Acute B lymphocytic leukemia noninvasive screening method based on peripheral blood CD3+CD19 + double positive cell subsets

Abstract

The invention belongs to the technical field of blood disease diagnosis, and provides a noninvasive screening method for acute B lymphocytic leukemia (B-ALL) based on a peripheral blood CD3 + CD19 + double positive cell subset, which comprises the following steps: detecting the proportion of CD3 + CD19 + double positive cells in peripheral blood through flow cytometry, and comparing the proportion with a proportion threshold in a positive judgment standard for judgment; when the detection result is greater than or equal to 0.20%, judging that the sample is positive; cD3 + CD19 + double positive cells exist in B-ALL peripheral blood, the detection rate of B-ALL peripheral blood in peripheral blood of healthy people and other types of leukemia patients is extremely low, and the B-ALL has high specificity; compared with bone marrow puncture and other invasive diagnosis methods, the non-invasive primary screening of B-ALL is realized, and the problem of insufficient specificity of the existing non-invasive detection technology is overcome; the kit is suitable for early screening of children B-ALL, dynamic monitoring of chemotherapy curative effect and prognosis evaluation, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of hematological diagnostic technology, and relates to the detection of acute B-lymphoblastic leukemia, specifically involving a method based on peripheral blood CD3. + CD19 + Non-invasive screening method for acute B-lymphoblastic leukemia based on double-positive cell subsets. Background Technology

[0002] B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common malignant tumors in children, accounting for more than 80% of childhood leukemias. Typical clinical symptoms include fever, anemia, bleeding, and hepatosplenomegaly and lymphadenopathy. A large number of primitive lymphocytes are visible in peripheral blood and bone marrow. Currently, the overall 5-year survival rate for childhood B-ALL has reached over 90%, making early screening and diagnosis crucial for early detection and treatment. Current diagnostic challenges include: early symptoms (such as fatigue and bone pain) are nonspecific and easily confused with infection or nutritional anemia; bone marrow aspiration is the gold standard for diagnosis, but its invasiveness leads to low compliance in some children, poses a risk of infection, and makes dynamic monitoring difficult. In recent years, the development of non-invasive liquid biopsy techniques (such as ctDNA and exosome detection) has provided new ideas for tumor screening, but its application in B-ALL still faces fundamental bottlenecks. ctDNA testing has low sensitivity in the initial low tumor burden stage and cannot distinguish B-ALL from other B-cell abnormal proliferation diseases; exosome biomarkers suffer from low standardization of exosome isolation techniques and poor specificity due to functional heterogeneity. Peripheral blood, as an important sample reflecting disease status, has been widely used in recent years for tumor biomarker discovery and precision medicine research. The development of non-invasive peripheral blood biomarkers for B-ALL screening can reduce trauma, improve early diagnosis rates, and is crucial for the early detection and treatment of B-ALL.

[0003] This invention utilizes flow cytometry to detect CD3 in the peripheral blood of newly diagnosed B-ALL children. + CD19 + The specific expression of a cell subset, which has an extremely low detection rate in non-leukemic and other types of leukemia, was confirmed through validation in 138 children, identifying CD3. + CD19 + Cell subsets can serve as non-invasive screening biomarkers for B-ALL. This provides a novel approach for the discovery of non-invasive screening biomarkers in peripheral blood for B-ALL, and has significant scientific and clinical translational value. Summary of the Invention

[0004] The purpose of this invention is to provide a method based on peripheral blood CD3. + CD19 +A non-invasive screening method for acute B-lymphoblastic leukemia using double-positive cell subsets involves detecting CD3+ in peripheral blood via flow cytometry. + CD19 + The proportion of double-positive cell subsets was determined based on the positive criteria to address the problem that existing technologies for acute B-lymphoblastic leukemia rely on invasive procedures such as bone marrow aspiration, while non-invasive screening lacks specific biomarkers.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: a seed based on peripheral blood CD3 + CD19 + A non-invasive screening method for acute B-lymphoblastic leukemia based on double-positive cell subsets, the steps of which are as follows:

[0006] S1. Immature cells were found in peripheral blood smears;

[0007] S2. Peripheral blood containing immature cells from S1 was analyzed by flow cytometry to detect CD3+ in the peripheral blood. + CD19 + The presence and proportion of double-positive cell subsets;

[0008] S3. Compare the test results in S2 with the positive judgment criteria. When the test result is greater than or equal to the proportion threshold in the positive judgment criteria, it is considered to be positive for acute B-lymphoblastic leukemia.

[0009] Preferably, the flow cytometry detection of CD3 in peripheral blood in step S2 is described. + CD19 + The method for determining the proportion of double-positive cell subsets is as follows:

[0010] a. Collect 1 mL of peripheral blood from the subject, i.e., EDTA-K2 anticoagulated blood;

[0011] b. Take 25 μL of peripheral blood from step a, add the fluorescent labeling combination, and incubate in the dark for 15 min.

[0012] c. Add 225 μL of hemolysin and incubate in the dark for 15 min;

[0013] d. No washing required; detection using flow cytometry.

[0014] Preferably, the fluorescent labeling combination in step b is CD3, CD19, and CD45.

[0015] Preferably, the positive criterion in step S3 is determined by calculating CD3. + CD19 + Double-positive cells in CD45 + The proportions in the cells are established, with a threshold of 0.20%.

[0016] The present invention further provides the above-mentioned CD3 + CD19 + Application of double-positive cell subsets.

[0017] Preferably, the CD3 + CD19 + Double-positive cell subsets serve as biomarkers for detecting acute B-lymphoblastic leukemia.

[0018] Preferably, the biomarker is used to prepare a product for detecting acute B-lymphoblastic leukemia.

[0019] Preferably, the product includes a kit for detecting acute B-lymphoblastic leukemia.

[0020] Preferably, the kit includes one or more of a nucleic acid probe, an antigen, or an antigen-binding fragment for identifying the biomarker.

[0021] Preferably, the biomarker is used to construct a system for detecting acute B-lymphoblastic leukemia.

[0022] The beneficial effects of this invention are:

[0023] 1. A highly specific biomarker: This study revealed and confirmed the presence of CD3 in peripheral blood. + CD19 + Double-positive cell subsets are highly specific to the occurrence of B-ALL. Compared with the cross-reactivity of liquid biopsy markers such as circulating tumor DNA (ctDNA) in B-ALL with other B-cell proliferative diseases, this marker has an extremely low detection rate in non-leukemia and other types of leukemia, showing excellent disease differentiation ability and effectively reducing the risk of misdiagnosis.

[0024] 2. Achieving non-invasive screening: This invention provides a non-invasive screening method that avoids the trauma, infection risk, and poor patient compliance associated with bone marrow aspiration by collecting peripheral blood.

[0025] 3. The detection sensitivity is high. For newly diagnosed patients with low tumor burden, this method can reliably detect abnormal cell subsets as low as 0.20%, which is more conducive to the early detection of acute B-lymphoblastic leukemia.

[0026] 4. It has clinical translation potential. Based on a mature flow cytometry platform, the detection process is highly standardized, easy to operate, and the results are stable and reliable, making it easy to promote and apply in clinical laboratories. Attached Figure Description

[0027] Figure 1 This is a diagram of the detection process and flow gating strategy in this invention;

[0028] Figure 2 CD3 in this invention + CD19 + Flow cytometry and ROC curve of double-positive cell subsets (A is CD3) + CD19 + Representative graph of cell subpopulation flow cytometry analysis, B is CD3 + CD19 + The cell proportion chart, where C is the AUC curve. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Example 1: Non-invasive screening method for acute B-lymphoblastic leukemia

[0033] (1) Blood smear test

[0034] If immature cells are found in the peripheral blood smear microscopy, proceed to the next step of flow cytometry.

[0035] (2) Cell flow cytometry detection

[0036] Peripheral blood was collected from the subjects, and 1 mL of EDTA-K2 anticoagulated solution was added. 25 μL of this peripheral blood was then added to a fluorescently labeled combination of CD3, CD19, and CD45, and incubated in the dark for 15 minutes. After adding 225 μL of hemolysin, the mixture was incubated again in the dark for 15 minutes. The samples were then analyzed by flow cytometry without washing (gating strategy as follows). Figure 1 (As shown).

[0037] (3) Establishment of positive criteria

[0038] Flow cytometry analysis of CD3 in different types of leukemia + CD19 + Double-positive cells in CD45 + The proportion in cells ( Figure 2 (A). A total of 28 cases of non-leukemia (NC), 31 cases of myeloid lymphoid leukemia (AML), 29 cases of acute T-lymphoblastic leukemia (T-ALL), and 50 cases of acute B-lymphoblastic leukemia (B-ALL) were included in the study. Figure 2(Middle B), compared with other types of leukemia, CD3 + CD19 + Double-positive cells are specifically expressed in B-ALL. A sample is considered positive for B-ALL when the proportion of this subset is higher than 0.20%. ROC curve analysis showed an AUC of 0.87. Figure 2 (C)

[0039] Application Example 1 Patient 1

[0040] Collect a blood smear containing immature cells detected by microscopic examination. Anticoagulated peripheral blood containing EDTA was collected. 50 μL of whole blood was washed twice with PBS, and 1.5 μL of CD45-V500 antibody, 5 μL of CD3-APC antibody, and 5 μL of CD19-PE-Cy7 antibody were added. The mixture was incubated in the dark for 15 minutes, then 2 mL of PBS was added, and the mixture was centrifuged at 500g for 5 minutes. The supernatant was discarded, and the blood was resuspended in 200 μL of PBS. Flow cytometry was used to detect CD3+. + CD19 + Cell ratio (gating strategy such as) Figure 1 (As shown).

[0041] The test results showed CD3 + CD19 + The cell percentage was 3.79%, which is greater than the positive threshold (0.20%). Preliminary screening of peripheral blood suggests that the patient may have B-ALL.

[0042] Application Example 2 Patient 2

[0043] Collect a blood smear containing immature cells detected by microscopic examination. Use EDTA-anticoagulated peripheral blood. Aspirate 50 μL of whole blood, wash twice with PBS, and add 1.5 μL CD45-V500 antibody, 5 μL CD3-APC antibody, and 5 μL CD19-PE-Cy7 antibody. Incubate in the dark for 15 minutes, add 2 mL PBS, and centrifuge at 500g for 5 minutes. Discard the supernatant, resuspend in 200 μL PBS, and analyze by flow cytometry. Detect CD3+. + CD19 + Cell ratio (gating strategy such as) Figure 1 (As shown).

[0044] The test results showed CD3 + CD19 + The cell percentage was 0.069%, which is less than the positive threshold (0.20%), and preliminary peripheral blood screening indicates that the patient is not B-ALL.

[0045] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method based on peripheral blood CD3 + CD19 + A non-invasive screening method for acute B-lymphoblastic leukemia based on double-positive cell subsets, characterized by: The method steps are as follows: S1. Immature cells were found in peripheral blood smears; S2. The peripheral blood from S1 was analyzed by flow cytometry to detect CD3 levels in the peripheral blood. + CD19 + The presence and proportion of double-positive cell subsets; S3. Compare the test results in S2 with the positive judgment criteria. When the test result is greater than or equal to the proportion threshold in the positive judgment criteria, it is considered to be positive for acute B-lymphoblastic leukemia.

2. The method according to claim 1, characterized in that: The flow cytometry detection of CD3 in peripheral blood described in step S2 + CD19 + The method for determining the proportion of double-positive cell subsets is as follows: a. Collect 1 mL of peripheral blood from the subject, i.e., EDTA-K2 anticoagulated blood; b. Take 25 μL of peripheral blood from step a, add the fluorescent labeling combination, and incubate in the dark for 15 min. c. Add 225 μL of hemolysin and incubate in the dark for 15 min; d. No washing required; detection using flow cytometry.

3. The method according to claim 2, characterized in that: The fluorescent labeling combination mentioned in step b is CD3, CD19, and CD45.

4. The method according to claim 1, characterized in that: The positive criterion mentioned in step S3 is determined by calculating CD3. + CD19 + Double-positive cells in CD45 + The proportions in the cells are established, with a threshold of 0.20%.

5. The CD3 method according to any one of claims 1-4 + CD19 + The application of double-positive cell subsets is characterized by: The CD3 + CD19 + Double-positive cell subsets serve as biomarkers for detecting acute B-lymphoblastic leukemia.

6. The application according to claim 5, characterized in that: The biomarker is used to prepare products for the detection of acute B-lymphoblastic leukemia.

7. The application according to claim 6, characterized in that: The product includes a kit for detecting acute B-lymphoblastic leukemia.

8. The application according to claim 7, characterized in that: The kit includes one or more of a nucleic acid probe, antigen, or antigen-binding fragment for recognizing the biomarker.

9. The application according to claim 5, characterized in that: The biomarkers are used to construct a system for detecting acute B-lymphoblastic leukemia.

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