Reagent composition for detecting whole-blood-method myeloid-derived suppressor cells by mass spectrometry flow cytometry and application of reagent composition

Abstract

The invention provides a reagent composition for detecting whole-blood-method myeloid-derived suppressor cells through mass spectrum flow cytometry and application of the reagent composition. The reagent composition comprises three groups of reagents and is a flow cytometry detection composition capable of being used for detecting whole-blood-method myeloid-derived suppressor cells through mass spectrometry flow cytometry, when the reagent composition is applied, 21 reagents are used in a single tube at the same time, and the three groups of reagents are added in sequence and used for a sample in the same tube. The reagent provided by the invention can be applied to mass spectrometry flow cytometry to detect whole-blood-method myeloid-derived suppressor cells and evaluate whether a detected sample has a myeloid suppression state or not.

Description

Technical Field

[0001] This invention relates to a reagent composition for detecting myeloid-derived suppressor cells in whole blood using mass flow cytometry and its application, belonging to the field of hematological disease detection technology. Background Technology

[0002] Monocytes and granulocytes are the dominant myeloid cell populations in bone marrow and peripheral blood, representing two major groups of innate immune cells. They play multiple roles in defending against pathogens and maintaining tissue integrity throughout the body. Classical immunological assays are limited by instrumental conditions, and generally only the dominant cell groups—classical monocytes and neutrophils—are mentioned, with little attention paid to the weaker myeloid subpopulations. The weaker myeloid subpopulations mainly refer to eosinophils, basophils, and myeloid-derived suppressor cells (MDSCs). Recent studies have found that in various conditions, especially in advanced cancers, different types of monocytes and granulocytes possess immunosuppressive and / or immunomodulatory properties, which have significant negative effects on prognosis or treatment response. In 2007, the international community recommended using the term "myeloid-derived suppressor cells (MDSCs)" to describe the heterogeneous, immature myeloid cell populations found in pathological conditions. Since then, the term MDSC has been used in a variety of situations, including autoimmune diseases, infectious diseases, obesity, and pregnancy, and has been further subdivided to distinguish three subgroups: polymorphonuclear MDSC (P-MDSC), monocytic MDSC (M-MDSC), and early-stage MDSC (E-MDSC).

[0003] MDSCs are negatively modulating immune cells that suppress the host's anti-tumor cellular immune response through various mechanisms, including inhibiting T cell activation and function, driving and recruiting regulatory T cells. In various tumors, the proportion and absolute number of peripheral blood MDSCs and their subsets are significantly elevated. Monitoring peripheral blood MDSC levels can help assess cellular immune function and prognosis. Furthermore, pre-treatment levels of peripheral blood MDSCs and their subsets can serve as companion diagnostic indicators for immunotherapy, aiding in the screening of potential beneficiaries. MDSCs are a significant cause of tumor resistance to immunotherapy, stemming from their strong ability to suppress cellular immune responses and directly promoting tumor angiogenesis and inducing hypoxia. Patients with low pre-treatment MDSC levels, especially low M-MDSC levels, tend to have better treatment outcomes and prognoses. Monitoring trends in MDSC and their subset levels before and after conventional treatment in cancer patients can help determine treatment efficacy and assess prognosis. High MDSC levels suggest a myeloid-suppressive state of the immune system.

[0004] Furthermore, clinical research at Hebei Yanda Lu Daopei Hospital showed that after granulocyte colony-stimulating factor mobilization, the absolute values ​​of total MDSCs, M-MDSCs, and P-MDSCs in the peripheral blood of allogeneic transplant donors, as well as their proportions in mononuclear cells, significantly increased. The absolute values ​​of MDSCs, M-MDSCs, and P-MDSCs, as well as the absolute values ​​of MDSCs, M-MDSCs, and P-MDSCs in the collected samples per kilogram of patient body weight, were negatively correlated with the incidence of graft-versus-host disease (GVHD) in transplant patients; that is, a higher number of MDSCs reinfused into the patient could reduce the occurrence of GVHD. The optimal cutoff value for P-MDSCs was calculated to be 14.5 × 10⁻⁶. 6 Cells per kilogram of body weight.

[0005] In summary, MDSCs are a heterogeneous population of immune cells discovered in recent years, representing an important component of the weakened myeloid subpopulation. They play a crucial role in the treatment of lymphohematopoietic system tumors and solid tumors, as well as in the prediction of complications. These cells possess characteristic immunophenotypes, which can be qualitatively and quantitatively detected using flow cytometry based on the unique phenotypic characteristics of different subpopulations.

[0006] There are two methods for detecting MDSCs: mononuclear cell extraction and whole blood analysis. The principle of mononuclear cell extraction is based on the different specific gravities of different cell types. It uses a cell separation solution, centrifugation, and physical separation. Mature neutrophils and eosinophils, with their higher specific gravities, are removed during separation, while monocytes, lymphocytes, immature cells, basophils, dendritic cells, and other cells with lower specific gravities are separated. Research often uses mononuclear cell extraction for MDSC detection because it eliminates interference from eosinophils (basophils are still present). However, it is more complex, time-consuming, and results in significant cell loss. Whole blood analysis is simpler and faster, making it more suitable for clinical testing. Its disadvantage is the need to eliminate interference from eosinophils and basophils. Therefore, regardless of the method, a major challenge in MDSC detection is eliminating interference from eosinophils and basophils, especially with whole blood analysis. Whole blood best represents the initial state of the sample, and is the only option for simultaneous eosinophil analysis. However, because these weak myeloid subpopulations are all negative for lymphoid markers (CD3, CD56, CD19), negative for CD16 and HLA-DR, and positive for CD33 and CD11b, and because in normal peripheral blood, eosinophils account for 0-5% of nucleated cells and basophils account for 0-1% of nucleated cells, with the proportion increasing in special disease states, and MDSCs are a weak myeloid subpopulation with a lower proportion than eosinophils and basophils, how to exclude the interference of these two cell populations is a matter that needs to be considered when detecting MDSCs by flow cytometry.

[0007] Spectroscopic flow cytometry includes conventional flow cytometry and full-spectrum flow cytometry. Identifying eosinophils and basophils is relatively simple because of side scatter (SSC), which can be effectively identified on the CD45 / SSC map. Basophils appear as CD45-positive cells with small SSCs, while eosinophils appear as strongly CD45-positive cells with very large SSCs. However, mass spectrometry flow cytometry, due to its different principle, does not have the SSC indicator, thus posing a significant challenge to detection. Basophils can be identified using HLA-DR-negative CD123-positive methods, but differentiating between MDSCs and eosinophils is more difficult. If only eosinophils are analyzed, some articles use a CD66b-positive, CD16-negative method for identification; however, this method cannot exclude interference from MDSCs, and activated eosinophils also express CD16. This may explain why, to date, only one or two articles have reported on the use of mass flow cytometry to detect MDSCs by extracting mononuclear cells, and there have been no reports on the use of whole blood for MDSC detection by mass flow cytometry. Research on eosinophils is also extremely limited. Therefore, finding a reliable method to address the challenge of using mass flow cytometry to detect myeloid-derived suppressor cells in whole blood, especially the simultaneous detection of MDSCs and eosinophils using whole blood, is a pressing issue in the fields of immunology and tumor diagnosis and treatment.

[0008] Furthermore, to date, because the vast majority of MDSCs are detected using traditional flow cytometry, it is very difficult to detect them at all, requiring multiple indicators. Therefore, there are no research reports on the differences and correlations between MDSCs and their mature monocyte and granulocyte phenotypes.

[0009] Therefore, there is an urgent need in this field to develop a highly sensitive, specific, and cost-effective method. Summary of the Invention

[0010] One object of the present invention is to provide a reagent composition that enables rapid and efficient detection of myeloid-derived suppressor cells (MDSCs) and their three subtypes using mass flow cytometry, while simultaneously detecting multiple related markers and analyzing the phenotypic correlation between MDSCs and mature granulocyte and monocyte populations.

[0011] Another object of the present invention is to provide the application of the reagent composition described herein in the detection of myeloid-derived suppressor cells in whole blood by mass spectrometry flow cytometry.

[0012] On one hand, the present invention provides a reagent composition comprising a first group of reagents, a second group of reagents, and a third group of reagents, wherein: The first set of reagents consists of the metal-labeled cell-activating dye cisplatin Pt194, which is added to the flow cytometry tube in which the sample to be tested is in a single-cell suspension state. The second group of reagents consists of metal-labeled CD3, CD4, CD11c, CD24, CD56, CD66b, CD45, CD19, CD16, CD14, CD123, CD9, HLA-DR, CD15, CD33, CD11b, CD38, and CD183 antibodies. The metal labels are in the following order: In115, Dy163, Tb159, Sm154, Pr141, Ho165, Tm169, Nd148, Lu175, Nd144, Dy162, La139, Yb176, Sm147, Eu151, Bi209, Yb172, and Er166. These reagents are added to flow cytometry tubes containing a single-cell suspension of the sample to be tested. The third set of reagents consists of metal-labeled DNA1 dye Ir191 and DNA2 dye Ir193, which are added to flow cytometry tubes containing single-cell suspensions of the sample to be tested.

[0013] The reagent composition of this invention can be used for mass spectrometry flow cytometry detection of myeloid-derived suppressor cells in whole blood. In specific application, a multi-color single-tube approach is used. First, a single live cell gate is established sequentially using TIME / cisplatin and DNA1 / DNA2. Within the single live cell, a mononuclear cell (PBMC) and granulocyte (Gra) gate is established using CD45 / CD66b, a mononuclear cell (mono) gate is established using CD14, a B cell gate is established using CD19-positive and HLA-DR-positive cells, and a sequential gate is established using CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, CD9-negative, CD11b-positive, and CD33-positive cells to obtain myeloid-derived suppressor cells (MDSCs). Within the MDSC group, three MDSC subsets were defined using CD15 / CD14: CD15-positive and CD14-negative cells were polymorphonuclear myeloid repressive cells (P-MDSC), CD14-positive and CD15-negative cells were mononuclear myeloid repressive cells (M-MDSC), and CD15-negative and CD14-negative cells were early myeloid repressive cells (E-MDSC). Within the CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, and CD11b-positive gates, a basophil (baso) gate is established using CD9-positive and CD123-positive criteria, and an eosinophil (eo) gate is established using CD9-positive and CD123-negative criteria. Furthermore, combined gates are established for three weak myeloid subpopulations: MDSC, eosinophils, and basophils; combined gates for M-MDSC, E-MDSC, and mono (ME and mono); and combined gates for P-MDSC, E-MDSC, and Gra (PE and Gra). Two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 are displayed for these combined gates. By analyzing the locations and expression patterns of these cell subpopulations, the authenticity of each cell population is determined, the expression characteristics of each subpopulation are elucidated, and the degree of difference in antigen expression with their respective major myeloid cell populations is described. This technology enables the addition of more immunological information while ensuring detection accuracy, something that traditional flow cytometry has not been able to do to date. It fills the gap in mass flow cytometry detection of myeloid-derived suppressor cells in whole blood, especially the simultaneous detection of MDSCs and eosinophils. It also helps to standardize the process, reduce error rates, and facilitates future automation and artificial intelligence.

[0014] According to a specific embodiment of the present invention, in the reagent composition of the present invention, cisplatin, DNA1 dye and DNA2 dye are dyes, and the remaining antibodies are monoclonal antibodies.

[0015] According to a specific embodiment of the present invention, in the reagent composition of the present invention, each dye and antibody is a metal-labeled reagent.

[0016] In this invention, by combining different dyes and antibodies with specific metal markers, the reagent composition of this invention can achieve excellent staining effects for all metal markers in each channel when used in rapid and efficient mass flow cytometry detection of myeloid-derived suppressor cells in whole blood.

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

[0018] According to a specific embodiment of the present invention, in the reagent composition of the present invention, the first group of reagents is added separately, and the second group of reagents is a mixture of CD3 antibody, CD4 antibody, CD11c antibody, CD24 antibody, CD56 antibody, CD66b antibody, CD45 antibody, CD19 antibody, CD16 antibody, CD14 antibody, CD123 antibody, CD9 antibody, HLA-DR antibody, CD15 antibody, CD33 antibody, CD11b antibody, CD38 antibody, and CD183 antibody in a volume ratio of 4:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1. The third group of reagents is a mixture of DNA1 dye and DNA2 dye in a volume ratio of 1:1. The mixing ratios of the antibodies mentioned above refer to the mixing ratios when the antibodies have substantially equivalent titers.

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

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

[0021] The kit of the present invention can be used for rapid and efficient mass flow cytometry detection of myeloid-derived suppressor cells in whole blood, including total MDSCs, as well as three subpopulations: P-MDSCs, M-MDSCs and E-MDSCs. It can also simultaneously detect eosinophils and basophils, and analyze the correlation and differences between the expression of major markers of MDSC subpopulations and granulocytes and monocytes.

[0022] Another aspect of the present invention provides the application of the reagent composition in the preparation of a rapid and efficient method for detecting whole blood-derived suppressor cells by mass flow cytometry.

[0023] According to a specific embodiment of the present invention, the process for preparing a flow cytometry sample for rapid and efficient mass cytometry detection of whole blood myeloid-derived suppressor cells includes the following steps: (1) Add the sample to be tested to a 15ml test tube 1 to make it into a single-cell suspension, and ensure that the cell count is 1×10⁻⁶. 6 / tube-1×10 7 / Tube; (2) Add 1× cell lysis buffer to test tube 1 and incubate at room temperature in the dark; (3) Centrifuge test tube 1 after incubation in step (2) and remove the supernatant; (4) Add FACS buffer to test tube 1 after removing the supernatant in step (3), wash, centrifuge, and remove the supernatant; (5) Add FACS buffer to test tube 1 after removing the supernatant in step (4), transfer to test tube 2, centrifuge and remove the supernatant; (6) Add the first group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 after removing the supernatant in step (5), and incubate at room temperature in the dark; (7) Add FACS buffer to test tube 2 after step (6) and wash, then centrifuge and discard the supernatant; (8) Add the blocking solution and the second group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 after removing the supernatant in step (7), mix well and incubate at room temperature in the dark; (9) Add FACS buffer to test tube 2 after incubation in step (8) and wash. After centrifugation, remove the supernatant. (10) Add fixative and the third group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 obtained in step (9), and incubate at room temperature in the dark; (11) Add FACS buffer to test tube 2 after incubation in step (10) and wash. After centrifugation, remove the supernatant. (12) Resuspend the cells in FACS buffer to obtain the flow cytometry sample.

[0024] In this invention, unless otherwise specified or clearly determined from the context, the order of the described operation steps is not intended to limit the actual order of these steps.

[0025] According to a specific embodiment of the present invention, the sample to be tested can be peripheral blood or bone marrow, or it can be any sample that can be prepared into a single live cell and is suitable for mass flow cytometry detection, such as tissue specimens or body fluid specimens.

[0026] According to a specific embodiment of the present invention, in step (1), the volume of added blood cells does not exceed 1.5 ml (if the patient has a low number of peripheral blood cells, the blood cells are divided into several test tubes for separate processing as needed, and then centrifuged to remove the supernatant and concentrated before being combined into one tube, or the cell lysis process is repeated until the blood is clear and transparent).

[0027] According to a specific embodiment of the present invention, the dosage of each reagent can be based on the conventional dosage in the art or the manufacturer's recommended dosage.

[0028] According to a specific embodiment of the present invention, the reagent composition of the present invention comprises a first group of reagents added in an amount of 1-4 μl / tube, a second group of reagents added in an amount of 11-42 μl / tube, and a third group of reagents added in an amount of 0.05-0.2 μl / tube.

[0029] According to a specific embodiment of the present invention, in step (2), the incubation time can be 10-30 minutes. The amount of 1× cell lysis buffer added is 8-10 ml / tube.

[0030] According to a specific embodiment of the present invention, in step (3), the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0031] According to a specific embodiment of the present invention, in step (4), the amount of FACS buffer added for washing is 8-10 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0032] According to a specific embodiment of the present invention, in step (5), the amount of FACS buffer added for washing is 1 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0033] According to a specific embodiment of the present invention, in step (6), the incubation time can be 2-6 minutes.

[0034] According to a specific embodiment of the present invention, in step (7), the amount of FACS buffer added for washing is 1 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0035] According to a specific embodiment of the present invention, in step (8), the amount of sealing liquid added is based on the manufacturer's recommended dosage, typically 50 μl / tube. The incubation time is 30 minutes.

[0036] According to a specific embodiment of the present invention, in step (9), the amount of FACS buffer added for washing is 1 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0037] According to a specific embodiment of the present invention, in step (10), the amount of fixative added is based on the manufacturer's recommended dosage, typically 200 μl / tube. The incubation time is 30 minutes.

[0038] According to a specific embodiment of the present invention, in step (11), the amount of FACS buffer added for washing is 1 ml / tube. The centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0039] According to a specific embodiment of the present invention, in step (12), the amount of FACS buffer added for resuspension is 0.5-1 ml / tube.

[0040] According to a specific embodiment of the present invention, when resuspending cells for mass cytometry analysis, the TIME / cisplatin setting is used to establish a TIME-stable / cisplatin-concentrated viable cell gate. Within the viable gate, DNA1 / DNA2 is used to establish a de-adhesion gate (single) to obtain single viable cells. Within the single gate, CD45 / CD66b is used to establish a CD45-positive / CD66b-negative mononuclear cell (PBMC) gate and a CD45-positive / CD66b-positive granulocyte (Gra) gate, and: Set up the gates as follows: Within the single gate, use a CD14 / CD4 2D dot plot, selecting CD14-positive cells as the Mono gate; within the single gate, use a CD19 / HLA-DR 2D dot plot, selecting CD19-positive and HLA-DR-positive cells as the B cell gate, and setting up the (19-DR-) gate for CD19-negative and HLA-DR-negative cells; within the (19-DR-) gate, set up a CD3 / CD56 2D dot plot, selecting CD3... A (3-56-) gate is set for CD56-negative cells. A two-dimensional scatter plot of CD16 / CD11b is set within the (3-56-) gate. A (16-11b+) gate is set for CD16-negative and CD11b-positive cells. A two-dimensional scatter plot of CD9 / CD33 is set within the (16-11b+) gate. CD9-positive cells are selected as the eosinophil and basophil (baso and eo) gates. CD123-negative, CD9-negative, and CD33-positive cells are selected as the myeloid-derived suppressor cells (MDSC) gate. Within the (baso and eo) gates, CD123 / CD183 is used to set up eosinophil and basophil gates. CD123-negative and CD183-positive cells are selected as the eosinophil (eo) gate, and CD123-positive and CD183-negative cells are selected as the basophil (baso) gate. Within the MDSC group, CD15-positive and CD14-negative cells are categorized as polymorphonuclear myeloid-derived suppressor cells (P-MDSC), CD14-positive and CD15-negative cells as monomorphonuclear myeloid-derived suppressor cells (M-MDSC), and CD15-negative and CD14-negative cells as early myeloid-derived suppressor cells (E-MDSC). A combined group of MDSC, baso, and eo cells (weak myeloid subsets) is also established. Combined groups of M-MDSC, E-MDSC, and monocytes (ME and Mono) are also established, as are combined groups of P-MDSC, E-MDSC, and granulocytes (PE and Gra).

[0041] In this invention, CD45 expression strength refers to the following: using normal peripheral blood or bone marrow as the standard, mature lymphocytes show strong positive CD45 expression, monocytes show strong positive CD45 expression, granulocytes show moderate to weak positive CD45 expression, and nucleated erythrocytes are CD45 negative.

[0042] In this invention, the strength of CD14 and CD11c expression refers to the following: using normal peripheral blood or bone marrow as the standard, CD14 and CD11c expression in monocytes is strongly positive, CD14 expression in M-MDSCs is weakly positive, CD14 expression in other cells is negative, and CD11c expression in neutrophils and E-MDSCs is moderate to weakly positive.

[0043] In the detection technology of this invention, a multicolor method using 21 markers in one tube is employed. Major lymphoid and myeloid cell markers are used to plot the major myeloid subsets of granulocytes and monocytes in peripheral blood and bone marrow. Simultaneously, a gating method is used to select cell populations that are CD11b positive but negative for CD19, HLA-DR, CD3, CD56, and CD16. Based on CD9 and CD33 expression, CD9-positive eosinophils and basophils (baso and eo) and CD9-negative, CD33-positive myeloid-derived suppressor cells (MDSCs) are defined. Within the total MDSC population, CD15 / CD14 analysis is used. CD15-positive, CD14-negative cells are classified as polymorphonuclear myeloid-derived suppressor cells (P-MDSCs), CD14-positive, CD15-negative cells are classified as mononuclear-like myeloid-derived suppressor cells (M-MDSCs), and CD15-negative, CD14-negative cells are classified as early myeloid-derived suppressor cells (E-MDSCs). See Table 1 for details. Figure 1 .

[0044] Table 1 Phenotypic identification of whole blood MDSC subsets by conventional flow cytometry

[0045] As the most abundant components in peripheral blood and bone marrow, the major myeloid subsets of neutrophils and monocytes are innate immune cell populations that have received attention in hematologic immunology since its inception, playing a crucial role in infection immunity. However, the recognition and attention given to weakened myeloid subsets, such as MDSCs, eosinophils, basophils, and other weakened subsets, is much less than that given to adoptive immune cells such as T cells, B cells, and NK cells. Research on MDSCs, in particular, only gained international recognition in 2007, but has long been limited by the development of immunological techniques, especially flow cytometry. This is because, as shown in Table 1, identifying and classifying MDSCs requires at least 10 biomarkers, not including the physical properties of lateral and forward light scattering. Therefore, although the importance of MDSC has been recognized in the past 20 years, especially its role in tumor prognosis and prevention of GVHD after transplantation, significant breakthroughs have been made. However, because the analysis requires the use of a large number of biomarkers, research on MDSC has long been limited to identifying MDSC with the 10 biomarkers in Table 1 and classifying them into three subgroups, and no further research has been conducted.

[0046] Since mass cytometry entered the global immunology field a decade ago, numerous research findings have been published. However, almost all of these studies focus on major immune cell subsets such as T, B, and NK lymphocytes and monocytes / dendritic cells, with no application to MDSCs. The primary reason is likely the lack of research on serotonin-rich eosinophils (SSCs) in mass cytometry. Even though some studies have found a correlation between WGA and SSCs, enabling the identification of eosinophils, this has been rarely used and has never been applied to the analysis of MDSCs. Our research indicates that WGA struggles to ideally distinguish between eosinophils, basophils, and MDSCs.

[0047] One of the most significant challenges in detecting MDSCs is excluding the influence of eosinophils and basophils. These cells are negative for lymphoid markers (CD3, CD56, CD19), negative for CD16 and HLA-DR, and positive for CD33 and CD11b. Furthermore, in normal peripheral blood, eosinophils comprise 0-5% of nucleated cells, and basophils comprise 0-1%, with the proportion increasing in specific disease states. MDSCs represent a low-proportion, weak myeloid cell population. Therefore, excluding interference from these two cell populations is a crucial consideration in flow cytometry detection of MDSCs. Traditional flow cytometry and full-spectrum flow cytometry, due to the presence of SSCs (Self-Sensitive Cells), can exclude basophils by showing a small CD45-positive SSC ratio on the CD45 / SSC map, while eosinophils can be excluded by showing a large CD45-positive SSC ratio.

[0048] Therefore, effectively eliminating the interference of eosinophils and basophils in mass flow cytometry is a major bottleneck in the analysis of MDSCs by mass flow cytometry. At the same time, the detection of MDSCs, eosinophils and basophils is an urgent problem in the fields of immunology and tumor diagnosis and treatment.

[0049] The lack of single-cell eosinophils (SSCs) poses a challenge for mass flow cytometry (MSC). In particular, eosinophil analysis requires whole blood; mononuclear cells cannot be extracted because eosinophils are denser and their extraction would result in eosinophil loss. The few existing MSC studies on eosinophil detection use CD45 / CD66b positivity to draw a granulocyte phylogenetic map, and then use CD66b-positive, CD16-negative eosinophils within this phylogenetic map. However, this approach introduces MDSCs into the eosinophils, and activated eosinophils can also acquire CD16. Therefore, a more efficient method is needed for analyzing both MDSCs and eosinophils, especially when analyzing both simultaneously.

[0050] We consulted a large amount of literature and made many attempts, including WGA ( Figure 2, Figure 3 Finally, it was found that using CD9, CD183, and CD123 can not only effectively identify eosinophils and basophils, but also analyze MDSCs and their three subsets, and their expression relationships with neutrophils and monocytes in related mature cell stages, as well as the expression of the weaker subsets of eosinophils and basophils within granulocytes. This important immunological information will break through the bottlenecks in mass cytometry analysis of myeloid immune cells, including weaker myeloid cells, thereby further promoting the widespread application of MDSCs in diagnosis, complication assessment, prognostic evaluation, and related therapeutic interventions.

[0051] Eosinophils, basophils, and MDSCs share the characteristic of not expressing lymphoid markers CD3, CD56, and CD19. Furthermore, HLA-DR and CD16 negativity are necessary to exclude major myeloid subsets such as monocytes, dendritic cells, and neutrophils. MDSCs, along with eosinophils and basophils, are weak myeloid cell populations that are positive for CD33 and CD11b. Based on the criteria of CD9 positivity, CD123 negativity, and CD183 positivity (eosinophils), CD9 positivity, CD123 positivity, and CD183 negativity (basophils), and the characteristic of being negative for all three (CD9, CD123, and CD183), MDSCs can be accurately identified. Further differentiation into three MDSC subsets is based on the differences in CD15 and CD14 expression: CD15 positivity and CD14 negativity (P-MDSCs), CD15 negativity and CD14 positivity (M-MDSCs), and both CD15 and CD14 negativity (E-MDSCs).

[0052] By cleverly adding CD9 to CD123 and CD183, markers frequently used in the study of fine immune cell subsets, and designing a flowchart, we can efficiently, economically, cost-effectively, and accurately obtain MDSCs and their subsets, while simultaneously detecting eosinophils and basophils.

[0053] In addition, we did something that no one had done before. We cleverly used the high parameter capacity of mass cytometry to set up three combined gates: a combined gate for weak myeloid subpopulations consisting of MDSC, baso, and eo; a combined gate for mononuclear cells (ME and Mono) consisting of M-MDSC, E-MDSC, and monocytes; and a combined gate for granulocytes (PE and Gra) consisting of P-MDSC, E-MDSC, and granulocytes. We then displayed two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 in the combined gate for weak myeloid subpopulations, showing the location of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations, and determining the authenticity and expression characteristics of each cell population. The principle is that real cells are distributed around a center, so cells are concentrated and have a center of gravity, while debris, dead cells, and non-specific staining do not have a center of gravity. The CD45 / CD66b ratio is used to determine the authenticity of each weak myeloid subset and the difference in CD45 expression. The graph shows that MDSCs can be distinguished from baso and eo cells. MDSCs have weak CD45, baso cells have moderate CD45 and are CD66b negative, and eo cells are strongly CD45 positive and CD66b positive. The CD123 / CD66b ratio can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), and P-MDSC (CD123 negative, CD66b positive) from other two types of MDSCs (CD123 and CD66b negative), but cannot distinguish E-MDSCs and M-MDSCs. The CD15 / CD24 two-dimensional scatter plot can distinguish eo (CD15 weakly positive, CD24 positive), P-MDSC (CD15 positive, CD24 weakly positive) from other three cell populations (CD15 negative, CD24 negative). The CD4 / CD11c two-dimensional scatter plot can distinguish E-MDSC (CD11c weakly positive) from other four cell populations (CD11c moderate intensity). Two-dimensional dot plots of CD123 / CD183 and CD9 / CD123 can distinguish between baso (CD123 positive, CD183 negative, CD9 positive), eo (CD123 negative, CD183 weakly positive, CD9 positive) and tri-group MDSC (CD123 negative, CD183 negative, CD9 negative).Two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, and CD38 / HLA-DR in cells within the ME and Mono phyla clearly show the evolution from E-MDSCs to M-MDSCs to monocytes (Mono). The only difference between E-MDSCs and M-MDSCs is the decreased CD11c expression in E-MDSCs; other expression levels are similar. M-MDSCs are highly similar to monocytes, but differ in that they lack CD4, and the expression of CD11c, CD45, CD38, and HLA-DR is decreased. Two-dimensional dot plots of CD24 / CD11c in cells within the PE and Gra phyla clearly show the evolution from E-MDSCs to P-MDSCs to granulocytes (Gra), with both CD11c and CD24 expression gradually increasing. The expression of the markers for the weakened myeloid subpopulations involved in this invention is shown in Table 2.

[0054] Table 2. Expression of markers of the weak myeloid subgroups involved in this invention.

[0055] On the other hand, the present invention also provides a device for rapid and efficient detection of myeloid-derived suppressor cells in whole blood by mass spectrometry flow cytometry. This device includes a detection unit and an analysis unit, wherein: The detection unit includes reagent materials for detecting samples from the individual to be tested by mass spectrometry flow cytometry, and for obtaining the detection results of the samples; the reagent materials include the reagent composition described in this invention; The analysis unit is used to analyze the detection results of the detection unit.

[0056] According to a specific embodiment of the present invention, a rapid and efficient method for detecting myeloid-derived suppressor cells in whole blood using mass spectrometry flow cytometry is provided. The application process of the detection unit includes: processing the sample to be tested using the reagent composition described in this invention to prepare a sample for mass spectrometry flow cytometry; and performing mass spectrometry flow cytometry detection. The analysis process of the analysis unit includes: analyzing the detection results to rapidly and accurately detect myeloid-derived suppressor cells in whole blood using mass spectrometry flow cytometry.

[0057] According to a specific embodiment of the present invention, when the device of the present invention is used for rapid and efficient mass flow cytometry detection of myeloid-derived suppressor cells in whole blood, gating analysis can be performed according to the aforementioned operation.

[0058] According to a specific embodiment of the present invention, in a rapid and efficient device for detecting myeloid-derived suppressor cells (MDSCs) in whole blood by mass spectrometry flow cytometry, the analysis unit may further include analyzing the expression differences of relevant myeloid cell subpopulations based on the gating analysis results. The present invention compares the displayed cell populations with the corresponding major cell populations within a multi-marker gating system to identify differences and expression correlations. In summary, the problem of difficulty in detecting MDSCs and eosinophils due to the lack of lateral light scattering in mass spectrometry flow cytometry is perfectly solved using this invention.

[0059] According to a specific embodiment of the present invention, when analyzing single-tube samples, the analysis of the predominantly MDSC-dominated weak myeloid subpopulation can be performed in the following manner: When performing flow cytometry analysis, gating is performed as follows: ① Fixed gating: Gating is performed sequentially to remove adherent cells and live cells; ② Major subpopulation gating: Starting with a single live cell, the major myeloid cell populations, such as granulocytes, monocytes, and reference mononuclear cells (PBMCs) and B cells, as well as CD19 and HLA-DR negative cells (19-DR-), are gated in parallel; ③ Sequential gating: Within the CD19 and HLA-DR negative cells (19-DR-) gating, common expression patterns of various marker combinations are displayed sequentially. Based on the expression characteristics of myeloid-derived suppressor cells (MDSCs), eosinophils (eo), and basophils (baso), each cell population is accurately identified through sequential gating, as well as the three subpopulations of MDSCs: polymorphonuclear MDSCs (P-MDSCs), mononuclear-like MDSCs (M-MDSCs), and early MDSCs (E-MDSCs). ④ Combined phylogenetic ... (1) Fixed gate: A single living cell gate is composed of a living cell gate and a de-adhesion gate. Because they are connected in series, the order of the gates can be changed.

[0060] Live cell gate: The principle is based on the usage time (TIME) / cisplatin setting TIME for stability / cisplatin concentration for the live cell gate. See [link / reference needed]. Figure 1 The first image from left to right in the top row features a door.

[0061] De-adhesion gate: Within the live gate, DNA1 / DNA2 are concentrated to set up a single de-adhesion gate, resulting in a single live cell. The principle is that cells are spherical, and DNA1 and DNA2 are expressed in a positively correlated manner. See... Figure 1 The second picture from left to right in the top row shows a door.

[0062] (2) Major subgroups are gated: within the single gate, CD45 / CD66b is used to set up a gate for mononuclear cells (PBMC) that are strongly positive for CD45 / CD66b and negative for CD66b; a gate for granulocytes (Gra) that are positive for CD45 / CD66b is set up; a CD14 / CD4 two-dimensional dot plot is used to select CD14 positive cells as the mononuclear cell (Mono) gate; a CD19 / HLA-DR two-dimensional dot plot is used to select CD19 positive cells and HLA-DR positive cells as the B cell (B cell) gate; and a (19-DR-) gate is set up for CD19 negative cells and HLA-DR negative cells. (3) Sequential gating: Set up a two-dimensional dot plot of CD3 / CD56 in the CD19 and HLA-DR negative cells (19-DR-) gate, set up the (3-56-) gate for CD3 negative and CD56 negative cells, set up a two-dimensional dot plot of CD16 / CD11b in the (3-56-) gate, set up the (16-11b+) gate for CD16 negative and CD11b positive cells, set up a two-dimensional dot plot of CD9 / CD33 in the (16-11b+) gate, select CD9+ as the eosinophil and basophil (baso and eo) gate, and CD9 negative and CD33 positive as the myeloid-derived suppressor cells (MDSC) gate. Within the MDSC phylogenetic group, CD15 / CD14 is used to establish phylogenetic groups for each MDSC subset. CD15-positive, CD14-negative cells belong to the polymorphonuclear myeloid-derived suppressor cell (P-MDSC) phylogenetic group; CD14-positive, CD15-negative cells belong to the mononuclear myeloid-derived suppressor cell (M-MDSC) phylogenetic group; and CD15-negative, CD14-negative cells belong to the early myeloid-derived suppressor cell (E-MDSC) phylogenetic group. Within the (baso and eo) phylogenetic groups, CD123 / CD183 is used to establish eosinophil and basophil phylogenetic groups. CD123-negative, CD183-positive cells belong to the eosinophil (eo) phylogenetic group; and CD123-positive, CD183-negative cells belong to the basophil (baso) phylogenetic group.

[0063] (4) Grouping: Set the grouping group of MDSC and eo, baso as the weak myeloid subgroup, set the grouping group of M-MDSC, E-MDSC and monocytes (ME and Mono), and set the grouping group of P-MDSC, E-MDSC and granulocytes (PE and Gra).

[0064] (5) Observe the expression relationships among the subgroups. Within the weak myeloid subpopulations, two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 are displayed, showing the location of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations. This helps determine the authenticity and expression characteristics of each cell population. The principle is that true cells are distributed around a center, thus cells are concentrated and have a center of gravity, while debris, dead cells, and non-specific staining do not have a center of gravity. CD45 / CD66b is used to determine the authenticity of each weak myeloid subpopulation and the difference in CD45 expression. The plot shows that MDSC can be distinguished from baso and eo. MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive. CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), and P-MDSC (CD123 negative, CD66b positive) from other two types of MDSCs (CD123 and CD66b negative), but cannot distinguish E-MDSCs and M-MDSCs. CD15 / CD24 two-dimensional dotted plots can distinguish eo (CD15 weakly positive, CD24 positive), P-MDSC (CD15 positive, CD24 weakly positive) from other three cell populations (CD15 negative, CD24 negative). CD4 / CD11c two-dimensional dotted plots can distinguish E-MDSCs (CD11c weakly positive) from other four cell populations (CD11c of moderate intensity). Two-dimensional dot plots of CD123 / CD183 and CD9 / CD123 can distinguish baso (CD123 positive, CD183 negative, CD9 positive), eo (CD123 negative, CD183 weakly positive, CD9 positive), and MDSC (CD123 negative, CD183 negative, CD9 negative), but cannot distinguish MDSC subgroups.

[0065] Two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, and CD38 / HLA-DR within the combined phyla of M-MDSCs, E-MDSCs, and monocytes (ME and Mono) clearly show the evolution from E-MDSCs to M-MDSCs to monocytes (Mono). The only difference between E-MDSCs and M-MDSCs is weak CD11c positivity; other expression is similar. M-MDSCs are highly similar to monocytes, but differ in that they lack CD4, and the expression of CD11c, CD45, CD38, and HLA-DR is weakened. Two-dimensional dot plots of CD24 / CD11c within the combined phyla of P-MDSCs, E-MDSCs, and granulocytes (PE and Gra) clearly show the evolution from E-MDSCs to P-MDSCs to granulocytes (Gra), with the expression of CD11c and CD24 gradually increasing.

[0066] In summary, using this method, the sensitivity, specificity, positive predictive value, and negative predictive value are all above 95%. Therefore, this invention represents an important detection and analysis solution for the entire field of hematology and immunology.

[0067] In summary, when the analysis unit of the present invention analyzes the detection results of the detection unit, it can output the detection results according to the following judgment method: The detection results will be output according to the following judgment method: Method 1: Myeloid-derived suppressor cells (MDSCs): Other cells were excluded by gating with CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, and CD11b-positive cells. CD9-negative and CD33-positive cells were identified as myeloid-derived suppressor cells (MDSCs).

[0068] Eosinophils and basophils: Using CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, CD11b-positive, and CD9-positive cells as a phylogenetic marker to exclude other cells, CD123-negative and CD183-positive cells are eosinophils, and CD123-positive and CD183-negative cells are basophils.

[0069] Method 2: Immunophenotypic differences among weak myeloid subsets: The immunophenotype of MDSCs is weakly positive for CD45, differentially expressed for CD66b, CD11c, CD24, CD15 and CD14, and negative for CD4, CD9, CD123, CD183 and CD38.

[0070] The P-MDSC immunophenotype is weakly positive for CD45, moderately positive for CD11c, positive for CD66b, CD24 and CD15, and negative for CD14, CD4, CD9, CD123, CD183 and CD38.

[0071] The M-MDSC immunophenotype is weakly positive for CD45 and CD38, moderately positive for CD11c, positive for CD14, and negative for CD66b, CD24, CD15, CD4, CD9, CD123, and CD183.

[0072] The E-MDSC immunophenotype is weakly positive for CD45 and CD11c, and negative for CD66b, CD24, CD15, CD14, CD4, CD9, CD123, CD183, and CD38.

[0073] The eosinophil immunophenotype is strongly positive for CD45, moderately positive for CD11c, positive for CD66b, CD9, CD24, CD15 and CD183, and negative for CD14, CD4, CD123 and CD38.

[0074] The basophil immunophenotype is moderately positive for CD45 and CD11c, positive for CD9, CD123 and CD38, and negative for CD66b, CD14, CD4, CD24, CD15 and CD183.

[0075] Method 3: The proportion of MDSCs in nucleated cells exceeds 1.54%, indicating that the body's immune system is in a state of myeloid suppression.

[0076] In summary, this invention provides a rapid and efficient reagent composition and its application for detecting myeloid-derived suppressor cells in whole blood using mass spectrometry flow cytometry. This invention offers the following advantages: ① Based on an understanding of the immunophenotypes of numerous myeloid cells, a comprehensive protocol has been designed to minimize interference from eosinophils and basophils during MDSC detection. ② In terms of application, it allows for rapid and accurate identification and further analysis. ③ This invention employs a rapid and efficient method for detecting myeloid-derived suppressor cells in whole blood using mass spectrometry flow cytometry. Premixed antibodies can significantly reduce workload, improve efficiency, and accelerate reporting time. Clinical patients can obtain data earlier to implement appropriate treatment, thereby improving remission and survival rates. ④ A significant factor hindering the development of flow cytometry for a long time has been the individualization of protocols and excessive manual operation, making automation, standardization, and normalization difficult. This invention can create conditions for subsequent sample preprocessing machines, automated sample loading in flow cytometers, data immobilization, and especially for the future development of artificial intelligence. ⑤ This invention accurately identifies major and minor myeloid subgroups based on biomarker combinations. It can further develop a scoring system by combining the proportions and absolute numbers of MDSCs and other myeloid subgroups across various diseases. Combined with computer software, accurate and rapid analysis can be performed. The detection and analysis methods of this invention meet the current clinical needs for MDSC analysis using mass spectrometry flow cytometry and are suitable for widespread application and promotion. Attached Figure Description

[0077] Figure 1 This invention displays the results of gating analysis of normal peripheral blood samples using flow cytometry in a specific embodiment of the present invention. Figure 2 This invention demonstrates that, during the experimental process, the WGA method was attempted to detect the gating analysis results of normal peripheral blood serum markers using flow cytometry. Figure 3 This demonstrates the effectiveness of the invention during the experimental process, in conjunction with... Figure 2 The same data, analyzed using gating techniques employed in a specific embodiment of this invention, showed the results of normal peripheral blood serum marker flow cytometry. Figure 1 The difference in the final solution of this invention is that it includes WGA, and because WGA uses Tm169, CD45 is replaced with Y89. Figure 4 Peripheral blood specimens from patients with elevated MDSC levels and myeloid-suppressive B-cell non-Hodgkin lymphoma (NHL) were analyzed using mass flow cytometry. Detailed Implementation

[0078] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the following detailed description, in conjunction with specific embodiments and the technical solutions of this invention, is provided. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this invention. In the embodiments, all original reagent materials are commercially available. Experimental methods not specifically specified are conventional methods and conditions well-known in the art, or are performed according to the conditions recommended by the instrument manufacturer.

[0079] Example 1: Preparation of Reagents The antibody combination used in this embodiment is: The first group of reagents consists of cisplatin, a metal-labeled cell-activating dye, labeled Pt194, and is added separately to the first container. The second group of reagents consists of metal-labeled antibodies against CD3, CD4, CD11c, CD24, CD56, CD66b, CD45, CD19, CD16, CD14, CD123, CD9, HLA-DR, CD15, CD33, CD11b, CD38, and CD183, labeled in the order In115. The first group of reagents consists of 18 monoclonal antibodies: Dy163, Tb159, Sm154, Pr141, Ho165, Tm169, Nd148, Lu175, Nd144, Dy162, La139, Yb176, Sm147, Eu151, Bi209, Yb172, and Er166. These 18 monoclonal antibody reagents are mixed in a volume ratio of 4:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1 in the second container. The second group of reagents consists of metal-labeled DNA1 dye and DNA2 dye, with the metal labeling sequence being Ir191 and Ir193. These two dyes are mixed in a volume ratio of 1:1 in the third container.

[0080] All antibodies used in this embodiment are commercially available and are products of Zhejiang Proton Health Technology Co., Ltd.

[0081] The cell lysis buffer is optionally placed in the fourth container, the FACS buffer in the fifth container, the blocking solution in the sixth container, and the fixative in the seventh container. The cell lysis buffer, FACS buffer, blocking solution, and fixative are all commercially available products of Zhejiang Proton Health Technology Co., Ltd.

[0082] Example 2: Specimen Processing Based on the cell count results, add 700 μl of peripheral blood sample anticoagulated with EDTA or heparin to test tube 1 (15 ml test tube), ensuring that the added cell quantity is approximately 2 × 10⁻⁶. 6 The cells were in a single-cell suspension state. Add 10 ml of 1× cell lysis buffer to tube 1, mix well, and incubate at room temperature in the dark for 10 minutes. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 10 ml of FACS buffer, wash, and centrifuge at 1500 rpm for 5 minutes, discard the supernatant. Add 1 ml of FACS buffer to tube 1, mix well, and transfer to tube 2 (1.5 ml EP tube). Centrifuge at 1500 rpm for 5 minutes, discard the supernatant. Add 2 μl of the first group of reagents to tube 2 according to Table 3, mix thoroughly with the cell suspension, and incubate at room temperature in the dark for 3 minutes. Add 1 ml of FACS buffer, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 21 μl of the second group of reagents, 29 μl of FACS buffer, and 50 μl of blocking solution to tube 2 according to Table 3, mix well, and incubate at room temperature for 30 minutes. Add 1 ml of FACS buffer, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 0.1 μl of the third group of reagents and 200 μl of fixative to test tube 2 according to Table 3. Mix well and fix at room temperature for 30 min. Add 1 ml of FACS buffer, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cells in 0.5 ml of FACS buffer. This is the processed specimen, ready for instrumental analysis.

[0083] Table 3: Reagent Compositions for Detecting Myeloid-Derived Suppressive Cells from Whole Blood by Mass Flow Cytometry .

[0084] Example 3: Specimen Detection The specimens processed according to the method in Example 2 were analyzed on a PLT-MC601 mass cytometer from Zhejiang Proton Health Technology Co., Ltd. It was preferred to obtain one tube containing 100,000 cells (at least 30,000-50,000 cells were recommended). The data were then analyzed using Kaluza software or other software such as Flowjo.

[0085] The flow cytometry analysis was performed using the following gate settings: ① Fixed gate: Adhesive cell removal gate and live cell gate were performed sequentially; ② Major subpopulation gate: Starting with a single live cell, the major myeloid cell populations, such as granulocytes and monocytes, as well as reference mononuclear cells (PBMCs) and B cells, and CD19 and HLA-DR negative cells (19-DR-), were performed in parallel; ③ Sequential gate: Within the 19-DR- group of cells, the common expression patterns of various marker combinations were sequentially displayed, based on myeloid-derived suppressor cells (MDSCs) and eosinophils. The expression characteristics of eo and basophils (baso) were analyzed, and sequential gating was performed using the CD3-CD56-CD16-CD11b+CD33+ method. CD9+ was used for eo and baso, and CD9-CD33+ for MDSCs, to accurately identify each cell population. Furthermore, the phenotypes of CD15+CD14-, CD15-CD14+, and CD15-CD14- were used to identify three MDSC subpopulations: polymorphonuclear MDSC (P-MDSC), monocytic MDSC (M-MDSC), and early-stage MDSC (E-MDSC). ④ Combined gating: A combined gating system was established between MDSC and eo / baso for weak myeloid subpopulations; a combined gating system was established between M-MDSC, E-MDSC, and monocytic cells (ME and Mono); and a combined gating system was established between P-MDSC, E-MDSC, and granulocytes (PE and Gra). Specifically: (1) Fixed gate: A single living cell gate is composed of a living cell gate and a de-adhesion gate. Because they are connected in series, the order of the gates can be changed.

[0086] Live cell gate: The principle is based on the usage time (TIME) / cisplatin setting TIME for stability / cisplatin concentration for the live cell gate. See [link / reference needed]. Figure 1 The first image from left to right in the top row features a door.

[0087] De-adhesion gate: Within the live gate, DNA1 / DNA2 are concentrated to set up a single de-adhesion gate, resulting in a single live cell. The principle is that cells are spherical, and DNA1 and DNA2 are expressed in a positively correlated manner. See... Figure 1 The second picture from left to right in the top row shows a door.

[0088] (2) Major subgroups are gated: within the single gate, CD45 / CD66b is used to set up a gate for mononuclear cells (PBMC) that are strongly positive for CD45 / CD66b and negative for CD66b; a gate for granulocytes (Gra) that are positive for CD45 / CD66b is set up; a CD14 / CD4 two-dimensional dot plot is used to select CD14 positive cells as the mononuclear cell (Mono) gate; a CD19 / HLA-DR two-dimensional dot plot is used to select CD19 positive cells and HLA-DR positive cells as the B cell (B cell) gate; and a (19-DR-) gate is set up for CD19 negative cells and HLA-DR negative cells. (3) Sequential gating: Set up a two-dimensional dot plot of CD3 / CD56 in the CD19 and HLA-DR negative cells (19-DR-) gate, set up the (3-56-) gate for CD3 negative and CD56 negative cells, set up a two-dimensional dot plot of CD16 / CD11b in the (3-56-) gate, set up the (16-11b+) gate for CD16 negative and CD11b positive cells, set up a two-dimensional dot plot of CD9 / CD33 in the (16-11b+) gate, select CD9+ as the eosinophil and basophil (baso and eo) gate, and CD9 negative and CD33 positive as the myeloid-derived suppressor cells (MDSC) gate. Within the MDSC phylogenetic group, CD15 / CD14 is used to establish phylogenetic groups for each MDSC subset. CD15-positive, CD14-negative cells belong to the polymorphonuclear myeloid-derived suppressor cell (P-MDSC) phylogenetic group; CD14-positive, CD15-negative cells belong to the mononuclear myeloid-derived suppressor cell (M-MDSC) phylogenetic group; and CD15-negative, CD14-negative cells belong to the early myeloid-derived suppressor cell (E-MDSC) phylogenetic group. Within the (baso and eo) phylogenetic groups, CD123 / CD183 is used to establish eosinophil and basophil phylogenetic groups. CD123-negative, CD183-positive cells belong to the eosinophil (eo) phylogenetic group; and CD123-positive, CD183-negative cells belong to the basophil (baso) phylogenetic group.

[0089] (4) Grouping: Set the grouping group of MDSC and eo, baso as the weak myeloid subgroup, set the grouping group of M-MDSC, E-MDSC and monocytes (ME and Mono), and set the grouping group of P-MDSC, E-MDSC and granulocytes (PE and Gra).

[0090] (5) Observe the expression relationships among the subgroups. Within the MDSC, baso, and eo phyla (weak myeloid subpopulations), two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 are displayed. This shows the location of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations, determining the authenticity and expression characteristics of each population. The principle is that true cells are distributed around a center, thus cells are concentrated and have a center of gravity, while debris, dead cells, and non-specific staining do not have a center of gravity. CD45 / CD66b is used to determine the authenticity of each weak myeloid subpopulation and the difference in CD45 expression. It can be seen that the plot can distinguish MDSC from baso and eo; MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive. CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), and P-MDSC (CD123 negative, CD66b positive) from other two types of MDSCs (CD123 and CD66b negative), but cannot distinguish E-MDSCs and M-MDSCs. CD15 / CD24 two-dimensional dotted plots can distinguish eo (CD15 weakly positive, CD24 positive), P-MDSC (CD15 positive, CD24 weakly positive) from other three cell populations (CD15 negative, CD24 negative). CD4 / CD11c two-dimensional dotted plots can distinguish E-MDSCs (CD11c weakly positive) from other four cell populations (CD11c of moderate intensity). Two-dimensional dot plots of CD123 / CD183 and CD9 / CD123 can distinguish baso (CD123 positive, CD183 negative, CD9 positive), eo (CD123 negative, CD183 weakly positive, CD9 positive), and MDSC (CD123 negative, CD183 negative, CD9 negative), but cannot distinguish MDSC subgroups.

[0091] Two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, and CD38 / HLA-DR within the combined phyla (ME and Mono) of M-MDSC, E-MDSC, and monocytes clearly show the evolution from E-MDSC to M-MDSC to monocytes (Mono). The only difference between E-MDSC and M-MDSC is the weakened CD11c expression in E-MDSC; other expression levels are similar. M-MDSC is highly similar to monocytes, but differs in that it lacks CD4, and the expression of CD11c, CD45, CD38, and HLA-DR is weakened. Two-dimensional dot plots of CD24 / CD11c within the combined phyla (PE and Gra) of P-MDSC, E-MDSC, and granulocytes clearly show the evolution from E-MDSC to P-MDSC to granulocytes (Gra), with both CD11c and CD24 expression gradually increasing.

[0092] Hebei Yanda Lu Daopei Hospital, Beijing Lu Daopei Hospital, and Henan Cancer Hospital began detecting MDSCs and their subpopulations in 2018. Initially, they used the traditional 3-laser 8-color BD CantoII flow cytometry method, which could only detect the proportion and absolute count of MDSCs in peripheral blood and other samples. Our research shows that MDSCs play a key role in the development of graft-versus-host disease (GVHD). A higher number of MDSCs in the peripheral blood samples transfused by patients is more conducive to reducing the incidence of GVHD, suggesting the potential of MDSCs as a target for prevention and treatment after transplantation. In lymphoma and other tumors, an elevated MDSC proportion indicates myeloid suppression, which is associated with treatment resistance and poor prognosis. Conversely, patients with low MDSC levels often benefit from various treatments. Starting in February 2025, mass cytometry combined with 21 metal biomarkers was used to detect MDSCs and their subsets. An initial study of 151 healthy individuals (78 men and 73 women, median age 44 years, range 22-60 years) was conducted. Twenty of these healthy individuals were compared with traditional flow cytometry, showing a good correlation. From May to September 30, 2025, samples from 154 patients were tested, including 150 peripheral blood samples and 4 bone marrow samples. Among these, 59 patients had undergone targeted therapy, 24 were patients following conventional chemotherapy for multiple myeloma (MM), and 71 were patients following hematopoietic stem cell transplantation. The 59 patients who underwent targeted therapy included 52 patients with B-NHL and 7 other patients; the 71 patients who were followed up after hematopoietic stem cell transplantation included 29 patients with acute myeloid leukemia (AML), 19 patients with B-cell acute lymphoblastic leukemia (ALL) / lymphoblastic lymphoma (LBL), 12 patients with T-ALL / LBL, 7 patients with chronic myeloid tumors, and 4 other patients. The results showed that among 52 B-NHL patients after targeted therapy, 22 patients in remission after 3 months of targeted therapy had a normal MDSC percentage, with a median MDSC percentage of 0.81% (0.12-1.78%) of single viable cells. However, the MDSC percentage was elevated in 26 patients in a specific treatment phase and 4 patients who did not achieve remission, with a median MDSC percentage of 2.77% (0.35-4.59%) of single nucleated cells (P<0.05). This is similar to previously reported results, possibly due to the small sample size and lack of significant differences in subgroup distribution. Other diseases have fewer cases and are still being analyzed.

[0093] In this invention, mass flow cytometry was used for multicolor detection of 21 biomarkers in a single tube. Four gating techniques were employed: TIME / cisplatin was used sequentially to set a live cell gating, and DNA1 / DNA2 was used to set a de-adhesion gating, to obtain single live cells. This was to eliminate interference from dead and adherent cells on biomarker expression intensity. Detection of the main cell populations, i.e., the first level of gating, all started with single live cells. Based on the expression of CD14 in monocytes, CD4 was used as an auxiliary method to set a CD14-positive monocyte gating. Based on the characteristic of granulocytes (including neutrophils and eosinophils, but excluding basophils) expressing CD66b, CD45 / CD66b was used to set a CD45-positive, CD66b-negative monocyte gating, and a CD45-positive, CD66b-positive granulocyte gating. Based on the characteristics of B cells expressing CD19 and HLA-DR, the remaining HLA-DR positive cells are mainly monocytes and dendritic cells, and MDSCs do not express HLA-DR and CD19, the first level of the initial gate is set up using CD19 and HLA-DR double positivity for B cells and HLA-DR and CD19 negative MDSCs (19-DR-) sequentially set up. Because MDSCs are a heterogeneous cell population, a functional definition of a weak myeloid subset with inhibitory effects among myeloid cells, it is necessary to use markers to establish gating layers to exclude interference from other cell populations. Therefore, after setting an initial (19-DR-) gate within a single live cell gate, a (3-56-) cell gate is further set sequentially, and a (16-11b+) cell gate is set within the (3-56-) cell gate. This is because CD19, CD3, and CD56 are mainly found in B, T, and NK cells, CD16 is mainly found in NK cells and neutrophils, and HLA-DR is mainly found in monocytes, dendritic cells, B cells, as well as activated T cells and activated NK cells. Therefore, by setting these layers of gating, common immune cells are basically excluded, but eosinophils and basophils cannot be excluded. Finally, CD9 and CD33 are used for identification. CD9-negative and CD33-positive cells are MDSCs, and CD9-positive cells are eosinophils and basophils. Within the MDSC cell population, further differentiation was made based on CD15 and CD14 expression: P-MDSC (CD15-positive, CD14-negative), M-MDSC (CD15-negative, CD14-positive), and E-MDSC (CD15-negative, CD14-negative). In fact, from the traditional myeloid development process, we can see that P-MDSCs are mainly cells between intermediate and late myeloid cells, M-MDSCs are cells in the stage preceding mature monocytes, and E-MDSCs are cells in the pre-MDSC stage, where both mature myeloid cells progress to granulocytic and monocytic MDSCs.Because granulocytes and monocytes differentiate from common myeloid stem and progenitor cells that are CD34 and CD117 positive, as they gradually mature, they lose early markers CD34, CD117, and CD38, acquire antigens for their respective differentiation stages, and begin to develop in different directions. CD33 is expressed in cells at all stages of the granulocyte-monocyte lineage, but its expression intensity changes with cell development. After entering the differentiation stage, both promyelocytes and monocytes highly express CD33. Subsequently, as cells mature, the expression intensity of CD33 in granulocytes weakens, while monocytes maintain high expression. CD15 expression begins in promyelocytes and remains strong throughout maturation. CD11b is expressed in neutrophils, basophils, eosinophils, and monocytes at all stages after metamyelocytes. CD16 is mainly expressed in mature neutrophils such as metamyelocytes, band cells, and segmented cells. Most eosinophils and basophils do not express CD16, which is why using CD16 in MDSC cannot satisfactorily exclude interference from these weak myeloid subsets. CD11c is very similar to CD11b, expressed in both granulocytes and monocytes at the maturation stage, appearing in granulocytes between the time of CD11b and CD16. Granulocytes do not express CD4, neutrophils do not express CD9, while eosinophils and basophils express CD9. CD123 is found in basophils and plasmacytic dendritic cells; HLA-DR can be used to distinguish between these two cell types. Basophils do not express HLA-DR, while plasmacytic dendritic cells do. CD183 is a chemokine that has long been used to analyze T cell subsets, but its expression in granulocytes has rarely been observed, and even the antigen tables of various reagent companies offer conflicting information regarding CD183 expression. Our study found that in the myeloid subset, CD183 is only found in eosinophils; therefore, using CD123 and CD183 can effectively distinguish between eosinophils and basophils. Because MDSCs in the neutrophil lineage are limited to a very specific stage from late promyelocyte to myeloblast, we created a composite diagram of E-MDSCs, P-MDSCs, and granulocytes to show this more clearly. We can clearly see the evolution from E-MDSCs to P-MDSCs to granulocytes, with the expression of CD11c and CD24 gradually increasing. The difference between MDSCs and more mature neutrophils is the absence of CD16, and the difference between MDSCs and eosinophils and basophils is the absence of CD9.

[0094] Monocyte differentiation antigens include CD4, HLA-DR, CD64, CD11b, CD11c, and CD14. Immature monocytes begin to express CD4, but its fluorescence intensity is significantly weaker than that of normal CD4-positive T cells. HLA-DR is also expressed in monocytes at all stages of the monocyte lineage. CD64, CD11b, and CD11c begin to be expressed in the immature monocyte stage. CD14 is strongly expressed in mature monocytes, weakly expressed in immature monocytes, and not expressed in primitive cells. Therefore, these monocyte expression markers are obtained from the time myeloid primitive cells begin to differentiate into monocytes, and their intensity increases with maturity. In our study, this regular trend was observed from E-MDSCs, M-MDSCs to classic monocytes. The only difference between E-MDSCs and M-MDSCs is that CD11c expression is weakened in E-MDSCs, while other expression is the same. M-MDSCs are very similar to monocytes, but the difference is that they do not express CD4, while the expression of CD11c, CD45, CD38, and HLA-DR is weakened.

[0095] In summary, this invention is the first to use mass flow cytometry to sequentially gating multiple markers to accurately identify eosinophils and basophils, while simultaneously detecting MDSCs and their subpopulations. Furthermore, it uses separate combined gating methods to reveal the expression differences and correlations among the three MDSC subpopulations and their associated mature stage cells.

[0096] This embodiment illustrates a historically continuous and gradually escalating process. Hebei Yanda Lu Daopei Hospital, Beijing Lu Daopei Hospital, and Henan Cancer Hospital began using traditional flow cytometry to detect MDSCs in 2018. They found that MDSCs have significant clinical value; in patients in complete remission after allogeneic hematopoietic stem cell transplantation, their suppressive function can effectively protect patients and reduce the incidence of GVHD. However, in other stages, especially in early-stage cancer patients, MDSCs are a major cause of tumor resistance to immunotherapy. Elevated MDSC levels have a myeloid-suppressive effect and are associated with specific treatment stages and poor prognosis. However, for a long time, the detection parameters of traditional flow cytometry have been limited, thus it has been restricted to detecting the proportion and quantity of MDSCs and their subsets. Since introducing mass flow cytometry in February 2025, we initially conducted a trial of refined immune cell subset detection using mass flow cytometry on 151 healthy adult volunteers. This included 21 biomarkers, and we found that refined detection of myeloid cell subsets was possible. This multicolor method not only accurately identifies traditional major myeloid subsets such as classic monocytes and mature neutrophils, but also overcomes the limitation of mass flow cytometry lacking lateral light scattering. By adding only CD9 as a biomarker and flexibly utilizing CD183, a commonly used biomarker for refined immune cell subset detection, it can differentiate between eosinophils, basophils, and MDSCs, perfectly solving the problem of detecting myeloid-derived suppressor cells in whole blood using mass flow cytometry. Subsequent tests were conducted on various other patients. This protocol utilizes other biomarkers essential for refined immune cell subset detection, enabling efficient, economical, and cost-effective detection of MDSCs and three subpopulations. Furthermore, it provides groundbreaking insights into the expression of more biomarkers in these weakened myeloid subpopulations, revealing their differences and correlations with the expression of major cell populations at relevant maturation stages. This protocol has significant practical value; in terms of detection, it allows for the development of artificial intelligence software, and in terms of treatment, the discovery of subpopulations with crucial protective effects against disease and complications allows for the extraction of these cell populations for cell therapy and targeted therapy.

[0097] This embodiment provides typical cases of healthy volunteers and patients. Figure 1 To develop an analytical method for detecting myeloid-derived suppressor cells in peripheral blood of healthy adults using mass flow cytometry, strict gating and observation were performed as described above. Figure 2 This invention demonstrates that, during the experimental process, the WGA method was attempted to detect the gating analysis results of normal peripheral blood serum markers using flow cytometry. Figure 3 This demonstrates the effectiveness of the invention during the experimental process, in conjunction with... Figure 2 The same data, analyzed using gating techniques employed in a specific embodiment of this invention, showed the results of normal peripheral blood serum marker flow cytometry. Figure 1The difference in the final solution of this invention is that it includes WGA, and because WGA uses Tm169, CD45 is replaced with Y89. Figure 4 To detect peripheral blood samples from B-NHL patients with elevated MDSC levels and myeloid suppression using mass flow cytometry.

[0098] Specifically, Figure 1 Peripheral blood samples from healthy adults were analyzed using mass flow cytometry, single-tube analysis. The following settings were executed sequentially: ① TIME / Cisplatin settings were configured for stable TIME / Cisplatin concentration to establish a live cell gate. ② Within the live gate, DNA1 / DNA2 concentration was used to establish a de-adhesion gate (single) to obtain single live cells. ③ Within the single gate, CD45 / CD66b were used to establish a CD45-positive / CD66b-negative mononuclear cell (PBMC) gate and a CD45-positive / CD66b-positive granulocyte (Gra) gate. ④ A CD14 / CD4 2D dot plot was set, selecting CD14 positivity as the mononuclear cell (Mono) gate. Figure 1 The middle section is predominantly light brown. Because the dark brown M-MDSCs also express CD14, a small population of dark brown M-MDSCs that are CD4-negative and CD14-positive can be observed. ⑤ Set up a CD19 / HLA-DR two-dimensional dot plot, selecting CD19-positive and HLA-DR-positive cells as the B cell phylum. Figure 1The diagram uses a deep blue color to represent the cells, with CD19 negative and HLA-DR negative cells grouped into the (19-DR-) gate. ⑥ Within the (19-DR-) gate, a two-dimensional scatter plot of CD3 / CD56 is created. Selecting CD3 negative and CD56 negative cells groups creates the (3-56-) gate. The diagram is predominantly sky blue, interspersed with different cell populations of various colors. ⑦ Within the (3-56-) gate, a two-dimensional scatter plot of CD16 / CD11b is created. Selecting CD16 negative and CD11b positive cells groups creates the (16-11b+) gate. The diagram contains different cell populations of various colors. ⑧ Within the (16-11b+) gate, a two-dimensional scatter plot of CD9 / CD33 is created. CD9+ cells represent eosinophils and basophils (baso and eo), while CD9 negative and CD33 positive cells represent myeloid-derived suppressor cells (MDSCs). ⑨ Within the MDSC group, CD15 / CD14 is used to define the groups for each MDSC subset. CD15-positive, CD14-negative cells represent the polymorphonuclear myeloid-derived suppressor cell (P-MDSC) group, represented by dark green. CD14-positive, CD15-negative cells represent the mononuclear myeloid-derived suppressor cell (M-MDSC) group, represented by dark brown. CD15-negative, CD14-negative cells represent the early myeloid-derived suppressor cell (E-MDSC) group, represented by red. ⑩ Within the (baso and eo) groups, CD123 / CD183 is used to define the groups for eosinophils and basophils. CD123-negative, CD183-positive cells represent the eosinophil (eo) group, represented by pink. CD123-positive, CD183-negative cells represent the basophil (baso) group, represented by light blue. ⑪ Set up MDSC, baso, and eo groupings (weak myeloid subpopulations) to display two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 in weak myeloid subpopulations. Different colors represent different cell populations; for example, pink represents eo, light blue represents baso, red represents E-MDSC, dark brown represents M-MDSC, and dark green represents P-MDSC. Therefore, the locations of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations can be clearly seen. This figure can distinguish MDSC from baso and eo. MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive. CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), P-MDSC (CD123 negative, CD66b positive) from other two types of MDSC (CD123 and CD66b negative), but cannot distinguish E-MDSC and M-MDSC.Two-dimensional dot mapping of CD15 / CD24 can distinguish eo (weakly positive CD15, positive CD24), P-MDSC (positive CD15, weakly positive CD24) from other three cell populations (CD15 negative, CD24 negative). Two-dimensional dot mapping of CD4 / CD11c can distinguish E-MDSC (weakly positive CD11c) from other four cell populations (moderate CD11c intensity). Two-dimensional dot mapping of CD123 / CD183 and CD9 / CD123 can distinguish baso (positive CD123, negative CD183, positive CD9), eo (negative CD123, positive CD183, positive CD9), and MDSC (negative CD123, negative CD183, negative CD9), but cannot distinguish MDSC subsets. ⑫ Set up combined phyla (ME and Mono) for M-MDSC, E-MDSC and monocytes, and display two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, CD38 / HLA-DR in cells within the (ME and Mono) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to M-MDSC (dark brown intensified) to monocytes (Mono, light brown) can be clearly seen. The only difference between E-MDSC and M-MDSC is that CD11c expression is weakened in E-MDSC, while other expression is the same. M-MDSC is very similar to monocytes, but the difference is that CD4 is lost, and the expression of CD11c, CD45, CD38 and HLA-DR is weakened. ⑬ Set up combined phyla (PE and Gra) of P-MDSC, E-MDSC and granulocytes, and display a two-dimensional dot plot of CD24 / CD11c in cells within the (PE and Gra) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to P-MDSC (dark green) to granulocytes (Gra, sky blue) can be clearly seen, and the expression of CD11c and CD24 gradually increases.

[0099] Specifically, Figure 2 This invention demonstrates that, during the experimental process, the WGA method was used to detect the results of gating analysis of normal peripheral blood sample chromatographic flow cytometry, with single-tube analysis. The following settings were executed sequentially: ① Using time (TIME) / cisplatin, the TIME / cisplatin concentration was set as the live cell gate. ② Within the live gate, DNA1 / DNA2 were used to set the de-adhesion gate (single), obtaining single live cells. ③ Within the single gate, CD45 / CD66b were used to set the CD45 strongly positive / CD66b negative mononuclear cell (PBMC) gate, and the CD45 positive / CD66b positive granulocyte (Gra) gate. ④ A CD14 / CD4 two-dimensional dot plot was set, selecting CD14 positivity as the mononuclear cell (Mono) gate. Figure 1The middle section is predominantly light brown. Because the dark brown M-MDSCs also express CD14, a small population of dark brown M-MDSCs that are CD4-negative and CD14-positive can be observed. ⑤ Set up a CD19 / HLA-DR two-dimensional dot plot, selecting CD19-positive and HLA-DR-positive cells as the B cell phylum. Figure 1The diagram uses a deep blue color to represent the cells, with CD19 negative and HLA-DR negative cells grouped into the (19-DR-) gate. ⑥ Within the (19-DR-) gate, a two-dimensional scatter plot of CD3 / CD56 is created. Selecting CD3 negative and CD56 negative cells groups creates the (3-56-) gate. The diagram is predominantly sky blue, interspersed with different cell populations of various colors. ⑦ Within the (3-56-) gate, a two-dimensional scatter plot of CD16 / CD11b is created. Selecting CD16 negative and CD11b positive cells groups creates the (16-11b+) gate. The diagram contains different cell populations of various colors. ⑧ Within the (16-11b+) gate, a two-dimensional scatter plot of CD123 / WGA is created. Selecting CD123 positive or WGA positive cells groups represents eosinophils and basophils (baso and eo), while CD123 negative and WGA positive cells group represents pre-myeloid-derived suppressor cells (pre-MDSC) cells. ⑨ Within the pre-MDSC gate, CD33 and CD66b are used to set up the MDSC gate, selecting CD33-positive cells as MDSCs. ⑩ CD15 / CD14 are used to set up the gates for each MDSC subset. CD15-positive and CD14-negative cells represent the polymorphonuclear myeloid suppressor cell (P-MDSC) gate, indicated by dark green. CD14-positive and CD15-negative cells represent the mononuclear myeloid suppressor cell (M-MDSC) gate, indicated by dark brown. CD15-negative and CD14-negative cells represent the early myeloid suppressor cell (E-MDSC) gate, indicated by red. ⑪ Within the (baso and eo) gates, CD123 / CD183 are used to set up the eosinophil and basophil gates. CD123-negative and CD183-positive cells represent the eosinophil (eo) gate, indicated by pink. CD123-positive and CD183-negative cells represent the basophil (baso) gate, indicated by light blue. ⑬ MDSC, baso, and eo groupings (weak myeloid subpopulations) are set up to display two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 in weak myeloid subpopulations. Different colors represent different cell populations; for example, pink represents eo, light blue represents baso, red represents E-MDSC, dark brown represents M-MDSC, and dark green represents P-MDSC. Therefore, the locations of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations can be clearly seen. This figure can distinguish MDSC from baso and eo. MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive. CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), P-MDSC (CD123 negative, CD66b positive) from other two types of MDSC (CD123 and CD66b negative), but cannot distinguish E-MDSC and M-MDSC.Two-dimensional dot mapping of CD15 / CD24 can distinguish eo (weakly positive CD15, positive CD24), P-MDSC (positive CD15, weakly positive CD24) from other three cell populations (CD15 negative, CD24 negative). Two-dimensional dot mapping of CD4 / CD11c can distinguish E-MDSC (weakly positive CD11c) from other four cell populations (moderate CD11c intensity). Two-dimensional dot mapping of CD123 / CD183 and CD9 / CD123 can distinguish baso (positive CD123, negative CD183, positive CD9), eo (negative CD123, positive CD183, positive CD9), and MDSC (negative CD123, negative CD183, negative CD9), but cannot distinguish MDSC subsets. ⑫ Set up combined phyla (ME and Mono) for M-MDSC, E-MDSC and monocytes, and display two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, CD38 / HLA-DR in cells within the (ME and Mono) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to M-MDSC (dark brown intensified) to monocytes (Mono, light brown) can be clearly seen. The only difference between E-MDSC and M-MDSC is that CD11c expression is weakened in E-MDSC, while other expression is the same. M-MDSC is very similar to monocytes, but the difference is that CD4 is lost, and the expression of CD11c, CD45, CD38 and HLA-DR is weakened. ⑭ Set up combined phyla (PE and Gra) of P-MDSC, E-MDSC and granulocytes, and display a two-dimensional dot plot of CD24 / CD11c in cells within the (PE and Gra) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to P-MDSC (dark green) to granulocytes (Gra, sky blue) can be clearly seen, and the expression of CD11c and CD24 gradually increases. Figure 2 and Figure 1 The differences are as follows: First, in setting up the MDSC, because WGA, unlike CD9, cannot simultaneously identify both eosinophils and basophils, two markers, CD123 and WGA, are required, thus necessitating an additional pre-MDSC gate. Second, because WGA expression intensity is not high enough, the boundary between it and the negative cell population is not as clear as that of CD9, requiring continuous adjustments and trial and error during the analysis. Third, to better analyze the advantages and disadvantages of CD9 versus WGA, in weak myeloid lineages, the degree to which CD9 / CD123, WGA / CD123, CD9 / CD183, and WGA / CD183 distinguish pink eosinophils was shown, revealing that CD9 expression was significantly higher than WGA. Fourth, because Tm169 was used in WGA, Y89 was used instead of CD45. Therefore, this protocol was abandoned after three trials.

[0100] Specifically, Figure 3This demonstrates the effectiveness of the invention during the experimental process, in conjunction with... Figure 2 The same data was analyzed using gating techniques employed in the specific implementation scheme of this invention for normal peripheral blood serum spectrometry flow cytometry, with single-tube analysis. The following settings were executed sequentially: ① The TIME / cisplatin setting was used to establish a stable TIME / cisplatin concentration for the live cell gate. ② Within the live gate, DNA1 / DNA2 were used to establish a de-adhesion gate (single) to obtain single live cells. ③ Within the single gate, CD45 / CD66b were used to establish a CD45-positive / CD66b-negative mononuclear cell (PBMC) gate and a CD45-positive / CD66b-positive granulocyte (Gra) gate. ④ A CD14 / CD4 two-dimensional dot plot was set, selecting CD14 positivity as the mononuclear cell (Mono) gate. Figure 1 The middle section is predominantly light brown. Because the dark brown M-MDSCs also express CD14, a small population of dark brown M-MDSCs that are CD4-negative and CD14-positive can be observed. ⑤ Set up a CD19 / HLA-DR two-dimensional dot plot, selecting CD19-positive and HLA-DR-positive cells as the B cell phylum. Figure 1The diagram uses a deep blue color to represent the cells, with CD19 negative and HLA-DR negative cells grouped into the (19-DR-) gate. ⑥ Within the (19-DR-) gate, a two-dimensional scatter plot of CD3 / CD56 is created. Selecting CD3 negative and CD56 negative cells groups creates the (3-56-) gate. The diagram is predominantly sky blue, interspersed with different cell populations of various colors. ⑦ Within the (3-56-) gate, a two-dimensional scatter plot of CD16 / CD11b is created. Selecting CD16 negative and CD11b positive cells groups creates the (16-11b+) gate. The diagram contains different cell populations of various colors. ⑧ Within the (16-11b+) gate, a two-dimensional scatter plot of CD9 / CD33 is created. CD9+ cells represent eosinophils and basophils (baso and eo), while CD9 negative and CD33 positive cells represent myeloid-derived suppressor cells (MDSCs). ⑨ Within the MDSC group, CD15 / CD14 is used to define the groups for each MDSC subset. CD15-positive, CD14-negative cells represent the polymorphonuclear myeloid-derived suppressor cell (P-MDSC) group, represented by dark green. CD14-positive, CD15-negative cells represent the mononuclear myeloid-derived suppressor cell (M-MDSC) group, represented by dark brown. CD15-negative, CD14-negative cells represent the early myeloid-derived suppressor cell (E-MDSC) group, represented by red. ⑩ Within the (baso and eo) groups, CD123 / CD183 is used to define the groups for eosinophils and basophils. CD123-negative, CD183-positive cells represent the eosinophil (eo) group, represented by pink. CD123-positive, CD183-negative cells represent the basophil (baso) group, represented by light blue. ⑪ Set up MDSC, baso, and eo groupings (weak myeloid subpopulations) to display two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 in weak myeloid subpopulations. Different colors represent different cell populations; for example, pink represents eo, light blue represents baso, red represents E-MDSC, dark brown represents M-MDSC, and dark green represents P-MDSC. Therefore, the locations of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations can be clearly seen. This figure can distinguish MDSC from baso and eo. MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive. CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), P-MDSC (CD123 negative, CD66b positive) from other two types of MDSC (CD123 and CD66b negative), but cannot distinguish E-MDSC and M-MDSC.Two-dimensional dot mapping of CD15 / CD24 can distinguish eo (weakly positive CD15, positive CD24), P-MDSC (positive CD15, weakly positive CD24) from other three cell populations (CD15 negative, CD24 negative). Two-dimensional dot mapping of CD4 / CD11c can distinguish E-MDSC (weakly positive CD11c) from other four cell populations (moderate CD11c intensity). Two-dimensional dot mapping of CD123 / CD183 and CD9 / CD123 can distinguish baso (positive CD123, negative CD183, positive CD9), eo (negative CD123, positive CD183, positive CD9), and MDSC (negative CD123, negative CD183, negative CD9), but cannot distinguish MDSC subsets. ⑫ Set up combined phyla (ME and Mono) for M-MDSC, E-MDSC and monocytes, and display two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, CD38 / HLA-DR in cells within the (ME and Mono) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to M-MDSC (dark brown intensified) to monocytes (Mono, light brown) can be clearly seen. The only difference between E-MDSC and M-MDSC is that CD11c expression is weakened in E-MDSC, while other expression is the same. M-MDSC is very similar to monocytes, but the difference is that CD4 is lost, and the expression of CD11c, CD45, CD38 and HLA-DR is weakened. ⑬ Set up combined phyla (PE and Gra) of P-MDSC, E-MDSC and granulocytes, and display a two-dimensional dot plot of CD24 / CD11c in cells within the (PE and Gra) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to P-MDSC (dark green) to granulocytes (Gra, sky blue) can be clearly seen, and the expression of CD11c and CD24 gradually increases. Figure 3 and Figure 1 The final difference in this invention lies in the following: First, the inclusion of WGA in the solution increases cost but does not add information. Second, because WGA uses TM169, CD45 is replaced with Y89. Third, compared to... Figure 2 Similarly, to better analyze the advantages and disadvantages of CD9 versus WGA, in a weak myeloid lineage, the degree to which CD9 / CD123, WGA / CD123, CD9 / CD183, and WGA / CD183 distinguish pink eosinophils was shown. CD9 expression was observed to be significantly higher than WGA. Therefore, this approach was abandoned. [The following was selected:] Figure 1 The final solution.

[0101] Figure 4Peripheral blood samples from B-NHL patients with elevated MDSC levels and myeloid suppression were analyzed using mass flow cytometry. Single-tube analysis was performed. The following settings were executed sequentially: ① Time of use (TIME) / cisplatin was set to a stable TIME / cisplatin concentration setting as the live cell gate. ② Within the live gate, DNA1 / DNA2 were used to set a de-adhesion gate (single) to obtain single live cells. ③ Within the single gate, CD45 / CD66b were used to set a CD45-positive / CD66b-negative mononuclear cell (PBMC) gate and a CD45-positive / CD66b-positive granulocyte (Gra) gate. ④ A CD14 / CD4 2D dot plot was set, selecting CD14 positivity as the mononuclear cell (Mono) gate. Figure 1 The middle section is predominantly light brown. Because the dark brown M-MDSCs also express CD14, a small population of dark brown M-MDSCs that are CD4-negative and CD14-positive can be observed. ⑤ Set up a CD19 / HLA-DR two-dimensional dot plot, selecting CD19-positive and HLA-DR-positive cells as the B cell phylum. Figure 1The image shows a 2D dot plot of CD3 / CD56 cells within the (19-DR-) gate, with CD3-negative and CD56-negative cells selected as the (3-56-) gate. The image is predominantly sky blue, interspersed with different cell populations of various colors. A 2D dot plot of CD16 / CD11b cells within the (3-56-) gate is shown, with CD16-negative and CD11b-positive cells selected as the (16-11b+) gate. The image contains different cell populations of various colors. A 2D dot plot of CD9 / CD33 cells within the (16-11b+) gate is shown, with CD9+ cells representing eosinophils and basophils (baso and eo), and CD9-negative and CD33-positive cells representing myeloid-derived suppressor cells (MDSCs). The proportion of MDSCs is 3.32% of single viable cells, higher than the highest value of 1.54% in healthy individuals. ⑨ Within the MDSC phylum, CD15 / CD14 is used to define the MDSC subpopulations. CD15-positive, CD14-negative cells represent the polymorphonuclear myeloid-derived suppressor cells (P-MDSC) phylum, indicated by dark green, accounting for 18.64% of MDSCs. CD14-positive, CD15-negative cells represent the mononuclear myeloid-derived suppressor cells (M-MDSC) phylum, indicated by dark brown, accounting for 73.40% of MDSCs. CD15-negative, CD14-negative cells represent the early myeloid-derived suppressor cells (E-MDSC) phylum, indicated by red, accounting for 7.86% of MDSCs. ⑩ Within the (baso and eo) phylum, CD123 / CD183 is used to define the eosinophil and basophil phylum phylum phylum. CD123-negative, CD183-positive cells represent the eosinophil phylum (eo), indicated by pink. CD123-positive, CD183-negative cells represent the basophil phylum (baso), indicated by light blue. ⑪ Set up MDSC, baso, and eo groupings (weak myeloid subpopulations) to display two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 in weak myeloid subpopulations. Different colors represent different cell populations; for example, pink represents eo, light blue represents baso, red represents E-MDSC, dark brown represents M-MDSC, and dark green represents P-MDSC. Therefore, the locations of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations can be clearly seen. This figure can distinguish MDSC from baso and eo. MDSC has weak CD45, baso has moderate CD45 and is CD66b negative, and eo is strongly CD45 positive and CD66b positive.CD123 / CD66b can distinguish baso (CD123 positive, CD66b negative), eo (CD123 negative, CD66b positive), and P-MDSC (CD123 negative, CD66b positive) from other two types of MDSCs (CD123 and CD66b negative), but cannot distinguish E-MDSCs and M-MDSCs. CD15 / CD24 two-dimensional dotted plots can distinguish eo (CD15 weakly positive, CD24 positive), P-MDSC (CD15 positive, CD24 weakly positive) from other three cell populations (CD15 negative, CD24 negative). CD4 / CD11c two-dimensional dotted plots can distinguish E-MDSCs (CD11c weakly positive) from other four cell populations (CD11c of moderate intensity). Two-dimensional dot plots of CD123 / CD183 and CD9 / CD123 can distinguish baso (CD123 positive, CD183 negative, CD9 positive), eo (CD123 negative, CD183 positive, CD9 positive), and MDSC (CD123 negative, CD183 negative, CD9 negative), but cannot distinguish MDSC subgroups. ⑫ Set up combined phyla (ME and Mono) for M-MDSC, E-MDSC and monocytes, and display two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, CD38 / HLA-DR in cells within the (ME and Mono) phyla. Since different colors represent different cell populations, the evolution from E-MDSC (red intensified) to M-MDSC (dark brown intensified) to monocytes (Mono, light brown) can be clearly seen. The only difference between E-MDSC and M-MDSC is that CD11c expression is weakened in E-MDSC, while other expression is the same. M-MDSC is very similar to monocytes, but the difference is that CD4 is lost, and the expression of CD11c, CD45, CD38 and HLA-DR is weakened. ⑬ A combined phylogenetic tree (PE and Gra) was created for P-MDSC, E-MDSC, and granulocytes, displaying a two-dimensional dot plot of CD24 / CD11c expression within each phylogenetic tree. Since different colors represent different cell populations, the evolution from E-MDSC (emphasized red) to P-MDSC (dark green) to granulocytes (Gra, sky blue) is clearly visible, with a gradual increase in CD11c and CD24 expression. The final results showed that the proportion of MDSCs in a single live cell was 3.32% in the patient, higher than the highest value of 1.54% in healthy individuals, indicating a myeloid-suppressive state. Specifically, P-MDSCs accounted for 18.64% of MDSCs, M-MDSCs for 73.40%, and E-MDSCs for 7.86%.

[0102] Clinical validation was conducted using the method described in this embodiment: Hebei Yanda Lu Daopei Hospital, Beijing Lu Daopei Hospital, and Henan Cancer Hospital, starting in 2018, used traditional flow cytometry to detect MDSCs and their subsets based on the expression of MDSCs as shown in Table 2. They found that MDSC cells in the peripheral blood of healthy donors had a mitigating effect on GVHD in patients after reinfusion, and could predict prognosis and benefit patients in cancer treatment. In February 2025, they began using this protocol, namely the mass flow cytometry 21-marker protocol. WGA was also tried, but it was found to be less effective than CD9, CD123, and CD183 in identifying eosinophils and basophils. See the comparison figure for details. Figure 2 and Figure 3 Simultaneously, 20 healthy volunteers were selected, and 2-3 ml of EDTA-anticoagulated peripheral blood was collected. The same specimen processing method was used (1 ml of whole blood was thawed first and then labeled to ensure a cell count of 1 x 10⁻⁶ cells per tube). 6 -1X10 7 The following were used for conventional flow cytometry to fluorescently label CD16 FITC / CD33 PE / CD3+CD19+CD56 PerCP-Cy5.5 / CD123PE-Cy7 / CD14 APC / HLA-DR APC-Cy7 / CD15 BV421 / CD45 V500 / CD11b. BV605, and a comparison of conventional flow cytometry and mass spectrometry flow cytometry, the proportions of MDSCs, P-MDSCs, M-MDSCs, and E-MDSCs. The proportion of MDSCs obtained by mass spectrometry flow cytometry to single viable cells was 0.77% (0.19-1.54%), of which P-MDSCs accounted for a median of 19.53% (5.47-83.40%), M-MDSCs accounted for a median of 72.58% (6.34%-89.01%), and E-MDSCs accounted for a median of 2.78% (0.06-43.23%). The proportion of MDSCs obtained by conventional cytology was 0.75% (0.17-1.63%) of single viable cells, of which P-MDSCs accounted for 18.61% (4.95-80.07%), M-MDSCs accounted for 74.53% (6.73%-90.90%), and E-MDSCs accounted for 2.18% (0.07-38.29%). There was no statistically significant difference between the two groups, and the correlation coefficient r for MDSCs was 98.40%.

[0103] Clinical validation was conducted using the method described in this embodiment: Starting in February 2025, mass cytometry combined with 21 metal biomarkers was used to detect MDSCs and their subsets. Initially, a study was conducted on 151 healthy individuals (78 males and 73 females, median age 44 years, range 22-60 years). Twenty of these individuals were selected for comparison with conventional flow cytometry, showing good correlation. From May 2025 to September 30, 2025, samples from 154 patients were tested, including 150 peripheral blood samples and 4 bone marrow samples. Among these, 59 patients had undergone targeted therapy, 24 patients were followed up after conventional chemotherapy for multiple myeloma (MM), and 71 patients were followed up after hematopoietic stem cell transplantation. The 59 patients who underwent targeted therapy included 52 patients with B-NHL and 7 other patients; the 71 patients who were followed up after hematopoietic stem cell transplantation included 29 patients with acute myeloid leukemia (AML), 19 patients with B-cell acute lymphoblastic leukemia (ALL) / lymphoblastic lymphoma (LBL), 12 patients with T-ALL / LBL, 7 patients with chronic myeloid tumors, and 4 other patients. The results showed that among 52 B-NHL patients after targeted therapy, 22 patients in remission after 3 months of targeted therapy had a normal MDSC percentage, with a median MDSC percentage of 0.81% (0.12-1.78%) of single viable cells. However, the MDSC percentage was elevated in 26 patients in a specific treatment phase and 4 patients who did not achieve remission, with a median MDSC percentage of 2.77% (0.35-4.59%) of single nucleated cells (P<0.05). This is similar to previously reported results, possibly due to the small sample size and lack of significant differences in subgroup distribution. Other diseases have fewer cases and are still being analyzed.

[0104] In summary, this method solves the challenge of detecting MDSCs and their subsets in whole blood samples using mass flow cytometry, filling the only current shortcoming of this high-end technology in detecting refined immune cell subsets. This method allows for the simultaneous detection of MDSCs and their subsets, as well as eosinophils and basophils, using whole blood samples. It also fully utilizes the multicolor capabilities of mass flow cytometry to simultaneously observe the expression differences and similarities between MDSCs and their subsets and other major related myeloid cell lines. Using this method, especially the modified version, fully leverages the advantages of mass flow cytometry and possesses enormous application potential.

Claims

1. A reagent composition for detecting myeloid-derived suppressor cells in whole blood by mass spectrometry flow cytometry and its application, characterized in that, The reagent composition comprises three groups of reagents, wherein: The first set of reagents consists of the metal-labeled cell-activating dye cisplatin Pt194, which is added to test tubes containing a single-cell suspension of the sample to be tested. The second group of reagents consists of metal-labeled CD3, CD4, CD11c, CD24, CD56, CD66b, CD45, CD19, CD16, CD14, CD123, CD9, HLA-DR, CD15, CD33, CD11b, CD38, and CD183 antibodies. The metal labels are in the following order: In115, Dy163, Tb159, Sm154, Pr141, Ho165, Tm169, Nd148, Lu175, Nd144, Dy162, La139, Yb176, Sm147, Eu151, Bi209, Yb172, and Er166. This reagent is used to add to test tubes containing a single-cell suspension of the sample to be tested. The third set of reagents consists of metal-labeled DNA1 dye Ir191 and DNA2 dye Ir193, which are added to test tubes in which the sample to be tested is in a single-cell suspension state. The first and third groups consist of metal-labeled dyes, while the second group contains monoclonal antibodies. All three groups of reagents are added to the same tube for use.

2. The reagent composition according to claim 1, characterized in that: The second group of reagents is a mixture of CD3 antibody, CD4 antibody, CD11c antibody, CD24 antibody, CD56 antibody, CD66b antibody, CD45 antibody, CD19 antibody, CD16 antibody, CD14 antibody, CD123 antibody, CD9 antibody, HLA-DR antibody, CD15 antibody, CD33 antibody, CD11b antibody, CD38 antibody, and CD183 antibody in a volume ratio of 4:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1 The third group of reagents is a mixture of DNA1 dye and DNA2 dye in a 1:1 volume ratio.

3. A reagent composition for detecting myeloid-derived suppressor cells by mass spectrometry flow cytometry, the kit comprising a first container, a second container, and a third container, each container containing a first group of reagents, a second group of reagents, and a third group of reagents of the reagent composition according to any one of claims 1-2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes one or more of the following: cell lysis buffer, FACS buffer, blocking solution, fixative, and flow cytometry tubes for use with a flow cytometer.

5. The use of the reagent composition according to any one of claims 1-2 in the preparation of flow cytometry samples for the detection of whole blood myeloid-derived suppressor cells by mass spectrometry.

6. The application according to claim 5, characterized in that, The process for preparing the reagent composition for detecting whole blood myeloid-derived suppressor cells by mass cytometry includes the following steps: (1) Add the sample to be tested to a 15ml test tube 1 to make it into a single-cell suspension, and ensure that the cell count is 1×10⁻⁶. 6 / tube-1×10 7 / Tube; (2) Add 1× cell lysis buffer to test tube 1 and incubate at room temperature in the dark; (3) Centrifuge test tube 1 after incubation in step (2) and remove the supernatant; (4) Add FACS buffer to test tube 1 after removing the supernatant in step (3), wash, centrifuge, and remove the supernatant; (5) Add FACS buffer to test tube 1 after removing the supernatant in step (4), transfer to test tube 2, centrifuge and remove the supernatant; (6) Add the first group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 after removing the supernatant in step (5), and incubate at room temperature in the dark; (7) Add FACS buffer to test tube 2 after step (6) and wash, then centrifuge and discard the supernatant; (8) Add the blocking solution and the second group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 after removing the supernatant in step (7), mix well and incubate at room temperature in the dark; (9) Add FACS buffer to test tube 2 after incubation in step (8) and wash. After centrifugation, remove the supernatant. (10) Add fixative and the third group of reagents in the reagent composition of any one of claims 1-2 to the test tube 2 obtained in step (9), and incubate at room temperature in the dark; (11) Add FACS buffer to test tube 2 after incubation in step (10) and wash. After centrifugation, remove the supernatant. (12) Resuspend the cells in FACS buffer to obtain the flow cytometry sample.

7. A device for detecting myeloid-derived suppressor cells in whole blood using mass spectrometry flow cytometry, characterized in that, The device includes a detection unit and an analysis unit, wherein: The detection unit includes reagent materials for detecting samples from the individual to be tested by mass spectrometry flow cytometry, and obtaining the detection results of the samples; the reagent materials include the reagent composition according to any one of claims 1-2; The analysis unit is used to analyze the detection results of the detection unit.

8. The apparatus according to claim 7, characterized in that, This device is used in mass flow cytometry to detect myeloid-derived suppressor cells in whole blood, wherein... The process of detecting samples from the individual being tested using mass flow cytometry includes: The sample to be tested is treated with the reagent composition according to any one of claims 1-2 to prepare a flow cytometry sample; Perform flow cytometry analysis; During mass cytometry analysis, the de-adhesion cell gate and the live cell gate are set sequentially to obtain single live cells. And the door should be installed in the following manner: Major subpopulation phylogenetics: Within the single viable cell phylogenetic group, a CD45 / CD66b-positive mononuclear cell phylogenetic group is established, along with a CD45 / CD66b-positive granulocyte phylogenetic group. A CD14 / CD4 two-dimensional dot plot is used, selecting CD14-positive cells as the monocyte phylogenetic group. A CD19 / HLA-DR two-dimensional dot plot is used, selecting CD19-positive and HLA-DR-positive cells as the B cell phylogenetic group, and a CD19-negative and HLA-DR-negative cell phylogenetic group is established. Sequential gating: A two-dimensional scatter plot of CD3 / CD56 is set within the 19-DR- gate. A 3-56- gate is set for CD3-negative and CD56-negative cells. A two-dimensional scatter plot of CD16 / CD11b is set within the 3-56- gate. A 16-11b+ gate is set for CD16-negative and CD11b-positive cells. A two-dimensional scatter plot of CD9 / CD33 is set within the 16-11b+ gate. CD9-positive cells are selected as eosinophils and basophils, named the baso and eo gates. CD9-negative and CD33-positive cells are designated as myeloid-derived suppressor cells (MDSC). Within the MDSC gate, CD... 15 / CD14 was used to establish gates for each subset of MDSCs. CD15-positive and CD14-negative cells were grouped into the P-MDSC gate for polymorphonuclear myeloid repressive cells, CD14-positive and CD15-negative cells were grouped into the M-MDSC gate for mononuclear myeloid repressive cells, and CD15-negative and CD14-negative cells were grouped into the E-MDSC gate for early myeloid repressive cells. Within the baso and eo gates, CD123 / CD183 were used to establish gates for eosinophils and basophils, respectively. CD123-negative and CD183-positive cells were grouped into the eosinophil gate, and CD123-positive and CD183-negative cells were grouped into the basophil gate. Grouping and phylogenetic designation: Grouping MDSC, eo, and baso is designated as the weak myeloid subpopulation; grouping M-MDSC, E-MDSC, and monocytes is designated as ME and Mono; grouping P-MDSC, E-MDSC, and granulocytes is designated as PE and Gra. This study demonstrates the phenotypic correlation between weak myeloid subpopulations and related major myeloid subpopulations: Within the weak myeloid subpopulation phylum, two-dimensional dot plots of CD45 / CD66b, CD123 / CD66b, CD15 / CD24, CD4 / CD11c, CD123 / CD183, and CD9 / CD123 are used to display the locations of P-MDSC, M-MDSC, E-MDSC, baso, and eo cell populations, determining the verifiability and expression characteristics of each population. Within the ME and Mono phyla, two-dimensional dot plots of CD4 / CD11c, CD45 / CD66b, and CD38 / HLA-DR are used to display the phenotypic evolution from E-MDSC to M-MDSC to monocytes. Within the PE and Gra phyla, a two-dimensional dot plot of CD24 / CD11c is used to display the phenotypic evolution from E-MDSC to P-MDSC to granulocytes.

9. The apparatus according to claim 7 or 8, characterized in that, When the analysis unit analyzes the detection results of the detection unit, it outputs the detection results according to the following judgment method: Method 1: Myeloid-derived suppressor cells (MDSCs): Other cells were excluded by phylogenetic analysis using CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, and CD11b-positive cells. CD9-negative and CD33-positive cells were identified as myeloid-derived suppressor cells (MDSCs). Eosinophils and basophils: Using CD19-negative, HLA-DR-negative, CD3-negative, CD56-negative, CD16-negative, CD11b-positive, and CD9-positive cells as a phylogenetic marker to exclude other cells, CD123-negative and CD183-positive cells are eosinophils, and CD123-positive and CD183-negative cells are basophils. Method 2: Immunophenotypic differences among weak myeloid subsets: The immunophenotype of MDSCs is weakly positive for CD45, differentially expressed for CD66b, CD11c, CD24, CD15 and CD14, and negative for CD4, CD9, CD123, CD183 and CD38. The P-MDSC immunophenotype is weakly positive for CD45, moderately positive for CD11c, positive for CD66b, CD24 and CD15, and negative for CD14, CD4, CD9, CD123, CD183 and CD38. The M-MDSC immunophenotype is weakly positive for CD45 and CD38, moderately positive for CD11c, positive for CD14, and negative for CD66b, CD24, CD15, CD4, CD9, CD123, and CD183. The E-MDSC immunophenotype is weakly positive for CD45 and CD11c, and negative for CD66b, CD24, CD15, CD14, CD4, CD9, CD123, CD183 and CD38. The eosinophil immunophenotype is strongly positive for CD45, moderately positive for CD11c, positive for CD66b, CD9, CD24, CD15 and CD183, and negative for CD14, CD4, CD123 and CD38. The basophil immunophenotype is moderately positive for CD45 and CD11c, positive for CD9, CD123 and CD38, and negative for CD66b, CD14, CD4, CD24, CD15 and CD183. Method 3: The proportion of MDSCs in nucleated cells exceeds 1.54%, indicating that the body's immune system is in a state of myeloid suppression.

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