Application of antibody combination in diagnostic kit for high-risk myelodysplastic syndrome

Abstract

This invention belongs to the field of medical biodiagnostic technology and relates to the application of an antibody combination in a diagnostic kit for intermediate- to high-risk myelodysplastic syndromes (MDS). The eight fluorescently labeled antibodies are as follows: PerCP-cy5.5 anti-human CD4 antibody, KO anti-human CD45 antibody, APC-H7 anti-human CD3 antibody, APC anti-human PD-1 antibody, PB anti-human CXCR5 antibody, PC7 anti-human CD19 antibody, PerCP-cy5.5 anti-human CD27 antibody, and FITC anti-human IgD antibody. This invention uses this antibody composition to detect the proportions of four cell subsets in the bone marrow of MDS patients, which can help clinicians quickly and efficiently screen intermediate- to high-risk MDS patients and guide subsequent treatment selection.

Description

Technical Field

[0001] This invention belongs to the field of medical biological diagnostic technology and relates to the application of an antibody combination in a diagnostic kit for intermediate- to high-risk myelodysplastic syndromes. Background Technology

[0002] Myelodysplastic neoplasm (MDS), a malignant clonal disease of hematopoietic stem cells, is one of the most common hematologic malignancies in the elderly, especially given the increasingly aging population. In recent years, its incidence has shown a trend towards affecting younger people. Clinically, based on indicators such as blood routine tests, chromosome analysis, and blast cell counts in bone marrow morphology, and referring to stratification systems such as IPSS, IPSS-R, and IPSS-M, MDS patients can be stratified into low-risk, intermediate-risk, and high-risk groups. Intermediate- and high-risk MDS patients have a poor prognosis and a high chance of developing acute leukemia, thus requiring more aggressive treatment measures, including targeted therapies such as demethylating agents. Delayed treatment can severely impact patient survival.

[0003] Traditional MDS risk stratification systems are based on bone marrow cell morphology, including the number of primitive cells and chromosome analysis. However, due to the subjectivity of laboratory personnel in interpreting results and the possibility of false negatives, the accuracy of these systems may be insufficient, failing to precisely distinguish between low-risk and intermediate-to-high-risk MDS patients. Furthermore, more accurate risk stratification for treatment planning often requires waiting for chromosome results, but chromosome analysis is time-consuming, frequently prolonging the onset of treatment. In addition, patients diagnosed as low-risk MDS using traditional stratification systems have a median survival of approximately 10 years. Some patients exhibit higher invasiveness and progress to high-risk MDS in a short period. If close monitoring is not maintained during follow-up, delaying treatment, and if the patient transforms into acute leukemia, the median survival after transformation is shortened to 5 to 12 months, resulting in a very poor prognosis. Therefore, accurate risk stratification for MDS patients is crucial. However, existing stratification systems still cannot fully meet clinical needs, and more sensitive, rapid, and specific biomarkers are currently lacking in technology. Summary of the Invention

[0004] This invention addresses the problems existing in traditional diagnostic kits for intermediate- and high-risk myelodysplastic syndromes by proposing a novel antibody combination for use in such kits.

[0005] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0006] This invention proposes the application of a combination of eight fluorescently labeled antibodies in a rapid screening diagnostic kit for high-risk myelodysplastic syndromes. The eight fluorescently labeled antibodies are as follows: PerCP-cy5.5 anti-human CD4 antibody, KO anti-human CD45 antibody, APC-H7 anti-human CD3 antibody, APC anti-human PD-1 antibody, PB anti-human CXCR5 antibody, PC7 anti-human CD19 antibody, PerCP-cy5.5 anti-human CD27 antibody, and FITC anti-human IgD antibody.

[0007] This invention proposes that the above-mentioned combination of eight fluorescently labeled antibodies be used to detect the proportions of Tfh, Tph, CD19+IgD-CD27+B cells, and CD19+IgD-CD27-B cells in human bone marrow fluid. An increased proportion of Tfh cells and CD19+IgD-CD27+B cells, and a decreased proportion of Tph cells and CD19+IgD-CD27-B cells, indicates that the tested subject has intermediate-to-high-risk MDS with high malignancy. If the proportions of Tfh cells, CD19+IgD-CD27+B cells, Tph cells, and CD19+IgD-CD27-B cells are not significantly different from those of normal individuals, it indicates that the tested subject has intermediate-to-high-risk MDS with low malignancy.

[0008] Furthermore, the proportion of Tfh cells among CD4+ T cells was higher than 16.2%, and the proportion of CD19+IgD-CD27+ B cells among CD19+ B cells was higher than 12.2%, while the proportion of Tph cells among CD4+ T cells was lower than 2.3%, and the proportion of CD19+IgD-CD27- B cells among CD19+ B cells was lower than 12.8%, indicating that the tested subjects had intermediate- to high-risk MDS with a high degree of malignancy.

[0009] Recent studies have shown that immune microenvironment dysregulation plays a crucial role in the pathogenesis, progression, and treatment of myasthenia gravis (MDS), exerting significant influence in regulating humoral immunity, inflammatory responses, and autoimmunity. This invention utilizes flow cytometry with various antibody compositions to discover that, compared to healthy individuals and those with low-risk MDS, patients with intermediate- and high-risk MDS exhibit specific immune cell population disturbances. Detection confirmed that patients with intermediate- and high-risk MDS showed increased proportions of follicular helper T cells (Tfh) and CD19+IgD-CD27+ B cells, and decreased proportions of peripheral helper T cells (Tph) and CD19+IgD-CD27- B cells. In contrast, there was no difference in cell proportions between healthy individuals and those with low-risk MDS. This information can aid in the rapid clinical screening of intermediate- and high-risk MDS and further guide subsequent patient treatment. Flow cytometry is a simple and rapid method with 10... -5With its advantages of high sensitivity and accuracy, this project is technically mature and stable, and is currently widely used in the diagnosis of malignant hematological diseases. This invention is based on flow cytometry and uses a new antibody composition to help clinicians quickly and efficiently screen intermediate- to high-risk MDS patients and guide patients in choosing subsequent treatment.

[0010] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0011] 1. This invention provides an antibody composition for rapidly detecting the proportions of specific T and B cell subsets in the bone marrow of MDS patients. This antibody composition can detect the proportions of four cell subsets in the bone marrow of MDS patients, helping to rapidly screen for intermediate- and high-risk MDS patients. It provides a rapid and sensitive screening method for clinical practice, and better guides the treatment of intermediate- and high-risk MDS patients.

[0012] 2. This invention only requires extracting the patient's bone marrow fluid, which can be collected at the same time as the patient undergoes bone marrow aspiration to confirm the diagnosis. The patient does not need to undergo additional invasive procedures, reducing additional pain and improving patient compliance. Moreover, the results are more accurate using mature flow cytometry technology, making it suitable for large-scale clinical application.

[0013] 3. This invention innovatively employs a combination of multiple antibodies, effectively avoiding the problem of repeated use of traditional gating antibodies, significantly increasing the types of effective antibodies that can be used, improving convenience, and ensuring the specificity and sensitivity of the diagnosis. It provides a new, rapid and efficient detection method for the accurate diagnosis and classification of high-risk MDS in clinical practice, and also provides an important basis for the subsequent selection of appropriate treatment plans. Attached Figure Description

[0014] Figure 1 This diagram illustrates the process of detecting the proportions of Tph and Tfh cells in bone marrow mononuclear cells using flow cytometry. In diagram a: FSC-A / FSC-H gate is used to enclose single cells, removing adherent cells. In diagram b: Active mononuclear cells are enclosed using FSC-A / SSC-A gate. In diagram c: Lymphocytes are enclosed using CD45 / SSC gate. CD3+ T cell populations are selected using CD3 / SSC expression. In diagram d: CD3+CD4+ T cells are selected using CD4 / CD3 antibody expression. In diagram e: Tfh and Tph cells are enclosed using PD-1 and CXCR5.

[0015] Figure 2 and Figure 3 To determine the proportions of Tfh, Tph, CD19+IgD-CD27+B, and CD19+IgD-CD27-B cells in the bone marrow of two patients with intermediate-to-high-risk MDS and normal individuals.

[0016] Figure 4 This is a ROC curve. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0019] The manufacturers or specifications of some reagents and instruments in the following examples are as follows:

[0020] PBS buffer: Commercially available 10×PBS buffer, diluted to 1×PBC for later use.

[0021] Flow cytometry-specific hemolysin: BD Corporation, catalog number 349202.

[0022] The Navios 10-color flow cytometer is equipped with three lasers (405nm, 488nm, and 635nm) and 10 fluorescence detectors. Example

[0023] This embodiment involves sorting bone marrow mononuclear cells from patients with myelodysplastic syndrome and performing flow cytometry analysis. The specific steps are as follows.

[0024] 1. Prepare cells

[0025] (1) Collect 3 mL of bone marrow fluid from newly diagnosed MDS patients and 3 mL of bone marrow fluid from normal controls (samples were obtained from the Department of Hematology, Qilu Hospital of Shandong University, and were anticoagulated with EDTA) for later use.

[0026] (2) Add 100 μL of bone marrow fluid to 150 μL of 1×PBS buffer, mix well, count the number of cells per microliter, and adjust the cell concentration to approximately 10×10⁻⁶ based on the test results. 6 / 100μL, take 50μL of cells and add them to the test tube.

[0027] (3) Prepare two flow cytometry tubes, with tube 1 used to detect Tph and Tfh cells. Add 50 μL of bone marrow fluid cells prepared in step (2) to tube 1, then add 20 μL of fluorescent antibody KO-CD45, 20 μL of PerCP-cy5.5-CD4, 5 μL of APC-H7-CD3, 5 μL of PB-CXCR5, and 5 μL of APC-PD-1. Mix well, incubate in the dark for 15 min, then centrifuge at 1300 rpm for 5 min, discard the supernatant, add 500 μL of PBS buffer and mix well for later use.

[0028] Flow cytometry tube No. 2 was used to detect CD19+IgD-CD27+B cells and CD19+IgD-CD27-B cells. 50 μL of bone marrow fluid cells prepared according to step (2) were added to tube No. 2, followed by 20 μL of fluorescent antibody KO-CD45, 5 μL of PC7-CD19, 5 μL of PerCP-cy5.5-CD27, and 5 μL of FITC-IgD. The mixture was then centrifuged at 1300 rpm for 5 min, the supernatant was discarded, and 500 μL of 1×PBS buffer was added and mixed thoroughly.

[0029] (4) The antibody combinations and fluorescein combinations used in the above steps for detecting cell populations are summarized in Table 1 below.

[0030] Table 1 Summary of Antibodies and Compatible Fluorescein Synthetic Agents

[0031]

[0032] (5) Hemolysis: Add 2 mL of 1×FACS hemolysin to each of the two flow cytometers, vortex at 80 rpm for 3 min, and then incubate at room temperature in the dark for 8-10 min. After incubation, centrifuge at 300 g for 5 min, discard the supernatant, and set aside.

[0033] (6) Add 1 mL of 1×PBS buffer to each of the two flow cytometry tubes, centrifuge at 300g for 5 min, discard the supernatant, and repeat the washing and centrifugation once more with 1×PBS buffer. Then add 200 μL of 1×PBS buffer to resuspend the cells and wait for the flow cytometry test.

[0034] (7) Transfer the cell suspension to a flow cytometer containing a filter cartridge, filter the cells, place on ice, and run on the machine within 24 hours.

[0035] 2. Navios instrument setup: Under the pre-set instrument conditions, obtain at least 50,000 CD45-positive white blood cells per flow cytometer. First, enclose the viable cells, adjust the compensation, and then perform gating. Use Kluza software to analyze the data.

[0036] (1) Set gates for FSC-A / FSC-H, circle individual cells, and remove adhering cells, such as Figure 1 As shown in Figure a.

[0037] (2) Gating of viable mononuclear cells: Using FSC-A / SSC-A as a gate, debris and dead cells are removed, such as... Figure 1 As shown in b.

[0038] (3) Gating with CD45 / SSC to select lymphocytes. CD3 / SSC expression is used to select CD3+ T cell populations, and CD19 / SSC expression is used to select CD19+ B cell populations, such as... Figure 1 As shown in c.

[0039] (4) Then, CD3+CD4+ T cells are selected by CD4 / CD3 antibody expression, such as... Figure 1 As shown in d.

[0040] (5) Tfh and Tph cells were circled using PD-1 (CD279) and CXCR5 (CD185). Figure 1 ), that is, Tfh: CD45+CD3+CD4+CXCR5+PD-1+; Tph: CD45+CD3+CD4+CXCR5-PD-1+, such as Figure 1 As shown in e.

[0041] (6) Circle CD19+IgD-CD27+B cells and CD19+IgD-CD27-B cells with IgD and CD27.

[0042] (7) Statistical analysis showed that the proportion of Tfh cells to CD4+ T cells in normal controls was 2.2%–16.2%, CD19+IgD-CD27+ B cells to CD19+ B cells was 6.2%–12.2%, Tph cells to CD4+ T cells was 2.3%–9.4%, and CD19+IgD-CD27- B cells to CD19+ B cells was 12.8%–29.7%. When the proportion of Tfh cells to CD4+ T cells in a patient is higher than 16.2%, the proportion of CD19+IgD-CD27+ B cells to CD19+ B cells is higher than 12.2%, and the proportion of Tph cells to CD4+ T cells is lower than 2.3% and the proportion of CD19+IgD-CD27- B cells to CD19+ B cells is lower than 12.8%, the patient is considered to have intermediate to high-risk MDS and needs to start demethylation therapy.

[0043] This invention proposes using the proportions of Tfh, CD19+IgD-CD27+B, Tph, and CD19+IgD-CD27-B cells in patients with myelodysplastic syndrome (MDS) as biomarkers to screen for intermediate- to high-risk MDS patients. To further verify the abnormal proportions of Tfh, CD19+IgD-CD27+B, Tph, and CD19+IgD-CD27-B cells in intermediate- to high-risk MDS patients, 30 MDS patients (selected based on blood routine tests, bone marrow aspiration, biopsy, and immunophenotyping) were collected and compared with 10 controls (3 mL of bone marrow fluid). Flow cytometry analysis was performed on both groups. The results showed significant differences between the two populations, indicating that these four indicators can be used to screen for intermediate- to high-risk MDS patients. Further statistical analysis showed that the area under the ROC curve for the diagnosis of these 30 intermediate- to high-risk MDS cases was 0.881 (…). Figure 4 ).

[0044] Accuracy verification: The method described in Example 1 was used to verify the accuracy of 20 clinically diagnosed MDS patients (sample data were obtained from Qilu Hospital of Shandong University, with informed consent from the patients). These 20 patients had been diagnosed as low-risk, intermediate-risk, or high-risk myelodysplastic syndromes using traditional clinical assessment methods. However, using the flow cytometry method proposed in this invention, the risk level can be determined in just 2 days, greatly shortening the diagnosis time. The statistical results are shown in Table 1 below.

[0045] Table 1. Statistical analysis of validation results in 20 patients.

[0046]

[0047] Table 1 shows that intermediate- and high-risk MDS patients (numbered 1-8) met the criteria of Tfh cells accounting for more than 16.2% of CD4+ T cells and CD19+IgD-CD27+ B cells accounting for more than 12.2%, while Tph cells accounting for less than 2.3% of CD4+ T cells and CD19+IgD-CD27- B cells accounting for less than 12.8%. The test values ​​for low-risk patients (numbered 9-20) differed significantly from those of intermediate- and high-risk patients.

[0048] To provide a more intuitive display, samples from two newly diagnosed high-risk MDS patients were randomly selected for testing, and the results are as follows: Figure 2 (Patient No. 1 in Table 1) and Figure 3 (Patient No. 3 in Table 1) As can be seen from the figure, the flow cytometry results showed that the proportion of Tfh cells and CD19+IgD-CD27+B cells was increased, while the proportion of Tph cells and CD19+IgD-CD27-B cells was decreased, which is consistent with the diagnosis.

[0049] In summary, the screening method proposed in this invention enriches the diagnostic methods for intermediate- and high-risk myelodysplastic syndromes, and the prepared kit can help to quickly and accurately determine the risk stratification of myelodysplastic syndromes, improving diagnostic efficiency and making it more operable and rapid.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of a combination of eight fluorescently labeled antibodies in a rapid screening diagnostic kit for high-risk myelodysplastic syndromes, characterized in that... The eight fluorescently labeled antibodies are as follows: PerCP-cy5.5 anti-human CD4 antibody, KO anti-human CD45 antibody, APC-H7 anti-human CD3 antibody, APC anti-human PD-1 antibody, PB anti-human CXCR5 antibody, PC7 anti-human CD19 antibody, PerCP-cy5.5 anti-human CD27 antibody, and FITC anti-human IgD antibody.

2. The application according to claim 1, characterized in that, The eight fluorescently labeled antibody combinations were used to detect the proportions of Tfh cells, Tph cells, CD19+IgD-CD27+B cells, and CD19+IgD-CD27-B cells in human bone marrow fluid. An increased proportion of Tfh cells and CD19+IgD-CD27+B cells, and a decreased proportion of Tph cells and CD19+IgD-CD27-B cells, indicated that the tested subject had intermediate- to high-risk MDS. If the proportions of Tfh cells, CD19+IgD-CD27+B cells, Tph cells, and CD19+IgD-CD27-B cells were not significantly different from those of normal individuals, it indicated that the tested subject did not have intermediate- to high-risk MDS.

3. The application according to claim 1, characterized in that, The presence of Tfh cells accounting for more than 16.2% of CD4+ T cells and CD19+IgD-CD27+ B cells accounting for more than 12.2% of CD19+ B cells, while the presence of Tph cells accounting for less than 2.3% of CD4+ T cells and CD19+IgD-CD27- B cells accounting for less than 12.8% of CD19+ B cells, indicates that the tested subjects have intermediate- to high-risk MDS.

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