CN117347633A - CD45 + CD8 + HLA-DRB5 + Application of T cells in preparation of kit for assisting in diagnosis of aplastic anemia and graft-versus-host disease - Google Patents
CD45 + CD8 + HLA-DRB5 + Application of T cells in preparation of kit for assisting in diagnosis of aplastic anemia and graft-versus-host disease Download PDFInfo
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Abstract
The invention belongs to the technical field of medical examination, and in particular relates to CD45 + CD8 + HLA‑DRB5 + Use of T cells in the preparation of a kit for aiding in the diagnosis of Aplastic Anemia (AA) and Graft Versus Host Disease (GVHD). The inventors have found for the first time that CD45 is compared to healthy volunteers and other blood disease patients + CD8 + HLA‑DRB5 + T cells are significantly elevated in the peripheral blood or bone marrow fluid of AA and GVHD patients. The invention can be used as one of the auxiliary diagnostic detection indexes of AA and GVHD. The invention provides a marker CD45 for diagnosing AA and GVHD + CD8 + HLA‑DRB5 + T cells can simply and efficiently diagnose AA and GVHD, and have the advantages of easy popularization and rapidness.
Description
Technical Field
The invention belongs to the technical field of medical examination, and in particular relates to CD45 + CD8 + HLA-DRB5 + The use of T cells in the diagnosis of aplastic anemia and in the adjuvant diagnosis of graft versus host disease.
Background
Aplastic Anemia (AA) is a symptom of bone marrow hematopoietic failure and is mainly manifested by anemia, hemorrhage and infection syndrome caused by low hematopoietic function of bone marrow and cytopenia of whole blood. The pathogenesis of the hematopoietic stem cell is related to primary or secondary hematopoietic stem cell defects, and the pathogenesis is not clear at present, but it is widely considered that bone marrow damage caused by abnormal activation and hyperfunction of T lymphocytes is one of the causes of aplastic anemia. The diagnostic criteria for aplastic anemia are as follows: (1) blood convention: whole blood cells (including reticulocytes) decrease and lymphocyte proportions increase. At least two of the following three are met: HGB (hybrid growth hormone receptor)<100g/L；PLT<50x10 9 L; absolute value of neutrophil (ANC)<1.5x 10 9 and/L. (2) bone marrow puncture: multiple site (different planes) myelodysplasia is reduced or severe; small granule void; non-hematopoietic cells (lymphocytes, reticulocytes, plasma cells, mast cells, etc.) are increased in proportion; megakaryocyte depletion or deficiency is evident; the erythroid cells and granulocyte cells are obviously reduced. (3) bone marrow biopsy (ilium): reduced proliferation of whole section, reduced hematopoietic cells, increased non-hematopoietic cells, and no abnormal cells. (4) It is necessary to exclude congenital and other acquired and secondary whole blood cytopenias, such as: firstNatural megakaryocytopenia-free thrombocytopenia, fanconi anemia, low proliferative MDS, primary immune thrombocytopenia, and the like.
Graft-versus-host disease (GVHD) is one of the most common life-threatening complications in allogeneic hematopoietic cell transplantation, mainly due to T cells in the donor's graft being activated by an immune response in the recipient after transplantation to gain cytolytic capacity, and then attacking cells carrying the foreign antigen. It is characterized by symptoms similar to autoimmune and other immune diseases, such as: scleroderma, S-syndrome, immune cytopenia, and the like. At present, the diagnosis of graft versus host disease is mainly based on pathological manifestations such as diarrhea, rash and the like after the transplantation of patients and laboratory related tests.
Human leukocyte antigens (human leukocyte antigen, HLA) are glycoproteins encoded by the histocompatibility complex (MHC), which specifically deliver short peptides to T cells, playing a key role in the immune defenses of the human body. HLA-DRB genes encoding the beta chain of HLA-DR molecules include: it has now been found that genetic variations of-DRB 1 are associated with autoimmune diseases and some inflammatory diseases and cancers, and that their association with the diseases is also confirmed, whereas-DRB 5 gene expression levels are low and research techniques are limited, and research on its association with the diseases is not clear, but there are few studies reporting that abnormal expression of-DRB 5 gene increases the risk of developing diseases such as type I diabetes, vitiligo and systemic scleroderma. CD45 has not yet been seen + CD8 + HLA-DRB5 + T cells in AA and GVHD related reports.
To date, 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.
Disclosure of Invention
The primary purpose of the invention is to make up for the defects in the prior art and provide CD45 + CD8 + HLA-DRB5 + The use of T cells as diagnostic markers in the preparation of a kit for aiding in the diagnosis of aplastic anemia and graft versus host disease.
In a first aspect, the present invention provides a diagnostic marker for aplastic anemia and graft versus host disease, the marker being CD45 + CD8 + HLA-DRB5 + T cells, human CD45 + CD8 + HLA-DRB5 + Flow cytometry analysis of T cells labeled CD45 + CD8 + HLA-DRB5 + Namely CD45 positive, CD8 positive, HLA-DRB5 positive.
A second aspect of the invention is to provide a CD45 + CD8 + HLA-DRB5 + The application of T cells as diagnostic markers in the preparation of detection products for diagnosing aplastic anemia and graft-versus-host disease.
In one embodiment, the test product is CD45 useful for aiding diagnosis + CD8 + HLA-DRB5 + T cell flow cytometry detection kit.
In one embodiment, the aplastic anemia and graft-versus-host disease related test product is a diagnostic kit, which may be used to aid in the diagnosis of aplastic anemia and graft-versus-host disease.
A third aspect of the present invention is to provide a CD45 + CD8 + HLA-DRB5 + T cell flow cytometry detection kit, wherein said CD45 + CD8 + HLA-DRB5 + The T cell flow cytometry detection kit comprises the following components: the kit is used for detecting CD45 by marking different fluorescence + CD8 + HLA-DRB5 + Monoclonal antibodies to T cell subsets of (c): anti-CD 45 antibodies, anti-CD 8 antibodies, anti-HLA-DRB 5 antibodies.
In one embodiment, the kit further comprises a fluorescent label: the fluorescent label of the anti-CD 45 antibody is PerCP-Cy5.5; the fluorescent label of the anti-CD 8 antibody is PE; the fluorescent label of the anti-HLA-DRB 5 antibody is FITC.
In one embodiment, the kit is used at a dosage recommended by the instructions for the purchased antibody.
In one embodiment, the kit test sample is peripheral blood or bone marrow fluid.
In one embodiment, the kit further comprises a red blood cell lysate and a Phosphate Buffered Saline (PBS) solution.
A fourth aspect of the invention is to provide the use of a kit as described above for the study of aplastic anemia and graft versus host disease for non-diagnostic purposes.
Compared with the prior art, the invention has the following advantages and effects:
the inventor discovers CD45 in bone marrow fluid of patients suffering from aplastic anemia and graft versus host disease for the first time + CD8 + HLA-DRB5 + T cell proportion was significantly increased. Wherein, the statistical analysis proves that CD45 + CD8 + HLA-DRB5 + T cells have significant differences between AA, GVHD and healthy volunteers and other blood disease patients (AA: P)<0.001，GVHD：P<0.05 Can be used as one of laboratory immune related detection indexes for auxiliary diagnosis of AA and GVHD, has simple and feasible operation, and has high clinical application value. In addition, the invention also provides CD45 + CD8 + HLA-DRB5 + The method for detecting the T cell subgroup can be used for quantitatively analyzing the phenotype of the T cell subgroup.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is a graph of flow cytometry analysis of healthy volunteers (Normal), aplastic anemia patients (AA), graft versus host disease patients (GVHD), and other hematological patients (AML patients herein).
FIG. 2 shows CD45 in bone marrow of aplastic anemia group and other blood diseases and healthy volunteers group + CD8 + HLA-DRB5 + Statistical analysis of T cell subpopulations, wherein, *** representing P<0.001, ns indicates that the difference is not statistically significant, P<0.05 has statistical significance.
FIG. 3 is a schematic representation of CD45 in bone marrow from the graft versus host disease group and other hematological disorders and healthy volunteers + CD8 + HLA-DRB5 + Statistical analysis of T cell subpopulations, wherein, * is P<0.05， ** Representing P<0.01, ns indicates that the difference is not statistically significant, P<0.05 has statistical significance.
Detailed description of the preferred embodiments
The invention will be further illustrated with reference to specific examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
The invention is illustrated below with reference to specific examples.
The main reagents used in the examples are as follows: HLA-DRB5 antibodies (ProSci, 64-133), PE anti-human CD8 (Biolegend, 980902), fluorescen (FITC) -conjugated affinity purified donkey anti-rabbit IgG (ProSci, JAC-711-095-152), perCP anti-human CD45 antibodies (Biolegend, 304025).
Examples
1. Selection of clinical cases
In this example 27 cases of aplastic anemia patients, 7 cases of graft-versus-host patients, 12 cases of other blood diseases (such as MDS, AML, etc.) other than aplastic anemia, 11 cases of healthy volunteers, and age and sex were selected randomly. After soliciting the subject's consent, an informed consent was signed.
Inclusion criteria for aplastic anemia patients:
(1) Hemogram (blood image): whole blood cell (including reticulocytes) depletion is at least in accordance with one of the following three factorsTwo of (3): HGB (hybrid growth hormone receptor)<100g/L；PLT<50x 10 9 L; absolute value of neutrophil (ANC)<1.5x 10 9 /L。
(2) Bone marrow puncture: multiple site (different planes) myelodysplasia is reduced or severe; small granule void; non-hematopoietic cells (lymphocytes, reticulocytes, plasma cells, mast cells, etc.) are increased in proportion; megakaryocyte depletion or deficiency is evident; the erythroid cells and granulocyte cells are obviously reduced.
(3) Bone marrow biopsy (ilium): reduced proliferation of whole section, reduced hematopoietic cells, increased non-hematopoietic cells, and no abnormal cells.
Graft versus host patient inclusion criteria:
(1) Receiving allogenic hematopoietic stem cell transplantation;
(2) After transplantation, the affected condition of each organ, such as continuous nausea, vomiting, diarrhea, rash and other skin and gastrointestinal reactions, and combined with laboratory result diagnosis, biopsy can be perfected if necessary.
Other hematological disease patients included criteria:
other hematological disorders that exclude aplastic anemia and that do not receive allogeneic hematopoietic stem cell transplantation, such as Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), multiple Myeloma (MM), etc.;
healthy volunteers were included in the standard:
(1) No acute or chronic systemic diseases, such as: diabetes, hypertension, liver dysfunction, renal dysfunction and other autoimmune-related diseases;
(2) Without mental illness.
2. The patient and healthy volunteers all collect bone marrow fluid samples for subsequent experiments according to the flow-type conventional operation flow.
3. Flow cytometry detection
(1) Isolation of mononuclear cells (PBMCs) from a bone marrow fluid sample: adding PBS (phosphate buffer solution) with the volume which is 3 times that of a blood sample into the sample, uniformly mixing to obtain a blood diluted sample, adding lymphocyte separation liquid with the volume which is equal to that of the blood diluted sample into a 50mL centrifuge tube, slowly adding the blood diluted liquid to the upper part of the lymphocyte separation liquid along the tube wall by using a suction tube, and centrifuging 600g (up to 5 and down to 0) of the centrifuge tube at 18 ℃ for 22min. After centrifugation, the middle buffy coat layer was carefully aspirated into a clean 15mL centrifuge tube, and then lysis was stopped by adding 10mL of pre-chilled PBS solution. Centrifuge the tube at 2000rpm for 5 minutes at room temperature and discard the supernatant to obtain a PBMC pellet.
(2) The resulting PBMCs were lysed and resuspended in PBS after lysis to form a single cell suspension, which was counted for flow detection.
(3) Monoclonal antibodies with different fluorescence labels are added into single cell suspension: 100 ten thousand PBMC were added to a flow tube, then 5mL of PBS was added and mixed well, the flow tube was centrifuged at 2000rpm for 5min, the supernatant was discarded, anti-CD 45 antibody, anti-CD 8 antibody, anti-HLA-DRB 5 antibody were added to PBS at the volume ratio of 1:200 according to the doses described in the purchased antibody instructions to obtain antibody dilutions, and 100. Mu.L of antibody dilutions were added to PBMC of each sample for staining: the flow tube was shaken on an shaker, thoroughly mixed and incubated at 4℃for 30min in the dark.
(4) After the staining was completed, 4mL of PBS was added to the flow tube, and centrifuged at 2000rpm for 5min, the supernatant was discarded, 300. Mu.LPBS was added to the cell pellet to resuspend the cells, the flow cytometer was used for on-machine detection, and analyzed with FlowJo10 software to obtain fluorescence-labeled CD45 + CD8 + HLA-DRB5 + Data of T cell subpopulations and median calculated for each group of data and P value calculated using GraphPad Prism9, statistically preferred rank sum test, P<0.05 is considered statistically significant.
4. Experimental results
The experimental results are shown in fig. 1 to 3. CD45 in bone marrow of aplastic anemia patients compared to healthy volunteers and other blood disease patients (as in FIG. 2) + CD8 + HLA-DRB5 + The proportion of T cells is increased, and P<0.001, indicating significant variability, suggesting CD45 + CD8 + HLA-DRB5 + T cells can be used as an auxiliary diagnostic index for clinical aplastic anemia.
CD45 in bone marrow of graft versus host patients compared to healthy volunteers (as in FIG. 3) + CD8 + HLA-DRB5 + The proportion of T cells is significantly increased, and P<0.01, secondly, P compared with other hematopathy patients<0.05, all of which suggest CD45 + CD8 + HLA-DRB5 + T cells can be used as an auxiliary diagnostic index for graft versus host disease clinic.
Taken together, these results indicate CD45 + CD8 + HLA-DRB5 + The T cells can be used as auxiliary diagnostic indexes of aplastic anemia and graft-versus-host disease, are simple and easy to operate, and have high clinical application value.
The above examples merely represent embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that several variations and modifications can be made by those skilled in the art without departing from the inventive concept, which fall within the protective scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims.
Claims (10)
1.CD45 + CD8 + HLA-DRB5 + Use of T cells as diagnostic markers in the preparation of a kit for aiding in the diagnosis of Aplastic Anemia (AA) and Graft Versus Host Disease (GVHD).
2. The use according to claim 1, characterized in that: the kit is a flow cytometry detection kit.
3. A flow cytometry detection kit for aiding in the diagnosis of aplastic anemia and graft versus host disease, characterized in that: the kit using CD45 of claim 1 + CD8 + HLA-DRB5 + T cells as diagnostic markers for aiding in the diagnosis of aplastic anemia and graft versus host disease, the kit comprising the detection of human CD45 + CD8 + HLA-DRB5 + Reagents for T cell content.
4. A test according to claim 3The kit is characterized in that: the reagent is used for detecting human CD45 + CD8 + HLA-DRB5 + T cell flow cytometry analyses of the labeled antibodies.
5. The kit of claim 4, wherein: the antibodies are the following monoclonal antibodies labeled with different fluorescence: anti-CD 45 antibodies, anti-CD 8 antibodies, and anti-HLA-DRB 5 antibodies.
6. The kit of claim 5, wherein: the fluorescent label of the anti-CD 45 antibody is PerCP-Cy5.5; the fluorescent label of the anti-CD 8 antibody is PE; the fluorescent label of the anti-HLA-DRB 5 antibody is FITC.
7. The kit of claim 5, wherein: the dosage of the antibody is recommended according to the instructions of the purchased antibody.
8. A kit according to claim 3, wherein: the detection sample of the kit is peripheral blood or bone marrow fluid.
9. A kit according to claim 3, wherein: the kit further comprises a red blood cell lysate and a Phosphate Buffered Saline (PBS) solution.
10. Use of a kit according to any one of claims 3 to 9 for the study of aplastic anemia and graft-versus-host disease for non-diagnostic purposes.
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| CN202311318830.4A CN117347633A (en) | 2023-10-12 | 2023-10-12 | CD45 + CD8 + HLA-DRB5 + Application of T cells in preparation of kit for assisting in diagnosis of aplastic anemia and graft-versus-host disease |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311318830.4A CN117347633A (en) | 2023-10-12 | 2023-10-12 | CD45 + CD8 + HLA-DRB5 + Application of T cells in preparation of kit for assisting in diagnosis of aplastic anemia and graft-versus-host disease |
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