A flow cytometry kit and method for monitoring minimal residual disease in acute myeloid 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 myeloid leukemia minimal residual disease (AML-MRD). The kit of the present application comprises 15 antibodies, which detect the developmental pattern and antigen expression intensity of myeloid cells, and detect the minimal residual disease of leukemia by comparing with the normal pattern. In combination 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 application relates to the field of flow cytometry, in particular, to a flow cytometry kit and a monitoring method for monitoring minimal residual disease of acute myeloid leukemia. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by clonal expansion of myeloid blast cells in blood and bone marrow. The self-renewal ability of leukemia cells is enhanced, and the proliferation is out of control, which can lead to differentiation disorders and apoptosis blockage, while inhibiting normal hematopoiesis in bone marrow and can infiltrate into various organs such as liver, spleen, lymph nodes, etc., showing clinical signs such as infection, bleeding, anemia, etc. AML is the most common type of adult leukemia, and the incidence rate increases with age. Although the clinical remission rate can be more than 80%, only 30% to 40% of young patients and less than 20% of elderly patients can achieve more than 5 years of disease-free survival. Relapse is still a difficult problem for leukemia cure. There are deficiencies in evaluating the efficacy and prognosis by morphological methods, and many patients who achieve complete remission after treatment will relapse at different time intervals. At this time, the emergence of minimal residual disease (MRD) detection provides a possibility to solve this clinical problem.

[0003] Minimal residual disease (MRD) refers to the state that a small amount of leukemia cells remains in the body after induction chemotherapy or bone marrow transplantation in patients with acute leukemia, which achieves clinical and hematological complete remission (morphological bone marrow primitive and immature cells less than 5%). MRD is the primary cause of leukemia relapse. Therefore, it is very important for leukemia patients to detect MRD: 1. It is beneficial to predict the relapse of leukemia earlier; 2. It guides the clinical treatment of leukemia, and decides whether to continue chemotherapy or stop treatment according to the amount of leukemia cells in the body, better to develop effective treatment plan and avoid over-treatment; 3. It is beneficial to find out whether leukemia cells are drug-resistant, and to choose more sensitive and more powerful treatment measures; 4. It is helpful to evaluate the stem cell purification effect of autologous hematopoietic stem cell transplantation patients.

[0004] In the past 20 years, with the development of MRD detection technology, various detection methods of MRD have been widely used in clinical evaluation of efficacy, such as flow cytometry (FCM), PCR (Polymerase chain reaction), FISH and mass spectrometry technology, and MRD technology needs to be sensitive, practical, accurate, reliable and fast. Bone marrow cell morphology, conventional karyotype analysis and FISH technology cannot meet the requirements of clinical MRD detection sensitivity due to low sensitivity. At present, the main detection methods of clinical MRD are FCM and polymerase chain reaction. Both have high sensitivity and good correlation, and the consistency of the two is 89.7%, but PCR-fusion gene can only be applied to part of AML patients, because not all AML patients have fusion genes or mutations, etc., and FCM mainly detects whether the development mode and specific marker expression of leukemia cells are normal, which can be used for almost all patients, and the detection depth can reach 10 4-5 However, the traditional flow detection channel is less, the compensation is difficult to adjust, and the commonly used detection antibodies are limited to 8-10 per tube, and the flow detection antibodies for MRD in the prior art are more than 20, so the previous detection can only select the first-line antibody and the necessary antibody closely related to the development mode of myeloid cells, and it is difficult to detect all residual leukemia cells, and if AML patients have MRD, the abnormal expression and development are mostly different from the initial diagnosis, and more extensive antibodies are needed for monitoring, therefore, the insufficient breadth of the detection antibodies is the main reason for the recurrence of some patients after MRD detection is negative.

[0005] In 2017, the full-spectrum flow cytometry was born, which increased the detection channels of three-laser flow cytometry from 4-13 of the traditional flow cytometry to 38, and can realize the monitoring of more than 25 colors per tube, greatly improving the detection information amount, the diagnostic precision and the depth of immunological detection.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] The first application purpose of the present application is to provide a flow cytometry kit for monitoring acute myeloid leukemia micro residual lesions.

[0008] The second application purpose of the present application is to provide a use method of the kit.

[0009] The third application purpose of the present application is to provide a monitoring method of acute myeloid leukemia micro residual lesions.

[0010] In order to achieve the application purpose of the present application, the technical scheme adopted is:

[0011] The present application provides a flow cytometry kit for monitoring acute myeloid leukemia minimal residual disease, the kit contains antibodies labeled with different fluorophores, the antibodies include: antibodies for detecting granulocyte development related antibodies include anti-CD117 antibody, anti-CD34 antibody, anti-CD33 antibody, anti-CD13 antibody, anti-CD11b antibody, anti-CD15 antibody, anti-CD64 antibody, anti-HLA-DR antibody, anti-CD45 antibody, anti-CD38 antibody, anti-CD14 antibody and anti-CD123 antibody; and antibodies for detecting abnormal expression related antibodies include anti-CD19 antibody, anti-CD7 antibody and anti-CD56 antibody.

[0012] Optionally, the anti-CD33 antibody is connected with fluorophore RB515, the anti-CD38 antibody is connected with fluorophore APC-cy7, the anti-CD15 antibody is connected with fluorophore FITC, the anti-CD34 antibody is connected with fluorophore PE-cy5, the anti-CD14 antibody is connected with fluorophore RV444, the anti-CD64 antibody is connected with fluorophore RR664, the anti-CD117 antibody is connected with fluorophore RB608, the anti-CD19 antibody is connected with fluorophore RB780, the anti-CD7 antibody is connected with fluorophore RV480, the anti-CD56 antibody is connected with fluorophore PE, the anti-CD45 antibody is connected with fluorophore RB697, the anti-CD11b antibody is connected with fluorophore RB730, the anti-HLA-DR antibody is connected with fluorophore APC, the anti-CD123 antibody is connected with fluorophore RV780, and the anti-CD13 antibody is connected with fluorophore RB710.

[0013] Optionally, the preparation of the antibody is a liquid preparation or a freeze-dried powder; in the liquid preparation, the concentration of the antibody is 80-120 μg / mL, preferably 100 μg / mL.

[0014] Optionally, all the antibodies are packaged in one container, preferably, the mass of the antibodies in the container is 80-120 μg; more preferably, the volume of the container is 5 mL.

[0015] The present application also relates to a method for using the kit, at least comprising the following steps:

[0016] (1) Take one empty flow tube and one antibody combination tube, mark as control and sample tube;

[0017] (2) Add the sample to be tested into the sample tube and the control tube respectively, mix, avoid light incubation for 15-20 min;

[0018] (3) Add red blood cell lysis solution into the sample tube and the control tube respectively, mix, lyse for 8-10 min;

[0019] (5) centrifugation, washing;

[0020] (6) re-homogenization, machine detection.

[0021] Optionally, the incubation time is 10-20 minutes, preferably 15 minutes.

[0022] Optionally, the lysis time is 10-15 minutes, preferably 10 minutes.

[0023] Optionally, the operations in the use method are all carried out at room temperature.

[0024] The application also relates to a monitoring method for monitoring acute B lymphocyte leukemia micro residual lesions, at least comprising the following steps:

[0025] (1) taking one empty flow tube and one antibody combination tube, and marking them as control and sample tubes;

[0026] (2) adding the sample to be detected into the sample tube and the control tube respectively, homogenizing, and avoiding light incubation for 15-20 minutes;

[0027] (3) adding red blood cell lysis solution into the sample tube and the control tube respectively, homogenizing, and lysis for 8-10 minutes;

[0028] (5) centrifugation, washing;

[0029] (6) re-homogenization, machine detection;

[0030] The application has at least the following beneficial effects:

[0031] The kit of the application comprises 15 antibodies, detects the development mode and antigen expression intensity of myeloid cells, detects the trace leukemia residual lesions by comparison with the normal mode, and combines the full-spectrum flow cytometer, so that all the above detections can be completed by one tube panel, which is convenient, fast, time-saving and labor-saving, and simple to operate. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 and Figure 2 is a two-dimensional dot plot of flow cytometry of Example 2;

[0033] Figure 3 is a two-dimensional dot plot of flow cytometry of the flow 6 color kit in Example 3;

[0034] Figure 4 and Figure 5 is a two-dimensional dot plot of flow cytometry of the kit of the application in Example 3; DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention is further illustrated below by means of embodiments and comparative examples. These embodiments are merely illustrative and the present invention is not limited to the following embodiments. Any modifications or equivalent substitutions to the technical solutions of the present invention that do not depart from the spirit and scope of the technical solutions of the present invention should be covered within the protection scope of the present invention.

[0038] This invention provides a flow cytometry kit for monitoring minimal residual disease (MRD) in acute myeloid leukemia. Utilizing full-spectrum flow cytometry, the kit employs a 15-color panel containing 15 antibodies. These antibodies target the developmental patterns and antigen expression intensities of myeloid cells. By comparing the results with normal patterns, trace amounts of residual leukemia lesions can be detected. Combined with a full-spectrum flow cytometer, all the above detections can be completed with a single panel, making it convenient, fast, time-saving, and simple to operate. The kit contains antibodies labeled with different fluorophores, including: antibodies for detecting myeloid development such as anti-CD117, anti-CD34, anti-CD33, anti-CD13, anti-CD11b, anti-CD15, anti-CD64, anti-HLA-DR, anti-CD45, anti-CD38, anti-CD14, and anti-CD123; and antibodies for detecting abnormal expression such as anti-CD19, anti-CD7, and anti-CD56. The significance of antibody detection is shown in Table 1:

[0039] Table 1

[0040]

[0041]

[0042] Specifically, the correspondence between the binding of fluorescein and antibody in the kit of this invention is shown in Table 2:

[0043] Table 2

[0044]

[0045]

[0046] The panel is optimized in experiments using a 3-laser 38-channel full-spectrum multi-color flow cytometer (NL-CLC) of the cytek company in the United States, and can be directly used in clinical practice without pre-experiment, and will not cause inaccurate detection results due to unsuitable expression of antibody intensity in detection.

[0047] Specifically, the antibody combination in the embodiment of the application is a tube mixed freeze-dried package, and the mass of the antibody combination is 80-120 μg. The packaging volume of each tube of the antibody combination is 5 mL. The freeze-dried powder can be prepared by a general method, and appropriate excipients and other auxiliary materials can be added.

[0048] Specifically, the antibody combination in the embodiment of the application is a tube liquid package, and in the liquid preparation, the concentration of the antibody is 80-120 μg / mL, preferably 100 μg / mL. The liquid preparation can be prepared by a general method, and appropriate stabilizers and other auxiliary materials can be added.

[0049] Other reagents required in the experiment are: Brilliant Stain Buffer (BD company, purchase link is https: / / www.bdbiosciences.com / cn / solrSearch?text=566349#); PBS; red blood cell lysis solution (Lyse Lysing Buffer, from BD Pharm, purchase link is https: / / www.bdbiosciences.com / cn / applications / research / stem-cell-research / stem-cell-buffers-and-ancillary-reagents / lysing-buffer / p / 555899).

[0050] The specific use method of the kit in the embodiment of the application is as follows:

[0051] I. Preparation of reagents:

[0052] (1) Take 1 empty flow tube and an antibody combination tube, and mark them as control and sample tubes;

[0053] (2) Add 100 ul of the sample to be tested into the sample tube and the control tube respectively, mix well, and incubate at room temperature for 15 min;

[0054] (3) After mixing again, add 3 mL of red blood cell lysate into the sample tube and the control tube respectively, and lyse at room temperature for 10 min;

[0055] (4) Centrifuge at 1050 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0056] (5) Add 3 mL of PBS into the sample tube and the control tube respectively, centrifuge at 1050 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0057] (6) After mixing again, filter through a 300-mesh filter, and then detect.

[0058] II. Detection on the instrument:

[0059] The prepared sample tube and the control tube are directly detected by a Cytek full-spectrum flow cytometer, and data analysis is performed by using the analysis system of the instrument. The target cells are drawn, and the development mode and antigen expression of each target cell are compared with the data (i.e. reference value) of normal people or the patient at the initial diagnosis, so as to determine whether the development and expression of the detected myeloid cells are normal, thereby evaluating the minimal residual disease of acute myeloid leukemia.

[0060] The data analysis is performed by using the software of the instrument, and the percentage data of each target cell in nucleated cells are compared with the data (i.e. reference value) of normal people or the patient at the initial diagnosis, so as to determine whether the proportion of the detected cell subpopulation is increased or decreased compared with the reference value, and whether the development mode of myeloid cells is abnormal, thereby evaluating the minimal residual disease of acute myeloid leukemia.

[0061] Specifically, the determination of the reference value refers to that a certain number of standard specifications are defined in a healthy population in a region, and the sample is investigated and determined in the whole population, and the average value (X) and the standard deviation (S) are obtained by statistical processing, and X is usually defined as the reference value, and X±2S is defined as the reference range.

[0062] Example 1

[0063] A flow cytometry kit for monitoring the minimal residual disease of myeloid leukemia, and the specific composition is shown in Table 3.

[0064] Table 3

[0065] Number Fluorescein Antibody Antibody type 1 RB515 CD33 Membrane antibody 2 APC-cy7 CD38 Membrane antibody 3 FITC CD15 Membrane antibody 4 PE-cy5 CD34 Membrane antibody 5 RV444 CD14 Membrane antibody 6 RR664 CD64 Membrane antibody 7 RB608 CD117 Membrane antibody 8 RB780 CD19 Membrane antibody 9 RV480 CD7 Membrane antibody 10 PE CD56 Membrane antibody 11 RB697 CD45 Membrane antibody 12 RB730 CD11b Membrane antibody 13 APC HLA-DR Membrane antibody 14 RB710 CD13 Membrane antibody 15 RV780 CD123 Membrane antibody

[0066] Example 2

[0067] The normal bone marrow sample is detected by using the kit of Example 1, and the two-dimensional points of flow cytometry are obtained by analyzing according to the following gate setting mode.Figure 1 and Figure 2 as shown in the following table:

[0068] P1 gate is set to remove the adherent cells; P2 gate is set in P1 gate to obtain single nucleus cells;

[0069] Multiple gates are set in P2 gate to detect the expression of different cell groups:

[0070] ①In P2 gate, CD45 / SSC gate is set to obtain lymphocytes, monocytes, granulocytes and nucleated red blood cells for detection.

[0071] ②In P2 gate, SSC / CD34 and SSC / CD117 are set to obtain CD34+ primitive cells and CD117+ immature myeloid cells, respectively, and the CD117+ cell and CD34+ cell groups are combined to observe the overall development and expression of immature myeloid cells.

[0072] ③In P2 gate, weak cell groups are detected, CD123 / HLA-DR gate is set to detect pDC and basophils (Ba), CD45 / SSC gate is set to detect eosinophils, CD45 / CD38 gate is set to detect CD38 strongly positive plasma cells, and SSC / CD117 gate is set to detect CD117 strongly positive mast cells.

[0073] ④In P2 gate, SSC / CD19 gate is set to detect B cell development.

[0074] ⑤In lymphocyte gate, CD7 / CD56 gate is set to detect T and NK cell groups.

[0075] When acute myelocytic leukemia microresidual disease is positive (i.e. AML MRD+), the abnormal conditions that may occur are:

[0076] 1. Cross-stage expression, i.e. early markers and developmental markers are co-expressed, or immature cells lose early markers, suggesting malignancy, CD34, CD117, CD38, CD45, CD10, CD11b, CD13, CD33, CD16, CD15, CD16, CD64 are set to observe the expression of myeloid immature cells at different developmental stages, and the specific reference is Table 1.

[0077] 2. Cross-line expression, i.e. target cell groups express lymphoid markers, suggesting malignancy, CD19, CD56, CD7 multiple gate combination is set to detect the expression of myeloid immature cells, and the specific reference is Table 1.

[0078] The instrument is equipped with software for data analysis, and the target cells are drawn to show the table as shown in Table 4. From top to bottom, the total number of cells obtained, the proportion of lymphocytes in total cells and the number of cells, the proportion of mononuclear cells in total cells and the number of cells, the proportion of neutrophils in total cells and the number of cells, and the proportion of CD34+ and CD117+ cells in total cells and the number of cells are shown.

[0079] Table 4

[0080] Population % parent count All events 100 835,568 P1 72.71 607,521 P2 89.56 544,075 P3 97.79 532,073 lym 4.05 21,562 mono 3.89 20,719 gly 68.01 361,862 CD34 1.20 6,390 CD117 0.65 3,484 CD34 OR CD117 1.55 8,272

[0081] The percentage of each target cell in nucleated cells is compared with the reference value to see if the proportion of the detected cell subpopulation (CD34+ cells, CD117+ cells, CD34+ or CD117+) is increased or decreased compared with the reference value, so that the myeloid cell leukemia microresidual lesion of the sample to be tested can be evaluated.

[0082] Example 3

[0083] The same bone marrow sample was detected as the sample, and the mature 8-color panel reagent was detected on the BD canto II flow cytometer. The fluorescence connected by the antibody in the 8-color panel is shown in Table 5, and the detection results are shown in Figure 3 and Table 6.

[0084] Table 5

[0085]

[0086]

[0087] Table 6

[0088] Population events % parent P8 10.211 2.69 117+ / P2 2.860 0.75 34+ 2.192 0.58

[0089] The same sample was detected on the spectral flow cytometer using the kit and detection method of the present application, and the detection results are shown in Figure 4 , Figure 5 and Table 7.

[0090] Table 7

[0091] Population % parent count All events 100.00 100,000 P1 77.64 77,642 P2 92.44 71,774 lym 6.21 4,454 NK 10.73 478 T 69.22 3,083 mono 3.97 2,846 gly 81.72 58,656 RBC 2.39 1,718 CD34+ 0.55 395 CD117+ 0.78 559 CD34+ OR CD117+ 1.07 765 CD19+ 0.98 704 CD19+ CD34+ 2.98 21 CD38hi+ 0.24 172 Ba 0.08 60 pDC 0.25 117

[0092] The proportion of CD34+ in the 8-color panel mononuclear cell gate is 0.58, and the proportion of CD117+ cells is 0.75%. The proportion and expression are not obviously abnormal, and the MRD detection result is negative.

[0093] The proportion of CD34+ cells in the single nucleus cell gate of the spectral flow 15 color panel kit of the embodiment of the present application is 0.55%, and the proportion of CD117+ cells is 0.78%. The proportion and expression are not obviously abnormal, and the MRD detection result is negative.

[0094] Figures 3-5 It can be seen that the proportion of target cell groups is basically consistent between the two, and the expression and development mode are not obviously abnormal, and the MRD detection result is negative.

[0095] In addition to CD45, CD19, CD34, CD117, CD33, CD11b and HLA-DR involved in the 8 color panel, the kit according to the embodiment of the present application is designed according to the guidelines at home and abroad, and CD38, CD123, CD15, CD13, CD7, CD56, CD64 and CD14 detection information are provided in the result. In addition to observing the expression of the above antibodies of the target cell group, the weak cell group: basophil (Ba, CD123 strongly positive HLA-DR-), plasmacytoid dendritic cell (pDC, CD123 strongly positive HLA-DR positive), plasma cell (CD19 positive CD38 strongly positive), T lymphocyte (CD7 positive CD56 negative) and NK cell (CD7 positive CD56 positive) can also be observed.

[0096] Although the above is disclosed in the preferred embodiment of the present application, it is not intended to limit the claims, and any person skilled in the art can make several possible changes and modifications without departing from the concept of the present application. Therefore, the protection scope of the present application should be limited by the scope defined by the claims of the present application.

Claims

1. The use of antibody combinations in the preparation of a flow cytometry kit for monitoring minimal residual disease in acute myeloid leukemia, characterized in that, The kit contains a combination of antibodies labeled with different fluorescent dyes, and the antibody combination is as follows: The antibodies used to detect granulocyte lineage development include anti-CD117, anti-CD34, anti-CD33, anti-CD13, anti-CD11b, anti-CD15, anti-CD64, anti-HLA-DR, anti-CD45, anti-CD38, anti-CD14, and anti-CD123; and the antibodies used to detect abnormal expression include anti-CD19, anti-CD7, and anti-CD56. All antibodies are packaged in a single antibody combination tube. The anti-CD33 antibody is linked to fluorescein RB515, the anti-CD38 antibody is linked to fluorescein APC-cy7, the anti-CD15 antibody is linked to fluorescein FITC, the anti-CD34 antibody is linked to fluorescein PE-cy5, the anti-CD14 antibody is linked to fluorescein RV444, the anti-CD64 antibody is linked to fluorescein RR664, the anti-CD117 antibody is linked to fluorescein RB608, the anti-CD19 antibody is linked to fluorescein RB780, the anti-CD7 antibody is linked to fluorescein RV480, the anti-CD56 antibody is linked to fluorescein PE, the anti-CD45 antibody is linked to fluorescein RB697, the anti-CD11b antibody is linked to fluorescein RB730, the anti-HLA-DR antibody is linked to fluorescein APC, the anti-CD123 antibody is linked to fluorescein RV780, and the anti-CD13 antibody is linked to fluorescein RB710.

2. The reagent kit according to claim 1, characterized in that, The antibody combination is formulated as a liquid or lyophilized preparation.

3. The reagent kit according to claim 2, characterized in that, In the liquid formulation, the concentration of the antibody is 80–120 μg / mL.

4. The reagent kit according to claim 2, characterized in that, In the liquid formulation, the concentration of the antibody is 100 μg / mL.

5. The reagent kit according to claim 2, characterized in that, In the lyophilized formulation, the antibody mass in the antibody combination tube is 80–120 μg.

6. The reagent kit according to claim 1, characterized in that, The volume of the antibody combination tube is 5 mL.

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