High-throughput 24-color flow detection kit for acute lymphoblastic leukemia

Abstract

The invention relates to a high-throughput 24-color flow type detection kit for acute lymphocytic leukemia, and belongs to the field of leukemia detection.16 ALL cell antigens are added on the basis of an original 8-color flow type, 24 antigens in the same cell can be detected at the same time, the detection precision of minimal residual focus (MRD) is greatly improved, the detection time is shortened, and the detection cost is reduced. The ALL cells are divided into 12 developmental stages, so that immunological typing during primary diagnosis and immunophenotype variation after bone marrow transplantation and CAR-T cell treatment can be more accurately recognized, and the method has very important guiding significance in evaluation of treatment effects of patients and adjustment of treatment schemes.

Description

Technical Field

[0001] The invention belongs to the field of leukemia detection, and particularly relates to a high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia. Background Art

[0002] Leukemia is a malignant clonal disease of hematopoietic stem and progenitor cells. Leukemic cells are arrested at various stages of development due to enhanced self-renewal, uncontrolled proliferation, impaired differentiation, and blocked apoptosis. Massive leukemic cell proliferation and accumulation suppress normal hematopoiesis and allow infiltration of other organs and tissues. In acute leukemia, cell differentiation is arrested at an early stage, often as blasts and early immature cells, leading to rapid progression. Therefore, there is an urgent need to improve the diagnosis and treatment of ALL to improve patient survival. For ALL patients who respond poorly to standard chemotherapy, despite being assigned to high-risk groups and receiving high-dose chemotherapy, a small number of residual leukemic cells (MRD) may remain in their bodies. The prognostic value and clinical significance of MRD in ALL were first established in the 1990s at multiple centers in Europe and the United States. Studies during this period have demonstrated that the level of residual leukemic cells strongly predicts disease relapse, and that remaining MRD levels at the end of induction therapy significantly increases the risk of relapse. MRD cells often show strong tolerance to chemotherapy drugs, and their number is rare. They are hidden in the patient's body for a long time and difficult to detect. They often proliferate again in large quantities after chemotherapy, causing disease recurrence. The survival rate of patients with relapsed ALL is generally less than 30%. Internationally, it is usually divided into one in ten thousand. An MRD of more than one in ten thousand usually means a higher cancer recurrence rate. Although there are various methods for detecting MRD, due to their limited sensitivity, true MRD negativity is actually difficult to achieve. A small amount of leukemia cells often remain in the patient's bone marrow. These residual leukemia cells (MRD cells) usually become the root cause of leukemia recurrence. Therefore, by monitoring the MRD level, while evaluating the efficacy, the recurrence of the disease can be predicted earlier and more accurately, thereby prompting interventional treatment before recurrence or in the early stage of recurrence to increase the patient's chance of survival and improve the patient's prognosis.

[0003] At present, clinical MRD monitoring in my country is mainly based on 8-12 flow cytometry, which monitors the MRD level during disease progression by detecting the expression of cell surface antigens with an accuracy of 10 -3 ~ -5. As the current gold standard for clinical MRD detection, the 8-color flow cytometry technology has the advantages of wide applicability and fast speed. It is clinically used to evaluate the early treatment response and prognostic risk assessment of patients with ALL. Its detection indicators mainly include surface proteins related to developmental stages such as CD10, CD19, CD20, CD33, CD34, and CD38. CD10 is a surface protein present in B precursor cells and mature neutrophils; CD38 is a multifunctional transmembrane glycoprotein, which is itself a cell membrane receptor that can affect the differentiation and proliferation of B cells and is expressed in B precursor cells. However, the expression of CD38 is reduced in most B-ALL cells. The above detection indicators have undergone long-term exploration and optimization in laboratories and clinical trials, and the combinations currently used by different research institutes are still not exactly the same. In addition, in addition to commonly detected antigens, ALL cells often express other important and complex surface antigens, and the phenotype of ALL cells often changes during treatment. Patients after bone marrow transplantation often have almost no circulating white blood cells until hematopoietic engraftment and reconstitution. Current flow cytometry tests are difficult to accurately assess immune phenotypic changes and precisely detect MRD levels in patients after bone marrow transplantation. What we commonly call the "leukemia-associated immunophenotype" (LAIP) is merely a combination of multiple leukemia subclones. The poor prognosis of relapsed and refractory patients has prompted the development of more accurate leukemia identification methods to help promptly identify patients with early relapse. Therefore, current flow cytometry is insufficient to judge the prognosis of ALL patients.

[0004] The phosphorylation levels of surface antigens and intracellular proteins related to leukemia differentiation are very closely related to the patient's prognosis. International research groups have used single-cell mass spectrometry technology to achieve parallel detection of the above-mentioned protein factors in the same cell, proving that these "developmentally dependent predictors associated with relapse (DDPR)" have important clinical value. Based on DDPR, a relapse prediction model for acute lymphoblastic leukemia was established, which significantly improved the existing risk stratification methods. In addition, the cell phenotype identified by DDPR can also provide reasonable drug targets for patients at risk of relapse, which has important guiding significance for clinical practice. However, single-cell mass spectrometry detection (CyTOF) has many problems such as high price (tens of thousands of yuan / sample) and long detection time (more than 12 hours), making it unsuitable for clinical application. Summary of the Invention

[0005] The technical problem addressed by the present invention is that ALL cells often express a large number of important and complex surface antigens, and their phenotype often changes during treatment. Eight-color flow cytometry is insufficient to accurately assess immunophenotypic changes and MRD levels in patients after bone marrow transplantation. The present invention can simultaneously detect 24 surface and intracellular antigens on the same cell, significantly improving the accuracy of MRD detection and classifying ALL cells into 12 developmental stages. Studying the developmental stages of leukemia cells is significant because drug resistance is often closely related to developmental stage. Numerous studies have shown that compared with adult ALL patients, pediatric ALL patients have better treatment responses and longer disease-free survival. One possible explanation is that the tumor cells in pediatric ALL patients are often at earlier developmental stages, such as pre-B cells or T cells, which are more sensitive to chemotherapy drugs. In contrast, adult ALL patients typically have a more mature B cell phenotype, and therefore their tumor cells may have already developed to a stage that is more resistant to chemotherapy drugs. Therefore, the detection of 24 antigens can more accurately identify immune typing at the time of initial diagnosis, as well as immune phenotypic changes after bone marrow transplantation and CAR-T cell therapy, which is of great guiding significance for patient prognosis assessment. In addition, the present invention incorporates surface antigens related to leukemia differentiation and intracellular protein phosphorylation levels that have been internationally verified to be related to leukemia prognosis, which is of great guiding significance for patient prognostic risk stratification and drug target selection.

[0006] In a first aspect, the present invention provides a high-throughput 24-color flow detection kit for acute lymphoblastic leukemia, the kit being detected based on flow cytometry, and its composition includes a cell suspension treatment solution, an antibody premix for cell membrane surface antigens, a solidification treatment solution, and an antibody premix for intracellular antigens; wherein the antibody premix for cell membrane surface antigens includes: anti-CD19 antibody, anti-CD10 antibody, anti-CD73 antibody, anti-CD58 antibody, anti-IgHs antibody, anti-CD45RB antibody, anti-CD95 antibody, anti-CD24 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-CD38 antibody, anti-CD45 antibody, anti-CD127 antibody, anti-CD20 antibody, anti-CD34 antibody, and anti-FVS620 antibody. The present invention can achieve signal compensation balance through precise surface antibody ratio dosage, thereby achieving simultaneous acquisition of 24 color signals.

[0007] Furthermore, the cell suspension treatment solution includes Stain Buffer and Brilliant Stain Buffer.

[0008] Furthermore, the solidification treatment solution includes Fix / Perm and Perm / Wash.

[0009] Furthermore, the intracellular antigen antibody premix includes: anti-p4EBP1 antibody, anti-ZAP70 antibody, anti-IgHi antibody, anti-CD179a antibody, anti-CD179b antibody, anti-TdT antibody, anti-PS6 antibody and anti-pCreb antibody.

[0010] In a second aspect, the present invention provides a method for using a high-throughput 24-color flow cytometry kit for acute lymphoblastic leukemia, comprising the following steps:

[0011] (1) Treat the cell suspension with Stain Buffer and Brilliant Stain Buffer;

[0012] (2) staining the cell suspension of step (1) using a premixed solution of antibodies against cell membrane surface antigens;

[0013] (3) Fixing and permeabilizing cells in suspension using Fix / Perm and Perm / Wash;

[0014] (4) staining the cell suspension from step (3) using a premixed solution of antibodies against intracellular antigens;

[0015] (5) Flow cytometry was used for detection and analysis.

[0016] Furthermore, in step (1), the 6 Add 2 ml of Stain Buffer to the cell suspension and wash the cells twice. Add 50 μl of Brilliant Stain Buffer and then add the antibody premix of cell membrane surface antigens.

[0017] Furthermore, in step (3), 1 ml of Fix / Perm was added to the cell suspension and incubated at 4°C for 40-50 min, the cells were washed twice with 2 ml of Perm / Wash, 50 μl of Perm / Wash was added, and then the antibody premix of the intracellular antigen was added.

[0018] Furthermore, in step (5), FACSymphony is used to first circle active monocytes, and then adjust the compensation and perform gating, so that abnormal B cells in acute lymphoblastic leukemia can be divided into 12 developmental stages.

[0019] In a third aspect, the present invention provides the use of the high-throughput 24-color flow cytometry kit for acute lymphoblastic leukemia according to the first aspect in the immunophenotyping and prognostic risk stratification of acute lymphoblastic leukemia. The 24-color flow cytometry kit provided by the present invention can achieve parallel detection of 24 surface antigens and intracellular proteins in the same cell, and divide abnormal B cells in acute lymphoblastic leukemia into 12 developmental stages. While significantly improving the accuracy of MRD detection, it can more accurately identify immunophenotyping at the time of initial diagnosis, as well as immune phenotypic changes after bone marrow transplantation and CAR-T cell therapy, and accurately guide the treatment and prognosis of acute lymphoblastic leukemia through the established acute lymphoblastic leukemia relapse prediction model.

[0020] Beneficial effects

[0021] ① The 24-color flow cytometry kit of the present invention can significantly improve the accuracy of MRD detection. The rich data provided by this technology can be combined with other molecular biology techniques (such as PCR, second-generation sequencing) to further improve the accuracy of ALL diagnosis and treatment. By integrating multi-dimensional data, the disease characteristics of ALL patients can be fully understood clinically and more personalized treatment plans can be formulated. For example, gene mutation and surface marker expression data can be combined to predict the patient's response to a specific drug, thereby selecting the most appropriate treatment plan. In addition, 24-color flow cytometry can identify leukemia cells at a lower detection limit, which is very important for discovering early relapse and evaluating treatment effects. Sensitive MRD detection can adjust treatment strategies for patients in a timely manner and improve patient survival.

[0022] ② The 24-color flow cytometry kit developed by the present invention adds ALL cell surface antigens to the original 8-color flow cytometry. By dividing ALL cells into 12 developmental stages, it can better evaluate immune phenotypic mutations and accurately detect the MRD level of patients after bone marrow transplantation, which has important guiding significance for the patient's prognosis. In addition, this multi-parameter analysis capability enables the detection platform to more comprehensively describe ALL cells and identify their heterogeneity, thereby improving the accuracy of diagnosis. For example, by simultaneously detecting multiple surface markers, different leukemia subtypes can be more accurately identified and distinguished, which is crucial for selecting appropriate treatment options.

[0023] ③ The 24-color flow cytometry kit developed by the present invention adds surface antigens and intracellular proteins related to leukemia differentiation to the original 8-color flow cytometry, which can achieve rapid and inexpensive parallel detection of 24 surface antigens and intracellular proteins in the same cell. Since ALL is a highly heterogeneous disease, the cell characteristics between different patients may vary significantly. 24-color flow cytometry can analyze the heterogeneity of leukemia cells in detail and provide richer cell phenotypic information. This helps to reveal the characteristics of cell populations in different patients and provide a basis for personalized treatment. For example, cell populations that are sensitive or resistant to specific treatment regimens can be identified, thereby optimizing treatment regimens and improving treatment efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Results of 24-color flow cytometry analysis of bone marrow specimens from acute lymphoblastic leukemia.

[0025] Figure 2 The results of 24-color flow cytometry detection of normal bone marrow specimens.

[0026] Figure 3 The results of 8-color flow cytometry detection of normal bone marrow specimens.

[0027] Figure 4 It is a 24-color flow cytometry test for the intracellular protein phosphorylation levels in normal bone marrow and acute lymphoblastic leukemia patients at the initial, MRD and relapse stages.

[0028] Figure 5 Abnormal B cells in different stages of acute lymphoblastic leukemia were divided into 12 developmental stages by 24-color flow cytometry. DETAILED DESCRIPTION

[0029] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0030] Example 1

[0031] This example provides a high-throughput 24-color flow cytometry kit for acute lymphoblastic leukemia, including 24 antibody combinations for surface antigens and intracellular proteins, corresponding flow channels, sample dosage (see the table below), and specific operating procedures.

[0032] markers Channel (A5 / S6) Surface / Intracellular Dosage (ul) CD19 BUV737 S 6.6 CD10 BV711 / BV750 S 1.5 CD73 BUV563 S 3.8 CD58 BV650 / BV711 S 8.8 IgHs BV786 S 9.6 CD45RB BUV805 S 5.5 CD95 BB630 S 5.1 CD24 BUV615 S 5.4 CD33 AF700 / A700 S 9.6 CD22 BV605 S 8.0 CD38 APC-CY7 S 9.8 CD45 BUV395 / BUV396 S 6.8 CD127 BB790 S 9.8 CD20 BUV496 S 8.6 CD34 PE-CY5 S 9.1 p4EBP1 PE-CY7 I 2.3 ZAP70 BB700 I 5.4 Ig BUV661 I 7.8 CD179a BV421 I 6.9 CD179b PE I 3.6 TdT BV510 I 5.2 PS6 APC-A I 9.1 pCreb BB515 I 4.2 FVS620 PE-CF594

[0033] 1. Prepare cells:

[0034] 1. The sample is fresh blood

[0035] 1) Add Ficoll-treated bone marrow sample or healthy PBMC sample to a 15 ml centrifuge tube.

[0036] 2) Resuspend the cells to 10 ml in 1640 medium, stain with trypan blue, and count.

[0037] 3) Take about 1x10 6 The cells were used for subsequent experiments, and the remaining cells were frozen.

[0038] 4) Centrifuge at 1000 rpm for 5 min and remove the supernatant.

[0039] 5) Wash the cells twice with 2 ml of DPBS, centrifuging at 1000 rpm for 5 min each time.

[0040] 6) Add 1 ml of DPBS to resuspend the cells.

[0041] 7) Add FVS620 dye solution at a ratio of 1:1000 and shake to mix.

[0042] 8) Incubate at room temperature in the dark for 10-15 minutes.

[0043] 9) Wash the cells twice with 2 ml of Stain Buffer, 5 min each time at 1000 rpm.

[0044] 10) Remove the supernatant and resuspend the cells in the remaining liquid by vortexing.

[0045] 11) Add 5 μl of Fc Blocking solution, mix well, and incubate at room temperature for 10-15 minutes.

[0046] 12) Prepare the cell membrane antibody premix in a flow cytometry tube.

[0047] 13) Add 50 μl of Brilliant Stain Buffer to the cells and transfer the cells to the flow cytometry tube containing the antibody premix.

[0048] 14) Mix well and incubate at room temperature in the dark for 15 min.

[0049] 15) Prepare Fix / Perm and Perm / Wash working solutions according to the instructions.

[0050] 16) Add 2 ml of pre-cooled Stain Buffer and wash the cells once at 1000 rpm for 5 min.

[0051] 17) Resuspend the cells in the remaining liquid by vortexing.

[0052] 18) Add 1 ml of Fix / Perm, mix well, and incubate at 4°C for 40-50 min.

[0053] 19) Prepare the intracellular antibody premix in a 1.5 ml centrifuge tube.

[0054] 20) Wash the cells twice with 2 ml of Perm / Wash, 1000 rpm for 5 min each time.

[0055] 21) Add 50 μl of Perm / Wash and resuspend the cells by vortexing.

[0056] 22) Add the premixed solution to the cells, mix well, and incubate at 4°C in the dark for 40-50 minutes.

[0057] 23) Wash the cells twice with 2 ml of Perm / Wash, 1000 rpm for 5 minutes each time.

[0058] 24) Resuspend the cells in 350-500 ml of Stain Buffer.

[0059] 25) Transfer the cell suspension to a flow cytometry tube containing a filter cartridge, filter the tube, place on ice, and load the tube within 24 hours.

[0060] 2. The sample is frozen cells

[0061] 1) Remove one tube of frozen cells from liquid nitrogen, heat in a 37°C water bath, and rapidly thaw (until only small ice crystals remain in the tube).

[0062] 2) Slowly add 5 ml of 1640 medium to the cells and transfer to a 15 ml centrifuge tube.

[0063] 3) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0064] 4) Add 10 ml of 1640 medium to resuspend the cells. Take 10 μl of the cell suspension for trypan blue staining and count.

[0065] 5) Take about 1x106 cells for subsequent experiments and freeze the remaining cells again.

[0066] 6) Centrifuge at 1000 rpm for 5 min and remove the supernatant.

[0067] 7) The subsequent steps are the same as 5)-25).

[0068] 2. FACSymphony

[0069] (1) Circumscribe active monocytes: Use FSC-A / SSC-A as the gate to exclude lymphocytes and remove cell debris; then use FSC-A / FSC-H as the gate to exclude single cells and remove adherent cells; then use FVS620 / SSC-A as the gate to exclude live cells. (2) After adjusting for compensation, perform developmental stage gating.

[0070] 3. Test results

[0071] Depend on Figure 1 It can be seen that 24-color flow cytometry can divide B-ALL cells into 12 developmental stages.

[0072] Depend on Figure 2 It can be seen that 24-color flow cytometry can divide normal bone marrow cells into 12 developmental stages.

[0073] Depend on Figure 3 It can be seen that 8-color flow cytometry can simply classify normal bone marrow cells.

[0074] Depend on Figure 4 It can be seen that there are significant differences in the phosphorylation levels of intracellular proteins between normal bone marrow and patients with acute lymphoblastic leukemia at the initial, MRD, and relapse stages, suggesting that these intracellular proteins have a high value for the prognosis of patients with acute lymphoblastic leukemia.

[0075] Depend on Figure 5 It can be seen that 24-color flow cytometry can divide abnormal B cells in acute lymphoblastic leukemia at the primary, MRD, and relapse stages into 12 developmental stages, and by comparing them with normal bone marrow, the developmental stage that is most valuable for patient prognosis can be found.

Claims

1. A high-throughput 24-color flow cytometry kit for acute lymphoblastic leukemia, characterized in that: The kit is based on flow cytometry for detection, and its composition includes a cell suspension treatment solution, an antibody premix solution for cell membrane surface antigens, a solidification treatment solution and an antibody premix solution for intracellular antigens; wherein the antibody premix solution for cell membrane surface antigens includes: anti-CD19 antibody, anti-CD10 antibody, anti-CD73 antibody, anti-CD58 antibody, anti-IgHs antibody, anti-CD45RB antibody, anti-CD95 antibody, anti-CD24 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-CD38 antibody, anti-CD45 antibody, anti-CD127 antibody, anti-CD20 antibody, anti-CD34 antibody and anti-FVS620 antibody.

2. The kit according to claim 1, characterized in that The cell suspension treatment solution includes Stain Buffer and Brilliant Stain Buffer.

3. The kit according to claim 1, characterized in that The solidification treatment solution includes Fix / Perm and Perm / Wash.

4. The kit according to claim 1, characterized in that The antibody premix of the intracellular antigen comprises: anti-p4EBP1 antibody, anti-ZAP70 antibody, anti-IgHi antibody, anti-CD179a antibody, anti-CD179b antibody, anti-TdT antibody, anti-PS6 antibody and anti-pCreb antibody.

5. The kit according to claim 1, characterized in that 24 flow cytometry antibodies are packaged in one antibody combination tube, enabling parallel detection of 24 surface antigens and intracellular proteins in the same cell in the same tube in leukemia.

6. A method for using the high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia according to any one of claims 1 to 4, comprising the following steps: (1) Treat the cell suspension with Stain Buffer and Brilliant Stain Buffer; (2) staining the cell suspension of step (1) using a premixed solution of antibodies against cell membrane surface antigens; (3) Fixing and permeabilizing cells in suspension using Fix / Perm and Perm / Wash; (4) staining the cell suspension of step (3) using a premixed solution of antibodies against intracellular antigens; (5) Detection and analysis using flow cytometry.

7. The method of use according to claim 6, characterized in that: In step (1), the 6 Add 2 ml of Stain Buffer to the cell suspension and wash the cells twice, then add 50 μl of Brilliant Stain Buffer and then add the antibody premix of the cell membrane surface antigen.

8. The method of use according to claim 6, characterized in that: In step (3), 1 ml of Fix / Perm is added to the cell suspension and incubated at 4°C for 40-50 min, the cells are washed twice with 2 ml of Perm / Wash, 50 μl of Perm / Wash is added, and then the antibody premix of the intracellular antigen is added.

9. The method of use according to claim 6, characterized in that: In the step (5), FACSymphony is used to first circle active monocytes, and then gate after adjusting compensation. This can divide abnormal B cells in acute lymphoblastic leukemia into 12 developmental stages.

10. Use of the high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia according to any one of claims 1 to 4 in immunophenotyping and prognostic risk stratification of acute lymphoblastic leukemia.

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