Flow cytometry kit and monitoring method for monitoring minimal residual disease of acute b-lymphocytic leukemia

Abstract

The present application relates to the field of flow cytometry, in particular to a flow cytometry kit and a monitoring method for monitoring acute B lymphocyte leukemia minimal residual disease, the kit comprises 15 antibodies, aiming at the development mode of bone marrow B cells and common abnormal expression of ALL-B, combined with a full spectrum flow cytometer, all the above detections can be completed by one tube panel, which is convenient, fast, time-saving and labor-saving, and simple to operate.

Description

Technical Field

[0001] The present invention relates to the field of flow cytometry, and in particular to a flow cytometry kit and a monitoring method for monitoring minimal residual lesions of acute B lymphocytic leukemia. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is a malignant tumor caused by the clonal proliferation of primitive and immature lymphocytes. It is more common in children aged 2 to 5 years (19%), among which B-ALL is more common.

[0003] The enhanced self-renewal capacity and uncontrolled proliferation of leukemia cells can lead to impaired differentiation and apoptosis, while also suppressing normal bone marrow hematopoiesis and infiltrating various organs such as the liver, spleen, and lymph nodes, manifesting as clinical signs such as infection, bleeding, and anemia. ALL progresses very rapidly and can lead to death within a few months, so treatment should be initiated as soon as possible after diagnosis. Currently, with the combined use of multiple drugs, risk stratification assessments, prevention of central nervous system invasion, and increasing understanding of the clinical, molecular, and therapeutic response characteristics of patients who fail chemotherapy, patient prognosis has significantly improved, with a 5-year overall survival (OS) rate of up to 90%. However, a small number of patients still relapse, and the secondary remission rate after relapse is low, making relapse the main cause of treatment failure.

[0004] Minimal residual disease (MRD) refers to the state in which a small number of leukemia cells remain in the body after acute leukemia patients achieve complete clinical and hematological remission (morphologically, primitive and immature cells in the bone marrow are less than 5%) after induction chemotherapy or bone marrow transplantation. MRD is the primary cause of leukemia relapse. Therefore, for leukemia patients, the significance of MRD detection is very important: 1. It helps to predict leukemia relapse earlier; 2. It guides the clinical treatment of leukemia, and decides whether to continue chemotherapy or stop treatment based on the number of leukemia cells in the body, so as to better formulate effective treatment plans and avoid overtreatment; 3. It helps to detect whether leukemia cells are drug-resistant earlier, so as to select clinically more sensitive and lethal treatment measures; 4. It helps to evaluate the stem cell purification effect of patients who have undergone autologous hematopoietic stem cell transplantation.

[0005] In the past 20 years, with the rapid development of MRD detection technology, various MRD detection methods have been widely used in clinical evaluation of therapeutic efficacy, such as flow cytometry (FCM), PCR (Polymerase chain reaction), FISH and mass spectrometry technology. MRD technology needs to be highly sensitive, practical, accurate, reliable and fast. Bone marrow cell morphology, conventional karyotype analysis and FISH technology are far from meeting the clinical requirements for MRD detection sensitivity due to their low sensitivity. Currently, the main clinical MRD detection methods are FCM and polymerase chain reaction. Both have high sensitivity and good correlation, with a consistency of 89.7%. However, PCR-fusion gene can only be applied to about 25% to 40% of ALL patients, because not all ALL patients have fusion genes or mutations, etc., while FCM mainly judges whether the development pattern of leukemia cells and the expression of specific markers are normal. It can be used for almost all patients, and the detection depth can reach 10 4-5 However, conventional flow cytometry currently has fewer channels, making compensation difficult to adjust. Commonly used detection antibodies are limited to 8 to 10 per tube, while existing technologies use more than 20 flow cytometry antibodies for MRD. Therefore, previous tests could only select first-line antibodies and essential antibodies closely related to B cell development patterns, making it difficult to detect all residual leukemia cells. Furthermore, if MRD is present in ALL-B patients, the abnormal expression and development are often different from those seen at initial diagnosis, requiring a wider range of antibodies for monitoring. Therefore, insufficient breadth of antibodies tested is the main reason why some patients relapse after a negative MRD test.

[0006] The advent of full-spectrum flow cytometry in 2017 has greatly increased the number of detection channels of three-laser flow cytometry from 4-13 in traditional flow cytometry to 38, enabling monitoring of more than 25 colors in one tube, greatly increasing the amount of detection information, and improving diagnostic accuracy and the depth of immunological testing.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The first invention object of the present invention is to provide a flow cytometry kit for monitoring minimal residual lesions of acute B lymphoblastic leukemia.

[0009] The second object of the present invention is to provide a method for using the kit.

[0010] The third invention object of the present invention is to provide a method for monitoring minimal residual lesions of acute B-lymphocytic leukemia using a flow cytometry kit.

[0011] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0012] The present invention relates to a flow cytometry kit for monitoring minimal residual lesions of acute B lymphocytic leukemia, wherein the kit contains antibodies for detecting B lineage development and antibodies for detecting abnormal expression.

[0013] The antibodies used to detect B lineage development include: anti-CD19 antibody, anti-CD10 antibody, anti-CD20 antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD81 antibody, anti-CD58 antibody, anti-CD45 antibody, anti-CD24 antibody, and anti-CD200 antibody; the antibodies used to detect abnormal expression include: anti-CD13 antibody, anti-CD33 antibody, anti-CD15 antibody, anti-CD66c antibody, and anti-CD123 antibody.

[0014] Optionally, the anti-CD33 antibody is connected to fluorescein RB515, the anti-CD38 antibody is connected to fluorescein APC-cy7, the anti-CD15 antibody is connected to fluorescein FITC, the anti-CD24 antibody is connected to fluorescein RV711, the anti-CD123 antibody is connected to fluorescein RV780, the anti-CD58 antibody is connected to fluorescein RB710, the anti-CD200 antibody is connected to fluorescein RV600, the anti-CD19 antibody is connected to fluorescein RB780, the anti-CD66c antibody is connected to fluorescein RR664, the anti-CD45 antibody is connected to fluorescein RB697, the anti-CD13 antibody is connected to fluorescein RB594, the anti-CD10 antibody is connected to fluorescein PE, the anti-CD81 antibody is connected to fluorescein RR710, the anti-CD20 antibody is connected to fluorescein RV570, and the anti-CD34 antibody is connected to fluorescein PE-cy5.

[0015] Optionally, the antibody preparation is a liquid preparation or a lyophilized powder; in the liquid preparation, the concentration of the antibody is 80 to 120 μg / mL, preferably 100 μg / mL.

[0016] Optionally, all the antibodies are packaged in one container. Preferably, the mass of the antibodies in the container is 80 to 120 μg; more preferably, the volume of the container is 5 mL.

[0017] The present invention also relates to a method for using the kit, which comprises at least the following steps:

[0018] (1) Take an empty flow cytometry tube and an antibody combination tube, and mark them as control and sample tubes;

[0019] (2) Add the sample to be tested to the sample tube and control tube respectively, mix well, and incubate in the dark for 15-20 minutes;

[0020] (3) Add red blood cell lysis buffer to the sample tube and control tube, mix well, and lyse for 8-10 minutes;

[0021] (5) Centrifugation and washing;

[0022] (6) Mix again and test on the machine.

[0023] Optionally, the incubation time is 10 to 20 minutes, preferably 15 minutes.

[0024] Optionally, the lysis time is 10 to 15 minutes, preferably 10 minutes.

[0025] Optionally, all operations in the method are performed at room temperature.

[0026] The present invention also relates to a method for monitoring minimal residual lesions of acute B-lymphocytic leukemia, comprising at least the following steps:

[0027] (1) Take an empty flow cytometry tube and an antibody combination tube, and mark them as control and sample tubes;

[0028] (2) Add the sample to be tested to the sample tube and control tube respectively, mix well, and incubate in the dark for 15-20 minutes;

[0029] (3) Add red blood cell lysis buffer to the sample tube and control tube respectively, mix well, and lyse for 8-10 minutes;

[0030] (5) Centrifugation and washing;

[0031] (6) Mix again and test on the machine;

[0032] The antibody combination tube contains antibodies for detecting B lineage development and antibodies for detecting abnormal expression. The antibodies for detecting B lineage development include: anti-CD19 antibody, anti-CD10 antibody, anti-CD20 antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD81 antibody, anti-CD58 antibody, anti-CD45 antibody, anti-CD24 antibody, and anti-CD200 antibody; the antibodies for detecting abnormal expression include: anti-CD13 antibody, anti-CD33 antibody, anti-CD15 antibody, anti-CD66c antibody, and anti-CD123 antibody; the anti-CD33 antibody is connected to fluorescein RB515, the anti-CD38 antibody is connected to fluorescein APC-cy7, the anti-CD15 antibody is connected to fluorescein FITC, The anti-CD24 antibody is connected to fluorescein RV711, the anti-CD123 antibody is connected to fluorescein RV780, the anti-CD58 antibody is connected to fluorescein RB710, the anti-CD200 antibody is connected to fluorescein RV600, the anti-CD19 antibody is connected to fluorescein RB780, the anti-CD66c antibody is connected to fluorescein RR664, the anti-CD45 antibody is connected to fluorescein RB697, the anti-CD13 antibody is connected to fluorescein RB594, the anti-CD10 antibody is connected to fluorescein PE, the anti-CD81 antibody is connected to fluorescein RR710, the anti-CD20 antibody is connected to fluorescein RV570, and the anti-CD34 antibody is connected to fluorescein PE-cy5.

[0033] The present invention has at least the following beneficial effects:

[0034] The kit of the present invention includes 15 antibodies targeting the developmental pattern of bone marrow B cells and the common abnormal expression of ALL-B. Combined with a full-spectrum flow cytometer, all the above tests can be completed in one panel, which is convenient, fast, time-saving, labor-saving and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 and Figure 2 is a two-dimensional dot plot of flow cytometry in Example 2;

[0036] Figure 3 2D dot plot of flow cytometry of the 6-color flow cytometry kit in Example 3;

[0037] Figure 4 and Figure 5 This is a two-dimensional dot plot of the flow cytometry of the kit of the present application in Example 3. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] The present invention combines domestic and international professional guidelines and consensus to select 15 detection antibodies, uses full-spectrum flow cytometry, screens fluorescein, and performs more reasonable matching and experimental optimization with antibodies to design a 15-color ALL-B MRD detection panel, which includes a total of 15 antibodies and can perform comprehensive leukemia minimal residual lesions detection in ALL-B patients. The kit contains antibodies for detecting B lineage development and antibodies for detecting abnormal expression. The antibodies for detecting B lineage development include: anti-CD19 antibody, anti-CD10 antibody, anti-CD20 antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD81 antibody, anti-CD58 antibody, anti-CD45 antibody, anti-CD24 antibody, and anti-CD200 antibody; the antibodies for detecting abnormal expression include: anti-CD13 antibody, anti-CD33 antibody, anti-CD15 antibody, anti-CD66c antibody, and anti-CD123 antibody.

[0042] The significance of antibody detection is shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] The fluorescein linked to the antibody is shown in Table 2:

[0047] Table 2

[0048]

[0049]

[0050] In the embodiment of the present invention, the intensity of the fluorescent dye corresponding to the strength of antibody expression is matched. This panel was optimized in experiments using the 3-laser 38-channel full-spectrum multicolor flow cytometer (NL-CLC) from Cytek Corporation in the United States. It can be used directly in clinical practice without the need for preliminary experiments, and will not lead to inaccurate test results due to inappropriate antibody expression intensity.

[0051] Specifically, the antibody combination in the embodiments of the present invention is packaged as a single tube of lyophilized mixed solution, with a mass of 80-120 μg. Each tube of the antibody combination has a packaging volume of 5 mL. The lyophilized powder can be prepared using conventional methods, and appropriate excipients and other auxiliary materials may be added.

[0052] Specifically, the antibody combination in the embodiments of the present invention is packaged in a liquid tube. The concentration of the antibody in the liquid preparation is 80-120 μg / mL, preferably 100 μg / mL. The liquid preparation can be prepared using general methods and can be supplemented with appropriate stabilizers and other excipients.

[0053] Other reagents required for the experiment include: Brilliant Stain Buffer (BD, purchase link: https: / / www.bdbiosciences.com / cn / solrSearch?text=566349#); PBS; red blood cell lysis buffer (LyseLysing Buffer, from BD Pharm, purchase link: https: / / www.bdbiosciences.com / cn / applications / research / stem-cell-research / stem-cell-buffers-and-ancillary-reagents / lysing-buffer / p / 555899).

[0054] The specific method of using the kit of the embodiment of the present invention is:

[0055] 1. Preparation of reagents:

[0056] (1) Take an empty flow cytometry tube and an antibody combination tube, and mark them as control and sample tubes;

[0057] (2) Add 100 μl of the sample to be tested to the sample tube and control tube respectively, mix well, and incubate at room temperature for 15 min;

[0058] (3) After mixing again, add 3 mL of red blood cell lysis buffer to the sample tube and control tube respectively, and lyse at room temperature for 10 min;

[0059] (4) Centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0060] (5) Add 3 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0061] (6) Add appropriate amount of PBS to the sample tube and control tube respectively, mix well, filter through a 300-mesh filter, and wait for testing on the machine.

[0062] 2. Machine testing:

[0063] The prepared sample tubes and control tubes were directly tested using a Cytek full-spectrum flow cytometer, and the data were analyzed using the instrument's built-in analysis system. The target cells were mapped, and their developmental patterns and antigen expression were compared with those of normal individuals or the patient's initial diagnosis (i.e., reference values) to determine whether the detected B cells developed and expressed normally. This allowed the assessment of whether the B cells in ALL-B patients were residual tumor cells.

[0064] Example 1

[0065] A flow cytometry kit for monitoring MRD in acute B-lymphoblastic leukemia, the specific composition of which is shown in Table 3.

[0066] Table 3

[0067]

[0068]

[0069] Example 2

[0070] Normal bone marrow specimens were used as test samples and the kit of Example 1 was used for detection. The analysis was performed according to the following gate setting method to obtain the two-dimensional points of flow cytometry as shown in FIG. Figure 1 and Figure 2 As shown:

[0071] Set the P1 gate to remove adherent cells; set the P2 gate within the P1 gate as the mononuclear cell gate;

[0072] Multiple gates can be set simultaneously within the mononuclear cell gate P2 to detect the expression of different cell populations:

[0073] ①In gate P2, set the CD45 / SSC gate to obtain the detection status of lymphocytes, monocytes, granulocytes and nucleated red blood cells;

[0074] ② Within gate P2, CD19 / SSC or SSC / CD19 / CD24 combined gates were set to detect the B cell development process.

[0075] Within the B cell gate (CD19+ gate), CD19 / CD34 was gated to detect the expression of primitive B cells, CD19 / CD10 was gated to detect the expression of immature B cells, and CD19 / CD20 was gated to detect the expression of mature B cells.

[0076] ③ Within the P2 gate, detect weak cell populations: set the CD123 / CD38 gate to detect basophils, set the CD45 / SSC gate to detect eosinophils, and set the CD45 / CD38 gate to detect CD38-strongly positive plasma cells.

[0077] Abnormalities that may occur when B-cell acute lymphoblastic leukemia is minimal residual disease positive (i.e., ALL-B MRD+) include:

[0078] 1. Cross-stage expression, i.e., co-expression of early markers and developmental markers, or loss of expression, indicates malignancy. It is necessary to set multiple combined gates for CD19, CD10, CD20, CD34, CD38, CD81, CD58, CD45, CD24, and CD200 to detect the expression of B cells at different developmental stages. For details, refer to Table 1.

[0079] 2. Cross-lineage expression, that is, the target cell population co-expresses myeloid markers, indicating malignancy. It is necessary to set multiple combination gates for CD15, CD13, CD33, CD66c, and CD123 to detect B cell expression. For details, please refer to Table 1.

[0080] The present invention uses the instrument's own software to perform data analysis, plot the target cells, and display the table shown in Table 5. From top to bottom, the total number of cells obtained, the proportion of lymphocytes in the total cells and the number of cells, the proportion of B cells in the lymphocytes and the number of cells, and the proportion of primitive and mature B cells in the B cells and the number of cells are displayed respectively.

[0081] Table 4

[0082] Population %parent count All events 100％ 1000000 Lym 2.27 20463 CD19+ 1.46 13168 CD19+CD34+ 6.13 807 CD19+CD20+ 60 7870 CD19+CD10+ 34.11 4491

[0083] The percentage of nuclear cells occupied by each target cell is compared with the reference value to see whether the proportion of the detected cell subpopulations (CD19+ naive B cells, CD19+CD34+, CD19+CD20+) is increased or decreased compared with the reference value, so as to evaluate the minimal residual lesions of acute B lymphoblastic leukemia in the test sample.

[0084] Example 3

[0085] The same bone marrow sample was used as the test sample and was tested on a BD canto II flow cytometer using a mature 6-color panel reagent. The fluorescent dyes linked to the antibodies in the 6-color panel are shown in Table 5. The test results are as follows: Figure 3 and as shown in Table 6.

[0086] Table 5

[0087] serial number Fluorescein Antibody 1 percp-cy5.5 CD10 2 V500 CD45 3 BV605 CD19 4 FITC CD81 5 APC CD13+CD33 6 APC-cy7 CD20

[0088] Table 6

[0089] Population events %parent 19 / P3 24.845 5.83 P5 21.783 87.68 P5 AND 19 / P3 AND P3 21.783 5.11

[0090] The same sample was tested on spectral flow cytometry using the kit and detection method of the present invention, and the test results were as follows: Figure 4 、 Figure 5 and as shown in Table 7.

[0091] Table 7

[0092] Population %parent count All events 100.00 200,000 P1 91.01 182.024 P2 84.71 154.191 lym 4.28 6.033 CD19+ 5.11 7879 CD19+CD10+ 50.24 4.431 CD19+CD34+ 17.84 1.436 CD19+CD20+ 25.91 2042 P4 77.51 4,477 P3 5.66 8,731 Ba 0.52 802

[0093] In the 6-color panel mononuclear cell gate, CD19+B cells accounted for 5.83%, with no obvious abnormalities in expression at each developmental stage, and the MRD test results were negative.

[0094] The CD19+B cells in the mononuclear cell gate of the spectral flow cytometry 15-color panel kit in the embodiment of the present application accounted for 5.11%, and there was no obvious abnormality in expression at each developmental stage, and the MRD test result was negative.

[0095] Figures 3 to 5 Comparison shows that the proportion of target cell populations between the two is basically the same, there are no obvious abnormalities in expression and development patterns, and the MRD test results are both negative.

[0096] In addition to CD45, CD19, CD20, CD81, CD10, CD13, and CD33 involved in the 6-color panel, the test kit of the embodiment of the present application is designed according to domestic and international guidelines, and the results also provide detection information of CD24, CD200, CD38, CD58, CD123, and CD66c. In addition to observing the expression of the above antibodies in the target cell population, basophils (strongly positive for CD123) and plasma cells (strongly positive for CD19 and CD38) can also be observed.

[0097] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any person skilled in the art may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Claims

1. A flow cytometry kit for monitoring minimal residual lesions in acute B lymphoblastic leukemia, comprising antibodies for detecting B lineage development and antibodies for detecting abnormal expression. The antibodies used to detect B lineage development are anti-CD19 antibody, anti-CD10 antibody, anti-CD20 antibody, anti-CD34 antibody, anti-CD38 antibody, anti-CD81 antibody, anti-CD58 antibody, anti-CD45 antibody, anti-CD24 antibody, and anti-CD200 antibody; The antibodies used to detect abnormal expression are anti-CD13 antibody, anti-CD33 antibody, anti-CD15 antibody, anti-CD66c antibody, and anti-CD123 antibody; The anti-CD33 antibody is connected to fluorescein RB515, the anti-CD38 antibody is connected to fluorescein APC-cy7, the anti-CD15 antibody is connected to fluorescein FITC, the anti-CD24 antibody is connected to fluorescein RV711, the anti-CD123 antibody is connected to fluorescein RV780, the anti-CD58 antibody is connected to fluorescein RB710, the anti-CD200 antibody is connected to fluorescein RV600, the anti-CD19 antibody is connected to fluorescein RB780, the anti-CD66c antibody is connected to fluorescein RR664, the anti-CD45 antibody is connected to fluorescein RB697, the anti-CD13 antibody is connected to fluorescein RB594, the anti-CD10 antibody is connected to fluorescein PE, the anti-CD81 antibody is connected to fluorescein RR710, the anti-CD20 antibody is connected to fluorescein RV570, and the anti-CD34 antibody is connected to fluorescein PE-cy5.

2. The kit according to claim 1, wherein The antibody preparation is a liquid preparation or a freeze-dried preparation; all the antibodies are packaged in one antibody combination tube.

3. The kit according to claim 2, wherein In the liquid preparation, the concentration of the antibody is 80 to 120 μg / mL.

4. The kit according to claim 2, wherein In the liquid preparation, the concentration of the antibody was 100 μg / mL.

5. The kit according to claim 2, wherein In the lyophilized preparation, the mass of the antibody in the antibody combination tube is 80-120 μg.

6. The kit according to claim 2, wherein The volume of the antibody combination tube is 5 mL.

Images