CD45 + CD8 + HLA-DRB5 + Application of T cells in the preparation of kits for auxiliary diagnosis of aplastic anemia and graft-versus-host disease

Abstract

The present invention belongs to the field of medical testing technology, and specifically relates to CD45 + CD8 + HLA‑DRB5 + The present inventors first discovered that compared with healthy volunteers and patients with other blood diseases, CD45 + CD8 + HLA‑DRB5 + The expression of T cells in the peripheral blood or bone marrow fluid of AA and GVHD patients is significantly increased. The present invention can be used as one of the auxiliary diagnostic detection indicators for AA and GVHD. The present invention provides a marker CD45 for the diagnosis of AA and GVHD. + CD8 + HLA‑DRB5 + T cells can diagnose AA and GVHD simply and efficiently, and have the advantages of being easy to promote and rapid.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing technology, and specifically relates to CD45 + CD8 + HLA-DRB5 + Application of T cells in auxiliary diagnosis of aplastic anemia and graft-versus-host disease. Background Art

[0002] Aplastic anemia (AA) is a bone marrow hematopoietic failure syndrome, mainly manifested by anemia, bleeding, and infection syndrome caused by low bone marrow hematopoietic function and pancytopenia. Its onset is related to primary or secondary hematopoietic stem cell defects. The pathogenesis is still unclear, but it is generally believed that abnormal activation and hyperfunction of T lymphocytes causing bone marrow damage is one of the causes of aplastic anemia. The diagnostic criteria for aplastic anemia are as follows: (1) Routine blood test: decreased total blood cells (including reticulocytes) and increased lymphocyte ratio. At least two of the following three items must be met: HGB <100g / L; PLT <50x 10 9 / L; absolute neutrophil count (ANC) <1.5x 10 9 / L. (2) Bone marrow puncture: hypoplasia or severe hypoplasia of bone marrow in multiple sites (different planes); empty granules; increased proportion of non-hematopoietic cells (lymphocytes, reticular cells, plasma cells, mast cells, etc.); significantly reduced or absent megakaryocytes; significantly reduced erythroid and granulocyte cells. (3) Bone marrow biopsy (iliac bone): hypoplasia of the whole section, decreased hematopoietic cells, increased non-hematopoietic cells, and no abnormal cells. (4) Congenital and other acquired and secondary pancytopenias must be excluded, such as congenital amegakaryocytic thrombocytopenia, Fanconi anemia, hypoproliferative MDS, primary immune thrombocytopenia, etc.

[0003] Graft-versus-host disease (GVHD) is one of the most common life-threatening complications of allogeneic hematopoietic cell transplantation. It is mainly caused by the T cells in the donor's graft being activated by immune response in the recipient's body after transplantation to acquire cytolytic ability and then attack cells carrying foreign antigens. Its characteristics are similar to the symptoms of autoimmune and other immune diseases, such as scleroderma, S-syndrome, immune cytopenia, etc. At present, the diagnosis of graft-versus-host disease is mainly based on pathological manifestations such as diarrhea and rash after transplantation and related laboratory tests.

[0004] Human leukocyte antigen (HLA) is a glycoprotein encoded by the major histocompatibility complex (MHC). It specifically delivers short peptides to T cells and plays a key role in the body's immune defense. The HLA-DRB genes encoding the β chain of the HLA-DR molecule include -DRB1, -DRB3, -DRB4, -DRB5, etc. It has been found that gene mutations in -DRB1 are associated with autoimmune diseases, some inflammatory diseases and cancers, and its correlation with diseases has also been confirmed. The expression level of the -DRB5 gene is low, and the research technology is limited. The research on its correlation with diseases is still unclear, but there are still a few studies reporting that abnormal expression of the -DRB5 gene can increase the risk of diseases such as type 1 diabetes, vitiligo and systemic sclerosis. CD45 has not yet been seen. + CD8 + HLA-DRB5 + Related reports on T cells in AA and GVHD.

[0005] So far, both aplastic anemia and graft-versus-host disease lack characteristic pathological diagnostic markers, and graft-versus-host disease lacks standard and widely used diagnostic guidelines. Summary of the invention

[0006] The primary purpose of the present invention is to make up for the deficiencies of the prior art and provide a CD45 + CD8 + HLA-DRB5 + The application of T cells as diagnostic markers in the preparation of a kit for auxiliary diagnosis of aplastic anemia and graft-versus-host disease.

[0007] The first aspect of the present invention is to provide a diagnostic marker for diagnosing aplastic anemia and graft-versus-host disease, wherein the marker is CD45 + CD8 + HLA-DRB5 + T cells, human CD45 + CD8 + HLA-DRB5 + Flow cytometric analysis of T cells labeled with CD45 + CD8 + HLA-DRB5 + , that is, CD45 positive, CD8 positive, and HLA-DRB5 positive.

[0008] The second aspect of the present invention is to provide a CD45 + CD8 + HLA-DRB5 +The application of T cells as diagnostic markers in the preparation of detection products related to the diagnosis of aplastic anemia and graft-versus-host disease.

[0009] In one embodiment, the detection product is a CD45 + CD8 + HLA-DRB5 + T cell flow cytometry detection kit.

[0010] In one embodiment, the aplastic anemia and graft-versus-host disease related detection product is a diagnostic kit, which can be used to assist in the diagnosis of aplastic anemia and graft-versus-host disease.

[0011] The third aspect of the present invention is to provide a CD45 + CD8 + HLA-DRB5 + T cell flow cytometry detection kit, wherein the CD45 + CD8 + HLA-DRB5 + The T cell flow cytometry detection kit includes the following components: The kit is used to detect CD45 + CD8 + HLA-DRB5 + Monoclonal antibodies for T cell subsets: anti-CD45 antibody, anti-CD8 antibody, anti-HLA-DRB5 antibody.

[0012] In one embodiment, the kit further comprises a fluorescent marker: the fluorescent marker of the anti-CD45 antibody is PerCP-Cy5.5; the fluorescent marker of the anti-CD8 antibody is PE; and the fluorescent marker of the anti-HLA-DRB5 antibody is FITC.

[0013] In one embodiment, the dosage of the antibody in the kit is based on the dosage recommended in the instructions of the purchased antibody.

[0014] In one embodiment, the sample detected by the kit is peripheral blood or bone marrow fluid.

[0015] In one embodiment, the kit further comprises red blood cell lysis solution and phosphate buffered saline (PBS) solution.

[0016] The fourth aspect of the present invention is to provide a use of the above-mentioned kit in studying aplastic anemia and graft-versus-host disease for non-diagnostic purposes.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] The inventors first discovered that CD45 in the bone marrow fluid of patients with aplastic anemia and graft-versus-host disease + CD8 + HLA-DRB5 + The proportion of T cells increased significantly. Among them, statistical analysis confirmed that CD45 + CD8 + HLA-DRB5 + T cells showed significant differences between AA, GVHD and healthy volunteers and other blood disease patients (AA: P < 0.001, GVHD: P < 0.05). ROC curve analysis showed that the AUCs for AA and GVHD were 0.9336 and 0.9221, respectively, with a sensitivity of 0.8182 and a specificity of 1. In the validation set, the AUCs for AA and GVHD were 0.9717 and 0.9524, respectively, with a sensitivity of 0.9333 and a specificity of 0.95 and 1, respectively. CD45 + CD8 + HLA-DRB5 + T cells have good accuracy in diagnosing AA and GVHD, indicating that they can be used as one of the laboratory immune-related detection indicators for auxiliary diagnosis of AA and GVHD, and the operation is simple and feasible, with high clinical application value. + CD8 + HLA-DRB5 + The detection method of T cell subsets can be used to quantitatively analyze the phenotype of the above T cell subsets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0020] In the attached picture:

[0021] Figure 1 Representative flow cytometry analysis results of healthy volunteers (Normal), patients with aplastic anemia (AA), patients with graft-versus-host disease (GVHD), and patients with other blood diseases (here, AML patients).

[0022] Figure 2 CD45 in bone marrow of aplastic anemia group, other blood diseases group and healthy volunteers group + CD8 + HLA-DRB5 + Statistical analysis of T cell subsets, including: ***means P < 0.001, ns means the difference is not statistically significant, and P < 0.05 is statistically significant.

[0023] Figure 3 CD45 + CD8 + HLA-DRB5 + ROC curve analysis of T cells in the diagnosis of AA.

[0024] Figure 4 CD45 in the validation set + CD8 + HLA-DRB5 + ROC curve analysis of T cells in the diagnosis of AA.

[0025] Figure 5 CD45 in bone marrow of GVHD group, other blood diseases group and healthy volunteers group + CD8 + HLA-DRB5 + Statistical analysis of T cell subsets, including: * P < 0.05, ** means P < 0.01, ns means there is no statistically significant difference, and P < 0.05 means there is statistical significance.

[0026] Figure 6 CD45 + CD8 + HLA-DRB5 + ROC curve analysis of T cells in the diagnosis of GVHD.

[0027] Figure 7 CD45 in the validation set + CD8 + HLA-DRB5 + ROC curve analysis of T cells in the diagnosis of GVHD. Specific implementation methods

[0028] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0029] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0031] The present invention is described below in conjunction with specific embodiments.

[0032] The main reagents used in the examples are as follows: HLA-DRB5 antibody (ProSci, 64-133), PE anti-human CD8 (Biolegend, 980902), Fluorescen (FITC)-conjugated affinity purified donkey anti-rabbit IgG (ProSci, JAC-711-095-152), and PerCP anti-human CD45 antibody (Biolegend, 304025).

[0033] Example

[0034] 1. Selection of clinical cases

[0035] In this example, 47 patients with aplastic anemia, 14 patients with graft-versus-host disease, 27 patients with other blood diseases (such as MDS, AML, etc.) other than aplastic anemia, and 26 healthy volunteers were randomly selected, with no age or gender restrictions. After obtaining the consent of the above subjects, an informed consent form was signed.

[0036] The above subjects were divided into a test set (27 cases of aplastic anemia, 12 cases of other blood diseases (such as MDS, AML, etc.), 7 cases of graft-versus-host disease, and 11 healthy volunteers) and a validation set (20 cases of aplastic anemia, 15 cases of other blood diseases (such as MDS, AML, etc.), 7 cases of graft-versus-host disease, and 15 healthy volunteers).

[0037] Inclusion criteria for patients with aplastic anemia:

[0038] (1) Blood picture: decrease in all blood cells (including reticulocytes), at least two of the following three items are met: HGB <100g / L; PLT <50x 10 9 / L; absolute neutrophil count (ANC) <1.5x 10 9 / L.

[0039] (2) Bone marrow puncture: decreased or severely decreased bone marrow proliferation in multiple sites (different planes); empty granules; increased proportion of non-hematopoietic cells (lymphocytes, reticular cells, plasma cells, mast cells, etc.); significantly reduced or absent megakaryocytes; and significantly reduced numbers of both erythroid and granulocyte cells.

[0040] (3) Bone marrow biopsy (iliac bone): The whole section showed hypoplasia, decreased hematopoietic cells, increased non-hematopoietic cells, and no abnormal cells.

[0041] Inclusion criteria for patients with graft-versus-host disease:

[0042] (1) Receiving allogeneic hematopoietic stem cell transplantation;

[0043] (2) Organ involvement after transplantation, such as persistent nausea, vomiting, diarrhea, rash, and other skin and gastrointestinal reactions, can be diagnosed in combination with laboratory results and, if necessary, a biopsy can be performed.

[0044] Inclusion criteria for patients with other blood diseases:

[0045] Patients with other blood system diseases, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma (MM), etc., who have excluded aplastic anemia and have not received allogeneic hematopoietic stem cell transplantation;

[0046] Inclusion criteria for healthy volunteers:

[0047] (1) No acute or chronic systemic diseases, such as diabetes, hypertension, liver insufficiency, renal insufficiency and other autoimmune diseases;

[0048] (2) No mental illness.

[0049] 2. Bone marrow fluid samples were collected from patients and healthy volunteers according to routine flow cytometry procedures for subsequent experiments.

[0050] 3. Flow cytometry

[0051] (1) Separation of mononuclear cells (PBMC) from bone marrow fluid samples: Add PBS 3 times the volume of the blood sample to the sample and mix well to obtain a diluted blood sample. Add a lymphocyte separation solution of the same volume as the diluted blood sample to a 50 mL centrifuge tube. Use a pipette to slowly add the blood dilution solution along the tube wall to the top of the lymphocyte separation solution. Then centrifuge the centrifuge tube at 18°C ​​and 600g (5 for up, 0 for down) for 22 minutes. After the centrifugation, carefully aspirate the middle white film layer into a clean 15 mL centrifuge tube, and then add 10 mL of pre-cooled PBS solution to terminate the lysis. Centrifuge the centrifuge tube at 2000 rpm for 5 minutes at room temperature, discard the supernatant, and obtain the PBMC precipitate.

[0052] (2) The obtained PBMCs were subjected to red blood cell lysis, and then resuspended in PBS to form a single cell suspension, which was counted and used for flow cytometry detection.

[0053] (3) Add monoclonal antibodies labeled with different fluorescence to the single cell suspension: take 1 million PBMCs and add them to the flow tube, then add 5 mL PBS to mix, centrifuge the flow tube at 2000 rpm for 5 min, discard the supernatant, add anti-CD45 antibody, anti-CD8 antibody, and anti-HLA-DRB5 antibody to PBS at a volume ratio of 1:200 according to the dosage specified in the instructions of the purchased antibodies to obtain antibody dilution solution, add 100 μL of antibody dilution solution to the PBMC of each sample for staining: shake the flow tube on an oscillator, mix thoroughly, and incubate at 4°C in the dark for 30 min.

[0054] (4) After staining, add 4 mL of PBS to the flow cytometer and centrifuge at 2000 rpm for 5 min. Discard the supernatant and add 300 μL of PBS to the cell pellet to resuspend the cells. Detect on a flow cytometer and analyze with FlowJo10 software to obtain the fluorescently labeled CD45 + CD8 + HLA-DRB5 + The data of T cell subsets were analyzed and GraphPad Prism9 was used to calculate the median of each group of data and the P value. The rank sum test was preferred for statistics, and P < 0.05 was considered statistically significant.

[0055] 4. Experimental Results

[0056] The experimental results are as follows Figures 1 to 7 shown.

[0057] In patients with aplastic anemia, the results were compared with healthy volunteers and patients with other blood disorders (eg Figure 2 ), CD45 in the bone marrow of patients with aplastic anemia + CD8 + HLA-DRB5 + The proportion of T cells increased, and P < 0.001, indicating a significant difference, suggesting that CD45 + CD8 + HLA-DRB5 + T cells can be used as an auxiliary diagnostic indicator for clinical aplastic anemia.

[0058] Non-parametric test: The median number of CD45+CD8+HLA-DRB5+T cells (%) of AA was 2.3, and the Z value was -5.361, P<0.001 compared with non-aplastic anemia patients (other blood diseases and healthy subjects).

[0059] ROC curve (such as Figure 3): The Youden index of CD45+CD8+HLA-DRB5+T cells (%) in AA patients was 0.8182, the cut-off value was 0.9550, the sensitivity was 0.8182, the specificity was 1, and the AUC was 0.9336, which was of diagnostic significance. The AUC of patients with other blood diseases was 0.5985, which was not of diagnostic significance.

[0060] In the validation set (such as Figure 4 ): The sensitivity of CD45+CD8+HLA-DRB5+T cells (%) in AA patients was 0.9333, the specificity was 0.95, and the AUC was 0.9717, which was of diagnostic significance. The AUC of patients with other blood diseases was 0.5689, which was not of diagnostic significance.

[0061] In patients with graft-versus-host disease, compared with healthy volunteers (eg Figure 5 ), CD45 in the bone marrow of patients with graft-versus-host disease + CD8 + HLA-DRB5 + The proportion of T cells was significantly increased, and P < 0.01. Secondly, compared with patients with other blood diseases, P < 0.05. The above results suggest that CD45 + CD8 + HLA-DRB5 + T cells can be used as an auxiliary diagnostic indicator for clinical graft-versus-host disease.

[0062] Non-parametric test: The median CD45+CD8+HLA-DRB5+T cells (%) of GVHD patients was 1.725, and the Z value was -3.388, P<0.05 compared with non-GVHD patients (other blood diseases and normal people).

[0063] ROC curve (such as Figure 6 ): The Youden index of CD45+CD8+HLA-DRB5+T cells (%) in patients with GVHD was 0.8182, the cut-off value was 1.105, the sensitivity was 0.8182, the specificity was 1, and the AUC was 0.9221, which was of diagnostic significance. The AUC of patients with other blood diseases was 0.5985, which was not of diagnostic significance.

[0064] In the validation set (such as Figure 7 ): CD45+CD8+HLA-DRB5+T cells (%) in patients with GVHD had a sensitivity of 0.9333, a specificity of 1, and an AUC of 0.9524, which was diagnostically significant. The AUC for patients with other blood diseases was 0.5689, which was not diagnostically significant.

[0065] In summary, the inventors first discovered that CD45+ CD8 + HLA-DRB5 + The proportion of T cells increased significantly. Among them, statistical analysis confirmed that CD45 + CD8 + HLA-DRB5 + T cells showed significant differences between AA, GVHD and healthy volunteers and other blood disease patients (AA: P < 0.001, GVHD: P < 0.05). ROC curve analysis showed that the AUCs for AA and GVHD were 0.9336 and 0.9221, respectively, with a sensitivity of 0.8182 and a specificity of 1. In the validation set, the AUCs for AA and GVHD were 0.9717 and 0.9524, respectively, with a sensitivity of 0.9333 and a specificity of 0.95 and 1, respectively. CD45 + CD8 + HLA-DRB5 + T cells have good accuracy in diagnosing AA and GVHD, indicating that they can be used as one of the laboratory immune-related detection indicators for auxiliary diagnosis of AA and GVHD. It is simple and feasible to operate and has high clinical application value.

[0066] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively detailed and specific, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. CD45 + CD8 + HLA-DRB5 + The use of T cells as diagnostic markers in the preparation of a kit for assisting the diagnosis of aplastic anemia (AA) and graft-versus-host disease (GVHD), characterized in that: The sample detected by the kit is bone marrow fluid.

2. The application according to claim 1, characterized in that: The kit is a flow cytometry detection kit.

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