An acute lymphoblastic leukemia high throughput 24-color flow cytometric test kit

Abstract

The present application relates to a kind of acute leukemia (ALL) high flux flow detection kit, belong to leukemia detection field, the present application is based on the original 8 color flow to increase 16 kinds of ALL cell antigen, can realize the simultaneous detection of 24 kinds of antigens in the same cell, not only greatly improve the detection precision of micro residual lesion (MRD), and by being divided into 12 development stages to ALL cell, can more accurately identify immunotyping when first diagnosis, and immunophenotypic aberration after bone marrow transplantation and after CAR-T cell therapy, this has very important guiding significance to the evaluation of patient treatment effect and the adjustment of treatment scheme.

Description

Technical Field

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

[0002] Leukemia is a malignant clonal disease of hematopoietic stem and progenitor cells. Due to enhanced self-renewal, uncontrolled proliferation, impaired differentiation, and inhibited apoptosis of leukemia cells, these cells are arrested at different stages of cell development. The massive proliferation and accumulation of leukemia cells suppresses normal hematopoiesis and infiltrates other organs and tissues. In acute leukemia, cell differentiation arrest occurs at an early stage, primarily consisting of primitive cells and early immature cells, leading to rapid disease progression. In China, leukemia ranks among the top ten in both new cancer cases and cancer-related deaths, with acute lymphoblastic leukemia (ALL) being the most common malignant tumor in people under 35 years of age. Although its cure rate has significantly improved (reaching over 80%), it remains a major cause of death among adolescents, and the prognosis for adult ALL patients remains poor—only about 40% of patients achieve long-term complete remission. Therefore, there is an urgent need to improve the diagnosis and treatment of ALL to improve patients' survival rates. Even when ALL patients respond poorly to standard chemotherapy regimens and are assigned to high-risk groups or receive high-dose chemotherapy, a small number of leukemia cells (MRD) may still remain in their bodies. In ALL, the prognostic value and clinical significance of MRD (metastatic leukemia) were first proposed by several centers in Europe and the United States in the 1990s. Studies during this period showed that the level of residual leukemia cells had a strong predictive effect on disease relapse; if the MRD level remained positive at the end of induction therapy, the risk of relapse increased significantly. MRD cells often exhibit strong resistance to chemotherapy drugs, are scarce, and remain hidden in the patient's body for a long time, making them difficult to detect. They often proliferate again in large numbers after chemotherapy, causing disease relapse. The survival rate of relapsed ALL patients is generally less than 30%. Internationally, a MRD rate exceeding 1 in 10,000 is generally considered a cutoff, and a rate exceeding 1 in 10,000 usually indicates a higher cancer relapse rate. Although there are various methods for detecting MRD, their limited sensitivity makes true MRD negativity difficult to achieve. Patients often have a small number of residual leukemia cells in their bone marrow, and these residual leukemia cells (MRD cells) are often the root cause of leukemia relapse. Therefore, by monitoring MRD levels, disease relapse can be predicted earlier and more accurately while assessing treatment efficacy. This allows for interventional treatment before or in the early stages of relapse, thereby improving the patient's survival rate and prognosis.

[0003] Currently, clinical MRD monitoring in my country primarily utilizes 8-12 flow cytometry, which detects cell surface antigen expression to monitor MRD levels during disease progression, achieving an accuracy of up to 10. -3 ~-5 Eight-color flow cytometry, currently the gold standard for clinical MRD (metastatic disease) detection, has advantages such as wide applicability and high speed. Clinically, it is used to assess early treatment response and prognostic risk in ALL patients. The main indicators it detects include developmental stage-related surface proteins such as CD10, CD19, CD20, CD33, CD34, and CD38. Here, we selected bone marrow samples from five clinical patients and performed eight-color flow cytometry results. Figure 1 (Example provided). CD10 is a surface protein present in B precursor cells and mature neutrophils; CD38 is a multifunctional transmembrane glycoprotein, itself a cell membrane receptor, capable of influencing B cell differentiation and proliferation and expressed in B precursor cells; however, CD38 expression is reduced in most B-ALL cells. These detection indicators have undergone long-term exploration and optimization in laboratories and clinical trials, and the combinations used by different research institutes are still not entirely the same. In addition to common detection antigens, ALL cells often express other important and complex surface antigens, and the ALL cell phenotype often changes during treatment. Patients after bone marrow transplantation usually have almost no circulating white blood cells until hematopoietic engraftment and reconstitution. Current flow cytometry is insufficient to accurately assess immunophenotypic changes and precisely detect MRD levels in patients after bone marrow transplantation. The commonly referred to "leukemia-associated immunophenotype" (LAIP) is merely a combination of multiple leukemia subclones. The poor prognosis of relapsed / refractory patients prompts the development of more precise leukemia identification methods, which can help identify early relapse in patients. Therefore, current flow cytometry is insufficient to assess the prognosis of ALL patients.

[0004] The phosphorylation levels of leukemia differentiation-related surface antigens and intracellular proteins are highly correlated with patient prognosis. Internationally, research groups have used single-cell mass spectrometry to achieve parallel detection of these protein factors within the same cell, demonstrating the significant clinical value of these "relapse-associated developmental dependent predictors (DDPRs)." Based on DDPRs, they have established a relapse prediction model for acute lymphoblastic leukemia, significantly improving existing risk stratification methods. Furthermore, DDPR-identified cell phenotypes can provide reasonable drug targets for patients at risk of relapse, offering important clinical guidance. However, single-cell mass spectrometry (CyTOF) suffers from numerous drawbacks, including high cost (tens of thousands of yuan per sample) and long processing time (over 12 hours), making it unsuitable for clinical application. Summary of the Invention

[0005] The technical problem to be solved by this invention is that ALL cells often express a large number of important and complex surface antigens, and the phenotype of ALL cells often changes during treatment. Eight-color flow cytometry is insufficient to accurately assess immunophenotypic changes and MRD levels in patients after bone marrow transplantation. This invention can simultaneously detect 24 surface and intracellular antigens of the same cell, which can not only significantly improve the accuracy of MRD detection, but also divide ALL cells into 12 developmental stages.

[0006] In a first aspect, the present invention provides a high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia. The kit is based on flow cytometry and comprises a cell suspension treatment solution, an antibody premix for cell membrane surface antigens, a solidification treatment solution, and an antibody premix for intracellular antigens. 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 achieves signal compensation balance through precise proportions of surface antibodies, thereby enabling the simultaneous acquisition of 24 color signals.

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

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

[0009] Furthermore, the antibody premix of the intracellular antigen 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 detection kit for acute lymphoblastic leukemia, comprising the following steps:

[0011] (1) Cell suspension was treated with Stain Buffer and Brilliant Stain Buffer;

[0012] (2) The cell suspension from step (1) was stained with an antibody premix of cell membrane surface antigens;

[0013] (3) Cells in suspension were fixed and permeated using Fix / Perm and Perm / Wash;

[0014] (4) Use an antibody premix of intracellular antigens to stain the cell suspension from step (3);

[0015] (5) Use flow cytometry for detection and analysis.

[0016] Furthermore, in step (1), the 1x10 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 cell membrane surface antigens.

[0017] Furthermore, in step (3), after adding 1 ml of Fix / Perm to the cell suspension and incubating at 4 degrees for 40-50 min, the cells are washed twice with 2 ml of Perm / Wash, and then 50 μl of Perm / Wash is added before adding the antibody premixed solution of the intracellular antigen.

[0018] Furthermore, in step (5), FACSymphony is used to first screen active mononuclear cells, adjust compensation, and then screen them. This can divide abnormal B cells in acute lymphoblastic leukemia 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 (ALL) according to the first aspect in the immunophenotyping and prognostic risk stratification of ALL. 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 classify abnormal B cells in ALL into 12 developmental stages. This significantly improves the accuracy of MRD detection while more accurately identifying the immunophenotyping at initial diagnosis, as well as immunophenotypic changes after bone marrow transplantation and CAR-T cell therapy. Furthermore, it accurately guides the treatment and prognosis of ALL through an established ALL relapse prediction model.

[0020] Beneficial effects

[0021] ① The 24-color flow cytometry kit of this 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 and next-generation sequencing) to further improve the accuracy of ALL diagnosis and treatment. By integrating multi-dimensional data, clinicians can gain a comprehensive understanding of the disease characteristics of ALL patients and develop more personalized treatment plans. For example, by combining gene mutation and surface marker expression data, the response of patients to specific drugs can be predicted, thereby selecting the most appropriate treatment plan. In addition, 24-color flow cytometry can identify leukemia cells at a lower detection limit, which is crucial for detecting early relapse and assessing treatment effectiveness. Sensitive MRD detection allows for timely adjustments to treatment strategies for patients, improving their survival.

[0022] ② The 24-color flow cytometry kit developed in this invention adds ALL cell surface antigens to the original 8-color flow cytometry kit. By dividing ALL cells into 12 developmental stages, it can better assess immunophenotypic variations and accurately detect MRD levels in patients after bone marrow transplantation, which has important guiding significance for patient prognosis. Furthermore, this multi-parameter analysis capability allows the detection platform to more comprehensively describe ALL cells and identify their heterogeneity, thereby improving diagnostic accuracy. For example, by simultaneously detecting multiple surface markers, different leukemia subtypes can be more accurately identified and differentiated, which is crucial for selecting appropriate treatment regimens.

[0023] ③ The 24-color flow cytometry kit developed in this invention, based on the original 8-color flow cytometry kit, adds leukemia differentiation-related surface antigens and intracellular proteins, enabling rapid and inexpensive parallel detection of 24 surface antigens and intracellular proteins in the same cell. Since ALL is a highly heterogeneous disease, cellular characteristics may differ significantly between different patients. 24-color flow cytometry can analyze the heterogeneity of leukemia cells in detail, providing richer cell phenotypic information. This helps reveal the cell population characteristics of different patients, providing a basis for personalized treatment. For example, it can identify cell populations that are sensitive to or resistant to specific treatment regimens, thereby optimizing treatment plans and improving treatment efficacy. Attached Figure Description

[0024] Figure 1 This is an example image of the 8-color flow cytometry method commonly used in clinical practice to detect bone marrow samples from patients with initial MRD and relapse.

[0025] Figure 2 The diagram shows an example of the 24-color flow cytometry developmental stage analysis strategy (a) and the 24-color flow cytometry result gating strategy developed for this invention (b).

[0026] Figure 3From a published article (GoodZ.Nat Med.2018), Figure a shows that bone marrow cells from healthy individuals can be divided into 12 developmental stages using mass cytometry; Figure b shows the differences in developmental stages between normal bone marrow cells and bone marrow cells from B-ALL patients; Figures c and d are experimental results from studies conducted using this kit. Figure c shows that the 24 flow cytometry antibodies in this kit can divide normal bone marrow cells into 12 developmental stages; Figure d shows the differences in developmental stages of B-ALL bone marrow cells at different stages (initial, MRD, relapse).

[0027] Figure 4 a is a flow cytometry analysis of different stages of B-ALL bone marrow (initial, MRD, relapse); Figure 4 b represents the clustering analysis results of the selected 24-color flow cytometry markers.

[0028] Figure 5 The changes in B-ALL cell development stages at each stage (initial onset, MRD, relapse) for each selected patient. Figure 6a Based on a published article (Good Z. Nat Med. 2018), the prognostic significance of its developmental stage classification is revealed; Figure 6b The results of detection and analysis using the reagent kit of this invention; Figure 6c The test results from the kit were analyzed in conjunction with RNA-seq. Figure 6d To combine the analysis results with the clinical information of relevant patients. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] Example 1

[0031] This embodiment provides a high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia, including antibody combinations of 24 surface antigens and intracellular proteins, corresponding flow cytometry channels, sample volume (Table 1), and specific operating procedures.

[0032] Table 1: Antibody Composition, Dosage, and Usage of the Kit

[0033] 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 IgHi 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

[0034] 1. Prepare cells:

[0035] I. The samples were fresh blood (derived from clinical ALL patients, as shown in Table 2).

[0036] 1) Add a Ficoll-treated bone marrow sample or a healthy PBMC sample to a 15ml centrifuge tube.

[0037] 2) Resuspend the cells in 1640 medium to 10 ml, stain with trypan blue and count them.

[0038] 3) Take approximately 1x10 6 The cells were used for subsequent experiments, and the remaining cells were cryopreserved.

[0039] 4) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0040] 5) Wash cells twice with 2ml DPBS, centrifuging at 1000rpm for 5min each time.

[0041] 6) Add 1 ml of DPBS to resuspend the cells.

[0042] 7) Add FVS620 dye solution at a ratio of 1:1000 and shake to mix.

[0043] 8) Incubate at room temperature in the dark for 10-15 minutes.

[0044] 9) Wash cells twice with 2ml of Stain Buffer, 1000rpm for 5min each time.

[0045] 10) Remove the supernatant and resuspend the cells by shaking with the remaining liquid.

[0046] 11) Add 5 μL of Fc Blocking, mix well, and incubate at room temperature for 10-15 minutes.

[0047] 12) Prepare cell membrane antibody premix in a flow cytometer.

[0048] 13) Add 50 μL of Brilliant Stain Buffer to the cells and transfer the cells to a flow cytometry tube containing antibody premix.

[0049] 14) Mix well and incubate at room temperature in the dark for 15 minutes.

[0050] 15) Prepare Fix / Perm and Perm / Wash working solutions according to the instructions.

[0051] 16) Add 2 ml of pre-cooled Stain Buffer to wash the cells once, 1000 rpm for 5 min.

[0052] 17) Resuspend the cells by shaking with the remaining liquid.

[0053] 18) Add 1 ml Fix / Perm, mix well, and incubate at 4 degrees Celsius for 40-50 minutes.

[0054] 19) Prepare an intracellular antibody premix in a 1.5 ml centrifuge tube.

[0055] 20) Wash cells twice with 2ml Perm / Wash, 1000rpm for 5min each time.

[0056] 21) Add 50ul Perm / Wash and shake to resuspend the cells.

[0057] 22) Add the premixed solution to the cells, mix well, and incubate at 4 degrees Celsius in the dark for 40-50 minutes.

[0058] 23) Wash cells twice with 2ml Perm / Wash, 1000rpm for 5min each time.

[0059] 24) Resuspend the cells in 350-500 ml of Stain Buffer.

[0060] 25) Transfer the cell suspension to a flow cytometer containing a filter cartridge, filter the solution, place on ice, and run on the machine within 24 hours.

[0061] II. The samples were cryopreserved cells (derived from peripheral blood or bone marrow of clinical ALL patients, as shown in Table 2).

[0062] 1) Take one tube of frozen cells from liquid nitrogen, heat it in a 37°C water bath, and quickly rewarm it to thaw (until only small ice crystals remain inside the tube).

[0063] 2) Slowly add 5 ml of 1640 culture medium to the cells and transfer it to a 15 ml centrifuge tube.

[0064] 3) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0065] 4) Add 10 ml of 1640 medium to resuspend the cells, take 10 μl of the cell suspension for trypan blue staining and counting.

[0066] 5) Take about 1x106 cells for subsequent experiments, and freeze the remaining cells again.

[0067] 6) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0068] 7) The subsequent steps are the same as in section 5)-25).

[0069] 2. FACSymphony (on-site)

[0070] (1) Gating active monocytes: Using FSC-A / SSC-A as a gate, lymphocytes are circled out and cell debris is removed; then using FSC-A / FSC-H as a gate, single cells are circled out and adherent cells are removed; then using FVS620 / SSC-A as a gate, live cells are circled out. (2) After regulation and compensation, developmental stage gates are performed.

[0071] 3. Test Results

[0072] Specific developmental stage differentiation strategies and flow cytometry result gating strategies, such as Figure 2 As shown.

[0073] This invention incorporates internationally validated surface antigens and intracellular protein phosphorylation level-related antigens associated with leukemia differentiation that are relevant to leukemia prognosis. This has important guiding significance for patient prognostic risk stratification and drug target selection. Figure 3 Figures a and b are from a published article (Good Z. in Nature Medicine, 2018), which has validated the clinical significance of leukemia differentiation-related surface antigens and intracellular protein phosphorylation levels-related antigens that are associated with leukemia prognosis. Firstly, mass cytometry can classify bone marrow cells from healthy individuals into 12 developmental stages. Figure 3 a), and demonstrated the differences in developmental stages between normal bone marrow cells and bone marrow cells from B-ALL patients ( Figure 3 (b) By linking developmental stages with associated clinical information, this study reveals for the first time the prognostic significance of staging ALL bone marrow cell developmental stages. This kit also represents the first domestic achievement of fully calibrated multicolor flow cytometry-mass spectrometry, and is the first to use the 24 flow cytometry antibody combinations in this kit to classify normal bone marrow cells into 12 developmental stages. Figure 3 c) The differences in the developmental stage of B-ALL bone marrow cells at different stages (initial onset, MRD, relapse) can be observed through the detection of different samples. Figure 3 d) This is completely consistent with mass cytometry, which shows that the developed kit can be fully benchmarked against the effects of mass cytometry.

[0074] In addition, bone marrow samples from healthy donors were examined. In healthy donors (HD), cells were distributed according to the natural developmental trajectory from HSC to Mature B. In newly diagnosed (DX) patients, this trajectory was significantly disrupted, with cells clustering in an early progenitor state rather than the normal differentiation pathway. This aberrant clustering persisted as the disease progressed to MRD and RE stages, differing from that at initial diagnosis, suggesting that the developmental stage of leukemia cells in the initial, MRD, and relapse stages may be related to prognosis (e.g., Figure 4 a). To further visualize the changes in surface markers at different stages of B-ALL, cluster analysis was performed on the flow cytometry results (e.g., Figure 4 b) High levels of biomarkers at the time of leukemia diagnosis (DX) decrease slightly as treatment progresses to the MRD stage, but some early biomarkers rise again in the RE (relapse) stage, indicating disease reactivation and recurrence of developmental arrest, reflecting the prognostic significance of these markers.

[0075] To support the above conclusions, we selected bone marrow samples from six patients at various stages (initial onset, MRD, relapse) for analysis. This revealed heterogeneity in the developmental stages of B-ALL cells among different patients. Patients were divided into two categories: a) Induced differentiation type: such as GJ, FF, and LCY, showing a clear trend towards differentiation into later-stage B cells during the relapse stage. This indicates that leukemia cells exhibit "differentiation escape" characteristics after relapse, suggesting a potentially reduced response to traditional early B-cell marker-targeted therapy, requiring monitoring of mature B-cell subsets. b) Developmental maintenance type: such as WCK, MFY, and DL, still predominantly consisting of early-stage B cells during the relapse stage. This indicates that leukemia cells remain immature, with continued blockage of the developmental process, suggesting a potential greater reliance on therapeutic targets that block early developmental pathways. Figure 5 This fully demonstrates the guiding significance of this kit for prognosis and treatment strategy selection.

[0076] Published articles (Good Z. Nat Med. 2018) have revealed that mass cytometry-based developmental stage staging has significant prognostic value and is superior to MRD and NCI risk prognostic staging. Figure 6a ); Figure 6b Based on the detection and analysis results of the kit of this invention, by integrating the detection and analysis results of this kit with the RNA-seq results of clinical patients, it can be seen that development-related signaling pathways are significantly activated, especially the CD20 (a marker of maturity) and BCR signaling pathways. Figure 6c Furthermore, CD20 expression is significantly correlated with prognosis; patients with positive / high CD20 expression have a significantly worse prognosis. Figure 6d This directly links the developmental stage in B-ALL with the prognosis, directly confirming the clinical application value of this kit.

[0077] Table 2. Clinical information of 6 B-ALL patients selected for this kit.

[0078]

Claims

1. A high-throughput 24-color flow cytometry detection kit for acute lymphoblastic leukemia, characterized in that, The kit is based on flow cytometry for detection and comprises a cell suspension treatment solution, an antibody premix for cell membrane surface antigens, a solidification treatment solution, and an antibody premix for intracellular antigens. 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 antibody premix for intracellular antigens 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.

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

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

4. The reagent kit according to claim 1, characterized in that, Twenty-four flow cytometry antibodies are packaged in a single antibody combination tube, enabling parallel detection of 24 surface antigens and intracellular proteins in the same cells within the same tube in leukemia.

5. The use of the high-throughput 24-color flow cytometry kit for acute lymphoblastic leukemia as described in any one of claims 1-4 in the preparation of a product for immunophenotyping and prognostic risk stratification of acute lymphoblastic leukemia.

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