CN118130798A - Antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cell and application thereof - Google Patents
Antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cell and application thereof Download PDFInfo
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Abstract
The invention provides an antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cells and application thereof, belonging to the technical field of antibody medicine. The primary antibody combination can realize single-tube detection of 20 antigens, can realize comprehensive detection of the phenotype of T cells, overcomes the defects of the conventional multi-tube detection, does not need to repeatedly use a gate-equipped antibody, can comprehensively analyze the interrelationship of 20 antigen expressions, greatly improves the analysis capability and the detection accuracy, reduces the demand and the operation quantity of specimens, and is beneficial to saving cells, saving cost, relieving labor and simplifying operation. The antibody combination can be used for T-ALL/LBL immunophenotype recognition, stage separation, ETP-ALL identification, trace residual disease marker screening, treatment target screening and the like.
Description
Technical Field
The invention belongs to the technical field of antibody medicines, and particularly relates to an antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cells and application thereof.
Background
Acute T-lymphoblastic leukemia/lymphoma (T-cell acute lymphoblastic leukemia/lymphoma, T-ALL/LBL) is a heterogeneous disease in which immature T-lymphocytes proliferate in bone marrow, peripheral blood, lymph, thymus and other extranodal tissues, accounting for 15% of pediatric Acute Lymphoblastic Leukemia (ALL), and 25% of adult ALL. At present, the treatment of T-ALL/LBL still has the challenges, the cure rate is lower, the recurrence rate is higher, and even if the allogeneic hematopoietic stem cell transplantation is carried out, the overall survival rate is still to be improved; the emerging immunotherapy has limited application of immunotherapy because the immune markers of normal cells and tumor cells are similar, and the cells of the immunotherapy are easy to kill while killing the tumor.
For definitive diagnosis and monitoring of immune residues after T-ALL/LBL treatment by screening appropriate antigen markers, early warning and countermeasures are taken in advance, immunophenotyping is required. While the combination of antibodies for immunophenotyping needs to meet the following requirements: (1) The method can identify the naive T cells, and the T cells originate from thymus, so that the marrow, peripheral blood or tissues except the thymus belong to abnormality as long as the naive T cells carrying the naive antigen markers are found; mediastinum penetrates tissue and normal and neoplastic thymocytes should also be distinguishable by combination. (2) Different maturation stages can be distinguished according to the immune markers and then staged, and T-ALL is classified into 4 stages according to WHO classification: T-I (CD7+pro-T), T-II (pre-T), T-III (CD1a+cortical T), T-IV (CD 4 or CD8 medullary T). (3) Being able to identify acute early precursor T-lymphoblastic leukemia (earlyT-cell precursor acute lymphoblastic leukemia, ETP-ALL), most literature reports show poorer prognosis of ETP-ALL, early identification facilitating early selection of appropriate treatments. (4) Screening for residual leukemia (minimal residual disease, MRD) after treatment monitors more sensitive immune markers. T-ALL is rarely associated with specific fusion gene abnormalities, so that determination of MRD status by Flow Cytometry (FCM) is particularly important for predicting recurrence, therapeutic intervention, when MRD tracking is performed. In theory, it is easy to detect T-ALL/LBL MRD after treatment using FCM because T cells originate from the thymus, only mature T cells in extrathymus tissue such as bone marrow, and the naive T cells at any stage are considered positive for MRD; in fact, the expression ratio of the naive antigen is low and the intensity is weak at the initial diagnosis of some tumor T cells, and the naive markers disappear or weaken to different degrees after treatments such as hormone and the like, so that the recognition of the naive T cells is difficult; on the other hand, some patients clinically have negative MRD tests, but patients relapse quickly, suggesting that it is necessary to find more sensitive antigen markers for MRD tests. (5) Screening can be used for targeting therapeutic targets and can distinguish normal T cells as much as possible. The inclusion of as many antigenic markers as possible at the time of initial typing helps to achieve the above objective.
Gating of naive T cells after treatment is also a challenge, usually using weak expression of CD45 (CD 45 dim), CD7 and/or cytoplasmic CD3 (CD 3) in combination with low SSC (sscow) to gate T cells, CD3 requires membrane disruption and perforation, and the markers are complex, some patients have stronger CD45 expression and stronger expression after treatment, resulting in indistinguishable naive from mature T cells. While some patients only partially express CD7, or have CD7 deleted following CAR-T treatment, these conditions can result in difficult precise identification of naive T cells. Therefore, other gating antigens need to be included in the antibody combination, and a simple and feasible T cell gating method with a wide application range needs to be studied.
Currently, several tens of antibodies need to be detected for the purpose of the above-mentioned several tests clinically. At present, after the conventional 10-color (10C) T-ALL/LBL combination is detected by the primary screen 1 pipe 10-color combination, the T-ALL/LBL is further analyzed to comprise 3 pipes 10-color combinations, and 30 antibodies are totally contained. Each tube included 3 repetitive scaffold antibodies: CD45, CD7 and CD5, only 21, resulting in more total antibodies, are ultimately detected efficiently, resulting in high costs. Moreover, more tube assays have difficulty in accurately analyzing the relationship between the detected antibodies in different tubes, affecting the judgment of cell line identity, differentiation and development pattern, and benign and malignant.
Therefore, there is an urgent need to design an effective multicolor antibody combination for detecting an acute T-lymphocyte leukemia/lymphoma immunophenotype, so as to increase the number of antibodies while reducing the number of detection tubes, improve the detection efficiency, avoid the problem that the conventional flow type requires repeated use of the gating antibody, and reduce the economic burden of patients; meanwhile, the problem that the antigen development mode of the conventional flow method is limited by different tubes is solved, the fineness of analysis is improved, the identification of tumor cells is increased, and the clinical detection level is improved.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an antibody combination for detecting an immunophenotype of acute T-lymphocyte leukemia/lymphoma by flow cytometry, which has a large number of antibodies and a small number of detection tubes, and to provide an application thereof, and which can improve the detection efficiency.
The invention provides an antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cells, which comprises a first antibody combination; the first antibody combination comprises the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody, and anti-CD 25 antibody; the primary antibody combination was used in 1 flow tube at the time of detection.
Preferably, the antibody is a fluorescein-labeled antibody; in the first antibody combination, the fluorescent labels of the anti-CD 99 antibody, the anti-CD 16 antibody, the anti-CD 45RA antibody, the anti-CD 5 antibody, the anti-CD 44 antibody, the anti-CD 57 antibody, the anti-CD 28 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 65 antibody, the anti-CD 4 antibody, the anti-HLA-DR antibody, the anti-CD 2 antibody, the anti-CD 1a antibody, the anti-CD 13 antibody, the anti-CD 3 antibody, the anti-CD 123 antibody, the anti-CD 45RO antibody and the anti-CD 25 antibody are ：BV421、eFluor V450、eFluor V547、BV570、BV605、BV650、BV711、BV785、FITC、eFluor B548、PE、PE/Dazzle594、PE/Cy5、PerCP/eFluor710、PE/Cy7、APC、eFluor R668、eFluor R720、APC/Cy7 and APC/Fire810, respectively, in order.
Preferably, the antibody combination further comprises a second antibody combination; the second antibody combination comprises an anti-cell membrane antigen antibody and an anti-cell intracellular antigen antibody; the anti-cell membrane antigen antibody consists of the following antibodies: anti-CD 38, anti-CD 3, anti-CD 10, anti-CD 33, anti-CD 5, anti-CD 19, anti-CD 45, anti-CD 7, anti-CD 117, anti-CD 34, anti-CD 56, and anti-TRBC 1 antibodies; the anti-intracellular antigen antibody consists of the following antibodies: anti-cd 79a, anti-cLambda, anti-cKappa, anti-cd 22, anti-cd 3, anti-cMPO antibodies, and anti-nTdT antibodies; the second antibody combination was used in 1 flow tube at the time of detection.
Preferably, the combination of antibodies is a first combination of antibodies; the first antibody combination consists of the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody and anti-CD 25 antibody.
Preferably, the antibodies in the antibody combination are monoclonal antibodies.
The invention also provides a kit for detecting the immunophenotype of the acute T lymphocyte leukemia/lymphoma by using the flow cell, which comprises the antibody combination according to the scheme; when the combination of antibodies includes a first combination of antibodies and a second combination of antibodies, the first combination of antibodies and the second combination of antibodies are packaged separately.
Preferably, the kit further comprises: erythrocyte lysate and buffer PBS.
The invention also provides a system for detecting the immunophenotype of the acute T lymphocyte leukemia/lymphoma by using the flow cell, which comprises a detection part and an analysis part; the detection part comprises the antibody combination or the kit of the scheme, and is used for detecting the antigen expression level of an individual to be detected through flow cytometry; and an analysis section for analyzing the detection result of the detection section.
Preferably, the antibody combination or the kit according to the above scheme is used for preparing a flow cytometry sample after treating a sample to be tested; performing flow cell on-machine detection; wherein the flow cell is provided with a gate during the on-machine detection according to the following mode: setting se:Sub>A disjunctor cell gate A by using the arese:Sub>A and the height of FSC, continuing to disbond by using SSC-A/SSC-B, setting se:Sub>A living cell gate R1 by using FSC-A/SSC-A, and removing fragments and dead cells; the interior of the R1 gate is conventionally provided with a hemocyte gate by using a CD45/SSC scatter diagram; the haemocyte gate includes lymphocytic, granulocytic, monocytic and nucleated erythrocytic gates.
The invention also provides an application of the antibody combination or the kit or the system in preparing a product related to acute T lymphocyte leukemia/lymphoma detection; the acute T lymphocyte leukemia/lymphoma detection comprises one or more aspects from (1) to (7): (1) Screening acute T lymphocyte leukemia/lymphoma treatment targets and/or abnormal phenotypes; (2) acute T-lymphocyte leukemia/lymphoma diagnosis; (3) naive T cell detection; (4) acute T lymphocyte leukemia stage; (5) screening for acute early precursor T-lymphoblastic leukemia; (6) Post-acute T-lymphoblastic leukemia/lymphoma treatment marker screening and/or residual leukemia cell monitoring; (7) lymphocyte grouping; the lymphocytes include one or more of mature T cells, B cells and NK cells.
The invention provides an antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cells, which comprises a first antibody combination; the first antibody combination includes 20 envelope antibodies. According to the traditional flow cytometer 8-10 color antibody combination, at least 3 tubes are needed to be detected in order to obtain the expression results of 20 antigens. The primary antibody combination can realize single-tube detection of 20 antigens, can realize comprehensive detection of the phenotype of T cells, overcomes the defects of the conventional multi-tube detection, does not need to repeatedly use a gate-equipped antibody, can comprehensively analyze the interrelationship of 20 antigen expressions, greatly improves the analysis capability and the detection accuracy, reduces the demand and the operation quantity of specimens, and is beneficial to saving cells, saving cost, relieving labor and simplifying operation. The first antibody combination of the invention comprises all stage antigens, and can definitely differentiate development stages according to the expression condition of the antigens for routine stage; in addition, the cells can be grouped according to the expression of CD2 and CD5, and the change process from early stage to late stage is divided into 3 stages according to the development mode of CD45RA/CD45RO, the first stage: CD2-/part+CD5-, CD2-/part+CD5+, second stage: cd2+cd5-, third stage: cd2+cd5+. Furthermore, the one-tube antibody combination of the invention comprises both CD99 and CD44, the expression of which is correlated, and the CD99/CD44 gating can be adopted to well distinguish the naive and mature T cells, and can be used as a sensitive antigen marker for MRD monitoring. The invention can flexibly gate T cells by adopting various antigens, and can gate naive T cells according to CD99/CD44, CD7/CD99 and the like besides the common CD7/SSClow and CD45 dim/SSClow. In addition, the antibody combination of the invention can effectively distinguish T, B, NK lymphocyte subsets according to the markers, distinguish mature and naive T cells according to CD3/CD44, and detect abnormal differentiation of mature T cell phenotype while analyzing the phenotype of the naive T cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a general gating approach for naive T cells;
FIG. 2 shows the expression of cell membrane antigens and their correlation in a two-dimensional dot map after gating naive T cells;
FIG. 3 shows that gating can be performed according to the scatter plot of CD3/CD44, CD99/CD44 or CD45RA/CD45RO, etc. according to the antigen expression characteristics of naive cells when gating cannot be performed according to CD 7;
FIG. 4 shows a strategy of gating naive cells together with lymphocytes and then gating naive T cells according to their antigen expression characteristics when the naive cells are not well demarcated from lymphocytes;
FIG. 5 shows gating strategy for differentiating naive T cells, mature T, B, NK cells, and abnormal cell subsets using the antibody combinations of the invention;
FIG. 6 shows 1 patient with clinical suspicion of acute leukemia, wherein A shows the selection of naive T cells using the antibody combination of the' 202111067074.3 patent; b is the combined detection result of the antibody, and the antigen mark suitable for MRD tracking and monitoring is screened out;
FIG. 7 shows the average fluorescence intensity results of 56T-ALL/LBL patients expressing CD 44.
Detailed Description
The invention provides an antibody combination for detecting acute T lymphocyte leukemia/lymphoma immunophenotype by using flow cells, which comprises a first antibody combination; the first antibody combination comprises the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody, and anti-CD 25 antibody; the primary antibody combination was used in 1 flow tube at the time of detection.
The primary antibody combination is a single-tube antibody combination, comprises 20 antibodies, mainly analyzes the immunophenotype of T cells through a single-tube detection technology, judges the T cells as the naive T cells according to the expression of early antigen markers, carries out stage according to different antigen expression results, judges whether the T P-ALL is ETP-ALL according to the immune markers, screens sensitive naive antigen markers, determines leukemia-related immunophenotype (LAIP), provides reference for MRD monitoring, can screen treatment targets, and provides basis for application of targeted drugs.
In the present invention, the first antibody combination preferably consists of the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody and anti-CD 25 antibody. In the present invention, the primary antibody combination includes only 20 envelope antibodies, which can simplify the operation steps. The primary diagnosis patients suspected of blood system diseases can be further classified and screened by comprehensively detecting the primary screening combination to determine that the specimens of the naive T cells exist.
In the present invention, the antibody is preferably a fluorescein-labeled antibody; in the first antibody combination, the fluorescent labels of the anti-CD 99 antibody, the anti-CD 16 antibody, the anti-CD 45RA antibody, the anti-CD 5 antibody, the anti-CD 44 antibody, the anti-CD 57 antibody, the anti-CD 28 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 65 antibody, the anti-CD 4 antibody, the anti-HLA-DR antibody, the anti-CD 2 antibody, the anti-CD 1a antibody, the anti-CD 13 antibody, the anti-CD 3 antibody, the anti-CD 123 antibody, the anti-CD 45RO antibody and the anti-CD 25 antibody are preferably ：BV421、eFluor V450、eFluor V547、BV570、BV605、BV650、BV711、BV785、FITC、eFluor B548、PE、PE/Dazzle594、PE/Cy5、PerCP/eFluor710、PE/Cy7、APC、eFluor R668、eFluor R720、APC/Cy7 and APC/Fire810, respectively, in this order.
In the present invention, the antibody combination preferably further comprises a second antibody combination. The second antibody combination of the invention is a reagent composition for flow cytometry leukemia/lymphoma typing disclosed in patent 202111067074.3, and is used for screening ALL primary patients with blood tumor, and can be used for screening T-ALL/LBL patients; the second antibody combination preferably comprises an anti-cell membrane antigen antibody and an anti-cell intracellular antigen antibody; the anti-cell membrane antigen antibody preferably consists of the following antibodies: anti-CD 38, anti-CD 3, anti-CD 10, anti-CD 33, anti-CD 5, anti-CD 19, anti-CD 45, anti-CD 7, anti-CD 117, anti-CD 34, anti-CD 56, and anti-TRBC 1 antibodies; the anti-intracellular antigen antibody preferably consists of the following antibodies: anti-cd 79a, anti-cLambda, anti-cKappa, anti-cd 22, anti-cd 3, anti-cMPO antibodies, and anti-nTdT antibodies; the second antibody combination was used in 1 flow tube at the time of detection.
The invention firstly performs primary screening of blood tumor, and when the naive T cells are screened, the combination of the invention is further adopted to perform secondary detection, and abnormal naive T cells are definitely detected to complete comprehensive immunophenotyping detection of patients. The specific detection steps are as follows: and a first step of detection: primary screening of hematological tumors using a tube of 19 antibody combinations of the second antibody combination, dividing hematological tumors into major classes AML, T-ALL/LBL, B-ALL, MPAL, B-NHL, T-NHL, NK-NHL, PCN, and chronic myeloid disease 9; samples in which cCD3+CD3-/+CD34+/-TdT+/-CD5+/-CD7+/-CD45dim+ did not express other series of markers were initially judged as T-ALL/LBL; and step two, detection: the method combines 20 antibodies in the first antibody combination to carry out accurate diagnosis of T-ALL/LBL, can carry out comprehensive immunophenotype analysis on T-ALL/LBL patients, utilizes 2-3 antibodies to gate T cells, simultaneously analyzes the expression of 20 antigens, carries out definite diagnosis, carries out stage division according to differentiation development stage, identifies whether ETP-ALL is the more sensitive MRD mark, screens the more sensitive MRD mark, saves antibodies and specimens, simultaneously can comprehensively analyze the interrelation of the expression of 20 antigens, and more clearly determines the distribution and development mode of the T cells.
In the present invention, the antibody combination is preferably a first antibody combination.
The primary antibody combination of the present invention can be used for immunophenotyping and marker analysis of ALL T-ALL/LBL patients, helps to clarify disease diagnosis, stage, screen for ETP-ALL types with poor prognosis, screen for more sensitive LAIP for MRD detection. The antibody combination reduces repeated application of the gated antibody, increases the use quantity of effective antibodies, and can observe the development modes and distribution of different antigen combinations at the same time.
In the present invention, the antibodies in the antibody combination are preferably monoclonal antibodies.
In the present invention, each antibody component of the antibody combination is commercially available. Each antibody should meet the requirements of the relevant industry standards.
The invention also provides a kit for detecting the immunophenotype of the acute T lymphocyte leukemia/lymphoma by using the flow cell, which comprises the antibody combination according to the scheme; when the combination of antibodies includes a first combination of antibodies and a second combination of antibodies, the first combination of antibodies and the second combination of antibodies are packaged separately.
In the present invention, the kit preferably further comprises: the red blood cell lysate and buffer PBS, more preferably, further comprises fluorescein compatible with the antibodies in the antibody combination.
In the present invention, the kit preferably comprises a first container for containing the first antibody combination of the present invention; the kit preferably further comprises a second container for housing a second antibody combination of the invention.
In the present invention, reagents and consumables in the kit are commercially available. Each reagent material may be contained in a different container.
The invention also provides a system for detecting the immunophenotype of the acute T lymphocyte leukemia/lymphoma by using the flow cell, which comprises a detection part and an analysis part; the detection part comprises the antibody combination or the kit of the scheme, and is used for detecting the antigen expression level of an individual to be detected through flow cytometry; and an analysis section for analyzing the detection result of the detection section.
In the invention, the antibody combination or the kit is used for preparing a flow cytometry sample after treating a sample to be detected; performing flow cell on-machine detection; wherein the flow cell is provided with a gate during the on-machine detection according to the following mode: setting se:Sub>A disjunctor cell gate A by using the arese:Sub>A (A) and the height (H) of the FSC, continuing to disbond by using SSC-A/SSC-B, setting se:Sub>A cell gate R1 by using the FSC-A/SSC-A, and removing fragments and dead cells; the interior of the R1 gate is conventionally provided with a hemocyte gate by using a CD45/SSC scatter diagram; the haemocyte gate includes lymphocytic, granulocytic, monocytic and nucleated erythrocytic gates.
After displaying R1 portal cells, the invention establishes a CD45/SSC diagram, gates lymphocytes, monocytes, granulocytes and nucleated erythrocytes according to different distributions of CD45 and SSC, namely lymphocyte portal R2, monocyte portal R5, granulocyte portal R4 and nucleated erythrocytes portal R6, and observes whether the proportion of each group of cells is normal; wherein, the lymphocyte: CD45 highest/SSC lowest; monocytes: CD45 is lower than lymphocytes/SSC is higher than lymphocytes than granulocytes; granulocytes: CD45 is lower than monocytes/SSC maximum; nucleated red blood cells: CD45 negative/SSC low is the same as lymphocytes. In normal bone marrow, each population of cells has a normal range of proportions: 20-40% of lymphocytes, 2-8% of monocytes, 40-60% of granulocytes and 2-15% of nucleated erythrocytes.
In the implementation process of the invention, the system is mainly used for identifying the naive T cells, when the detection result of the detection part is analyzed, the R1 gate is firstly used for setting the gate R3 for the cells in the naive cell holes according to the CD45/SSC, then the naive T cells are further purified according to the CD7/SSC, the CD7/CD99, the CD7/CD44 or the CD99/CD44, and the like, and whether the expression of other membrane antigens is abnormal, the positive expression rate and the expression intensity are analyzed.
In the present invention, the gate is set for cells in the immature cell holes: CD45 is lower than lymphocytes and higher than nucleated erythrocytes, SSC is similar to lymphocytes; in the R3 gate, the expression characteristics of a plurality of antigens and a plurality of methods can be utilized to accurately gate and quantify the naive T cells.
After gating the cells in the wells of naive cells with R3 according to CD45/SSC, the present invention preferably comprises further gating CD7+ cells according to CD7/SSC, creating a series of two-dimensional dot patterns of two antigens, and observing the antigen expression in these patterns.
When CD7 is expressed partially positive at initial diagnosis, attenuated after treatment or CD7 deleted after CAR-T treatment, the present invention preferably gates naive T cells according to CD3/CD44, CD99/CD44 or CD45RA/CD45RO etc.
When the expression of CD45 in the naive cells is strong and indistinguishable from lymphocytes, the lymphocytes are included when the first step is required when the cells are not in the naive cell wells in the CD45/SSC map, and then the cells are further clustered according to the antigen expression characteristics, such as strong expression of CD7 and CD99, weak expression of CD44, positive expression of CD45RO, negative or weak expression of CD2, and the like.
When the system of the present invention is used for analyzing naive T cells, if the cell and lymphocyte population in the CD45/SSC naive cell hole are not clearly clustered, mature and naive T cells cannot be distinguished only according to the CD7/SSC, and the naive T cells need to be determined after being clustered according to multiple gates such as the CD7/CD99, the CD99/CD44, the CD3/CD44, and the like.
In the present invention, CD7 may be deleted and CD99/CD44 may be used as an alternative gating marker to identify naive T cells in refractory T-ALL/LBL patients treated with CAR-T.
The invention also provides an application of the antibody combination or the kit or the system in preparing a product related to acute T lymphocyte leukemia/lymphoma detection; the acute T lymphocyte leukemia/lymphoma detection comprises one or more aspects from (1) to (7): (1) Screening acute T lymphocyte leukemia/lymphoma treatment targets and/or abnormal phenotypes; (2) acute T-lymphocyte leukemia/lymphoma diagnosis; (3) naive T cell detection; (4) acute T lymphocyte leukemia stage; (5) screening for acute early precursor T-lymphoblastic leukemia; (6) Post-acute T-lymphoblastic leukemia/lymphoma treatment marker screening and/or residual leukemia cell monitoring; (7) lymphocyte grouping; the lymphocytes include one or more of mature T cells, B cells and NK cells.
In the present invention, the naive T cell assay preferably comprises a residual naive T cell assay following acute T lymphocyte leukemia/lymphoma treatment.
The invention can evaluate the phenotype of mature T cells through the distribution of the CD7/CD5 of the T cells, and can detect other abnormal cell subsets.
The first antibody combination is matched with the second antibody combination, so that the abnormal phenotype of a patient suspected to exist in the naive T cells can be further clarified, the marker which is obviously different from the expression of the mature T cells and has high incidence rate is suitable for MRD tracking detection after treatment, for example, the naive T cells are found by primary screening combination of the patient suspected to be in acute leukemia, the proportion of the naive T cells in the nucleated cells can be calculated and the abnormal phenotype can be clarified through the first antibody combination, reliable support is provided for clinical diagnosis, a treatment target is provided for subsequent treatment, and sensitive antigen markers for MRD detection are screened.
The antibody combination can be used for T-ALL/LBL immunophenotype recognition, stage separation, ETP-ALL identification, trace residual disease marker screening, treatment target screening and the like.
For further explanation of the present invention, an antibody combination for flow cytometric detection of acute T-lymphoblastic leukemia/lymphoma immunophenotype and its use are provided in the following detailed description with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the invention.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The experimental methods used in the examples below are conventional methods unless otherwise specified. All materials, reagents, etc. in the examples described below are commercially available unless otherwise specified.
According to the embodiment of the invention, the flow cytometry is adopted to carry out immunophenotype analysis on samples such as marrow, hydrothorax and ascites, peripheral blood and the like of clinical patients, and the combination is applied to carry out comprehensive phenotypic detection on samples of the primarily screened suspicious naive T cells; the 19 antibody combinations used for the primary screening are described in the 202111067074.3 patent application. For patients with defined T-ALL/LBL, detection of naive T cells after treatment can also be flexibly configured using the combination of the present invention to assess the number and phenotype of naive T cells.
Example 1 formulation of reagents
The antibody combination provided by the invention comprises 20 antibodies and compatible fluorescein, and comprises the following components: anti-CD 99 BV421, anti-CD 16 eFluor V450, anti-CD 45 eFluor V547, anti-CD 45RA BV570, anti-CD 5 BV605, anti-CD 44 BV650, anti-CD 57 BV711, anti-CD 28 BV785, anti-CD 7 FITC, anti-CD 8 eFluor B548, anti-CD 65 PE, anti-CD 4 PE/Dazzle594 54, anti-HLA-DR PE/Cy5, anti-CD 2 PerCP/eFluor710, anti-CD 1a PE/Cy7, anti-CD 13 APC, anti-CD 3 eFluor R668, anti-CD 123 eFluor R720, anti-CD 45RO APC/Cy7, and anti-CD 25 APC/Fire810.
Configuration of antibody combinations: the antibodies were mixed in 1 container for the phenotypic labelling of naive T cells according to the amount determined by the pre-experiment.
The antibodies described above are all commercially available, and the antibodies of the examples of the present invention are purchased from BD company, biolegend and Cytek company.
The antibodies are combined together to prepare a detection kit for screening abnormal phenotype and treatment targets of the naive T cells. The kit also comprises a red blood cell lysate and a buffer PBS, wherein the red blood cell lysate can be self-prepared or can be purchased commercially.
Example 2 flow cytometer detection analysis of immunophenotype of naive T cells
Experimental main material and instrument
Materials: 10 XPBS buffer, flow cytometer specific erythrocyte lysate (FACS hemolysin, BD Co.).
Instrument: cytekNL-3000 model full spectrum flow cytometer equipped with 405nm,488nm, 635nm three lasers, 38 fluorescence detectors. A desk type low-speed centrifuge and a vortex mixer.
Method of
1. Sample collection:
1-3 mL of the obtained liquid specimen (bone marrow, peripheral blood and the like) is immediately placed in a heparin anticoagulation tube and is rapidly reversed for several times to prevent various cells such as hydrothorax and ascites, lavage liquid and the like from coagulating the specimen, and the specimen is sent to a laboratory as soon as possible after being collected and is placed in a 4 ℃ refrigeration storage. The flow cytometer detection must be completed within 48 hours, operating in accordance with instructions.
2. Sample preparation process:
Cell count: taking 10 μl of liquid sample, adding 150 μl of PBS, mixing, counting each microliter of cells by utilizing Michael FCM, adjusting the cell concentration to 2× 6/100 μl according to the detection result, and respectively taking 50 μl and 100 μl of cells into the flow tube by the patient after initial diagnosis and treatment.
3. Cell membrane surface antigen staining:
Each tube is added with the antibody premix solution for cell membrane marking, which is marked by the corresponding fluorescein, and the marrow specimen, which are used for cell membrane marking, respectively, and are fully and evenly mixed, and incubated for 15 min at room temperature and in a dark place.
4. Hemolysis: and adding 2ml of 1 XFACS hemolysin, uniformly mixing by low-speed vortex, and standing at room temperature in a dark place for 8-10 min. Centrifuge wash at 300g for 5min and discard supernatant.
5. Washing: 1ml of 1 XPBS buffer containing 0.1% NaN3 and 1% -2% BSA was added, and the supernatant was discarded after centrifugation at 300g for 5 min. 200-300 mu l of PBS (phosphate buffer solution) is added to the suspension cells for on-line detection.
6. And (3) detecting: determining an optimum voltage and compensation: setting voltage according to the conventional operation method of the spectrum flow cytometer, preparing a single-dye sample by referring to fluorescent color matching of the kit, and setting the single-dye sample by using the single-dye sample.
Instrument setup, calibration and quality control: cytekNL-3000 starting the preheating machine for more than 20min, washing with deionized water, detecting quality control products, and ensuring that each detection value is within a control range. And respectively establishing antibody combination templates, taking and loading samples each time, and collecting data.
According to the set instrument conditions, the initial patient acquires 10 ten thousand cells per tube, and the patient acquires 100 ten thousand cells per tube after treatment. If the detection can not be carried out on the machine in time, 0.5ml of 1% paraformaldehyde is added, and the mixture is uniformly mixed and then is stored in a refrigerator at 4 ℃ for 24 hours to finish the detection.
7. Data analysis: analysis of data using Kaluza software
Firstly, setting se:Sub>A double-body cell gate A by using FSC-A/FSC-H, continuing setting se:Sub>A gate B by SSC-A/SSC-B to remove adhesion, removing fragments and dead cells by using an FSC-A/SSC-A diagram, and setting se:Sub>A gate R1 for se:Sub>A single living cell.
R1 portal cells are shown, a CD45/SSC map is established, and lymphocytes, monocytes, granulocytes and nucleated erythrocytes are gated according to the difference of the distribution of CD45 and SSC. Lymphocytes (R2): CD45 highest/SSC lowest, monocytes (R5): CD45 is lower than lymphocytes/SSC is higher than lymphocytes than granulocytes, granulocytes (R4): CD45 is lower than monocytes/SSC maximum, nucleated red blood cells (R6): CD45 negative/SSC low is the same as lymphocytes. In normal bone marrow, each population of cells has a normal range of proportions: 20-40% of lymphocytes, 2-8% of monocytes, 40-60% of granulocytes and 2-15% of nucleated erythrocytes. The cell proportion of each group was observed to be normal.
Naive T cell gating analysis: in the R1 gate, the cells in the immature cell well are gated (R3): CD45 is lower than lymphocytes and higher than nucleated erythrocytes, SSC is similar to lymphocytes; in the R3 gate, the expression characteristics of a plurality of antigens and a plurality of methods can be utilized to accurately gate and quantify the naive T cells. Table 1 shows the expression of different antigens in normal mature T cells, naive T cells and their major role in combination.
TABLE 1 expression of antigens on Normal mature T cells, naive T cells and their effects in the antibody combinations of the invention
(1) The general door setting method comprises the following steps: the cells in the wells of naive cells were initially gated with R3 using CD45/SSC, and further gated with CD7+ cells according to CD7/SSC (FIG. 1). A series of two-dimensional dot patterns of antigens were created and the expression of the antigens in these patterns was observed (FIG. 2).
(2) When CD7 is partially positive for expression at initial diagnosis, attenuated after treatment, or CD7 is deleted after CAR-T treatment, gating on cd7+ cells according to CD7/SSC underestimates naive T cell numbers, and this combination does not contain cytoplasmic CD3 (CD 3), thus making gating problematic. Naive T cells can be gated according to the characteristics of strong CD99 expression (CD99st+), weak CD44 expression (CD 44 dim+), negative mCD3 expression, and uniform single positive expression of CD45RA or CD45RO, etc., as described for CD3/CD44, CD99/CD44, or CD45RA/CD45RO (FIG. 3).
(3) When the naive cells were strongly expressed in CD45 and indistinguishable from lymphocytes, the lymphocytes were included when the first step was required when not in the naive cell well in the CD45/SSC map, and further clustered according to antigen expression characteristics such as strong expression of CD7 and CD99, weak expression of CD44, positive expression of CD45RO, negative or weak expression of CD2, etc. (FIG. 4).
(4) In addition to recognizing naive T cells, the combination can also utilize antigen expression characteristics to group mature T cells (CD3+CD44st+), B cells (CD 3-CD7-CD 16-), NK cells (CD7+CD16+), and by distribution of T cells CD7/CD5, the phenotype of mature T cells can be assessed; other abnormal cell subsets, such as X cells CD99 which are slightly weaker and CD44 which are slightly stronger, CD7st+, but CD16-CD3-, were found, but other antigens expressed similarly to CD99st+CD44dim+naive T cells, such as CD5-CD2 part+CD45RO+CD45RA-CD 4-CD8-, should also be a population of naive T cells.
(5) In combination with the primary screening, the abnormal phenotype was further defined for patients suspected of having naive T cells, and markers that were clearly distinguished from mature T cell expression and had high incidence were suitable for post-treatment MRD follow-up detection (Table 1). The combination of primary screening for naive T cells in patients with clinical suspicion of acute leukemia as shown in FIG. 6 allows calculation of the proportion of naive T cells in nucleated cells and definition of abnormal phenotypes, provides reliable support for clinical diagnosis, provides therapeutic targets for subsequent treatment, and screens for sensitive antigenic markers for MRD detection by the combination of antibodies of the invention.
Results:
1. The patient is constituted by:
A total of 58T-ALL/LBL patient bone marrow samples were tested using the antibody combination of the present invention, 39 men and 19 women, with a median age of 30 (1-69). According to detection, the ratio of the naive T cells of 43 patients is higher than 20%, the ratio of the naive T cells of 13 patients is between 5% and 20%, and the ratio of the naive T cells of 2 patients is lower than 5%. According to the stage, CD7+CD2-pro-T25, CD2+CD1a-pre-T20, CD1a+cortex T10, medulla T3. According to the ETP-ALL judgment standard, 19 cases are ALL obtained.
2. Comparison of the antibody combinations of the invention with 10-color (10C) conventional flow cytometer detection results:
The ratio of the naive T cells after initial diagnosis and treatment of the T-ALL/LBL patient is detected by adopting the antibody combination of the invention, the median ratio of the naive T cells is 63.31 percent (0.46-95.17 percent), and the median ratio of the 10C combination detection is 64.43 percent (2.71-95.82 percent), which are not obviously different. The stage is consistent with the judgment of ETP-ALL by using two methods.
The combined detection result of the antibody shows (Table 2) that almost ALL the naive T cells of the T-ALL/LBL patient highly express CD45, CD7 and CD99, the positive expression proportion is higher, and the positive expression proportion is more than 90% between 80% and 100%; the detection of the antibody combination of the invention shows that although the positive expression proportion of CD7 has no statistical difference, 52 cases with the positive expression proportion of the antibody combination of the invention between 80% and 100% are slightly higher than 46 cases with the detection of 10C, and the detection result of 10C of 6 patients with phase difference shows that the partial expression of CD7, the positive expression proportion is 40% to 80%, and the possibility that the fluorescein FITC marked by CD7 in the antibody combination of the invention is slightly stronger than the PerCP-Cy5.5 in the 10C combination. The positive expression rates of CD5, CD2 and HLA-DR are 67.24%, 56.90% and 41.38% respectively, which are slightly higher than that of 10C combination, but have no statistical difference, and the positive expression proportion distribution of the two antigens is similar. Both methods showed that the phenotype of the vast majority of naive T cells was CD45RA+CD45RO-or CD45RA-CD45RO+, and the combined detection of antibodies of the invention showed that 53 of 58 patients were single positive for CD45RA or CD45RO, 4 were CD45RA-CD45RO-, and 1 was CD45RA+CD45RO+. The positive expression rate of CD4, CD8, CD3 and CD1a is about 20%, the positive expression rate of the marrow antigen CD13 is lower, the positive expression rate of CD123, CD16 and CD65 is lower by about 15%, and the detection results of the two methods are indistinguishable (P > 0.05). In addition, there were 3 antigens CD28, CD57 and CD25 in the antibody combinations of the invention, which were not included in 10C, with positive expression rates of 63.79%,1.75% and 5.17%, respectively. The antibody combinations and 10C combinations of the present invention also include the antigen CD44, which is specifically analyzed below because CD44 is positively expressed.
Table 2 comparison of expression of common antigen for the antibody combinations of the invention and 10C combinations (n=58)
Remarks: * Representing the positive proportion of antigen expression in naive cells
Relationship between differentiation and development patterns of CD45ra and CD45RO and CD2 and CD5 expression:
CD45RA is a marker for naive T cells, while CD45RO is a marker for memory T cells. The expression of CD45RA and CD45RO in ALL-T is rarely reported. Our results show that cells with CD2 negative (-) or partial expression (part+) are predominantly expressed (73% -80%) with CD45RA positive either CD5+ (Table 3), 13% -15% expressing CD45RO; while CD2+CD5-is still predominantly CD45RA, CD2+CD5+ is predominantly CD45 RO. CD 2-naive T cells are considered to be the earliest naive T stage, and the phenotype after T cell maturation is cd2+cd5+. Our results indicate that during the CD2-/part+ phase, the expression of CD5 does not reflect the differentiation of the cells. CD2+CD5-unlike CD2+CD5+ appears to be an intermediate stage. Based on these several markers, the cells can be divided into 3 phases, the first phase: CD2-/part+CD5-, CD2-/part+CD5+, second stage: cd2+cd5-, third stage: cd2+cd5+, reflecting the early to late progression.
TABLE 3 relationship between differentiation patterns of CD45RA and CD45RO and CD2 and CD5 expression
The CD99/CD44 combination can be used as a sensitive MRD tracking marker:
CD44 is believed to play an important role in the pathogenesis of T-ALL, where immature forms (mCD 3/CD1a -) express less CD44, whereas the NOTCH1 mutation and STIL-TAL1 are associated with low CD44 expression. In this example, the positive expression rate was initially evaluated for CD44 expression, and as a result, it was found that the positive expression rate was 100%, but the fluorescence intensity expression was different, and the CD44 expression was also different for different lymphocyte subsets, mature T cells, B cells and NK cells, so that when the results were summarized in the antibody combination of the present invention, the CD44 MFI ratio of naive T cells to mature T cells was calculated as CD44 correction value for naive T cells, with the average fluorescence intensity (MFI) of mature T cell CD44 expression as a control. Expression of CD99 was also categorized into str+, + and dim+ with normal mature T cells as a reference.
The results of the 56 patients showed that the expression of CD99 was correlated with that of CD44, and that the CD99dim+/+ patients were 23 and that of CD99str+ expressing patients were 33, the former showed significantly higher expression of CD44 than the latter (P < 0.0001), as shown in FIG. 7: relationship between differential expression of CD99 and CD44 MFI ratio. Meanwhile, the strong expression of the CD99 of the naive T cells is easy to be accompanied with the weak expression of the CD44, which is obviously different from the mature T cells, and can be used as a very sensitive MRD detection mark.
In summary, the 20-color antibody combination can reach the examination result of the 10C 4 tube combination, can carry out comprehensive immunophenotype analysis, stage and ETP-ALL identification on T-ALL/LBL, is better than the 10C combination, and the CD7 marked fluorescein FITC in the invention is slightly stronger than PerCP-Cy5.5 in the 10C combination; the co-tube detection finds the differentiation development mode of CD45RA/CD45RO in the differentiation process of the naive T cells CD2 and CD 5; the correlation between the expression of CD99 and CD44 is found for the first time, the CD99/CD44 gating can well distinguish the naive T cells from the mature T cells, and can be used as a sensitive antigen mark for MRD tracking detection; in addition, the antibody combination of the invention can flexibly gate T cells by adopting a plurality of antigens and divide lymphocytes by utilizing a plurality of antigen markers, overcomes the limitation of being dependent on the gating of CD7 and cCD3, and is particularly suitable for targeting or patients with the deletion of the gated antigen expression after CAR-T treatment. The combination of antibodies above 10 colors requires consideration of the choice and collocation of antibody clones and luciferin, and requires a combination of expertise and clinical experience for those skilled in the art.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.
Claims (10)
1. An antibody combination for flow cytometric detection of acute T-lymphoblastic leukemia/lymphoma immunophenotype comprising a first antibody combination; the first antibody combination comprises the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody, and anti-CD 25 antibody; the primary antibody combination was used in 1 flow tube at the time of detection.
2. The combination of antibodies of claim 1, wherein the antibodies are fluorescein-labeled antibodies; in the first antibody combination, the fluorescent labels of the anti-CD 99 antibody, the anti-CD 16 antibody, the anti-CD 45RA antibody, the anti-CD 5 antibody, the anti-CD 44 antibody, the anti-CD 57 antibody, the anti-CD 28 antibody, the anti-CD 7 antibody, the anti-CD 8 antibody, the anti-CD 65 antibody, the anti-CD 4 antibody, the anti-HLA-DR antibody, the anti-CD 2 antibody, the anti-CD 1a antibody, the anti-CD 13 antibody, the anti-CD 3 antibody, the anti-CD 123 antibody, the anti-CD 45RO antibody and the anti-CD 25 antibody are ：BV421、eFluor V450、eFluor V547、BV570、BV605、BV650、BV711、BV785、FITC、eFluor B548、PE、PE/Dazzle594、PE/Cy5、PerCP/eFluor710、PE/Cy7、APC、eFluor R668、eFluor R720、APC/Cy7 and APC/Fire810, respectively, in order.
3. The antibody combination according to claim 1 or 2, wherein the antibody combination further comprises a second antibody combination; the second antibody combination comprises an anti-cell membrane antigen antibody and an anti-cell intracellular antigen antibody; the anti-cell membrane antigen antibody consists of the following antibodies: anti-CD 38, anti-CD 3, anti-CD 10, anti-CD 33, anti-CD 5, anti-CD 19, anti-CD 45, anti-CD 7, anti-CD 117, anti-CD 34, anti-CD 56, and anti-TRBC 1 antibodies; the anti-intracellular antigen antibody consists of the following antibodies: anti-cd 79a, anti-cLambda, anti-cKappa, anti-cd 22, anti-cd 3, anti-cMPO antibodies, and anti-nTdT antibodies; the second antibody combination was used in 1 flow tube at the time of detection.
4. The antibody combination according to claim 1 or 2, wherein the antibody combination is a first antibody combination; the first antibody combination consists of the following antibodies: anti-CD 99 antibody, anti-CD 16 antibody, anti-CD 45RA antibody, anti-CD 5 antibody, anti-CD 44 antibody, anti-CD 57 antibody, anti-CD 28 antibody, anti-CD 7 antibody, anti-CD 8 antibody, anti-CD 65 antibody, anti-CD 4 antibody, anti-HLA-DR antibody, anti-CD 2 antibody, anti-CD 1a antibody, anti-CD 13 antibody, anti-CD 3 antibody, anti-CD 123 antibody, anti-CD 45RO antibody and anti-CD 25 antibody.
5. The antibody combination according to claim 1 or 2, wherein the antibodies in the antibody combination are monoclonal antibodies.
6. A kit for flow cytometry detection of an acute T-lymphoblastic leukemia/lymphoma immunophenotype, comprising an antibody combination according to any one of claims 1-5; when the combination of antibodies includes a first combination of antibodies and a second combination of antibodies, the first combination of antibodies and the second combination of antibodies are packaged separately.
7. The kit of claim 6, further comprising: erythrocyte lysate and buffer PBS.
8. A system for flow cytometry detection of an acute T-lymphoblastic leukemia/lymphoma immunophenotype comprising a detection portion and an analysis portion; the detection part comprises the antibody combination according to any one of claims 1-5 or the kit according to claim 6 or 7, and is used for detecting the antigen expression level of an individual to be detected by flow cytometry; and an analysis section for analyzing the detection result of the detection section.
9. The system according to claim 8, wherein the sample to be tested is processed by the antibody combination according to any one of claims 1 to 5 or the kit according to claim 6 or 7 to prepare a flow cytometry sample; performing flow cell on-machine detection; wherein the flow cell is provided with a gate during the on-machine detection according to the following mode: setting se:Sub>A disjunctor cell gate A by using the arese:Sub>A and the height of FSC, continuing to disbond by using SSC-A/SSC-B, setting se:Sub>A living cell gate R1 by using FSC-A/SSC-A, and removing fragments and dead cells; the interior of the R1 gate is conventionally provided with a hemocyte gate by using a CD45/SSC scatter diagram; the haemocyte gate includes lymphocytic, granulocytic, monocytic and nucleated erythrocytic gates.
10. Use of an antibody combination according to any one of claims 1-5 or a kit according to claim 6 or 7 or a system according to claim 8 or 9 for the preparation of a product related to acute T-lymphoblastic leukemia/lymphoma detection; the acute T lymphocyte leukemia/lymphoma detection comprises one or more aspects from (1) to (7): (1) Screening acute T lymphocyte leukemia/lymphoma treatment targets and/or abnormal phenotypes; (2) acute T-lymphocyte leukemia/lymphoma diagnosis; (3) naive T cell detection; (4) acute T lymphocyte leukemia stage; (5) screening for acute early precursor T-lymphoblastic leukemia; (6) Post-acute T-lymphoblastic leukemia/lymphoma treatment marker screening and/or residual leukemia cell monitoring; (7) lymphocyte grouping; the lymphocytes include one or more of mature T cells, B cells and NK cells.
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| CN118130798A true CN118130798A (en) | 2024-06-04 |
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