signal transduction genes. Peripheral blood monocytes can be divided into three subsets based on CD14 and CD16 expression levels: classical monocytes (cMo) highly express CD14 and do not express CD16; intermediate monocytes (iMo) express both CD14 and CD16; and non-classical monocytes (ncMo) express CD16 at levels similar to iMo, but have low or no CD14 expression. In healthy individuals, cMo accounts for approximately 85%, iMo approximately 5%, and ncMo approximately 9%. The 2022 WHO Fifth Edition diagnostic guidelines use abnormal distribution of peripheral blood monocyte subsets as a supporting diagnostic criterion for CMML, particularly a significantly increased proportion of cMo (threshold 94%).

[0003] Currently, there is no standardized international testing protocol for CMML. Clinically, monocytes are typically detected using side-scattered light (SSC) and CD45 (human leukocyte common antigen), with CD45high / SSCint used as the marker for the monocyte population. However, because granulocytes and monocytes are not significantly different in size, and lymphocytes and monocytes show little difference in CD45 expression intensity, this method cannot accurately distinguish the monocyte population, leading to an abnormally high proportion of the cMo subset. Furthermore, the expression of the ncMo subset is weakened on many classic monocyte markers, such as CD33, CD64, and CD36, which results in an underestimation of the ncMo proportion when using these markers.

[0004] Multiparameter flow cytometry (MFC) has become the preferred method for detecting cell subpopulations due to its advantages such as speed, cost, reliable results, and precise quantification. To obtain accurate results while avoiding difficulties in instrument compensation and interpretation caused by using too many antibodies, antibody combinations should be sufficiently concise to obtain necessary information without redundancy. In practice, there is an urgent need to develop an antibody composition and a precise method for obtaining monocyte populations that can be applied and widely used on commonly used flow cytometers to solve the aforementioned technical challenges, reduce inter-laboratory and intra-laboratory variability, and achieve accurate detection of monocyte subpopulations. Summary of the Invention

[0005] The primary technical problem to be solved by this invention is to provide a method for accurately and rapidly identifying monocyte subsets on a commonly used flow cytometer, and for immunophenotyping chronic myelomonocytes using the identified monocyte subsets.

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

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] According to a first aspect of the present invention, a method for identifying chronic myelomonocyte immunophenotypic cells using flow cytometry is provided for the diagnosis of non-disease conditions, comprising the following steps:

[0009] (1) The sample to be tested is incubated with a fluorescently labeled antibody composition and then purified;

[0010] (2) Perform flow cytometry analysis on the resuspended cells in step (1): Using Time / CD45, FSC INT / PEAK and FSC INT / SSC INT scatter plots, first circle the stable flow region and remove adhesions and cell debris; circle all white blood cell populations using CD45 combined with SSC, and circle the monocyte populations in all white blood cell gates using 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 using CD16 / CD24 gate; exclude CD14-CD16- basophils and residual NK cells. The remaining cells are monocyte populations. Divide the cells into cross gates to determine the proportion of CD14+CD16-cMo cells, CD14+CD16+iMo cells and CD16+CD14ncMo cells; circle CD34+ cells using CD34 / SSC gate.

[0011] The antibody composition comprises 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 fluorescein: 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 step (1), the cell concentration of the sample to be tested is 5–10 × 10⁻⁶. 6 / mL.

[0014] Preferably, the purification method involves adding hemolysin to a mixture of the sample to be tested and a fluorescein-labeled antibody composition, mixing and allowing it to stand, then centrifuging and washing to remove the supernatant, and resuspending the cells in phosphate-buffered saline.

[0015] According to a second aspect of the present invention, a method for identifying chronic myelomonocyte immunophenotyping using flow cytometry is provided. The method is based on the above-described subpopulation identification method and involves calculating the number of CD34+ cells identified by the above method by dividing the total number of cells in the FSC INT / SSC INT scatter plot.

[0016] According to a third aspect of the present invention, an antibody composition for identifying chronic myelomonocyte immunophenotyping using flow cytometry is provided, comprising 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] The application of the above antibody composition in the preparation of immunophenotyping products that identify chronic myeloid monocytes.

[0018] According to a fourth aspect of the present invention, a kit for identifying chronic myelomonocyte immunophenotypic cells using flow cytometry is provided, the kit comprising the antibody composition described above.

[0019] According to a fifth aspect of the present invention, an apparatus for identifying chronic myelomonocyte immunophenotyping using flow cytometry is provided, comprising: a detection unit and an analysis unit; the detection unit includes reagent materials for detecting a sample to be tested by flow cytometry, for obtaining detection results of the sample; the reagent materials include the above-described antibody composition; the analysis unit is used to analyze the detection results of the detection unit.

[0020] All antibodies described in this invention are monoclonal antibodies.

[0021] Compared with existing technologies, this invention proposes an innovative exclusion gating strategy to exclude granulocytes, T cells, B cells, and NK cells from CD45+ cells to obtain a more accurate monocyte population and calculate the percentage of each subset. This method uses a single-tube antibody combination to achieve immunophenotyping detection of chronic myelomonocytic leukemia. Unlike traditional methods, this invention uses only one tube containing a combination of eight antibodies to accurately detect the percentage and subsets of monocytes. Peripheral blood is recommended as the specimen, and a "no-wash" method is employed, eliminating the traditional washing step. This method not only reduces the required specimen volume and number of operations, lowers the requirements for specimen collection, reduces labor intensity, but also saves operation time. Compared with the traditional forward gating strategy, the exclusion gating strategy adopted in this invention can more accurately delineate the monocyte population, improving the accuracy, specificity, and sensitivity of monocyte subset detection results, and effectively reducing intra- and inter-laboratory variability caused by previous methods using side-scattered light (SSC) / CD45 gating to delineate monocytes. In addition, this invention utilizes SSC / CD34 gating to delineate CD34+ primitive cells and calculate the percentage, providing effective assistance for the clinical diagnosis of chronic myelomonocytic leukemia. Attached Figure Description

[0022] Figures 1A to 1G This is a flowchart illustrating the analysis process of detecting monocyte subsets using a six-color scheme (CD 14 / CD16 / HLA-DR / CD33 / CD36 / CD45) during the early stages of the research and development of this invention.

[0023] Figures 2A to 2J The flowchart illustrates the process of analyzing monocyte subsets in the same sample using the six-color scheme (CD24 / CD7 / CD14 / CD56 / CD16 / CD45) published by the European Leukemia Network Flow Cytometry Working Group in 2024.

[0024] Figures 3A to 3K The flowchart illustrates the process of analyzing mononuclear cell subsets in the same sample using the eight-color scheme (CD2 / CD7 / CD14 / CD16 / CD19 / CD24 / CD34 / CD45 antibodies) as described in this invention.

[0025] Figures 4A to 4K Flowchart for analyzing mononuclear cell subsets using this invention in healthy human samples;

[0026] Figure 5 Receiver operating characteristic (ROC) curves were established for different patient groups (CMML, Co, Non-CMML, and Reactive patient groups) to analyze the sensitivity and specificity of the percentage of cMo cells in peripheral blood mononuclear cells in diagnosing CMML.

[0027] Figures 6A to 6DThis is a graph showing the difference between the results obtained by using the washing method and omitting the washing step;

[0028] Figures 7A to 7D To validate the results of monocyte subset detection using MFC in different patient groups (CMML patient group, healthy group, non-CMML other hematologic malignancy patient group, reactive mononucleosis patient group). Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such processes, methods, products or devices.

[0031] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. All materials, reagents, etc. in the following embodiments are commercially available unless otherwise specified.

[0032] The samples used in this embodiment of the invention are CMML patients (patients with a persistent absolute peripheral blood mononuclear cell count ≥0.5×10⁻⁶). 9 Peripheral blood samples with an increase of 1 / L and a relative increase (≥10%).

[0033] Example 1: Screening of Antibody Combination Regimens

[0034] (1) In the early stages of research and development, this invention applied a six-color scheme, namely,

[0035] CD 14 / CD16 / HLA-DR / CD33 / CD36 / CD45, such as Figures 1A to 1G As shown. Among them, Figures 1A to 1CThe time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots were used to delineate the stable fluid flow range and remove adhesions and cell debris. Figure 1D To identify all leukocyte populations using the human leukemia common antigen CD45 combined with SS, monocytes were roughly identified by phylogenetic phylum CD45 / SSC. Figure 1E To delineate the HLA-DR+ / CD33+ cell population using the HLA-DR / CD33 dual-parameter map; Figure 1F After excluding CD36-negative cells, the population consisted of mononuclear cells. Figure 1G To classify the phylum, the proportions of CD14+CD16-cMo cells were determined to be 96.87%, CD14+CD16+iMo cells to be 3.26%, and CD16+CD14(- / dim)ncMo cells to be 0.05%. This case was a specimen from a healthy population.

[0036] Subsequently, in clinical applications, a defect was found in this six-color scheme: due to the downregulation of CD33, CD36, and HLA-DR expression in monocytes of patients with hematologic malignancies or reactive diseases, monocyte gating was inaccurate, the proportion of cMo cells was too high, the proportion of iMo cells was too low, and the ncMo cell population was almost undetectable, thus affecting the clinical diagnosis of CMML.

[0037] (2) Subsequently, the inventors used the six-color scheme (CD24 / CD7 / CD14 / CD56 / CD16 / CD45) published by the European Leukemia Network (ELN) Flow Cytometry Working Group in 2024 to analyze the same sample, which was a healthy human sample. Figures 2A to 2C The time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots were used to delineate the stable fluid flow range and remove adhesions and cell debris. Figure 2D To identify all leukocyte populations using the human leukemia common antigen CD45 combined with SS, monocytes were roughly identified by phylogenetic phylum CD45 / SSC. 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 phylogenetic gate; Figure 2I After excluding CD14-CD16- basophils and residual NK cells, the remaining cells were mononuclear cells, accounting for 6.75%. Figure 2JTo classify the phylum, the proportions of CD14+CD16-cMo cells were determined to be 94.67%, CD14+CD16+iMo cells to be 2.62%, and CD16+CD14(- / dim)ncMo cells to be 2.68%.

[0038] The inventors found that the above scheme and combination still have defects: on the one hand, due to the pathological upregulation of CD56 expression in monocytes of patients with CMML or reactive mononucleosis, some monocytes are excluded by the F gate; on the other hand, due to the possible downregulation of CD24 expression in B lymphocytes of patients with hematologic malignancies or reactive diseases, the G gate cannot completely exclude B lymphocytes, resulting in an abnormally high cMo cell ratio and a low iMo / ncMo cell ratio.

[0039] (3) In a further improved scheme, the inventors replaced CD7 as the initial marker for T and NK cells for exclusion, and then used CD2 to exclude potentially missed CD7-terminal T and NK cells. CD2 and CD7 are expressed on both NK and T cells, which can corroborate each other and reduce errors under specific pathological conditions. Simultaneously, CD19 replaced CD24. CD19 is stably expressed on B lymphocytes, thus avoiding interference caused by CD24 downregulation under pathological or physiological conditions. Increasing the proportion of CD34 markers in detecting primitive cells helps in the diagnosis of CMML and other hematologic malignancies. Figures 3A to 3K As shown, Figures 3A to 3C The time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots were used to delineate the stable fluid flow range and remove adhesions and cell debris. Figure 3D To identify all leukocyte populations using the human leukemia common antigen CD45 combined with SS, monocytes were roughly identified by phylogenetic phylum CD45 / SSC. 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 by phylogenetic gate; Figure 3I After excluding CD14-CD16- basophils and residual NK cells, the remaining cells were mononuclear cells, accounting for 7.11%. Figure 3J To classify the phylum, the proportions of CD14+CD16-cMo cells were determined to be 86.85%, CD14+CD16+iMo cells to be 4.77%, and CD16+CD14(- / dim)ncMo cells to be 8.20%. Figure 3KTo achieve gate selection via CD34 / SSC, the proportion of CD34+ blast cells was 0.02%. Because T cells in their terminal stage exhibit CD2+CD7-, the inventors achieved more precise gate selection by first excluding CD7+ T / NK cells and then excluding CD2+ T / NK cells. Figure 3F and Figure 3G The order cannot be reversed. Analysis results showed that the proportions of each monocyte subset were normal, consistent with the differentiation patterns of healthy individuals. This improved protocol, an enhancement of the ELN protocol, can more accurately identify monocyte subsets for detection, significantly improving specificity and sensitivity.

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

[0041] 1. Preparation of reagents

[0042] Antibody combinations for CMML immunophenotyping: Antibody combinations were prepared according to the combinations in Table 1. The antibodies were mixed separately in the specified proportions and packaged into individual containers for determining the mononuclear cell immunophenotypic markers of CMML. All the above antibodies are commercially available and are monoclonal antibodies. The antibodies used in this embodiment were purchased from BD (Becton, Dickinson and Company), Biolegend, and Beckman Coulter. The antibody types, fluorescein, and volume compatibility of the eight antibody compositions are shown in Table 1.

[0043] Table 1

[0044]

[0045] The above antibody combinations were used to prepare CMML immunophenotyping kits. These kits also include necessary erythrocyte lysis buffer, which can be prepared in-house or purchased commercially (e.g., from BD Biosciences).

[0046] 2. Flow cytometry detection of CMML immunophenotype using a combination of 2.8 antibodies

[0047] 2.1 Main materials and instruments used in the experiment

[0048] 2.1.1 Materials: 10×PBS (Phosphate-Buffered Salin) buffer (prepared in-house), flow cytometry-specific hemolysin (from BD); 2.1.2 Instruments:

[0049] The Navi os 10-color flow cytometer is equipped with three lasers (405nm, 488nm, and 635nm) and 10 fluorescence detectors. It also includes a benchtop low-speed centrifuge and a vortex mixer.

[0050] 2.2 Methods:

[0051] 2.2.1 Sample Collection:

[0052] Immediately place 1-2 mL of the collected peripheral venous blood into an EDTA anticoagulant tube and quickly invert it several times to prevent coagulation. After collection, the sample should be sent to the laboratory as soon as possible and stored at room temperature. Flow cytometry (FCM) analysis must be completed within 48 hours, following the instructions.

[0053] 2.2.2 Sample preparation process:

[0054] (1) Cell count: Count the number of white blood cells per microliter. Adjust the cell concentration to 5–10 x 10⁻⁶ cells / μL based on the test results. 6 Add 200–400 μL of cell solution to a flow cytometer tube at a ratio of 100 mL.

[0055] (2) Antigen staining:

[0056] a) Add the corresponding fluorescently labeled monoclonal antibody premix and sample to each tube, mix thoroughly, and incubate at room temperature in the dark for 15 min;

[0057] b) Hemolysis: Add 2 mL of 1×FACS hemolysin, vortex at low speed to mix, and incubate 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 suspend the cells, and wait for instrumental analysis. If immediate analysis is not possible, add 0.5 mL of 1% paraformaldehyde, mix well, and store at 4°C. Analyze within 24 hours.

[0058] (3) On-machine testing and data analysis:

[0059] a) Determine the optimal voltage and compensation: Set the voltage and compensation according to the standard operating procedures for flow cytometers.

[0060] b) On-machine testing and data acquisition.

[0061] According to the set instrument conditions, at least 200,000 CD45-positive white blood cells were obtained from each tube, and the data were analyzed using Kluza software.

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

[0063] (1) Add the sample to be tested into a flow cytometry tube and adjust the cell concentration to 5–10 x 10⁻⁶.6 / mL;

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

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

[0066] (4) Perform flow cytometry analysis on the resuspended cells from step (3). Use Time / CD45, FSC INT / PEAK, and FSC INT / SSC INT scatter plots to circle the stable flow zone and remove adhesions and cell debris. Use CD45 (human leukocyte common antigen) combined with SSC to circle all leukocyte populations. Use CD45 / SSC to roughly circle the monocyte population among all leukocyte phyla. Exclude CD19+ B lymphocytes; exclude CD7+ T and NK cells; exclude CD2+ T and NK cells; use CD16 / CD24 to exclude CD16(hi)CD24+ immature and mature granulocytes; exclude CD14-CD16- basophils and residual NK cells. The remaining cells are the monocyte population. Use cross-gates to identify CD14+CD16-cMo cells, CD14+CD16+iMo cells, and CD14+CD16-iMo cells. The proportion of 16+CD14(- / dim)ncMo cells; the proportion was calculated by gated CD34 / SSC, selecting CD34+ primitive cells, and the calculation method was to divide the number of CD34+ cells by the total number of cells in the FSC INT / SSC INT scatter plot. Figure 3J For example, the number of CD34+ blast cells divided by the total number of cells in Figure C is 79 / 461893 = 0.017%. If the number of CD34+ blast cells in peripheral blood is <20%, then the characteristics of CMML are met.

[0067] 2.3 Experimental Results:

[0068] Figures 4A to 4K This diagram shows the flowchart of monocyte subset analysis using MFC detection in a healthy individual sample from a hospital's health management center. (The flowchart is not included in the provided text.) Figures 4A to 4C The time / CD45, FSC INT / PEAK, and FSC INT / SSCINT scatter plots were used to delineate the stable fluid flow range and remove adhesions and cell debris. Figure 4D All white blood cell populations were identified by combining the human leukemia common antigen CD45 with SS, and monocyte populations were roughly identified by phylogenetic grouping using CD45 / SSC. 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 CD16 / CD24 phylogenetic analysis; Figure 4I Excluding CD14-CD16- basophils and residual NK cells, the remaining cells are mononuclear cell populations; Figure 4J The proportions of CD14+CD16-cMo cells were determined by dividing the cells into three phyla: 84.62%, CD14+CD16+iMo cells were 7.07%, and CD16+CD14(- / dim)ncMo cells were 8.17%. Figure 4K : Gating is performed using CD34 / SSC to identify CD34+ primitive cells. The calculation method is as follows: Figure 4J The number of CD34+ cells divided by Figure 4C The total number of cells in the sample was 42 / 224910 = 0.018%. This method can effectively detect the proportion of monocyte subsets and their corresponding differentiation patterns in healthy individuals, and can differentiate monocyte subsets from CMML by detecting the proportion of monocyte subsets.

[0069] like Figure 5 As shown, for 40 patients clinically suspected of having CMML (40 patients ultimately diagnosed with CMML and 40 patients without CMML; the diagnostic criteria for CMML were: persistent absolute (≥0.5×10⁹ / L) and relative (≥10%) increase in peripheral blood mononuclear cells, consistent bone marrow morphology, <20% of peripheral blood or bone marrow blasts, and clonal cytogenetic or molecular evidence), receiver operating characteristic (ROC) curves were established to analyze the sensitivity and specificity of cMo cell percentage in peripheral blood mononuclear cells in diagnosing CMML. In samples analyzed using this invention, a cMo cell percentage >94% supported a CMML diagnosis with a specificity of 95.0% and a specificity of 97.5% (80 samples). The AUC of the ROC curve was 0.992 (95% confidence interval: 0.979–0.999).

[0070] Comparative Example

[0071] Figures 6A to 6D This figure shows the difference in results between a healthy person sample from a hospital's health management center and the sample after omitting the washing step. Figure 6A and Figure 6B All images are results obtained by using the operating steps provided by this invention, specifically the results of antigen staining where no washing step was performed during the hemolysis process and the antigen was directly fed into the instrument. Figure 6A The CD16 expression intensity clusters at the point indicated by the arrow are clear; Figure 6BThe detection results shown 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 For comparison, i.e., the same as the rest of the steps in this invention, the traditional washing step is added to the hemolysis process in the antigen staining part before the machine is run. Figure 6C The CD16 expression at the location indicated by the arrow is not clustered. Figure 6D The detection results shown are as follows: CD14+CD16-cMo cells accounted for 90.56%, CD14+CD16+iMo cells accounted for 3.50%, and CD16+CD14(- / dim)ncMo cells accounted for 5.72%. Figure 6B The results from the no-wash procedure were largely consistent. Therefore, omitting the washing step not only makes the results more accurate but also saves operation time.

[0072] Verification Example

[0073] Verification was performed using the eight-color scheme provided by this invention. Figures 7A to 7D This study presents the proportions of monocyte subsets in different groups, including the cMo, iMo, and ncMo subsets in CMML patients, healthy controls (Co), patients with other hematologic malignancies (Non-CMML), and patients with reactive mononucleosis. Specifically, Figure 7A The study showed that the proportion of cMo cells in the CMML patient group exceeded 94%, while Figure 7B (Control group of healthy individuals) Figure 7C (other hematologic malignancies patient group) and Figure 7D The proportion of cMo cells in the (reactive mononucleosis patient group) was below 94%. This result is consistent with the significant increase in the proportion of cMo cells in CMML patients (threshold 94%). Therefore, the eight-color scheme provided by this invention can effectively distinguish CMML patients, patients with other hematologic disorders, and healthy individuals, providing an effective tool for clinical differential diagnosis.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for identifying chronic myelomonocytes using flow cytometry for immunophenotyping, used for non-disease diagnosis, characterized in that... Includes the following steps: (1) The sample to be tested is incubated with a fluorescently labeled antibody composition and then purified; (2) Perform flow cytometry analysis on the resuspended cells in step (1): Using Time / CD45, FSC INT / PEAK and FSC INT / SSC INT scatter plots, first circle the stable flow region and remove adhesions and cell debris; circle all white blood cell populations using CD45 combined with SSC, and circle monocytes in all white blood cell gates using 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 using CD16 / CD24 gate; exclude CD14-CD16- basophils and residual NK cells. The remaining cells are monocyte populations. Divide the cells into cross gates to determine the proportion of CD14+CD16-cMo cells, CD14+CD16+iMo cells and CD16+CD14ncMo cells; circle CD34+ cells using CD34 / SSC gate. The antibody composition comprises 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.

2. The method for identifying chronic myelomonocytes using flow cytometry for immunophenotyping as described in claim 1, characterized in that: The antibody composition is labeled with the following fluorescein: 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.

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

4. The method for identifying chronic myelomonocyte immunophenotyping using flow cytometry as described in claim 1, characterized in that: The purification method involves adding hemolysin to the sample to be tested and the fluorescein-labeled antibody composition, mixing and allowing it to stand, then centrifuging to remove the supernatant, and resuspending the cells in phosphate-buffered saline.

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

6. An antibody composition for recognizing chronic myelomonocytes (CMBs) using flow cytometry, characterized in that... Composed 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.

7. The use of the antibody composition of claim 6 in the preparation of an immunophenotyping product for recognizing chronic myeloid monocytes.

8. A kit for immunophenotyping chronic myelomonocytes using flow cytometry, characterized in that... Includes the antibody composition according to claim 6.

9. A device for identifying chronic myelomonocyte immunophenotypic markers using flow cytometry, characterized in that... Includes a detection unit and an analysis unit; The detection unit includes reagent materials for detecting the sample to be tested by flow cytometry, and for obtaining the detection results of the sample; wherein, the reagent materials include the antibody composition according to claim 6; The analysis unit is used to analyze the detection results of the detection unit.

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