CN112552407A

CN112552407A - Antibody composition and application thereof in detecting acute B lymphocyte leukemia

Info

Publication number: CN112552407A
Application number: CN202110202574.7A
Authority: CN
Inventors: 王卉; 陈曼; 王爱先; 傅旻婧
Original assignee: Xinnake Beijing Biochemical Marker Detection Medical Research Co ltd
Current assignee: Xinnake Beijing Biochemical Marker Detection Medical Research Co ltd
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2021-03-26
Anticipated expiration: 2041-02-24
Also published as: CN112552407B

Abstract

The invention provides an antibody composition and application thereof in detecting acute B lymphocyte leukemia. The antibody composition comprises four groups of antibodies, the first group of antibodies comprising a CD38 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody, an anti-CD 19 antibody, an anti-CD 20 antibody, and an anti-CD 45 antibody; the second group of antibodies includes anti-cytoplasmic CD79a antibodies; the third group of antibodies comprises anti-CD 81 antibodies, anti-CD 34 antibodies, anti-CD 10 antibodies, and anti-CD 45 antibodies; the fourth set of antibodies included anti-TdT antibodies and anti-cytoplasmic CD79a antibodies. The antibody composition can be applied to flow cytometry for detecting acute B lymphocyte leukemia and/or bone marrow minimal residual disease, and can effectively select B cells including CD 19-B cells and CD19+ B cells, so that the B cells cover the most complete immature B cell group or even the B cell group, and the diagnosis omission probability is reduced.

Description

Antibody composition and application thereof in detecting acute B lymphocyte leukemia

Technical Field

The invention relates to an antibody composition and application thereof in detecting acute B lymphocyte leukemia, belonging to the technical field of hematopathy detection.

Background

In recent years, the incidence of leukemia/lymphoma has increased significantly and has become one of the important diseases affecting human health, Acute Lymphocytic Leukemia (ALL) accounts for 15% of ALL leukemias, about 30% -40% of acute leukemias, of which 80-85% are B-cell type (B-ALL). In the united states, the incidence of ALL is 1.38/10 million per year, with about 5930 new cases and 1500 deaths in 2019. Domestic research shows that the incidence rate of male ALL in Shanghai city in 2008-2012 is 0.86/10 ten thousand, the incidence rate of female ALL is 0.76/10 ten thousand, and the incidence rate is increased by 0.69/10 ten thousand compared with that in 1986-1988.

With the continuous emergence of new therapies such as targeted therapy and the like, leukemia and lymphoma with high mortality rate have curable hopes, and the current treatment method becomes a new development direction for cellular immunotherapy besides traditional chemotherapy and targeted drugs. Chimeric Antigen receptor T cells (CAR-T) realize specific recognition of tumor-associated antigens by genetically modifying T cells, so that effector T cells can fully play an anti-tumor role, the Chimeric Antigen receptor T cells are the key points of current cellular immunotherapy and one of the most important medical achievements in the beginning of the century, particularly have very obvious effects on the treatment of blood system diseases such as acute B lymphocyte leukemia (B-ALL), and the remission rate of refractory relapsing cases which are ineffective in conventional treatment can reach 90%. However, for a number of reasons, this approach has achieved less than 50% of long-term remission, and it is important to monitor post-treatment care, detect Minimal Residual Disease (MRD) early, and perform bridge grafting or secondary CAR-T therapy early.

Flow Cytometry (FCM) is a detection means capable of realizing quantitative analysis of a single cell, has the advantages of rapidness, high precision, multiple parameters and the like, and is one of the most advanced cell quantitative analysis methods at present. FCM plays an important role in clinical and scientific research fields, has been widely and deeply applied particularly in clinical in vitro diagnosis and becomes one of the most mainstream detection means, and the detection result of FCM is the gold standard for clinical diagnosis of malignant diseases such as leukemia, lymphoma and the like at present.

The FCM is used for carrying out bone marrow MRD detection on B-ALL patients and is an important prognostic variable, a specific antibody composition is used, a multicolor flow cytometer is matched, prognosis and relapse can be effectively evaluated, a treatment scheme can be guided to be selected, and the FCM is an indispensable clinical evaluation index of B-ALL. However, the correct selection of antibody compositions is critical to achieve this goal, for example, the most common assay protocol is the gating of B cells with the cell membrane pan B cell marker CD19 supplemented with SSC or CD45 (dual parameters CD19/SSC or CD45/CD 19), but in patients treated with CD19-CAR-T cell infusion, gating with CD19 is no longer applicable because partially normal or abnormal B cell surface CD19 antigen exhibits loss or attenuation of expression after treatment, and there is a great need to find new effective protocols to gate B cells of interest.

To solve this problem, researchers have conducted research studies, including the Washington university study, using CD22 in combination with CD24+/CD66 c-gated B cells to detect MRD following CAR-T cell infusion therapy, but they still suffer from the following drawbacks: 1. CD24 is easy to express and lose CD24 in the ALL-B with 80.6% of B-ALL expression rate, especially in MLL gene rearranged ALL-B, thereby affecting the cycle gate efficiency of B cells, 2 and CD22 are expressed or weakly expressed in basophils, plasmacytoid dendritic cells and mast cells, so the specificity is not good, and CD22 antigen gradually increases with the expression intensity of mature CD22 of cells in the maturation process of normal B cells, while in B-ALL, the expression of original B cell CD22 is reduced, thereby affecting the sensitivity of CD22 gating B cells. 3. Is not suitable for patients treated by CD22-CAR-T cells or CD19-CD22-CAR-T double-target cell infusion.

Disclosure of Invention

It is an object of the present invention to provide a novel technique for detecting acute B-lymphocyte leukemia and/or bone marrow minimal residual disease.

One aspect of the invention provides an antibody composition comprising a first set of antibodies, a second set of antibodies, a third set of antibodies, and a fourth set of antibodies, wherein:

the first group of antibodies comprises: CD38, anti-CD 10, anti-CD 34, anti-CD 19, anti-CD 20, and anti-CD 45 antibodies;

the second group of antibodies includes: anti-cytoplasmic (c) CD79a antibodies;

the third group of antibodies includes: anti-CD 81, anti-CD 34, anti-CD 10, and anti-CD 45 antibodies;

the fourth set of antibodies comprises: anti-TdT antibodies and anti-cytoplasmic CD79a antibodies.

The antibody composition can be applied to flow cytometry detection to detect acute B lymphocyte leukemia and/or bone marrow minimal residual disease, especially to cases that more B cell markers such as CD19 and/or CD22 and even BAFFR are targeted for treatment, and the B cell markers can be weakened or lost. In specific application, a 2-tube parallel scheme is used, each tube uses four antibodies, namely CD10, CD34, cytoplasmic CD79a, CD19, CD10, TdT, CD34 and cytoplasmic CD79a, and the antibodies are respectively combined with SSC to set immature B cells and all B cell gates, so that B cells including CD 19-B cells and CD19+ B cells can be effectively gated to cover the most complete immature B cell groups and even B cell groups, the missed diagnosis probability is reduced, and particularly, the missed diagnosis probability is reduced for follow-up cases after targeted therapy of B cell markers such as CD19 and CD22 or the cases with negative or weak expression of the markers.

According to a particular embodiment of the invention, in the antibody composition of the invention, each antibody is a monoclonal antibody.

According to a particular embodiment of the invention, in the antibody composition of the invention, each antibody is a fluorescein-labeled antibody. Preferably, in the first group of antibodies, the fluorescein markers of the CD38 antibody, the anti-CD 10 antibody, the anti-CD 34 antibody, the anti-CD 19 antibody, the anti-CD 20 antibody and the anti-CD 45 antibody are respectively: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, V500. In the second group of antibodies, the fluorescein of the anti-cytoplasmic CD79a antibody was labeled APC. In the third group of antibodies, the fluorescein labels of the anti-CD 81 antibody, the anti-CD 34 antibody, the anti-CD 10 antibody and the anti-CD 45 antibody are respectively: PE, PerCP-Cy5.5, PE-Cy7, V500. In the fourth group of antibodies, the fluorescein labels of the anti-TdT antibody and the anti-cytoplasmic CD79a antibody were FITC and APC, respectively, in that order. According to the invention, different antibodies are matched with specific fluorescein, so that when the antibody composition is applied to flow cytometry for detecting acute B lymphocyte leukemia and/or bone marrow minimal residual disease, all the fluorescein in each channel can achieve an excellent dyeing effect.

According to a particular embodiment of the invention, each antibody component of the antibody composition of the invention is commercially available. Each antibody should meet relevant industry standard requirements.

According to a particular embodiment of the invention, in the antibody composition of the invention, the first group of antibodies is CD38 antibodies, anti-CD 10 antibodies, anti-CD 34 antibodies, anti-CD 19 antibodies, anti-CD 20 antibodies and anti-CD 45 antibodies according to 5:5:5:3:2:3 (in the case of substantially equivalent titers) are mixed. The third group of antibodies is anti-CD 81 antibody, anti-CD 34 antibody, anti-CD 10 antibody and anti-CD 45 antibody according to 5:5:3:3 (in the case of substantially equivalent titers) are mixed. The fourth set of antibodies was anti-TdT and anti-cytoplasmic CD79a antibodies according to 2:3 (in the case of substantially equivalent titers) are mixed.

Another aspect of the invention provides a kit comprising a first container, a second container, a third container, and a fourth container, each container holding a first set of antibodies, a second set of antibodies, a third set of antibodies, or a fourth set of antibodies, respectively, of an antibody composition of the invention.

According to a particular embodiment of the invention, the kit of the invention may further comprise: one or more of erythrocyte lysate, membrane breaking agent, buffer solution and flow tube matched with the flow cytometer. These reagents and consumables are commercially available. Wherein the film breaking agent is preferably a film breaking agent comprising solution A and solution B. The reagent materials can be respectively contained in different containers.

The kit can be used for detecting acute B lymphocyte leukemia and/or bone marrow minimal residual disease.

The invention also provides application of the antibody composition in preparing a kit for detecting acute B lymphocyte leukemia and/or bone marrow minimal residual disease.

According to a specific embodiment of the present invention, the process for detecting acute B-lymphocyte leukemia and/or bone marrow minimal residual disease comprises the steps of:

treating a sample to be detected by using the antibody composition to prepare a flow cytometry on-machine sample;

performing flow cytometry on the machine for detection;

the detection result is analyzed to judge (including auxiliary judgment) the acute B lymphocyte leukemia and/or the bone marrow minimal residual disease.

(1) respectively adding the sample to be detected into two flow tubes of tube A and tube B to make it be in single cell suspension state and ensure cell quantity to be 1X 10⁶pipe-1X 10⁷A pipe; the sample to be detected is bone marrow or peripheral blood;

(2) adding a first group of antibodies in the antibody composition into the sample obtained by the step (1), adding a third group of antibodies in the antibody composition into the tube B, uniformly mixing, and incubating at room temperature in a dark place;

(3) adding the membrane breaking agent A solution into the flow tube incubated in the step (2), and incubating at room temperature in a dark place;

(4) adding 1 Xhemolysin into the flow tube incubated in the step (3), and incubating at room temperature in a dark place;

(5) centrifuging the flow tube incubated in the step (4), removing supernatant, adding the membrane breaking agent B liquid and a second group of antibodies in the antibody composition into the tube A, adding the membrane breaking agent B liquid and a fourth group of antibodies in the antibody composition into the tube B, and incubating at room temperature in a dark place;

(6) adding PBS buffer solution into the flow tube incubated in the step (5) for washing, centrifuging, removing supernatant, and resuspending cells by using the PBS buffer solution;

(7) and (3) performing flow cytometry detection on the resuspended cells in the step (6), gating SSC for selecting antibodies CD10, CD34, CD19 and cytoplasmic CD79a in the first tube sample, gating SSC for selecting antibodies TdT, CD10, CD34 and cytoplasmic CD79a in the second tube sample, screening B cells and immature B cells, and analyzing.

According to a particular embodiment of the invention, the reagents other than the antibody composition of the invention may be used in amounts conventionally used in the art or in accordance with the recommendations of the manufacturer.

According to a specific embodiment of the invention, the antibody composition of the invention comprises a first group of antibodies added in an amount of 10-30. mu.l/tube, a second group of antibodies added in an amount of 2-5. mu.l/tube, a third group of antibodies added in an amount of 10-20. mu.l/tube, and a fourth group of antibodies added in an amount of 5-20. mu.l/tube.

According to a specific embodiment of the present invention, in step (1), the sample is added in a volume of not more than 160. mu.l per tube (if the patient has a small amount of peripheral blood cells, a volume of more than 160. mu.l is added first, and the supernatant is centrifuged to concentrate the blood).

According to a specific embodiment of the present invention, in the step (2), the incubation time may be 10 to 30 minutes.

According to a specific embodiment of the present invention, in the step (3), the incubation time may be 5 to 20 minutes. The addition amount of the film breaking agent A solution is required according to the recommended dose of a merchant, and is usually 100 mul/tube.

According to a specific embodiment of the present invention, in the step (4), the incubation time may be 5 to 30 minutes. The amount of 1 Xhemolysin added is 2-3 ml/tube.

According to a specific embodiment of the present invention, in step (5), the incubation is usually carried out for about 10 to 30 minutes. The centrifugation conditions may be 1000-2000rpm (or 300-450 g) for 5 minutes. The addition amount of the film breaking agent B solution is required according to the recommended dose of a merchant, and is usually 50 mul/tube.

According to a specific embodiment of the present invention, in step (6), the amount of PBS buffer for washing is 2 to 3ml per tube. The centrifugation conditions may be 1000-2000rpm (or 300-450 g) for 5 minutes. The amount of PBS buffer added for resuspension was 0.5-1 ml/tube.

Another aspect of the present invention provides an apparatus for detecting acute B-lymphocyte leukemia and/or myeloid minimal residual disease, the apparatus comprising a detection unit and an analysis unit, wherein:

the detection unit comprises a reagent material for detecting a sample from an individual to be detected by flow cytometry, and is used for obtaining a detection result of the sample; the reagent material comprises an antibody composition of the invention;

the analysis unit is used for analyzing the detection result of the detection unit.

According to a particular embodiment of the invention, the device of the invention is used for detecting acute B-lymphoblastic leukemia and/or myeloid minimal residual disease, wherein the process of detecting a sample from a test subject by flow cytometry comprises:

after the antibody composition is used for processing a sample to be detected, a flow cytometry sample is prepared (the specific processing process can refer to the above record);

performing flow cytometry on the machine for detection;

wherein the first tube is provided with a door according to the following mode during flow cytometry on-machine detection: setting a debonding cell gate P1 and a living cell gate P2 to obtain a single living cell; setting each blood cell gate within P2 using CD45/SSC antibody; p2 multiple markers in gate combination B cells were observed: the B cell gate is set by using CD19/SSC and cytoplasmic CD79a/SSC antibodies, and the immature B cell marker is set by selecting CD10/SSC and CD34/SSC antibodies;

wherein, the second tube is provided with a door according to the following mode when the flow cytometry is used for detecting: the procedures of setting a detachment cell gate P1 and a live cell gate P2 are sequentially carried out, then setting each blood cell gate by using a CD45/SSC antibody in a P2 gate, and setting the gates by using a cytoplasmic CD79a/SSC, CD10/SSC, CD34/SSC and TdT/SSC antibody multi-marker combination (the B cell gate is set by using a cytoplasmic CD79a/SSC antibody, and the immature B cell marker is set by selecting CD10/SSC, CD34/SSC and TdT/SSC antibodies).

According to the specific embodiment of the invention, when the device is used for detecting acute B lymphocyte leukemia and/or bone marrow minimal residual disease, and the analysis unit analyzes the detection result of the detection unit, the displayed B cell development pattern is compared with normal cells in the multi-marker combination set door to find out tumor cells.

In some embodiments of the invention, the door is provided as follows:

the debonding cell gate (commonly designated P1) was first set using the area (area, a) and height (height, H) of Forward angle light scattering (FSC), then the cells within P1 were visualized, and the living cell gate (commonly designated P2) was set using FSC/side angle light scattering (SSC), resulting in a single living cell.

The conventional method for setting each blood cell gate in a single living cell gate (P2 gate) firstly uses CD45/SSC to roughly observe lymphocytes, monocytes, granulocytes and whether obvious tumor cells or abnormal cells exist.

Multiple marker combinations gated in the single viable cell gate (gate P2) to observe B cells, especially immature B cells: the first vessel sample is gated by cytoplasmic CD79a/SSC (FIG. 1, FIG. 2, FIG. 7), CD19/SSC (FIG. 3, FIG. 8), CD10/SSC (FIG. 4, FIG. 9), CD34/SSC (FIG. 5, FIG. 10) antibody, and the second vessel sample is gated by cytoplasmic CD79a/SSC (FIG. 1, FIG. 7), CD10/SSC (FIG. 4, FIG. 9), CD34/SSC (FIG. 5, FIG. 10), TdT/SSC (FIG. 6, FIG. 11) antibody.

In some embodiments of the invention, the analyzing unit, when analyzing the result of the detecting unit, compares the displayed B cell development pattern with the normal cell in the multi-marker combination setting gate to find out the tumor cell, and specifically judges according to at least one of the following ways: the MRD mainly comprises two methods for scheme design and result judgment: leukemia Associated Immunophenotyping (LAIP), a marker that is not normally expressed or that is normally expressed by a tumor cell is lost; compared with Normal differential expression (Di ﬀ energy-from Normal, DFN), i.e., a reasonable scheme is designed to make several normally expressed antigens form a developmental pattern, and if the expression intensity or composition pattern of the antigens in the sample to be tested is changed compared with that of cells at the Normal stage, the cells are DFN. The currently accepted method is a combination of the two. The normal B progenitor development process can be divided into 4 stages: first, cells express high expression (high, hi) of CD38+/CD34+/CD 10/low expression (low) of CD 45/low expression of CD19 low/cytoplasmic CD79a +/TdT +/CD81 moderate strength/CD 20 (FIG. 1, T1). (the antigen expression change described here refers to the normal change trend in the development process, and the change in the development intensity of normal cells is represented by hi and low in order to distinguish bri and dim used when the difference in the expression intensity of malignant cells from normal cells at that stage is described in MRD), SSC is almost the same as that of mature cells. ② with the mature cell development, the expression of CD34 is lost, the expression of CD10 is weakened, the expression of CD45 and CD19 is strengthened compared with the first stage, the expression of CD81 reaches the strongest stage, and SSC is reduced. The second phase phenotype is: CD38+/CD34-/CD10+/CD45 medium strength/CD 19 +/cytoplasmic CD79a +/TdT-/CD81hi/CD20- (FIG. 1, T2). Third stage cells, as the cells further mature, CD20 begins to appear, CD45 and CD19 expression increases, CD81 expression begins to decrease, SSC begins to increase, and the expression pattern is: CD38+/CD34-/CD10+/CD45+/CD19 +/cytoplasmic CD79a +/TdT-/CD81+/CD20+ (FIG. 1, T3). Mature stage B cell, the expression mode is: CD38-/CD34-/CD10-/CD45hi/CD19 +/cytoplasmic CD79a +/TdT-/CD81 low/CD20+ (FIG. 1, T4).

If the development pattern is different from the normal development pattern, the suspicious pattern is malignant, and the MRD is diagnosed to be positive after the influence of other factors is eliminated.

In summary, the present invention provides an antibody composition and its application in flow cytometry for detecting acute B-lymphocyte leukemia and/or bone marrow minimal residual disease. The invention has the advantages that: (1) by using multiple markers in combination, missed diagnosis caused by the reduction or loss of CD19 expression after CD19 treatment can be avoided. The research of the invention shows that the negative cases of CD10, CD34 and TdT all express cytoplasmic CD79a, and the four marks can cover 100% of cases, so that the immature B cell gate covers the most complete B cell population; (2) the combination of multiple marks is designed, besides the prevention of missed diagnosis, the combination is also helpful for finding special clones and weak clones, although most tumors are single clones, about 5 percent of the tumors consist of 2-3 clones, and the weak clones are easy to ignore under the general condition. The weak clones have great significance for future relapse, phenotype change and even evolution of main clones at a certain moment, whether secondary tumor is diagnosed or not, and revealing the occurrence and development rules of tumor and treating the secondary tumor in a targeted manner. (3) The combined gating of the invention is different from the traditional CD22 gating, and can not be interfered by basophils, plasmacytoid dendritic cells, mast cells, granulocytes and the like; (4) the combined gate of the invention is not influenced by biological target treatment such as cell treatment. At present, the clinical treatment aiming at B-ALL mostly adopts a CD19-CAR-T treatment scheme, a CD19-CAR-T, CD22-CAR-T cell sequential treatment scheme and a CD19/CD22 combined double CAR-T scheme, and a plurality of hospitals try various and continuously advanced new targeted treatments such as BAFFR and the like, and ALL the schemes cause normal and neoplastic B cells of patients to lose antigens aiming at other targeted treatments such as CD19 antigen and/or CD22 antigen, so the detection analysis aiming at the antigens has inherent limitations. However, in the combined gate, CD34 is expressed in ALL early cells, CD10 is expressed in mature granulocytes, TdT and cytoplasmic CD79a are expressed in cells, targeted therapy aiming at the antigens has foreseeable toxic and side effects, or targeted cytoplasmic antigens have great technical difficulty, and the antigens are the marks which cannot be selected or are difficult to select in the existing B-ALL therapy, even in the future targeted biological therapies such as CAR-T aiming at a new target point, so that the combined gate is hardly influenced by the selection or change of a treatment scheme, is urgently needed by the existing B-ALL targeted therapy, and is suitable for MRD detection and analysis schemes which are widely applied and popularized.

Drawings

FIGS. 1-6 show the results of flow cytometry gating analysis of normal bone marrow specimens according to one embodiment of the present invention. Wherein, fig. 1 and fig. 2: except for the conventional P1, P2 and CD45/SSC gating, cytoplasmic CD79a/SSC gating is used; FIG. 3: setting a gate for CD 19/SSC; FIG. 4: setting a gate for CD 10/SSC; FIG. 5: setting a gate for CD 34/SSC; FIG. 6: TdT/SSC is provided with a gate.

FIGS. 7-11 show bone marrow sample flow cytometric analysis of relapsed cases after CD19-CAR-T treatment in one embodiment of the invention. Wherein, fig. 7: except for the conventional P1, P2 and CD45/SSC gating, cytoplasmic CD79a/SSC gating is used; FIG. 8: setting a gate for CD 19/SSC; FIG. 9: setting a gate for CD 10/SSC; FIG. 10: setting a gate for CD 34/SSC; FIG. 11: TdT/SSC is provided with a gate.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

EXAMPLE 1 formulation of reagents

The combination of antibodies used in this example was,

a1 is: CD38-FITC, CD10-PE, CD34-PerCP-Cy5.5, CD19-PE-Cy7, CD20-APC-Cy7 and CD45-V500, and the above six monoclonal antibody reagents are mixed according to the volume ratio of 5:5:5:3:2:3 and are filled in a first container;

a2 is: cytoplasmic CD79a-APC in a second container;

b1 is: CD81-PE, CD34-PerCP-Cy5.5, CD10-PE-Cy7 and CD45-V500, and the four monoclonal antibody reagents are mixed according to the volume ratio of 5:5:3:3 and filled in a third container;

b2 is: TdT-FITC and cytoplasmic CD79a-APC, and the two monoclonal antibody reagents are mixed according to the volume ratio of 2:3 and filled in a fourth container. The antibodies in this example were commercially available, wherein TdT-FITC was Beckman Coulter, USA, cytoplasmic CD79a-APC was Tetraselaginella, and the other fluorescein-labeled antibodies were Becton Dickinson, USA.

And (3) optionally adding a red blood cell lysate into a fifth container, adding a membrane breaking agent A into a sixth container, adding a membrane breaking agent B into a seventh container, adding a PBS buffer solution into an eighth container, wherein the red blood cell lysate, the membrane breaking agent and the PBS buffer solution are commercially available, the cell lysate and the membrane breaking agent are both products of Becton Dickinson company in the United states, and the PBS buffer solution is a product of Beckman Coulter company.

EXAMPLE 2 treatment of specimens

According to the cell counting result, heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added into the first tube of the flow tube to ensure that the added cell amount is about 2 x 10⁶Adding 23 mu l of six cell membrane monoclonal antibody reagents marked by different fluorescein into the flow tube according to the table 1, fully mixing the six cell membrane monoclonal antibody reagents with the cell suspension, incubating for 15 minutes at normal temperature in the dark, adding 100 mu l of the solution A of the membrane breaking agent, incubating for 5 minutes at room temperature in the dark, and adding 3ml of the solution A1 Xhemolysin, incubating for 10 minutes in the dark to lyse red blood cells, centrifuging for 5 minutes at 1500rpm to remove supernatant, adding 50 mul of membrane breaking agent B liquid and 3 mul of monoclonal antibody reagent cytoplasm CD79a-APC (cCD 79 a), incubating for 15 minutes in the dark at room temperature, finally adding 3ml of PBS buffer solution to wash, centrifuging to remove supernatant, and resuspending cells with 0.5ml of PBS buffer solution to obtain the processed sample which can be used for on-machine detection.

TABLE 1

According to the cell counting result, heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added into a flow tube B tube to ensure that the added cell amount is about 2 multiplied by 10⁶And then adding 16 mu l of four cell membrane monoclonal antibody reagents marked by different fluorescein into the flow tube according to the table 2, fully mixing the four cell membrane monoclonal antibody reagents with the cell suspension, incubating for 15 minutes at normal temperature in the dark, adding 100 mu l A liquid, incubating for 5 minutes at room temperature in the dark, adding 3ml of 1 Xhemolysin, incubating for 10 minutes in the dark to lyse red blood cells, centrifuging for 5 minutes at 1500rpm, removing supernatant, adding 50 mu l B liquid, 2 mu l of cytoplasmic monoclonal antibody reagent TdT-FITC and 3 mu l of cytoplasmic antibody CD79a-APC, incubating for 15 minutes at room temperature in the dark, finally adding 3ml of PBS buffer solution for washing, centrifuging, removing supernatant, using 0.5ml of PBS buffer solution to resuspend cells, and obtaining the processed sample which can be used for machine detection.

TABLE 2

EXAMPLE 3 detection of specimens

The specimens treated according to example 2 were tested on a 3-laser 8-color FACS Canto II flow cytometer (Becton Dickinson, Inc., USA) preferably after acquiring 100 million cells per tube (at least 30 million suggested), and the data was analyzed using diva 2.8 software or other software such as kaluza.

Wherein, set up the door according to following mode when flow cytometry is gone up quick-witted detection:

firstly, a door is fixedly arranged: sequentially removing a sticky cell gate, a living cell gate and a blood cell gate; secondly, a door is arranged in a multi-mark combination mode: starting from a single living cell, in order to prevent tumor cells from being missed, the marking and gating of the B line and the immature B cell are carried out under the condition of being parallel to a blood cell gate; and thirdly, in the multi-marker combination setting gate, displaying the common B cell development modes of various marker combinations, and finding out the tumor cells according to the difference from normal cells.

1. Fixedly arranging a door: consists of a desmokinetic, a living, a haematopoietic phylum. In a serial relationship.

Desmoking of the cytogate: adherent cells can be removed by FSC-Area (Area, A)/Height (Height, H) on the principle that the cells are spherical and A is positively correlated with H. (see FIG. 1).

The phylum of living cells: the common method for clinical examination of specimens is FSC/SSC, which is based on the principle that the size and granularity of living cells are approximately normally distributed, the living cells are clustered around a center, and the clustering has obvious limits on dead cells, apoptotic cells, debris and background noise.

Blood cell gate: the principle of roughly differentiating the groups of blood cells by CD45/SSC is that the hematopoietic cells CD45 express different fluorescence intensities (mature lymphocytes > monocytes > granulocytes > nucleated red blood cells) and SSC differ in size (granulocytes > monocytes > mature lymphocytes > nucleated red blood cells) (see FIG. 1). The aim is to prevent large tumor cell populations from being missed after misdiagnosis or tumor evolution, and to set internal controls.

2. The door is established in the combination of many marks: in synchronism with the gating of CD45/SSC, starting from a single live cell (P2).

B cell gate: in addition to the conventionally used CD19/SSC, the present invention adds a cytoplasmic CD79a (cCD 79 a)/SSC gating.

Immature B cell gate: the first tube uses CD34/SSC, CD10/SSC, and the second tube increases TdT/SSC to identify immature B cells. Can prevent cases of reduced or lost cytoplasmic CD79a expression without missing immature B cells; in addition, vulnerable clones can be precisely found.

3. Precise targeting of target cells based on combinations of observation signatures

Because there is an indefinite proportion of normally proliferating B progenitor cells in normal bone marrow, these proliferating early cells are significantly elevated after chemotherapy or when stimulated by other factors, interfering with the MRD determination (fig. 1-6).

The marker combination of the invention can accurately lock target cells.

4. The expression of normal CD 19-negative B cells was also different from that of common CD 19-positive B cells, which are most early stage B progenitor cells, and were characterized by reduced CD10 expression, large SSC, and easy misdiagnosis as ALL-B MRD in unfamiliar cases (fig. 2). This population of cells is also present normally, but is overlooked because of the extremely low proportion of cases, plus the previous gating with CD 19. The method of the invention may render this population of cells evident.

In this example, 20 normal bone marrow specimens were selected for testing, all normal bone marrow B cells had different ratios and different B cell ratios in different differentiation stages, but the antigen appearance time, expression intensity and combination of two was in a regular differentiation pattern (fig. 1), which was divided into 4 stages, and some of them had CD19 negative B cells as shown in fig. 2. Based on the above, the tumor cells will show more or less difference from normal cells in various antigens and antigen combination expression patterns, and the difference is judged to be MRD positive, and the rate of malignant cells exceeds that of nucleated cells by 5%, so that relapse is considered.

This example provides an example of each of normal and post B-ALL CD19-CAR-T relapse. FIGS. 1-6 show normal specimens, except for conventional P1, P2, and CD45/SSC gates, cytoplasmic CD79a/SSC gate (FIG. 1, FIG. 2), CD19/SSC gate (FIG. 3), CD10/SSC gate (FIG. 4), CD34/SSC gate (FIG. 5), and TdT/SSC gate (FIG. 6), respectively. FIGS. 7-11 show tumor specimens, gated for analysis by the same method: except for the conventional P1, P2 and CD45/SSC gates, cytoplasmic CD79a/SSC gate (FIG. 7), CD19/SSC gate (FIG. 8), CD10/SSC gate (FIG. 9), CD34/SSC gate (FIG. 10) and TdT/SSC gate (FIG. 11) are used.

Specifically, fig. 1: normal bone marrow specimens were analyzed together with tube A and tube B. Sequentially setting: FSC-A/H setting P1 as the door of the cell de-adhesive, resulting in single cells in P1; FSC/SSC was shown in P1 setting P2 as the viable cell gate, resulting in a single viable cell in P2; the P2 gate was gated with CD45/SSC and cytoplasmic CD79a/SSC, respectively. CD45/SSC set the blood cell gate, and lymphocyte (lym), granulocyte (Gra) and monocyte (mono) cell gate are respectively obtained; b cell gates were set using cytoplasmic CD79a/SSC (P3) to generate B cell populations. A common combination of normally proliferating B progenitor cell antigen expression patterns is shown separately. Normal B cell development can be seen in four stages (gradually lightening as the cells mature): t1: the earliest stages of normally proliferating B cells, the expression pattern is: CD34+/TdT +/CD10hi/CD20-/CD38+/CD81 medium strength/CD 45low/CD19 low/cytoplasmic CD79a +; t2: the second phase cell expression pattern was: CD34-/TdT-/CD10+/CD20-/CD38+/CD81hi/CD45 low/CD19 +/cytoplasmic CD79a +; t3: a third stage cell, the expression pattern being: CD34-/TdT-/CD10+/CD20 +/CD38 +/CD81+/CD45 +/CD19 +/cytoplasmic CD79a +; t4: mature stage B cells, the expression pattern is: CD34-/TdT-/CD 10-/CD 20+/CD38-/CD81low/CD45hi/CD19 +/cytoplasmic CD79a +.

Specifically, fig. 2: normal bone marrow specimen, same as in fig. 1, was subjected to nail tube analysis. By gating with cytoplasmic CD79a/SSC (P3), a small number of CD 19-negative B cells (P9, dark black cell population) were found, which were dominated by the CD 34-positive early component, with larger SSC and reduced CD10, compared to the CD 19-positive component (gray cell population).

Specifically, fig. 3: normal bone marrow specimen, same as in fig. 1, was subjected to nail tube analysis. Sequentially setting: the door for the detachment cell (P1), the living cell (P2) and the P2 was gated with CD45/SSC and CD19/SSC, respectively. CD45/SSC set the blood cell gate, and CD19/SSC set the B cell gate (P4) to obtain a B cell population. The same four developmental stages of normal B cells as the cytoplasmic CD79a/SSC gate can be seen, but no CD19 negative B cells can be seen. Since only the nail tube had CD19, CD19 was not analyzed for CD81, TdT expression.

Specifically, fig. 4: the same normal bone marrow specimen as in FIG. 1 was analyzed by both A-channel and B-channel. Sequentially setting: the door for the detachment cell (P1), the living cell (P2) and the P2 was gated with CD45/SSC and CD10/SSC, respectively. CD45/SSC set the blood cell gate, and CD10/SSC low set the immature B cell gate (P5), resulting in a CD10+ immature B cell population. It can be seen that although there are four developmental stage normal B cells gated similarly to cytoplasmic CD79a/SSC in FIG. 2, there is only a significant reduction in T4. CD19 negative B cell population (P9, dark black bold dots) was seen.

Specifically, fig. 5: the same normal bone marrow specimen as in FIG. 1 was analyzed by both A-channel and B-channel. Sequentially setting: the door for the detachment cell (P1), the living cell (P2) and the P2 was gated with CD45/SSC and CD34/SSC, respectively. CD45/SSC set the blood cell gate, and CD34/SSC low set the primary B cell gate (P6), resulting in a CD34+ primary B cell population. See only stage T1 cells. CD19 negative primary B cell population (P9, dark black bold dots) can be seen.

Specifically, fig. 6: normal bone marrow specimen, same as in fig. 1, was analyzed by tube b. Sequentially setting: the adhesion-free phylum (P1), the viable phylum (P2) and the P2 are gated with CD45/SSC and TdT/SSC, respectively. CD45/SSC set the blood cell gate, and TdT/SSC set the primary B cell gate (P7), resulting in a TdT + primary B cell population. Only T1 stage cells were analyzed briefly. Since the tube was CD 19-free, CD 19-cells could not be identified.

Specifically, fig. 7: relapse cases after CD19-CAR-T treatment were co-analyzed by tube A and tube B. Sequentially setting: FSC-A/H setting P1 as the door of the cell de-adhesive, resulting in single cells in P1; FSC/SSC was shown in P1 setting P2 as the viable cell gate, resulting in a single viable cell in P2; the P2 gate was gated with CD45/SSC and cytoplasmic CD79a/SSC, respectively. CD45/SSC set the blood cell gate, and lymphocyte (lym), granulocyte (Gra) and monocyte (mono) cell gate are respectively obtained; b cell gates were set using cytoplasmic CD79a/SSC (P3) to generate B cell populations. As can be seen in comparison to fig. 1: this includes the tumor cell population of CD 45-/cytoplasmic CD79a + (MRD, black), as well as the normal B cell population (grey). The tumor cell population was mostly CD19 negative cells, and a few CD19 positive cells. The malignant basis is as follows: loss of CD45, loss of CD34, reduced expression of CD38, reduced expression of CD81, enhanced expression of CD10, and co-expression of CD20 maturation stage markers with strong expression of these early markers CD38, TdT, and CD 10.

Specifically, fig. 8: bone marrow was returned after CD19-CAR-T treatment, and analyzed in the nail canal, in the same manner as in FIG. 7. Sequentially setting: the door for the detachment cell (P1), the living cell (P2) and the P2 was gated with CD45/SSC and CD19/SSC, respectively. CD45/SSC set the blood cell gate, CD19/SSC set the B cell gate (P4), including a small number of CD19+ normal B cells (grey) and a small number of CD19+ tumor cells (MRD, black bold dots), but missed a large number of CD 19-negative tumor cells (black bold dots represent only a small proportion of the cytoplasmic CD79a + MRD population P8).

Specifically, fig. 9: the same specimens as in FIG. 7, bone marrow relapsed after CD19-CAR-T treatment, were co-analyzed by tube A and tube B. Sequentially setting: the door for the detachment cell (P1), the living cell (P2) and the P2 was gated with CD45/SSC and CD10/SSC, respectively. CD45/SSC set the blood cell gate, and CD10/SSC set the immature B cell gate (P5), similar to the cytoplasmic CD79a/SSC set gate shown in FIG. 7, tumor cells (MRD, black) were mostly CD19 negative cells, and a few CD19 positive tumor cells.

Specifically, fig. 10: the same specimens as in FIG. 7, bone marrow relapsed after CD19-CAR-T treatment, were co-analyzed by tube A and tube B. Sequentially setting: the debonding cell gate (P1), the Living cell gate (P2), and the P2 gate were gated with CD45/SSC and CD34/SSClow, respectively. CD45/SSC set the blood cell gate and CD 34/sscow set the immature B cell gate (P6), although CD34 gated found only a very few tumor cells (MRD, thick black dots) and missed a large number of CD19 negative tumor cells (thick black dots in the cytoplasmic CD79a + MRD population P8, which is a minor proportion, because CD34 is mainly negative in this case. However, CD34+ tumor cells are weak clones different from the main clone and, in addition to expressing CD34, appear CD38 negative and CD81 negative.

Specifically, fig. 11: bone marrow relapse after CD19-CAR-T treatment was analyzed by tube B analysis in the same specimens as in FIG. 7. Sequentially setting: the adhesion-free phylum (P1), the viable phylum (P2) and the P2 are gated with CD45/SSC and TdT/SSC, respectively. CD45/SSC set the blood cell gate, and TdT/SSC set the immature B cell gate (P7), similar to the cytoplasmic CD79a/SSC set shown in FIG. 7, with tumor cells (MRD, black) mostly being CD19 negative cells and a few CD19 positive tumor cells.

Clinical validation was performed using the method of this example: the continental culture hospital started the CAR-T clinical trial in 2015, and completed 1000 CD19-CAR-T treatment refractory/relapsed B-ALL cases to date with 91.3% clinical remission. FCM is started to detect MRD at the same time, after more than half a year of exploration and attempt, the scheme is started to be used for detection in 2016, and 980 patients are detected so far, and the bone marrow MRD detection is carried out 4000 times. The sensitivity for detecting MRD is 10 by using the simultaneous morphological, genetic, clinical manifestation and other methods for synchronous verification^-4Coverage and specificity approach 100%. The false positive rate and the false negative rate are both close to 0. 86% of patients were negative in FCM MRD after CD19-CAR-T, with a median percentage of MRD of 1.00% (0.0031% -45.3%). 80% of patients with CD19-CAR-T have been post-engraftment with allogeneic hematopoietic stem cell transplantation and, as a result, it was found that, using the present invention to detect MRD, prognosis can be assessed at various time points: post-CD 19-CAR-T, pre-transplant MRD status correlates with transplant prognosis; post-transplant MRD status was also strongly correlated with the post-transplant prognosis (P values P < 0.0001, P =0.000, respectively).

Claims

1. An antibody composition comprising a first set of antibodies, a second set of antibodies, a third set of antibodies, and a fourth set of antibodies, wherein:

the second group of antibodies includes: anti-cytoplasmic CD79a antibody;

2. The antibody composition of claim 1, wherein each antibody is a monoclonal antibody.

3. The antibody composition of claim 1, wherein each antibody is a fluorescein-labeled antibody;

in the first group of antibodies, the fluorescein markers of the CD38 antibody, the anti-CD 10 antibody, the anti-CD 34 antibody, the anti-CD 19 antibody, the anti-CD 20 antibody and the anti-CD 45 antibody are respectively as follows in sequence: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7 and V500;

in the second group of antibodies, the fluorescein label of the anti-cytoplasmic CD79a antibody was APC;

in the third group of antibodies, the fluorescein labels of the anti-CD 81 antibody, the anti-CD 34 antibody, the anti-CD 10 antibody and the anti-CD 45 antibody are respectively: PE, PerCP-Cy5.5, PE-Cy7, V500;

in the fourth group of antibodies, the fluorescein labels of the anti-TdT antibody and the anti-cytoplasmic CD79a antibody were FITC and APC, respectively, in that order.

4. The antibody composition of claim 1, wherein:

the first group of antibodies is CD38 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 19 antibody, anti-CD 20 antibody and anti-CD 45 antibody according to 5:5:5:3:2:3 in a volume ratio;

the third group of antibodies is anti-CD 81 antibody, anti-CD 34 antibody, anti-CD 10 antibody and anti-CD 45 antibody according to 5:5:3:3 in a volume ratio;

the fourth set of antibodies was anti-TdT and anti-cytoplasmic CD79a antibodies according to 2:3 volume ratio of the mixture.

5. A kit comprising a first container, a second container, a third container and a fourth container, each container holding a first set, a second set, a third set or a fourth set of antibodies, respectively, of the antibody composition of any one of claims 1-4.

6. The kit of claim 5, further comprising: one or more of erythrocyte lysate, membrane breaking agent, buffer solution and flow tube matched with the flow cytometer.

7. Use of an antibody composition according to any one of claims 1 to 4 for the preparation of a kit for the detection of acute B-lymphocyte leukemia and/or bone marrow minimal residual disease.

8. The use according to claim 7, wherein the process for detecting acute B-lymphoblastic leukemia and/or myeloid minimal residual disease comprises the steps of:

(2) adding the first group of antibodies in the antibody composition of any one of claims 1 to 4 into a tube A and adding the third group of antibodies in the antibody composition of any one of claims 1 to 4 into a tube B of the sample obtained by the treatment of the step (1), uniformly mixing, and incubating at room temperature in a dark place;

(5) centrifuging the flow tube incubated in the step (4), removing supernatant, adding the membrane breaking agent B liquid and a second group of antibodies in the antibody composition of any one of claims 1 to 4 into the tube A, adding the membrane breaking agent B liquid and a fourth group of antibodies in the antibody composition of any one of claims 1 to 4 into the tube B, and incubating at room temperature in a dark place;

9. Device for the detection of acute B-lymphocyte leukemia and/or myeloid minimal residual disease, comprising a detection unit and an analysis unit, wherein:

the detection unit comprises a reagent material for detecting a sample from an individual to be detected by flow cytometry, and is used for obtaining a detection result of the sample; the reagent material comprises an antibody composition of any one of claims 1-4;

10. The device according to claim 9, wherein the device is used for detecting acute B-lymphocyte leukemia and/or bone marrow minimal residual disease, wherein:

the process of detecting a sample from an individual to be tested by flow cytometry comprises:

preparing a flow-cytometric sample after treating a test sample with the antibody composition of any one of claims 1 to 4;

performing flow cytometry on the machine for detection;

wherein, the second tube is provided with a door according to the following mode when the flow cytometry is used for detecting: sequentially setting a detachment cytogate P1 and a living cytogate P2, then setting each blood cytogate by using a CD45/SSC antibody in a P2 gate, and setting the gates by using a multi-mark combination of cytoplasmic CD79a/SSC, CD10/SSC, CD34/SSC and TdT/SSC antibodies;

when the analysis unit analyzes the detection result of the detection unit, the displayed B cell development pattern is compared with the normal cells in the multi-mark combination setting door to find out the tumor cells.