Reagent composition capable of being used for detecting erythrocyte line cell differentiation through flow cytometry and application of reagent composition

Abstract

The invention discloses a reagent composition capable of being used for flow cytometry detection of erythrocyte line cell differentiation and application of the reagent composition. The reagent composition disclosed by the invention is used for being added into a flow type tube in a single cell suspension state of a sample to be detected, and comprises an anti-CD71 antibody, an anti-CD117 antibody, an anti-CD34 antibody, an anti-CD105 antibody, an anti-CD36 antibody and an anti-CD45 antibody. The reagent composition disclosed by the invention can be applied to flow cytometry for detecting the cell differentiation condition of the red blood cell line, screening and / or diagnosing and follow-up visit, so that high-sensitivity and specific diagnosis is realized, follow-up visit after treatment can be realized, a standardized scheme is formed, the efficiency and the accuracy are improved, and convenience is provided for realizing automation and artificial intelligence in the future.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a reagent composition that can be used for flow cytometry to detect erythrocyte differentiation and its application. Background Technology

[0002] Erythroid cell differentiation is a crucial component of the hematopoietic process, beginning with hematopoietic stem / progenitor cells and progressing through stages such as erythroid progenitor cells, proerythroblasts, early erythroblasts, intermediate erythroblasts, and late erythroblasts, ultimately differentiating into mature erythrocytes. Abnormalities in erythroid cell differentiation are closely related to the occurrence and development of various diseases. For example, impaired or disordered erythroid differentiation is observed in hematological diseases such as myelodysplastic syndromes, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia. Therefore, accurate identification and detection of erythroid cell differentiation stages can be used for the auxiliary diagnosis, disease monitoring, treatment evaluation, and screening of these diseases, possessing significant clinical value and application prospects.

[0003] Currently, the main techniques used to detect and identify erythroid cell differentiation include cell morphology observation and flow cytometry. However, these existing methods have significant limitations: 1. Cell morphology observation: highly dependent on the experience of the examiner, subjective, poor reproducibility, and difficult to accurately identify cells in the early stages of differentiation with atypical morphological characteristics. 2. Traditional flow cytometry analysis: although providing an objective quantitative method, it usually uses only a single or a few cell surface markers, most commonly CD235a and CD71. This simple combination cannot accurately distinguish the continuous intermediate stages of erythroid cell differentiation, especially for early and mid-stage cells with overlapping phenotypes, resulting in limited identification ability, insufficient resolution, and a need to improve sensitivity and specificity. It also limits the types of diseases that can be screened and diagnosed. Therefore, there is an urgent need in this field to develop a detection tool that can highly sensitively and specifically distinguish different stages of erythroid cell differentiation and has multi-disease coverage. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, one of the objectives of the present invention is to provide a reagent composition that can be used for flow cytometry to detect erythroid cell differentiation. It is suitable for screening or assisting in the diagnosis of, but not limited to, myelodysplastic syndromes, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia, with or without any clinical information, by detecting erythroid cell differentiation through highly sensitive, specific, and universal flow cytometry.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a reagent composition for flow cytometry detection of erythrocyte differentiation. The composition is added to a flow cytometry tube containing a single-cell suspension of the sample. It comprises anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody. All antibody components in this antibody composition are commercially available. Each antibody should meet relevant industry standards.

[0007] In some embodiments of the present invention, each antibody is a monoclonal antibody.

[0008] In some embodiments of the present invention, each antibody is a fluorescently labeled antibody; the fluorescent labeling order of the anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody is FITC (Fluorescein Isothiocyanate), PE (Phycoerythrin), ECD (Phycoerythrin-Texas), PC7 (Phycoerythrin-Cyanin 7), APC (Allophycocyanin), and KO (Krome Orange). By combining different antibodies with specific fluorescent dyes, the antibody compositions of the present invention can achieve excellent staining effects for all fluorescent dyes in each channel when applied to screening and / or diagnosis and follow-up of conditions including but not limited to myelodysplastic syndromes, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0009] In some embodiments of the present invention, the volume ratio of anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody is 8:4:6:3:4:4. The mixing ratio of each antibody mentioned above refers to the mixing ratio when the titers of each antibody are substantially equivalent.

[0010] A second objective of this invention is to provide a kit comprising a container for containing the reagent composition described above, which can be used for flow cytometry detection of erythroid cell differentiation. This kit can be applied to flow cytometry detection of erythroid cell differentiation for screening and / or diagnosis and follow-up, including but not limited to myelodysplastic syndromes, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia. It achieves highly sensitive and specific diagnosis, enables post-treatment follow-up, establishes a standardized protocol, improves efficiency and accuracy, and facilitates future automation and artificial intelligence implementation.

[0011] In some embodiments of the present invention, the kit includes multiple containers, each containing the antibody, hemolysin, buffer solution, and flow cytometry tubes for use with a flow cytometer. These reagents and consumables are commercially available.

[0012] In some embodiments of the present invention, the amount of antibody added to the reagent composition of the present invention is 14-35 μL / tube.

[0013] A third objective of this invention is to provide the application of the reagent composition described above, which can be used for flow cytometry detection of erythrocyte differentiation, in the preparation of flow cytometry samples for detecting erythrocyte differentiation. In this invention, the detection includes screening, diagnosis, and / or follow-up detection.

[0014] In some embodiments of the present invention, the erythroid cells differentiate into abnormal erythroid cell differentiation caused by myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0015] In some embodiments of the present invention, the preparation of flow cytometry samples for detecting erythroid cell differentiation specifically includes the following steps:

[0016] (1) Add the sample to be tested into a flow cytometry tube to form a single-cell suspension, ensuring that the number of nucleated cells is 5 x 10^6 cells / mL. 9 / pipe-20X10 9 / Tube;

[0017] (2) Add the reagent composition described above, which can be used for flow cytometry to detect erythrocyte differentiation, to the flow cytometer obtained in step (1), and incubate at room temperature in the dark.

[0018] (3) Add 1X hemolysin to the flow cytometer after incubation in step (2) and incubate at room temperature in the dark;

[0019] (4) Wash the flow cytometry tube after incubation in step (3) with PBS buffer, centrifuge and remove the supernatant, then resuspend the cells with PBS buffer to obtain the flow cytometry sample for detecting erythrocyte differentiation.

[0020] In some embodiments of the present invention, the sample to be tested may be bone marrow or peripheral blood, or it may be any sample that can be prepared into a single live cell and is suitable for flow cytometry detection, such as tissue sample or body fluid sample.

[0021] In some embodiments of the present invention, in step (1), the volume of each sample added does not exceed 100 μL. If the patient has a low number of peripheral blood cells, the volume can be increased to more than 160 μL if necessary, and the sample is centrifuged to remove the supernatant and concentrated.

[0022] In some embodiments of the present invention, the amount of PBS added each time is 2-3 ml / tube, and the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes.

[0023] In some embodiments of the present invention, the incubation time in step (2) may be 10-30 minutes.

[0024] In some embodiments of the present invention, in step (3), the incubation time may be 10-30 minutes. The amount of 1X hemolysin added is 2-3 ml / tube.

[0025] In some embodiments of the present invention, in step (4), the amount of PBS buffer added for washing is 2-3 ml / tube; the centrifugation conditions can be 1000-2000 rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspending is 0.5-1 ml / tube.

[0026] In some embodiments of the present invention, when resuspending cells for flow cytometry analysis, CD36 / CD71 and CD45 / SSC samples are selected to screen nucleated erythrocytes, CD117 and CD105 are combined to stage nucleated erythrocytes, and CD71 and CD36 are combined with CD105 to view the erythrocyte differentiation trajectory for analysis.

[0027] In some embodiments of the present invention, the dosage of each reagent may refer to the conventional dosage in the art or the manufacturer's recommended dosage.

[0028] The present invention also provides an apparatus for detecting erythroid cell differentiation, comprising a detection unit and an analysis unit; wherein: the detection unit is used to perform flow cytometry detection on reagent materials of a sample from a test individual to obtain the test results; the reagent materials include the reagent composition described above that can be used for flow cytometry detection of erythroid cell differentiation; the analysis unit is used to analyze the test results of the sample to identify (including auxiliary identification) conditions including but not limited to myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0029] In some embodiments of the present invention, during flow cytometry analysis, the following gates are set: a de-adhesion cell gate P1 is set, and a live cell gate P2 is set within P1 to obtain single live cells; within gate P2, CD45 / SSC antibodies are used to view cell populations; within gate P2, a P3 gate is set using CD45 / CD34 antibodies to detect CD34+ cell populations; within gate P3, a K gate is set using CD45 / SSC antibodies to detect non-lymphatic progenitor cells; within gate P2, a P4 gate is set using CD71 / CD36 to detect CD36+CD71+ cells; within gate P4, an ErBs gate is set using CD45 / SSC antibodies to circle nucleated erythrocytes; and within the ErBs display gate, gates are set according to CD105 expression. The system uses several gates. Gate P5 encloses CD105-nucleated erythrocytes as late erythroblasts. Within gate P5, further gates are set based on CD117 expression: gate P6 encloses CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 encloses CD117-nucleated erythrocytes as intermediate erythroblasts. A combined ErBs and K gate is used to visualize the expression of non-lymphatic blasts and nucleated erythrocytes in CD105 / CD71. A combined ErBs and K gate is also used to visualize the expression of non-lymphatic blasts and nucleated erythrocytes in CD105 / CD36. Gate P6, based on CD105 expression, categorizes cells into CD105dim early erythroblasts and CD105+ early erythroblasts.

[0030] In this invention, a combined ErBs and K gate logic gate is used to compare the normal differentiation trajectory of nucleated erythrocytes. Patients with myelodysplastic syndrome show decreased CD71 and / or CD36 expression and increased CV; while megaloblastic anemia shows increased CD36 expression in the intermediate erythroblast population; in typical pure red cell leukemia patients, the proportion of CD36+CD71+ nucleated erythrocytes in the bone marrow is significantly increased, accompanied by a significant increase in the proportion of early erythroblasts among all nucleated erythrocytes; the P6 gate is divided into CD105dim early erythroblasts and CD105+ early erythroblasts based on CD105 expression, mainly used for the diagnosis of chronic myeloid leukemia. It can be seen that the proportion of CD105dim early erythroblasts among all early erythroblasts is significantly increased (in the data statistics of this invention, >50% is a critical value with high sensitivity and specificity).

[0031] In the apparatus for detecting erythroid cell differentiation of the present invention, the analysis unit may further include a module for further determining the disease based on the gating analysis results (the determination described in the present invention includes screening, indication, auxiliary diagnosis, diagnosis, and follow-up of minimal residual disease). The present invention compares the displayed cell populations with corresponding normal cells within a multi-marker gating system to identify abnormally expressing cells and determine the nature of these abnormally expressing cells.

[0032] Specifically, the present invention can be used for screening and / or diagnosis and screening of myelodysplastic syndromes, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia, including but not limited to myelodysplastic syndromes.

[0033] In this invention, when analyzing samples, the universally applicable CD45 / SSC and CD45 / CD34 are used to select non-lymphocytic progenitor cells, including progenitor erythrocytes. Then, CD36 / CD71 and CD45 / SSC are used to select nucleated erythrocytes. Subsequently, the expression of CD105 / CD117 is used to further group early erythroblasts, intermediate erythroblasts, and late erythroblasts. Specifically, the determination of various diseases can be performed as follows:

[0034] (1) Identification of pure red cell leukemia: In normal samples, when using a general protocol to select nucleated red blood cells based on CD36 / CD71 and CD45 / SSC, the proportion of nucleated red blood cells is generally <10% of all nucleated cells. In hemolytic anemia, the proportion of nucleated red blood cells may reach 20% of all nucleated cells, but the proportions of early erythroblasts, intermediate erythroblasts, and late erythroblasts are normal when combined with CD105 / CD117 expression, with intermediate and late erythroblasts being the predominant group. In typical patients with pure red cell leukemia, the proportion of CD36+CD71+ nucleated red blood cells in the bone marrow usually exceeds 30%, even reaching 50%-80% or more, and is accompanied by a significant increase in the proportion of early erythroblasts among all nucleated red blood cells.

[0035] (2) Identification of Myelodysplastic Syndromes: Myelodysplastic syndromes often involve one or more lineage abnormalities, among which erythroid abnormalities are very common. Based on the expression of nucleated erythrocytes in CD105 / CD36 and CD105 / CD71, common erythroid differentiation abnormalities in myelodysplastic syndromes include decreased CD36 expression intensity, increased CV of CD36 expression, decreased CD71 expression intensity, and increased CV of CD71 expression, which may be accompanied by one or more abnormalities.

[0036] (3) Identification of megaloblastic anemia: Increased expression intensity of CD36 in intermediate erythroblast populations is a characteristic expression of megaloblastic anemia. This is applicable to the auxiliary diagnosis of untreated megaloblastic anemia. If the patient has received folic acid and B12 supplementation therapy beforehand, the increase in CD36 expression intensity in intermediate erythroblast populations may not be obvious.

[0037] (4) Identification of Chronic Myeloid Leukemia (CML): A standard protocol was used to select nucleated erythrocytes based on CD36 / CD71 and CD45 / SSC, with a normal percentage of nucleated erythrocytes. Based on CD105 expression, cells were divided into CD105dim erythroblasts and CD105+ erythroblasts. The proportion of CD105dim erythroblasts among all erythroblasts was significantly increased (in the statistical data of this invention, >50% is a critical value with high sensitivity and specificity).

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) High resolution and accuracy: The reagent composition of the present invention achieves high-precision identification of the entire erythroid differentiation lineage, especially the early and middle transitional cells that are difficult to distinguish by traditional methods, by carefully screening and combining multiple erythroid differentiation stage-specific markers. Its resolution is significantly better than traditional schemes that rely on a single or a few markers.

[0040] (2) Objectivity and quantifiability: The detection methods based on this reagent composition (such as multicolor flow cytometry) can achieve rapid, automated and quantitative analysis, and the results are objective and reliable. This overcomes the shortcomings of strong subjectivity and poor repeatability of cell morphology examination, and is conducive to standardization and result comparison between laboratories.

[0041] (3) Convenient and efficient operation: This invention provides a ready-to-use multicolor antibody combination, which simplifies the experimental process. A comprehensive erythroid differentiation map can be obtained with one staining, which improves the detection efficiency and is suitable for clinical testing and large-scale sample screening.

[0042] (4) Strong clinical application potential: The application scope of this reagent composition includes, but is not limited to, myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, chronic myeloid leukemia, etc. It provides a powerful new tool for screening, prompting, assisting diagnosis, diagnosis and follow-up of diseases, and has important clinical translational value.

[0043] (5) After the scheme is standardized and universalized, it can promote the pipeline work of sample processing, machine acquisition and data analysis, realize automation, and create conditions for the future development of artificial intelligence.

[0044] (6) Filling a gap in the application of flow cytometry: Chronic myeloid leukemia was once identified by the Bethesda consensus as one of the diseases unsuitable for diagnosis by flow cytometry, mainly due to the lack of characteristic features in flow cytometry. However, the application of this invention has high sensitivity and high specificity for the diagnosis of chronic myeloid leukemia. Megaloblastic anemia is generally diagnosed by detecting folic acid and vitamin B12 and by artificial microscopy of bone marrow. It also has characteristic manifestations in erythroid cell differentiation and can be used as an auxiliary diagnostic tool. Attached Figure Description

[0045] Figure 1 This invention displays the results of flow cytometry gating analysis of normal bone marrow samples according to a specific embodiment of the present invention. (a) to (1) are respectively: (a) FS-PEAK / INT sets P1 as the de-adhesion cell gate; (b) within P1, FS / SS sets P2 as the live cell gate; (c) within P2, CD45 / SS is used to set the blood cell gate, allowing for preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; (d) within P2, CD45 / CD34 antibody is used to set P3 to detect CD34+ cell populations; (e) within P3, CD45 / SSC antibody is used to set the K gate to circle non-lymphatic progenitor cells; (f) within P2, CD71 / CD36 is used to set the P4 gate to circle CD36+CD71+ cells; (j) within P4, CD45 / SSC antibody is used to set the ErBs gate to circle nucleated erythrocytes; (h) ErBs is displayed. (i) Within the Bs gate, gates are set up based on CD105 expression. Gate P5 circles CD105- nucleated erythrocytes as late erythroblasts; (i) Within the P5 gate, gates are set up based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; (j) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD71 is displayed; (k) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD36 is displayed; (l) Within the P6 gate, based on CD105 expression, cells are divided into CD105dim early erythroblasts and CD105+ early erythroblasts.

[0046] Figure 2This invention displays the results of flow cytometry gating analysis of bone marrow samples from myelodysplastic syndrome according to a specific embodiment of the present invention. (a) to (1) are respectively: (a) FS-PEAK / INT sets P1 as the de-adhesion cell gate; (b) within P1, FS / SS sets P2 as the live cell gate; (c) within P2, CD45 / SS is used to set the blood cell gate, allowing preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; (d) within P2, CD45 / CD34 antibody is used to set P3 to detect CD34+ cell populations; (e) within P3, CD45 / SSC antibody is used to set the K gate to circle non-lymphatic progenitor cells; (f) within P2, CD71 / CD36 is used to set the P4 gate to circle CD36+CD71+ cells; (j) within P4, CD45 / SSC antibody is used to set the ErBs gate to circle nucleated erythrocytes; (h) ErBs is displayed. (i) Within the Bs gate, gates are set up based on CD105 expression. Gate P5 circles CD105- nucleated erythrocytes as late erythroblasts; (i) Within the P5 gate, gates are set up based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; (j) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD71 is displayed; (k) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD36 is displayed; (l) Within the P6 gate, based on CD105 expression, cells are divided into CD105dim early erythroblasts and CD105+ early erythroblasts.

[0047] Figure 3This invention presents the results of flow cytometry gated analysis of bone marrow samples from megaloblastic anemia according to a specific embodiment of the present invention. (a) to (1) are respectively: (a) FS-PEAK / INT sets P1 as the de-adhesion cell gate; (b) within P1, FS / SS sets P2 as the live cell gate; (c) within P2, CD45 / SS is used to set the blood cell gate, allowing preliminary observation of the grouping of lymphocytes, granulocytes, monocytes, and nucleated erythrocytes; (d) within P2, CD45 / CD34 antibody is used to set P3 to detect CD34+ cell populations; (e) within P3, CD45 / SSC antibody is used to set the K gate to circle non-lymphatic progenitor cells; (f) within P2, CD71 / CD36 is used to set the P4 gate to circle CD36+CD71+ cells; (j) within P4, CD45 / SSC antibody is used to set the ErBs gate to circle nucleated erythrocytes; (h) ErBs is displayed. (i) Within the Bs gate, gates are set up based on CD105 expression. Gate P5 circles CD105- nucleated erythrocytes as late erythroblasts; (i) Within the P5 gate, gates are set up based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; (j) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD71 is displayed; (k) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD36 is displayed; (l) Within the P6 gate, based on CD105 expression, cells are divided into CD105dim early erythroblasts and CD105+ early erythroblasts.

[0048] Figure 4This invention displays the gating results of flow cytometry analysis of bone marrow samples from pure red cell leukemia according to a specific embodiment of the present invention. (a) to (1) are respectively: (a) FS-PEAK / INT sets P1 as the de-adhesion cell gate; (b) within P1, FS / SS sets P2 as the live cell gate; (c) within P2, CD45 / SS is used to set the blood cell gate, allowing preliminary observation of the grouping of lymphocytes, granulocytes, monocytes, and nucleated erythrocytes; (d) within P2, CD45 / CD34 antibody is used to set P3 to detect CD34+ cell populations; (e) within P3, CD45 / SSC antibody is used to set the K gate to circle non-lymphatic progenitor cells; (f) within P2, CD71 / CD36 is used to set the P4 gate to circle CD36+CD71+ cells; (j) within P4, CD45 / SSC antibody is used to set the ErBs gate to circle nucleated erythrocytes; (h) ErBs is displayed. (i) Within the Bs gate, gates are set up based on CD105 expression. Gate P5 circles CD105- nucleated erythrocytes as late erythroblasts; (i) Within the P5 gate, gates are set up based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; (j) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD71 is displayed; (k) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD36 is displayed; (l) Within the P6 gate, based on CD105 expression, cells are divided into CD105dim early erythroblasts and CD105+ early erythroblasts.

[0049] Figure 5This invention displays the gating analysis results of flow cytometry on bone marrow samples from chronic myeloid leukemia according to a specific embodiment of the present invention. (a) to (1) are respectively: (a) FS-PEAK / INT sets P1 as the de-adhesion cell gate; (b) within P1, FS / SS sets P2 as the live cell gate; (c) within P2, CD45 / SS is used to set the blood cell gate, allowing preliminary observation of the grouping of lymphocytes, granulocytes, monocytes, and nucleated erythrocytes; (d) within P2, CD45 / CD34 antibody is used to set P3 to detect the CD34+ cell population; (e) within P3, CD45 / SSC antibody is used to set the K gate to circle non-lymphatic progenitor cells; (f) within P2, CD71 / CD36 is used to set the P4 gate to circle CD36+CD71+ cells; (j) within P4, CD45 / SSC antibody is used to set the ErBs gate to circle nucleated erythrocytes; (h) ErBs is displayed. (i) Within the Bs gate, gates are set up based on CD105 expression. Gate P5 circles CD105- nucleated erythrocytes as late erythroblasts; (i) Within the P5 gate, gates are set up based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; (j) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD71 is displayed; (k) Using a logic gate combining ErBs and K gates, the expression of non-lymphatic blast cells and nucleated erythrocytes in CD105 / CD36 is displayed; (l) Within the P6 gate, based on CD105 expression, cells are divided into CD105dim early erythroblasts and CD105+ early erythroblasts. Detailed Implementation

[0050] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0051] Example 1

[0052] This embodiment provides a reagent composition for flow cytometry detection of erythrocyte differentiation, comprising: anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody. The fluorescein labeling order of each antibody is FITC (Fluorescein Isothiocyanate), PE (Phycoerythrin), ECD (Phycoerythrin-Texas), PC7 (Phycoerythrin-Cyanin 7), APC (Allophycocyanin), and KO (Krome Orange). The above six monoclonal antibody reagents are mixed in a volume ratio of 8:4:6:3:4:4 and packaged in a container.

[0053] This embodiment also provides a kit containing the above-described reagent composition.

[0054] All antibodies used in this embodiment are commercially available. CD36-APC (clone number 5-271) is a product of BioLegend, Inc., USA, while the other fluorescently labeled antibodies are products of Beckman Coulter, Inc., USA.

[0055] Example 2

[0056] The test sample was treated using the reagent composition of Example 1:

[0057] According to the cell counting results, add the sample to tube A of the flow cytometer, ensuring that the number of cells added is approximately 2 x 10⁶. Then, add 29 ml of six different fluorescently labeled monoclonal antibody reagents to the flow cytometer according to Table 1. After thoroughly mixing with the cell suspension, incubate at room temperature in the dark for 15 minutes. Add 3 ml of 1X hemolysin and incubate in the dark for 10 minutes to lyse the red blood cells. Centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 3 ml of PBS, mix well, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and resuspend the cells in 0.5 ml of PBS buffer. This is the processed flow cytometry sample, ready for flow cytometry analysis.

[0058] In this embodiment, hemolysin and PBS buffer are commercially available. Hemolysin is a product of Tiangen Biotech (Beijing) Co., Ltd., and PBS buffer is a product of Beckman Coulter.

[0059] Table 1. Details of Antibodies in Reagent Compositions and Recommended Dosage

[0060] fluorescein Antibody Dosage FITC CD71 8ml PE CD117 4ml ECD CD34 6ml PC7 CD105 3ml APC CD36 4ml KO CD45 4ml

[0061] Example 3

[0062] The samples processed according to the method in Example 2 were analyzed on a Beckman Coulter 3-laser 10-color Navios flow cytometer in the United States. It is preferred to obtain 1 million cells per tube (at least 300,000 is recommended). The data were then analyzed using other software such as Kaluza.

[0063] The gating method for flow cytometry analysis is as follows:

[0064] ① Fixed gating: The adhesion cell gating and live cell gating were performed sequentially; ② Multi-marker combination gating to delineate non-lymphoid primitive cells and nucleated erythrocytes: Starting from a single live cell, the gating and delineation of all cells were performed with reference to biological controls; ③ Within the multi-marker combination gating, the normal erythrocyte differentiation trajectory was constructed as a reference by statistically analyzing the expression of a large number of normal samples, which facilitates the visual detection of weakened erythrocyte population expression; ④ Based on the expression of CD117 and CD105, nucleated erythrocytes at various common stages were distinguished.

[0065] 1. Fixed phylum: consisting of de-adhesion cell phylum and live cell phylum, which are connected in series.

[0066] 2. De-adhesion cell gate: First, the de-adhesion cell gate (commonly represented by P1) is set using the integral (INT) and peak (PEAK) of forward scatter (FS). FS-INT represents the total area of ​​the pulse signal, and FS-PEAK represents the peak of the pulse signal. Adhesive cells can be removed by using FS-INT / FS-PEAK, based on the principle that cells are spherical, and the integral and peak are positively correlated. (See...) Figures 1-5 (a) in the middle.

[0067] 3. Viable cell phylogenetics: Cells within P1 are phylogenized using FS / side scatter (SS) (commonly represented as P2) to obtain single viable cells. The principle of FS / SS is that viable cells exhibit a near-normal distribution in size and granularity, clustering around a central point and clearly demarcated from dead cells, apoptotic cells, debris, and background noise (see...). Figures 1-5 (b) in the middle.

[0068] 4. Blood Cell Phylogenetics: Within the single viable cell phylum (P2 phylum), CD45 / SSC is routinely used to establish each blood cell phylum, roughly observing lymphocytes, monocytes, granulocytes, nucleated erythrocytes, and the presence of obvious abnormal cells. CD45 / SSC is used to roughly distinguish different blood cell phyla based on the differences in CD45 expression fluorescence intensity of hematopoietic cells (mature lymphocytes > monocytes > granulocytes > nucleated erythrocytes) and the differences in SSC size (eosinophils > granulocytes > monocytes > mature lymphocytes > nucleated erythrocytes) (see...). Figures 1-5 (c) in the middle.

[0069] 5. Multi-marker gating to identify non-lymphocytic blasts and nucleated erythrocytes: This is performed simultaneously with CD45 / SSC gating, starting with a single live cell (P2). Within P2, a P3 gate is established using CD45 / CD34 antibodies to detect CD34+ cell populations; within P3, a K gate is established using CD45 / SSC antibodies to detect non-lymphocytic blasts; within P2, a P4 gate is established using CD71 / CD36 to detect CD36+CD71+ cells. In typical erythroleukemia patients, the proportion of CD36 and CD71 double-positive nucleated erythrocytes in the bone marrow is usually abnormally high, often exceeding 30%, and even reaching 50%-80% or more; within P4, a CD45 / SSC antibody is used to detect CD34+CD71+ cells. The 45 / SSC antibody was used to enclose nucleated erythrocytes using an ErBs gate. Within the ErBs gate, gates were set based on CD105 expression, with CD105- nucleated erythrocytes classified as late erythroblasts. Further gates were set within the P5 gate based on CD117 expression, with CD117- nucleated erythrocytes classified as intermediate erythroblasts and CD117+ nucleated erythrocytes as early erythroblasts (P6 gate). Using a combined ErBs and K gate logic gate, the expression of CD105 / CD71 in non-lymphocytic blasts and nucleated erythrocytes was visualized. Figures 1-5 Arrow (j) in the tenth inset shows the normal differentiation trajectory of nucleated erythrocytes. Patients with myelodysplastic syndrome exhibit decreased CD71 expression and increased CV. Using a combined ErBs and K gate logic, the expression of non-lymphoid blasts and nucleated erythrocytes in CD105 / CD36 is shown. Figures 1-5 In the eleventh small figure, (k) shows the normal differentiation trajectory of nucleated erythrocytes. Patients with myelodysplastic syndrome show decreased CD36 expression and increased CV, while megaloblastic anemia shows increased CD36 expression in the intermediate erythroblast population. In the P6 phylum, based on CD105 expression, they are divided into CD105dim erythroblasts and CD105+ erythroblasts, which are mainly used for the diagnosis of chronic myeloid leukemia. It can be seen that the proportion of CD105dim erythroblasts in all erythroblasts is significantly increased (in the data statistics of this invention, >50% is a critical value with high sensitivity and specificity).

[0070] This invention compares the displayed erythrocyte differentiation trajectory with the corresponding normal erythrocyte differentiation trajectory within a multi-marker combination gate to identify abnormal erythrocyte differentiation groups, thus aiding in the diagnosis of diseases such as myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0071] In short, this invention, within a multi-marker combination gate, aims to showcase the complete erythrocyte differentiation trajectory as much as possible, covering multiple diseases. It cleverly uses these markers of erythrocyte differentiation trajectory to screen and / or diagnose and assist in the diagnosis of diseases such as myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0072] Specifically, Figure 1 This displays the analysis of a normal bone marrow sample. The settings are as follows: ① In FS-PEAK / INT, set P1 to the de-adhesion cell gate to obtain single cells within P1; ② Within P1, display FS / SS and set P2 to the live cell gate to obtain single live cells within P2; ③ Within P2, use CD45 / SS to set the blood cell gate to preliminarily observe the populations of lymphocytes, granulocytes, monocytes, and nucleated erythrocytes; ④ Within P2, use CD45 / CD34 antibodies to set P3 to detect CD34+ cell populations. ⑤ Within the P3 gate, non-lymphatic progenitor cells were circled using the K gate with CD45 / SSC antibody; ⑥ Within the P2 gate, CD36+CD71+ cells were circled using the P4 gate with CD71 / CD36. In this sample, CD36+CD71+ cells accounted for 2.39% of single nucleated viable cells, a normal proportion; ⑦ Within the P4 gate, nucleated erythrocytes were circled using the ErBs gate with CD45 / SSC antibody; ⑧ The ErBs gate was set according to CD105 expression. ⑨ Gate P5 is used to enclose CD105- nucleated erythrocytes as late erythroblasts; ⑨ Gate P5 is further configured based on CD117 expression, with gate P6 enclosing CD117+ nucleated erythrocytes as early erythroblasts and gate P7 enclosing CD117- nucleated erythrocytes as intermediate erythroblasts; ⑩ Using a combined ErBs and K gate, the expression of non-lymphoid blasts and nucleated erythrocytes in CD105 / CD71 is shown. This sample shows normal nucleated erythrocyte expression. The differentiation trajectory was observed; using a combined ErBs and K gate, the expression of non-lymphoid blast cells and nucleated erythrocytes in CD105 / CD36 was displayed. This sample showed the normal differentiation trajectory of nucleated erythrocytes; in the P6 gate, based on CD105 expression, they were divided into CD105dim erythroblasts and CD105+ erythroblasts. In this sample, CD105dim erythroblasts accounted for 13.88% of erythroblasts, which is a normal proportion.

[0073] Specifically, Figure 2This demonstrates the analysis of bone marrow samples from patients with myelodysplastic syndrome. The following settings were executed sequentially: ① FS-PEAK / INT was set to P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FS / SS was used to set P2 as the live cell gate, resulting in single live cells within P2; ③ Within P2, CD45 / SS was used to set the blood cell gate, allowing for preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; ④ Within P2, CD45 / CD34 antibodies were used to set the P3 gate to detect CD34+ cell populations; ⑤ Within the P3 gate, non-lymphatic progenitor cells were enclosed using a K gate set with CD45 / SSC antibody; within the P2 gate, CD36+CD71+ cells were enclosed using a P4 gate set with CD71 / CD36. In this sample, CD36+CD71+ cells accounted for 1.67% of single nucleated viable cells, a normal proportion; within the P4 gate, nucleated erythrocytes were enclosed using an ErBs gate set with CD45 / SSC antibody; the ErBs gate was set based on CD105 expression, with P... Gate 5 circles CD105- nucleated erythrocytes as late erythroblasts; ⑨ Further gates are set within gate P5 based on CD117 expression: gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts; ⑩ Using a combined ErBs and K gate, the expression of CD105 / CD71 in non-lymphoid blasts and nucleated erythrocytes is shown. This sample contains intermediate erythroblasts with CD71 expression CV. Increased expression; using a combined ErBs and K gate, the expression of CD105 / CD36 in non-lymphoid blast cells and nucleated erythrocytes was shown. This sample showed decreased CD36 expression intensity and increased CV in all nucleated erythrocytes; in the P6 gate, based on CD105 expression, early erythroblasts were divided into CD105dim early erythroblasts and CD105+ early erythroblasts. In this sample, CD105dim early erythroblasts accounted for 8.11% of early erythroblasts, which is a normal proportion.

[0074] Specifically, Figure 3This demonstrates the analysis of bone marrow samples from patients with megaloblastic anemia. The following settings were executed sequentially: ① FS-PEAK / INT was used to set P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FS / SS was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SS was used to set the blood cell gate, allowing for preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; ④ Within P2, CD45 / CD34 antibodies were used to set P3 to detect CD34+ cell populations; ⑤ Within P3... Using CD45 / SSC antibody, a K-gate was set to circle non-lymphoid blast cells; within the P2 gate, a P4 gate was set using CD71 / CD36 to circle CD36+CD71+ cells. In this sample, CD36+CD71+ cells accounted for 6.18% of single nucleated viable cells, a normal proportion; within the P4 gate, an ErBs gate was set using CD45 / SSC antibody to circle nucleated erythrocytes; ⑧ The ErBs gate was set based on CD105 expression, with a P5 gate to circle CD105- cells. Nucleated erythrocytes are late erythroblasts; ⑨ Further gates are set within P5 based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts. The distribution ratio of early and intermediate erythroblasts in this sample is normal; ⑩ Using a combined ErBs and K gate, the expression of non-lymphoid blasts and nucleated erythrocytes in CD105 / CD71 is shown. This sample contains intermediate erythroblasts with CD71 expression. 1. Decreased expression intensity and increased CV: Using a logic gate combining ErBs and K gates, the expression of CD105 / CD36 in non-lymphoid blast cells and nucleated erythrocytes was shown. This sample showed increased CD36 expression intensity in all nucleated erythrocytes. In the P6 gate, based on CD105 expression, early erythroblasts were divided into CD105dim early erythroblasts and CD105+ early erythroblasts. In this sample, CD105dim early erythroblasts accounted for 12.54% of early erythroblasts, which is a normal proportion.

[0075] Specifically, Figure 4This demonstrates the analysis of bone marrow samples from patients with erythroleukemia. The settings are as follows: ① In FS-PEAK / INT, set P1 to the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, display FS / SS and set P2 to the live cell gate, resulting in single live cells within P2; ③ Within P2, use CD45 / SS to set the blood cell gate, allowing for preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; ④ Within P2, use CD45 / CD34 antibodies to set P3 to detect CD34+ cell populations; ⑤ Within P3, use... ⑥ Within the P2 gate, CD36+CD71+ cells were enclosed using the P4 gate with CD71 / CD36. In this sample, CD36+CD71+ cells accounted for 47.74% of single nucleated viable cells, a significantly increased proportion. ⑦ Within the P4 gate, nucleated erythrocytes were enclosed using the ErBs gate with CD45 / SSC antibody. ⑧ The ErBs gate was set based on CD105 expression; CD105- nucleated erythrocytes were late erythroblasts. Cells; ⑨ Further gates are set within the P5 gate based on CD117 expression: CD117- nucleated erythrocytes are intermediate erythroblasts, and CD117+ nucleated erythrocytes are early erythroblasts (P6 gate). This sample shows a significantly higher proportion of early erythroblasts, with an approximate ratio of early to intermediate erythroblasts of 5:1; ⑩ Using a combined ErBs and K gate, the expression of non-lymphoid blasts and nucleated erythrocytes in CD105 / CD71 is shown. This sample exhibits weakened CD71 expression in early erythroblasts. And increased CV; using a logic gate combining ErBs and K gates, the expression of CD105 / CD36 in non-lymphoid blast cells and nucleated erythrocytes was shown. In this sample, all nucleated erythrocytes showed increased CD36 expression CV; in the P6 gate, based on CD105 expression, they were divided into CD105dim erythroblasts and CD105+ erythroblasts. In this sample, CD105dim erythroblasts accounted for 74.05% of erythroblasts, which was increased, but the boundary was blurred and the trajectory was abnormal.

[0076] Specifically, Figure 5This demonstrates the analysis of bone marrow samples from patients with chronic myeloid leukemia. The following settings were executed sequentially: ① FS-PEAK / INT was set to P1 as the de-adhesion cell gate, resulting in single cells within P1; ② Within P1, FS / SS was used to set P2 as the live cell gate, again resulting in single live cells within P2; ③ Within P2, CD45 / SS was used to set the blood cell gate, allowing for preliminary observation of lymphocyte, granulocyte, monocyte, and nucleated erythrocyte populations; ④ Within P2, CD45 / CD34 antibodies were used to set P3 to detect CD34+ cell populations. ⑤ Within the P3 gate, non-lymphatic progenitor cells were circled using the K gate with CD45 / SSC antibody; ⑥ Within the P2 gate, CD36+CD71+ cells were circled using the P4 gate with CD71 / CD36 antibody. In this sample, CD36+CD71+ cells accounted for 2.50% of single nucleated viable cells, a normal proportion; ⑦ Within the P4 gate, nucleated erythrocytes were circled using the ErBs gate with CD45 / SSC antibody; ⑧ The ErBs gate was set based on CD105 expression. CD105- nucleated erythrocytes are late erythroblasts; ⑨ Further gates are set within P5 based on CD117 expression: CD117- nucleated erythrocytes are intermediate erythroblasts, and CD117+ nucleated erythrocytes are early erythroblasts (P6 gate). This sample shows a significantly increased proportion of early erythroblasts; ⑩ Using a combined ErBs and K gate, the expression of non-lymphoid blasts and nucleated erythrocytes in CD105 / CD71 is shown. This sample shows early erythroblast C... D71 expression was reduced; using a combined ErBs and K gate, the expression of CD105 / CD36 in non-lymphoid blast cells and nucleated erythrocytes was shown. This sample showed reduced CD36 expression in early erythroblasts; based on CD105 expression, the P6 gate divided early erythroblasts into CD105dim early erythroblasts and CD105+ early erythroblasts. In this sample, CD105dim early erythroblasts accounted for 66.43% of early erythroblasts, a significantly increased proportion.

[0077] The development process of this invention and the clinical validation using the method described in this embodiment: Dongyang People's Hospital in Zhejiang Province began exploring this work in 2020, and a definitive protocol was established in October 2023. As of October 2025, 1916 samples were screened, including 812 males and 1104 females. Of the 1916 samples, 1898 were bone marrow samples and 18 were peripheral blood samples. Among these, 115 samples showed MDS, 77 showed MA, 3 cases of erythroleukemia, and 55 cases of CML.

[0078] Of the 115 MDS samples, 70% reported abnormal erythrocyte differentiation as a diagnostic criterion. The main abnormal erythrocyte differentiation phenotypes were increased CD71 CV (78%), increased CD36 CV (78%), decreased CD71 expression (45%), and decreased CD36 expression (68%). Of the 77 MA samples, 95% reported abnormal erythrocyte differentiation with a positive rate of CD36highCD105+. Of the 3 erythroleukemia samples, 100% reported a significantly increased proportion of CD36+CD71+ erythroblasts. Of the 55 CML samples, 100% reported an abnormal erythrocyte differentiation phenotype with a significantly increased proportion of CD117+CD105dim erythroblasts.

[0079] To further verify the efficiency of the proposed erythrocyte differentiation method in the auxiliary diagnosis of megaloblastic anemia, this invention selected 133 anemia samples from the flow cytometry laboratory of Dongyang People's Hospital for immunophenotyping diagnosis between January 1, 2024 and October 30, 2025. These included 42 males and 91 females, with a median age of 43 years (range 15-78 years). The basic diagnostic criteria for megaloblastic anemia were: clinical manifestations (pale skin and mucous membranes, smooth tongue (mirror tongue), etc.) + macrocytic anemia / bone marrow megaloblastic changes + biochemical evidence of serum folic acid or vitamin B12 deficiency. The final clinical diagnoses of the 133 samples are shown in Table 2. The clinical diagnostic accuracy rate for megaloblastic anemia reached 95%. Therefore, this invention demonstrates its potential as an auxiliary diagnostic tool for anemia.

[0080] Table 2

[0081] diagnosis quantity Megaloblastic anemia 22 Iron deficiency anemia 37 Myelodysplastic syndrome 39 hemolytic anemia 5 Anemia of chronic disease 30

[0082] To further verify the efficiency of the erythrocyte differentiation method proposed in this invention for the specific diagnosis of chronic myeloid leukemia (CML), this invention selected 105 samples from the flow cytometry laboratory of Dongyang People's Hospital, collected from May 1, 2024 to October 30, 2025, for immunophenotyping to conduct a flow cytometry diagnostic study on suspected CML cases. These included 92 males and 88 females, with a median age of 55 years (range 16-87 years). The basic diagnostic criteria for CML were: peripheral blood neutrophilia; detection of the Ph chromosome and / or BCR::ABL by cytogenetic and / or other molecular genetic techniques. The final clinical diagnoses of 180 samples are shown in Table 3. The clinical diagnostic accuracy for CML reached 100%. Therefore, this invention represents a highly comprehensive, practical, highly sensitive, and highly specific detection and analysis scheme for the clinical flow cytometry diagnostic field, capable of reducing the rate of missed diagnoses, and has the potential to serve as an auxiliary diagnostic and monitoring tool for this disease.

[0083] Table 3

[0084] diagnosis quantity Chronic myeloid leukemia 15 Chronic myelomonocytic leukemia 3 Essential thrombocytosis 48 Polycythemia vera 25 Increased reactivity 12 Myelofibrosis 2

[0085] Compared with existing technologies, a key breakthrough of this invention is that, based on the characteristics of erythrocyte differentiation trajectory, it has found an antibody combination that can screen and differentiate multiple diseases, including but not limited to myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

[0086] Those skilled in the art will readily understand that the above description is merely an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A reagent composition for flow cytometry detection of erythroid cell differentiation, for addition to a flow cytometry tube containing a single-cell suspension of the sample to be tested, characterized in that, This includes anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody.

2. The reagent composition according to claim 1, which can be used for flow cytometry detection of erythroid cell differentiation, is characterized in that, All antibodies are monoclonal antibodies.

3. The reagent composition according to claim 1, which can be used for flow cytometry detection of erythroid cell differentiation, is characterized in that, All antibodies were fluorescently labeled; the fluorescent labeling order of anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody was FITC (Fluorescein Isothiocyanate), PE (Phycoerythrin), ECD (Phycoerythrin-Texas), PC7 (Phycoerythrin-Cyanin 7), APC (Allophycocyanin), and KO (Krome Orange).

4. The reagent composition according to claim 1, which can be used for flow cytometry detection of erythroid cell differentiation, is characterized in that, The volume ratio of anti-CD71 antibody, anti-CD117 antibody, anti-CD34 antibody, anti-CD105 antibody, anti-CD36 antibody, and anti-CD45 antibody is 8:4:6:3:4:

4.

5. A reagent kit, comprising a container, characterized in that, The container is used to contain the reagent composition according to any one of claims 1-4, which can be used for flow cytometry detection of erythrocyte differentiation.

6. The use of the reagent composition according to any one of claims 1-4 for detecting erythrocyte differentiation by flow cytometry in the preparation of flow cytometry samples for detecting erythrocyte differentiation.

7. The application according to claim 6, characterized in that, The erythroid cell differentiation is an abnormal differentiation of erythroid cells caused by myelodysplastic syndrome, megaloblastic anemia, pure red cell leukemia, and chronic myeloid leukemia.

8. The application according to claim 6, characterized in that, The preparation of flow cytometry samples for detecting erythrocyte differentiation specifically includes the following steps: (1) Add the sample to be tested into a flow cytometry tube to form a single-cell suspension, ensuring that the number of nucleated cells is 5 x 10^6. 9 / pipe-20X10 9 / Tube; (2) Add the reagent composition of any one of claims 1-4 that can be used for flow cytometry detection of erythroid cell differentiation to the flow cytometer obtained in step (1) and incubate at room temperature in the dark. (3) Add 1X hemolysin to the flow cytometer after incubation in step (2) and incubate at room temperature in the dark; (4) Wash the flow cytometry tube after incubation in step (3) with PBS buffer, centrifuge and remove the supernatant, then resuspend the cells with PBS buffer to obtain the flow cytometry sample for detecting erythrocyte differentiation.

9. A device for detecting erythroid cell differentiation, characterized in that, The device includes a detection unit and an analysis unit; wherein: the detection unit is used to perform flow cytometry detection on reagent materials from a sample of the individual to be tested, and obtain the detection results of the sample; the reagent materials include the reagent composition according to any one of claims 1-4 that can be used for flow cytometry detection of erythroid cell differentiation; the analysis unit is used to analyze the detection results of the sample.

10. The apparatus for detecting erythroid cell differentiation according to claim 9, characterized in that, During flow cytometry analysis, gating was performed as follows: a de-adhesion cell gate P1 was set, and a live cell gate P2 was set within P1 to obtain single live cells; within gate P2, CD45 / SSC antibodies were used to examine cell populations; within gate P2, a CD45 / CD34 antibody gate P3 was set to detect CD34+ cell populations; within gate P3, a CD45 / SSC antibody gate K was set to detect non-lymphatic progenitor cells; within gate P2, a CD71 / CD36 gate P4 was set to detect CD36+CD71+ cells; within gate P4, a CD45 / SSC antibody ErBs gate was set to circle nucleated erythrocytes; within the ErBs display gate, gating was set based on CD105 expression, and a gate P5 was set to circle CD105- nucleated erythrocytes as late erythroblasts; gate P5... The model continues to use gates based on CD117 expression. Gate P6 circles CD117+ nucleated erythrocytes as early erythroblasts, and gate P7 circles CD117- nucleated erythrocytes as intermediate erythroblasts. A combined ErBs and K gate is used to visualize the expression of non-lymphatic blasts and nucleated erythrocytes in CD105 / CD71. Arrows in the figure show the normal differentiation trajectory of nucleated erythrocytes. Similarly, a combined ErBs and K gate is used to visualize the expression of non-lymphatic blasts and nucleated erythrocytes in CD105 / CD36. Arrows in the figure show the normal differentiation trajectory of nucleated erythrocytes. Gate P6, based on CD105 expression, categorizes cells into CD105dim early erythroblasts and CD105+ early erythroblasts.

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