Application of substance for detecting ratio of lymphocyte subgroups in preparation of reagent or kit for diagnosis or auxiliary diagnosis of Graves disease

Abstract

The invention provides application of a substance for detecting the ratio of lymphocyte subgroups in preparation of a reagent or a kit for diagnosis or auxiliary diagnosis of Graves disease, and belongs to the technical field of preparation of disease diagnosis reagents. A research shows that compared with a healthy sample, the proportion of B cells in a lymphocyte subpopulation of a to-be-detected sample is remarkably increased, the proportion of NK cells is remarkably reduced, the proportion of activated T cells is remarkably reduced, the proportion of memory T cells is remarkably reduced, the proportion of CD4 + memory T cells is remarkably reduced, the proportion of Th1 cells is remarkably reduced, and the proportion of Tc1 cells is remarkably reduced, so that the to-be-detected sample is a GD patient; the substance for detecting the ratio of lymphocyte subgroups is high in sensitivity and specificity when being used for diagnosis or auxiliary diagnosis of Graves disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease diagnosis reagent preparation, and particularly relates to application of a substance for detecting lymphocyte subpopulation ratio in preparation of a reagent or kit for diagnosis or auxiliary diagnosis of Graves disease. BACKGROUND

[0002] Graves disease (GD) is also known as toxic diffuse goiter, which is an autoimmune disease caused by abnormal secretion of antibodies against thyroid follicular cell membrane thyrotropin receptor (TSHR). As for the immunological mechanism in the etiology, it is recognized to be related to the destruction of self-tolerance, but the process of how B cells and T cells break the self-tolerance to produce autoantibodies lacks in-depth research. At present, the research on the immune mechanism of GD mainly focuses on CD4 + T lymphocyte subpopulation function and the immune regulation of cytokines secreted by the T lymphocyte subpopulation.

[0003] The treatment options for GD include drugs, surgery and 131 I. Since the introduction of antithyroid drugs (ATD) into clinical application, it has been one of the main methods for treating hyperthyroidism. However, ATD treatment is to control thyroid function rather than to treat the cause, and the treatment course is long, and it is easy to relapse after drug withdrawal and may cause adverse reactions such as leukopenia and liver damage. 131 I is one of the main treatment methods for adult GD and the first-line treatment method for elderly GD, but the hypothyroidism caused after treatment needs to be taken for life. The surgical option is not the preferred treatment method, and the indications and contraindications must be strictly controlled. In recent years, how to induce immune tolerance again has become a hot spot in clinical research, and the antigen-specific immunotherapy targets include thyrotropin receptor, CD20, CD40 and B cell activating factor (BAFF) for regulating B cells, and cytokines such as TNF-alpha and IL-6, but the T cell sites are relatively few. At present, the evaluation of the efficacy of GD patients still relies on thyroid function detection, the method is single, and there is less research on the deep etiology, especially the use of substances for detecting lymphocyte subpopulation ratio for diagnosis or auxiliary diagnosis of Graves disease has not been reported. SUMMARY

[0004] In view of this, the purpose of the present application is to provide application of a substance for detecting lymphocyte subpopulation ratio in preparation of a reagent or kit for diagnosis or auxiliary diagnosis of Graves disease.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides application of a substance for detecting lymphocyte subpopulation ratio in preparation of a reagent or kit for diagnosis or auxiliary diagnosis of Graves disease, wherein the lymphocyte subpopulation is B cells, NK cells, activated T cells, memory T cells, CD4+ One or more of the memory T cells, Th1 cells, and Tc1 cells.

[0006] Preferably, the lymphocyte subsets are B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells.

[0007] Preferably, the substance is a fluorescent monoclonal antibody for detecting the lymphocyte subsets.

[0008] Preferably, the reagent or kit also includes a sample of a healthy person.

[0009] This invention provides a system for diagnosing or assisting in the diagnosis of Graves' disease. The system includes a data processing device and a detection device. The detection device detects the proportion of lymphocyte subsets in a test sample and a healthy sample. The data processing device includes a conclusion output module. The conclusion output module compares the results of the lymphocyte subset proportion detection between the test sample and the healthy sample, and determines the comparison results according to predetermined judgment conditions to diagnose or assist in the diagnosis of whether the test sample has Graves' disease.

[0010] Preferably, the determination criterion is that, compared with a healthy sample, the lymphocyte subsets of the test sample show a higher proportion of B cells, NK cells, activated T cells, memory T cells, and CD4+. + If there is a significant difference in the proportions of memory T cells, Th1 cells, and Tc1 cells, the sample to be tested will be diagnosed with Graves' disease.

[0011] Preferably, the ratio refers to B cells, NK cells, activated T cells, memory T cells, and CD4+. + The percentage of memory T cells in the total number of lymphocytes, and the percentage of Th1 cells in the total number of CD3 cells. + CD4 + The percentage of cells or the number of Tc1 cells in CD3 + CD8 + Percentage of cell count.

[0012] The present invention provides a kit for diagnosing or assisting in the diagnosis of Graves' disease, the kit comprising a substance for detecting the proportion of the aforementioned lymphocyte subsets.

[0013] Preferably, the substance is a fluorescent monoclonal antibody for detecting lymphocyte subsets.

[0014] Preferably, the kit also includes a sample of a healthy person.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of a substance for detecting the proportion of lymphocyte subsets in the preparation of reagents or kits for the diagnosis or auxiliary diagnosis of Graves' disease. Studies have found that, compared to healthy samples, the proportion of B cells, NK cells, activated T cells, and memory T cells in the lymphocyte subsets of the tested samples was significantly increased, while the proportion of CD4 cells was significantly decreased. + If the proportion of memory T cells, Th1 cells, and Tc1 cells is significantly reduced, then the sample to be tested is a patient with Graves' disease. This invention utilizes a substance that detects the proportion of lymphocyte subsets for the diagnosis or auxiliary diagnosis of Graves' disease with high sensitivity and specificity. Attached Figure Description

[0016] Figure 1 ROC curves for detecting B cells alone in the diagnosis of GD; Figure 2 ROC curves of NK cells were measured separately for the diagnosis of GD. Figure 3 ROC curves of activated T cells were measured separately for the diagnosis of GD. Figure 4 ROC curves of memory T cells were measured separately for the diagnosis of GD. Figure 5 CD4 testing alone for the diagnosis of GD + ROC curve of memory T cells; Figure 6 ROC curves of Th1 cells were measured separately for the diagnosis of GD. Figure 7 ROC curves of activated Tc1 cells were measured separately for the diagnosis of GD. Figure 8 Comprehensive testing of B cells, NK cells, activated T cells, memory T cells, and CD4+ is used to diagnose Gram-D. + ROC curves of memory T cells, Th1 cells, and Tc1 cells. Detailed Implementation

[0017] This invention provides the application of a substance for detecting the proportion of lymphocyte subsets in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of Graves' disease, wherein the lymphocyte subsets are one or more of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells.

[0018] In this invention, as a preferred embodiment, the lymphocyte subsets are B cells, NK cells, activated T cells, memory T cells, and CD4+. +Memory T cells, Th1 cells, and Tc1 cells. The substance is a fluorescent monoclonal antibody for detecting these lymphocyte subsets, specifically B cells, NK cells, activated T cells, memory T cells, and CD4+. + Fluorescent monoclonal antibodies for memory T cells, Th1 cells, and Tc1 cells. This invention does not specifically limit the source of each fluorescent monoclonal antibody; preparation methods known in the art or commercially available products are acceptable.

[0019] In this invention, the reagent or kit further includes a healthy human sample. The sample is a peripheral blood sample. This invention uses the healthy human sample as a control group. If the lymphocyte subsets in the test sample include B cells, NK cells, activated T cells, memory T cells, and CD4+... + Significant differences in the proportions of memory T cells, Th1 cells, and Tc1 cells indicate Graves' disease in the tested sample, either diagnostically or as a supplementary diagnostic criterion. This invention utilizes the detection of B cells, NK cells, activated T cells, memory T cells, and CD4+ cells in the lymphocyte subsets of peripheral blood from GD patients. + The area under the curve for the ratio of memory T cells, Th1 cells, and Tc1 cells, after ROC analysis, reached 0.8989. Therefore, the substance used in this invention to detect the ratio of the above lymphocyte subsets has high sensitivity and strong specificity for the diagnosis or auxiliary diagnosis of Graves' disease.

[0020] This invention provides a system for diagnosing or assisting in the diagnosis of Graves' disease. The system includes a data processing device and a detection device. The detection device detects the proportion of lymphocyte subsets in a test sample and a healthy sample. The data processing device includes a conclusion output module. The conclusion output module compares the results of the lymphocyte subset proportion detection between the test sample and the healthy sample, and determines the comparison results according to predetermined judgment conditions to diagnose or assist in the diagnosis of whether the test sample has Graves' disease.

[0021] In this invention, the determination criterion is that, compared with a healthy sample, the lymphocyte subsets of the test sample contain more B cells, NK cells, activated T cells, memory T cells, and CD4+. + If there is a significant difference in the proportions of memory T cells, Th1 cells, and Tc1 cells, the sample to be tested is diagnosed with Graves' disease.

[0022] In this invention, the ratio refers to B cells, NK cells, activated T cells, memory T cells, and CD4+. + The percentage of memory T cells in the total number of lymphocytes, and the percentage of Th1 cells in the total number of CD3 cells. + CD4 + The percentage of cells or the number of Tc1 cells in CD3 + CD8 + Percentage of cell count.

[0023] The present invention provides a kit for diagnosing or assisting in the diagnosis of Graves' disease, the kit comprising a substance for detecting the proportion of the aforementioned lymphocyte subsets.

[0024] In this invention, the substance is a fluorescent monoclonal antibody for detecting lymphocyte subsets. The kit also includes a sample from a healthy individual, specifically a peripheral blood sample.

[0025] Changes in the proportion and function of lymphocyte subsets lead to an imbalance in the immune system, directly affecting the thyroid gland's responsiveness to its own tissue antigens, particularly changes in cell function. In other words, changes in lymphocyte subset proportions precede changes in thyroid function. Therefore, compared to methods for detecting thyroid function, this invention, by detecting the aforementioned changes in lymphocyte subset proportions, can diagnose or assist in the diagnosis of whether a sample has Graves' disease at an earlier stage.

[0026] In this invention, a substance for detecting the proportion of lymphocyte subsets is provided for use in the preparation of reagents or kits for evaluating the curative effect of drugs on Graves' disease. The lymphocyte subsets are one or more types of B cells and NK cells. The reagents or kits also include samples from healthy individuals and samples from Graves' disease patients. The samples are peripheral blood samples. The substance is a fluorescent monoclonal antibody for detecting lymphocyte subsets. When there is no significant difference in the proportion of B cells and / or NK cells between the test sample and the healthy individual sample (…),… P >0.05), which is significantly different from the proportion of B cells and / or NK cells in GD patient samples. P If the value is less than 0.05, it indicates that the sample has been cured after drug treatment.

[0027] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 The use of a substance for detecting the proportion of lymphocyte subsets in the preparation of reagents or kits for the diagnosis or auxiliary diagnosis of Graves' disease.

[0030] I. Research Methods (S1) Reagents and Instruments: Hemolysin and membrane-breaking agent were purchased from Beckman Coulter, USA; staining antibodies were purchased from BD Biosciences, USA; the instrument was a BD CANTO; and thyroid function test reagents were from Siemens, USA. Flow cytometry data acquisition software was FACSDiva version 8.0, and analysis software was Kaluza 2.3.

[0031] (S2) Thyroid function test: 2 mL of fasting venous blood was collected, serum was extracted by centrifugation, and thyroid function was measured by electrochemiluminescence method, including free triiodothyronine (FT3), free thyroxine (FT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroid-stimulating hormone (TSH), TSH receptor antibody (TRAb), thyroglobulin antibody (TgAb), and thyroid peroxidase antibody (TPOAb).

[0032] (S3) Refined lymphocyte subset detection: Collect peripheral blood samples from patients, add fluorescent monoclonal antibodies or combinations, and add reagents according to the standard dosage in the product instructions. Mix 100 μL of peripheral blood, react at room temperature in the dark for 15 min, add 1 mL of phosphate buffer, centrifuge and wash twice, and then perform the detection. In this embodiment, a total of 112 refined lymphocyte subset samples were collected, including 85 samples from newly diagnosed patients. Flow cytometry was used to detect cell membrane surface antigens with fluorescent monoclonal antibodies CD3-FITC / CD16&CD56-PE, CD19-APC, CD4-Percp cy5.5, CD8-PECY7, HLA-DR-APC-Cy7, CD28-BV421, CD3-APC, CD25-PE, CD8-APC-cy7, CD45RA-FITC, CD45RO-PECY7, and CD127-BV421. Cytoplasmic antigens were detected using an IFN-γ-FITC / IL-4-PE two-color reagent. The detection methods for B cells included CD19-APC and CD3-FITC antibodies; CD3-FITC / CD16 & CD56-PE antibodies; HLA-DR-APC-Cy7 and CD3-FITC antibodies for activated T cells; CD45RO-PECY7 and CD3-APC antibodies for memory T cells; CD4+ memory T cells for CD45RO-PECY7, CD4-Percp cy5.5, and CD3-APC antibodies; and CD8-APC-cy7, CD45RA-FITC, CD45RO-PECY7, and CD3-APC antibodies for detecting CD8+ naive T cells or CD8+ memory T cells. CD3-APC, CD8-PECY7, and IFN-γ-FITC / IL-4-PE dual-color reagents were used to detect Tc1 cells and Th1 cells. 10,000 lymphocytes were obtained, and CD3+ was calculated. + CD4 + Percentage of helper / inducing T cells (Th)1 and Th2 cells in cells, CD3 + CD8 +The percentages of cytotoxic / suppressive T cells (Tc1) and Tc2 cells in the cells, with the percentages of other subtypes all expressed as lymphocytes. The detailed lymphocyte typing scheme is shown in Table 1.

[0033] Table 1. Refined Lymphocyte Subpopulation Analysis Protocol

[0034] Note: CD, cluster differentiation antigen; NK, natural killer; Th, helper T cells; Tc, cytotoxic T cells; RA, quiescent; RO, memory; cIL, cytoplasmic interleukin; cIFN, cytoplasmic interferon; HLA-DR, human leukocyte differentiation antigen DR subtype.

[0035] III. Statistical Analysis Statistical analysis was performed using Graphpad Prism 8.0. The Shapiro-Wilk test was used to verify whether the data conformed to a normal distribution. Normally distributed continuous data were expressed as mean ± standard deviation. s Description: One-way ANOVA was used for comparisons among multiple groups, and independent samples t-test was used for comparisons between two groups. Non-normally distributed continuous data were analyzed using... M ( Q 1, Q 3) Description: Rank-sum tests were used for comparisons between groups. Pearson correlation analysis was used to analyze the correlation between thyroid function results and refined lymphocyte subsets. P <0.05 indicates a statistically significant difference (two-sided).

[0036] This invention, based on the 2008 "Guidelines for the Diagnosis and Treatment of Thyroid Diseases in China," used the aforementioned research methods to conduct refined lymphocyte subset detection in patients diagnosed with Graves' disease (GD) and healthy individuals (control group). The study found that, compared with healthy individuals, the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells in the lymphocyte subsets of GD patients were significantly different (p < 0.05). Specifically, compared with healthy individuals, the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells were significantly increased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), and decreased (p < 0.05) in the lymphocyte subsets of GD patients.

[0037] Example 2 The diagnostic efficacy of GD was evaluated by detecting the proportions of lymphocyte subsets: B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells.

[0038] Compared with healthy individuals, the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells in the lymphocyte subsets of the tested samples were significantly different (p < 0.05). Specifically, compared with healthy individuals, the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells in the lymphocyte subsets of the tested samples were significantly increased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), decreased (p < 0.05), and decreased (p < 0.05) in the lymphocyte subsets of the tested samples, indicating that the tested samples were GD patients.

[0039] I. Research Subjects (1) GD group: 97 GD patients hospitalized and treated at the Department of Endocrinology, Fifth Medical Center of the PLA General Hospital from October 2018 to July 2021. The diagnostic criteria were based on the 2008 Chinese Guidelines for the Diagnosis and Treatment of Thyroid Diseases. Patients using immunosuppressants, hyperthyroidism complicated with Hashimoto's thyroiditis, secondary hyperthyroidism, and iatrogenic hyperthyroidism were excluded. Peripheral blood samples were sent for detailed lymphocyte subset analysis of all patients. There were 38 males and 59 females, with an age of 37 (28, 48) years. GD patients were divided into a complication group (35 cases) and a simple GD group (54 cases) without complications according to their clinical manifestations. In the complication group, based on the characteristics of ocular lesions, there were 17 cases of GO (thyroid-associated ophthalmopathy); 7 cases of thyrotoxicosis-related cardiomyopathy, 4 cases of periodic paralysis, and 7 cases of other autoimmune diseases. There were 68 non-GO patients (non-GO subgroup) without ocular symptoms. Based on the results of routine blood tests, the patients were divided into a granulocytopenic subgroup (21 cases) and a granulocytopenic subgroup (59 cases).

[0040] In this embodiment, the GD group is used as the sample to be tested.

[0041] (2) GD treatment group: receiving conventional treatment (drugs such as methimazole, propylthiouracil, or...) 131 (I) Treatment: 27 patients with normal thyroid function and hypothyroidism, aged 46 (32, 59) years, including 12 males and 15 females. Of these, 17 received drug treatment. 131 I treated 10 cases.

[0042] (3) Control group: 31 healthy people who underwent physical examinations during the same period served as the control group, including 12 males and 19 females, aged 48, (43, 53) years.

[0043] All patient case data and test data were obtained and collected with the approval of the Ethics Committee of the Fifth Medical Center of the General Hospital of the Chinese People's Liberation Army, approval number KY-2024-11-180-1.

[0044] II. Research Methods Same as Example 1.

[0045] III. Statistical Analysis Same as Example 1.

[0046] IV. Results (A1) Comparison of T cells, B cells and NK cells in the GD group, GD treatment group and control group The main lymphocytes are T cells, B cells, and NK cells. The proportion of B cells in the GD group was higher than that in the control group [16.2%, (11.8%, 21.8%) vs. 10.2%, (8.1%, 13.6%)]; the proportion of NK cells in the GD group was lower than that in the control group [9.4%, (4.9%, 13.6%) vs. 14.6%, (12.1%, 18.8%)], and the differences were statistically significant. P <0.01), but there was no statistically significant difference in the proportion of these two cell types between the GD treatment group and the control group [9.7% (7.1%, 12.3%) vs. 10.2% (8.1%, 13.6%), 15.8% (10.8%, 20.8%) vs. 14.6% (12.1%, 18.8%), P>0.05]. There was no statistically significant difference in T cell changes (F=0.521, P>0.05), see Table 2.

[0047] Table 2 Comparison of T, B, and NK cell ratios in the GD group, GD treatment group, and control group

[0048] Note: GD, Graves' disease; NK, Natural Killer. a Compared with the control group, P <0.05; b Compared to the GD group, P <0.05; c , F Value, all other statistical values ​​are H value.

[0049] (A2) Comparison of T cell subsets among different groups The results in Table 3 indicate that, compared with the control group, although there was no statistically significant difference in the proportions of helper / inducing T cells (CD4+ T cells) and cytotoxic / suppressive T cells (CD8+ T cells) in the GD group ( P>0.05), but the proportions of activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells were all lower than those in the control group, statistical value Z The values ​​were -2.69, -2.35, -2.77, -2.34, and -3.28, respectively. P The values ​​are 0.013, 0.012, 0.017, 0.025, and 0.0004, respectively.

[0050] Table 3 Comparison of T lymphocyte subsets between the GD group and the control group

[0051] Note: GD, Graves' disease; NK, natural killer; CD, cluster differentiation antigen; Th, helper T cells; Tc, cytotoxic T cells. a Compared with the control group, P <0.05.

[0052] III. Correlation Analysis between Thyroid Function and Refined Lymphocyte Subsets TT3 levels were positively correlated with the proportion of B cells (r = 0.356, P < 0.01) and negatively correlated with the proportion of NK cells (r = -0.417, P < 0.01). TT4 levels were negatively correlated with the proportions of NK cells and activated T cells (r = -0.318 and -0.336, respectively, P < 0.01). TSH levels were positively correlated with the proportion of CD8+ naive T cells (r = 0.382, P < 0.01).

[0053] This invention analyzes individual indicators of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells, as well as combined indicators (B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells), to observe the accuracy of lymphocyte subset indicators in the auxiliary diagnosis of GD. The results are shown in Table 4.

[0054] Table 4. ROC analysis of lymphocyte subset indices

[0055] Table 4 and Figures 1-8The results showed that detecting the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells individually can be used to diagnose or assist in the diagnosis of Graves' disease. The area under the ROC curve was above 0.6465. Among them, the area under the ROC curve for comprehensively detecting the proportions of B cells, NK cells, activated T cells, memory T cells, CD4+ memory T cells, Th1 cells, and Tc1 cells reached 0.8989, with a 95% CI of 0.8359~0.9620. The use of substances that detect the proportions of lymphocyte subsets for the diagnosis or auxiliary diagnosis of Graves' disease has high sensitivity and specificity, and has high diagnostic efficacy for GD. This invention provides a new diagnostic method for the clinical diagnosis of GD.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a substance for detecting the proportion of lymphocyte subsets in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of Graves' disease, characterized in that, The lymphocyte subsets include B cells, NK cells, activated T cells, memory T cells, and CD4+. + One or more of the memory T cells, Th1 cells, and Tc1 cells.

2. The application according to claim 1, characterized in that, The lymphocyte subsets include B cells, NK cells, activated T cells, memory T cells, and CD4+. + Memory T cells, Th1 cells, and Tc1 cells.

3. The application according to claim 1, characterized in that, The substance is a fluorescent monoclonal antibody for detecting the lymphocyte subset.

4. The application according to claim 1, characterized in that, The reagents or kits also include samples from healthy individuals.

5. A system for diagnosing or assisting in the diagnosis of Graves' disease, characterized in that, The system includes a data processing device and a detection device. The detection device is used to detect the proportion of lymphocyte subsets as described in claim 1 in a test sample and a healthy sample. The data processing device includes a conclusion output module. The conclusion output module is used to diagnose or assist in the diagnosis of whether the test sample has Graves' disease by comparing the results of the lymphocyte subset proportion detection of the test sample and the healthy sample and judging the comparison results according to predetermined judgment conditions.

6. The system according to claim 5, characterized in that, The determination criterion is that, compared with a healthy sample, the lymphocyte subsets of the test sample show a higher proportion of B cells, NK cells, activated T cells, memory T cells, and CD4+. + If there is a significant difference in the proportions of memory T cells, Th1 cells, and Tc1 cells, the sample to be tested will be diagnosed with Graves' disease.

7. The system according to claim 5, characterized in that, The ratio refers to B cells, NK cells, activated T cells, memory T cells, and CD4+. + The percentage of memory T cells in the total number of lymphocytes, and the percentage of Th1 cells in the total number of CD3 cells. + CD4 + The percentage of cells or the number of Tc1 cells in CD3 + CD8 + Percentage of cell count.

8. A kit for diagnosing or assisting in the diagnosis of Graves' disease, characterized in that, The kit includes a substance for detecting the proportion of lymphocyte subsets as described in claim 1 or 2.

9. The reagent kit according to claim 8, characterized in that, The substance is a fluorescent monoclonal antibody for detecting lymphocyte subsets.

10. The reagent kit according to claim 8, characterized in that, The kit also includes samples from healthy individuals.

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