CN113933513A - Reagent composition for detecting acute T lymphocyte leukemia after targeted therapy and application thereof - Google Patents
Reagent composition for detecting acute T lymphocyte leukemia after targeted therapy and application thereof Download PDFInfo
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Abstract
The invention provides a reagent composition for detecting acute T lymphocyte leukemia after targeted therapy and application thereof. The reagent composition comprises a first group of antibodies, a second group of antibodies and a third group of antibodies, wherein the first group of antibodies comprise an anti-CD 99 antibody, an anti-CD 1a antibody, an anti-CD 34 antibody, an anti-CD 3 antibody, an anti-CD 4 antibody, an anti-CD 5 antibody, an anti-CD 8 antibody, an anti-CD 7 antibody, an anti-CD 45 antibody and an anti-CD 2 antibody; the second group of antibodies includes anti-CD 3 antibodies, anti-CD 7 antibodies, anti-CD 16 antibodies, anti-CD 56 antibodies, anti-CD 45 antibodies; the third group of antibodies includes anti-nuclear TdT antibodies, anti-cytoplasmic CD3 antibodies. The reagent composition can be used for detecting acute T lymphocyte leukemia after targeted therapy by flow cytometry, reduces the missed diagnosis probability and can also perform immune evaluation simultaneously.
Description
Technical Field
The invention relates to a reagent composition for detecting acute T lymphocyte leukemia after targeted therapy and application thereof.
Background
Acute leukemia always becomes a disease seriously influencing human health due to high malignancy, fast disease progression, high recurrence rate and high mortality, and Acute Lymphoblastic Leukemia (ALL) accounts for about 30-40% of acute leukemia, wherein acute T-lymphoblastic leukemia (T-ALL) is one of acute leukemias with poor current treatment effect, and the evaluation of the effect after treatment is particularly important.
At present, Flow Cytometry (FCM) is an important means for evaluating the effect of various acute leukemias after treatment by detecting Minimal Residual Disease (MRD). Multiple studies also indicate that FCM detection of MRD is of great significance for prognosis and for guiding therapy, regardless of the time point after chemotherapy, before and after transplantation, before and after CAR-T, and before and after CAR-T bridge transplantation. However, although the technology of flow cytometry is rapidly developed for a long time, the clinical application scale is difficult to expand, mainly because the whole process from specimen processing to data analysis is manually operated, and the popularization of the technology is severely limited due to the lack of standardization, automation and intellectualization.
MRD of T-ALL, which is the earliest developed item in the FCM detection MRD, is not mature in the bone marrow unlike Acute Myeloid Leukemia (AML) and B-ALL, and thus naive T cells are seen in the bone marrow, i.e., highly suspicious to malignant. However, in recent years, it has been found that MRD detection of T-ALL is not as simple as theoretically, and MRD detection of FCM is severely restricted for the following reasons: the early marker loss rate is high: the follow-up after the treatment of T-ALL has high antigen drift rate, especially early markers (such as CD99 strong expression and TdT) with high coverage rate in the initial treatment have 70-95% of expression rate in the initial treatment, but the expression intensity of the markers can be weakened or lost along with the treatment, even the marker cCD3 can be lost, which brings great difficulty to the MRD detection with high sensitivity; acquisition of a mature mark: T-ALL is often treated, the phenotype is more mature, not only expressed by the loss of early markers, but also expressed by the possibility of obtaining some mature markers, and the detection scheme of the prior art does not carry out systematic research on the mature markers and has a high rate of case missed diagnosis; ③ immunophenotypic diversity of mature lymphocytes: although mature T cells are theoretically classified into CD4+ and CD8+ cell populations, some non-tumor patients or other tumor patients other than T-ALL can have an increased proportion of CD4+/CD8+ or CD4-/CD8-T cells (mainly gamma delta T cells) due to virus infection, individual difference and the like, which seriously interfere with the MRD detection of T-ALL; phenotypic heterogeneity of T-ALL: T-ALL phenotype consistency is not strong, and the difficulty of analysis is increased due to phenotype diversity; the prior vast majority of T-ALL MRD gating adopts CD7 to set a T cell gate, however, in recent years, successful cases of CAR-T technology for treating T-ALL basically focus on gene technology knockout of CD 7-expressed CD7-CAR-T on CAR-T cells, so that the treated cases lose CD7 with high probability in 2-3 months, and thus, the MRD detection of T-ALL is challenged. Sixthly, the MRD analysis of T-ALL cannot be seen by dividing B cells into 3-4 cells in different stages like the panel of B-ALL because there are no naive T cells in the bone marrow. Also, MRD analysis of T-ALL also presented interference with NK cells.
In view of the above, the detection of T-ALL is more difficult and more subjective, and therefore, a detection and analysis technique which is relatively standardized, normalized, has high coverage rate and is relatively less affected by the treatment of CD7-CAR-T is urgently needed.
Disclosure of Invention
It is an object of the present invention to provide a novel technique for detecting acute T-lymphocyte leukemia following targeted therapy.
One aspect of the present invention provides a reagent composition for flow cytometry detection of acute T lymphocyte leukemia after targeted therapy, the reagent composition comprising a first set of antibodies, a second set of antibodies and a third set of antibodies, wherein the flow cytometry detection is performed using a two-tube parallel protocol, wherein:
the first group of antibodies comprises: anti-CD 99 antibody, anti-CD 1a antibody, anti-CD 34 antibody, anti-CD 3 antibody, anti-CD 4 antibody, anti-CD 5 antibody, anti-CD 8 antibody, anti-CD 7 antibody, anti-CD 45 antibody, anti-CD 2 antibody; each antibody in the first group of antibodies is used for being added into a first flow tube of which the sample to be detected is in a single cell suspension state;
the second group of antibodies includes: anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody, anti-CD 45 antibody; each antibody in the second group of antibodies is used for being added into a second flow tube of which the sample to be detected is in a single cell suspension state;
the third group of antibodies includes: anti-nuclear TdT antibody, anti-cytoplasmic CD3 antibody; each antibody in the third set of antibodies is used to add to the second flow tube after the second set of antibodies has been added and subjected to membrane rupture treatment.
The reagent composition can be applied to flow cytometry for detecting acute T lymphocyte leukemia after targeted therapy, mature markers CD4, CD8 and CD3 are included for T-ALL disease with very strong heterogeneity, especially for the case that CD7 cell markers are possibly weakened or lost after CD7-CAR-T, and original cell markers and T cell markers are used in combination for gating detection, so that various T cell subsets can be effectively gated and selected, and each subtle change can be superposed by reasonably arranging multidimensional parameter space positions, so that multiple parameter combinations can be rapidly observed in one step, and the diagnosis omission probability is reduced. And can be simultaneously evaluated immunologically.
According to a particular embodiment of the invention, each antibody in the reagent composition of the invention is a monoclonal antibody.
According to a particular embodiment of the invention, each antibody in the reagent composition of the invention is a fluorescein-labeled antibody. Preferably, the fluorescein label of each antibody in the first set of antibodies can be: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, BV421, V500, BV 605. Wherein, CD34 and CD1a share a common fluorescence channel, which can play a role in reducing fluorescence channels. The respective antibodies fluorescein can be exchanged with each other. More preferably, in the first group of antibodies, the fluorescein markers of the anti-CD 99 antibody, the anti-CD 1a antibody, the anti-CD 34 antibody, the anti-CD 3 antibody, the anti-CD 4 antibody, the anti-CD 5 antibody, the anti-CD 8 antibody, the anti-CD 7 antibody, the anti-CD 45 antibody and the anti-CD 2 antibody are respectively: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, BV421, V500, BV 605.
According to a specific embodiment of the present invention, in the reagent composition of the present invention, the fluorescein label of each antibody in the second set of antibodies may be: PerCP-Cy5.5, APC-Cy7, BV421, V500. The respective antibodies fluorescein can be exchanged with each other. More preferably, in the second group of antibodies, the fluorescein labels of the anti-CD 3 antibody, the anti-CD 7 antibody, the anti-CD 16 antibody, the anti-CD 56 antibody and the anti-CD 45 antibody are, in order: PerCP-Cy5.5, APC-Cy7, BV421, V500.
According to a particular embodiment of the invention, in the reagent composition of the invention, in the third group of antibodies, the fluorescein label of the anti-TdT antibody is FITC and the fluorescein label of the anti-cytoplasmic CD3 antibody is PE or APC.
According to the invention, by matching different antibodies with specific fluorescein, all fluorescein in each channel can achieve an excellent dyeing effect when the reagent composition is applied to detecting acute T lymphocyte leukemia minimal residual disease and lymphocyte subpopulation after targeted therapy.
According to a particular embodiment of the invention, each antibody component of the reagent composition of the invention is commercially available. Each antibody should meet relevant industry standard requirements. Among them, the anti-TdT antibody is preferably selected from the antibodies with the clone numbers HT6 or HT NK 1+ HT4+ HT8+ HT 9.
According to a particular embodiment of the invention, in the reagent composition of the invention, the first group of antibodies is anti-CD 99 antibody, anti-CD 1a antibody, anti-CD 34 antibody, anti-CD 3 antibody, anti-CD 4 antibody, anti-CD 5 antibody, anti-CD 8 antibody, anti-CD 7 antibody, anti-CD 45 antibody, anti-CD 2 antibody according to 5: 5: 5: 5: 3: 2: 3: 3: 3: 3 (in the case of substantially equivalent titers);
the second group of antibodies is anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody, anti-CD 45 antibody according to 5: 2: 3: 3: 3 (in the case of substantially equivalent titers);
the third group of antibodies was anti-TdT and anti-cytoplasmic CD3 antibodies according to 2: 5 (in the case of substantially equivalent titers) are mixed.
In another aspect of the present invention, a kit, in particular for detecting acute T-lymphocyte leukemia minimal residual disease and lymphocyte subpopulations after targeted therapy, is provided, which comprises a first container, a second container and a third container, each container respectively containing a first group of antibodies, a second group of antibodies and a third group of antibodies of the reagent composition according to the present 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 T lymphocyte leukemia after targeted therapy and can also be used for immune evaluation.
The invention also provides application of the reagent composition in preparing a flow cytometry on-machine sample for detecting acute T lymphocyte leukemia after targeted therapy.
According to a specific embodiment of the present invention, the process for preparing a flow cytometric sample for detecting acute T-lymphocyte leukemia after targeted therapy comprises the steps of:
(1) respectively adding the sample to be tested into the first and second flow tubes (tubes A and B) to make it be in single cell suspension state and ensure cell amount to be 1 × 106pipe-1X 107A pipe; the sample to be detected is bone marrow or peripheral blood;
(2) adding a first group of antibodies in the reagent composition into the sample obtained by the step (1) in a first flow tube, adding a second group of antibodies in the reagent composition into a second flow tube, uniformly mixing, and incubating at room temperature in a dark place;
(3) adding the membrane breaking agent A solution into the second flow tube incubated in the step (2), and incubating at room temperature in a dark place;
(4) adding 1 Xhemolysin into the first flow tube incubated in the step (2) and the second 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) and removing supernatant;
(6) then adding the solution B of the membrane breaking agent and a third group of antibodies in the reagent composition into the second flow tube after the supernatant is removed in the step (5), and incubating at room temperature in a dark place;
(7) and (4) adding a PBS buffer solution into the first flow tube after the supernatant is removed in the step (5) and the second flow tube after the room-temperature light-shielding incubation in the step (6) for washing, removing the supernatant after centrifugation, and resuspending the cells by using the PBS buffer solution to obtain the flow cell on-machine sample.
In the present invention, the operation steps are described in a sequence without any specific indication or obvious determination of the sequence relationship from the context, and the sequence is not used to limit the actual operation sequence of the steps.
According to a particular embodiment of the invention, the reagents other than the reagent composition of the invention may be used in amounts conventionally used in the art or recommended by the supplier.
According to a particular embodiment of the invention, the reagent composition of the invention, the first group of antibodies is added in an amount of 17-68. mu.l/tube, the second group of antibodies is added in an amount of 7-26. mu.l/tube and the third group of antibodies is added in an amount of 4-14. 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 the specific embodiment of the present invention, in the step (5), the centrifugation conditions may be generally 1000-2000rpm (or 300-450 g) for 3-10 minutes.
According to a specific embodiment of the present invention, in step (6), the incubation is performed for about 10-30 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 (7), 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 3-10 minutes. The amount of PBS buffer added for resuspension was 0.5-1 ml/tube.
Another aspect of the invention provides a device for detecting acute T-lymphocyte leukemia after targeted therapy, the device 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 a reagent composition of the present invention;
the analysis unit is used for analyzing the detection result of the detection unit.
According to a specific embodiment of the invention, the device of the invention is used for detecting acute T-lymphocyte leukemia after targeted therapy, wherein the process of detecting a sample from a test subject by flow cytometry comprises:
after a sample to be detected is processed by using the reagent composition, a flow cytometry sample is prepared (the specific processing process can refer to the above record);
and (5) performing flow cytometry detection.
According to a particular embodiment of the invention, at least 30 million, preferably 100 million cells are recommended for flow cytometric machine detection.
According to a specific embodiment of the present invention, the analysis unit in the device of the present invention is used for analyzing the detection result to discriminate (including auxiliary discrimination) acute T lymphocyte leukemia after the targeted therapy.
According to a particular embodiment of the invention, the device of the invention is used for the detection of acute T-lymphocyte leukemia after targeted therapy, wherein the gating analysis is performed on flow cytometric machines in the following manner:
a first flow tube: setting a debonding cell gate P1 and a living cell gate P2 in a P1 gate to obtain a single living cell; setting each blood cell gate within P2 using CD45/SSC antibody; performing four-parameter four-dimensional gating by using CD7, CD2, CD5 and SSC in a P2 gate, and circling a T NK1 cytogate which is CD7 positive, CD2 positive and/or CD5 positive (namely, a cytogate which is CD7 positive and/or CD2 positive and/or CD5 positive and is called a T NK1 cytogate in the invention); setting an NK cell gate by using CD45 strong positive/CD 3 negative in the T NK1 cell gate; a CD3 cytogate is arranged in a T NK1 cytogate by using CD45 strong positive/CD 3 positive, and a CD4CD8 double-negative cytogate is arranged in a CD3 cytogate; dividing T NK1 cells into NK and CD4CD8 double negative cells and the T cell gate; and/or
A second flow tube: sequentially arranging a cell gate P1 for removing adhesion and a cell gate P2 for living cells; setting each blood cell gate within P2 using CD45/SSC antibody; in a P2 gate, performing three-parameter three-dimensional gating by using CD7, cytoplasmic CD3 and SSC, and delineating any marker-positive cell T NK 2 gate (namely any marker-positive cell gate is called T NK 2 cell gate in the invention); in the T NK 2 gate, CD3/CD16/CD56 three-parameter three-dimensional setting is used for setting CD16+ CD56 positive/CD 3 negative NK cell gates, and the T NK 2 gate is divided into an NK cell gate and a T cell gate.
According to a specific embodiment of the present invention, when the apparatus of the present invention is used for detecting acute T lymphocyte leukemia after targeted therapy, the gating analysis of the first flow tube further comprises:
the proportion of each subgroup of T cells of CD4 and CD8 is analyzed in a CD3 cell gate to carry out immune evaluation; and/or
After dividing the T NK1 cells into an NK and CD4CD8 double negative cell gate and a T cell gate, performing seven-parameter seven-dimensional gating on the T NK1 cell gate, the T cell gate, the NK and CD4CD8 double negative cell gate by adopting CD45/CD99/CD34+ CD1a/CD3/CD4/CD8/CD5 respectively, so that the normal T cells are distributed in different regions according to the subsets.
According to a specific embodiment of the present invention, when the apparatus of the present invention is used for detecting acute T lymphocyte leukemia after targeted therapy, the gating analysis of the second flow tube further comprises:
in NK cell gate, analyzing NK cell subgroup of CD56briCD16neg, CD56dimCD16pos and CD56negCD16pos, and carrying out immune evaluation; and/or
After dividing the T NK 2 gate into an NK cell gate and a T cell gate, adopting CD 45/cytoplasmic CD 3/nuclear TdT/CD3/CD16/CD56 to respectively carry out six-parameter six-dimensional gating in the T NK 2 cell gate, the NK cell gate and the T cell gate, so that normal T cells and NK cells are distributed in different regions according to subgroups.
According to a specific embodiment of the present invention, when the apparatus of the present invention is used for detecting acute T lymphocyte leukemia after targeted therapy, the gating analysis of the first flow tube further comprises: if the seven-parameter seven-dimension gate shows a cell group except normal cells, the seven-dimension gate is defined as MRD1 gate; the two-dimensional dot diagram analysis of CD4/CD34+ CD1a and CD5/CD7 is carried out in MRD1 door, so that the expression of CAR-T targets (such as CD4, CD1a, CD5 and CD 7) commonly used at present can be analyzed.
According to a specific embodiment of the present invention, when the apparatus of the present invention is used for detecting acute T lymphocyte leukemia after targeted therapy, the gating analysis of the second flow tube further comprises: if the six-parameter six-dimension gate shows a cell group except normal cells, the cell group is defined as MRD2 gate, and whether MRD is positive is further judged.
According to the specific embodiment of the invention, when the device is used for detecting acute T lymphocyte leukemia after targeted therapy, and the analysis unit analyzes the detection result of the detection unit, the displayed distribution pattern of each subgroup of T cells is compared with normal cells in the multi-symbol combination setting gate to find out tumor cells.
More specifically, when using multi-parameter multi-dimensional analysis, when the angles of the coordinate axes can be defined in the order of 0 degree in the direction of 0 point and 360 degrees clockwise, it is preferable to perform the following operations:
setting a T cell gate:
the first flow tube, CD7, CD2, CD5 and SSC four parameters are displayed at proper angles (preferably: SSC is vertical axis, three parameters of CD7, CD2 and CD5 have 5-10 angle difference, form about similar horizontal axis, the whole can be taken as a group, namely any parameter is positive and SSC forms T NK1 cell group), T NK1 cell gate can be selected through the union (any marker is expressed) of three T cell marker expressions, and whether a certain marker is weakened or lost before or after targeted therapy or not can be observed to select proper targeted therapy; NK cell and CD4CD8 double negative cell (mainly TCR γ δ T) interference can also be excluded by CD45 and CD3 gating; furthermore, the immunosuppressive status can be assessed by analyzing the CD4, CD8 cell population within the CD3 gate;
the second flow tube, CD7, CD3, SSC, shown at appropriate angles, can select the T NK 2 cell gate by the union of the two marker expressions (either marker expression); NK cell gates can also be set through CD3, CD16 and CD56, NK interference is eliminated, and each NK cell subset can be further analyzed.
Secondly, observing T cell immunophenotype in a T cell gate, and arranging a multidimensional map, arranging normal cells according to the sequence of CD4+ T cells, CD8+ T cells, CD4CD8 double-negative cells (mainly TCR gamma delta T cells) and NK cells (CD 3 negative CD45 strong positive, CD3 negative/CD 56+ CD16 positive), thereby distributing normal subgroups of cells in the upper half area, and leaving out areas (CD 7 and/or CD99 are enhanced, CD45 and/or CD5 are weakened, CD4, CD3 and CD8 are weakened or lost, and CD34+ CD1a and/or TdT positive) where MRD is easy to appear, so that any abnormality appears, even the abnormality which is difficult to find when two parameters of the conventional two-dimensional map is combined, can be amplified through a superposition effect, and the multidimensional superposition effect is displayed.
The invention can compare the distribution pattern of each subgroup of the displayed T and NK cells with normal cells in the combination set door with multiple marks and multiple dimensions at proper angles, and efficiently find out the tumor cells with high sensitivity.
In some embodiments of the invention, the door is provided as follows:
the first flow tube is gated as follows: setting a debonding cell gate P1 using FSC-A/FSC-H and a viable cell gate P2 using FSC/SSC to obtain a single viable cell; setting each blood cell gate within P2 using CD45/SSC antibody; within the P2 gate, T and NK cells were observed using a four-dimensional combination of CD7, CD5, CD2, SSC parameters to set the T NK1 cell gate. When multidimensional picture analysis is carried out, the center of a coordinate axis is positioned at the lower left position, SSC is a vertical axis, CD7, CD2 and CD5 form a small-angle horizontal axis, any mark positive cell population is clearly displayed, and a T NK1 cytogate is set; the NK cell gate and the CD3 gate are set by using CD3/CD45 in the T NK1 cell gate, and the CD4CD8DN (namely CD4 and CD8 are double negative) cell gate is set in the CD3 gate; the T NK1 gate is divided into NK cells, a CD4CD8DN cell gate and a T cell gate; the seven-parameter seven-dimensional radar chart respectively shows T NK1 cell gate, NK cell and CD4CD8DN cell gate and T cell gate, CD99/CD34+ CD1a/CD3/CD4/CD8/CD5/CD45 shows each subgroup of T and NK cells, and the angle is adjusted to enable the T and NK cells to be distributed in different regions according to each subgroup. Therefore, the invention can improve the detection sensitivity and prevent the unclear influence of door arrangement caused by weak fluorescein or tumor cell over-expression and over-low instrument condition. More specific operations may be: CD45 was located at a small angle at the upper right (about 350-20 degrees), CD4 was located at the upper left (about 300-330 degrees), CD3 was located at the left (about 255-285 degrees), CD5 was located at the lower left (about 220-250 degrees), CD8 was located at the lower left (about 190-220 degrees), CD99 was located at the right (about 75-105 degrees), CD34+ CD1a was located at the lower right (about 120-150 degrees), so that mature T cells were at the upper left, CD4+ T cells were above, CD8+ T cells were below, NK cells were at the upper right, and CD4CD8DN cells (TCR. gamma. delta. T cells were dominant) at the upper left position immediately adjacent to the center. The lower side is a neutral zone. Since T-ALL high probability events are CD45 attenuated, CD3, CD4, CD8, CD5 lost or attenuated, CD7 and/or CD99 expression enhanced, CD34+ CD1a acquired abnormally, and therefore the lower, either central, directly below, or below left or below right, is a region in which MRD is prone to occur.
The second flow tube is gated in the following manner: sequentially arranging a cell gate P1 for removing adhesion and a cell gate P2 for living cells; then, within the P2 gates, each blood cell gate was set using the CD45/SSC antibody; in the P2 gate, a T NK 2 cell gate is set by using a SSC and cCD3 and CD7 three-parameter three-dimensional radar chart, the center of a coordinate axis is positioned at the lower left position, the SSC is a vertical axis, the CD7 and the cCD3 form a small-angle horizontal axis, any mark positive cell group is clearly displayed, and the T NK 2 cell gate is circled. In a T NK 2 cell gate, setting an NK cell gate by three-parameter three-dimensional radar charts of CD3, CD16 and CD 56; the T NK 2 cell gate is divided into an NK cell gate and a T cell gate; in T NK 2, NK and T cell compartments, TdT/CD45/cCD3/CD3/CD56/CD16 six-dimensional radar maps are respectively used for displaying each subgroup of T and NK cells, and angles are adjusted to enable each subgroup of T and NK cells to be distributed in different areas. T cells can be detected as sensitively as possible. More specific operations may be: CD45 is positioned at the upper left position (about 315-345 ℃), CD3 is positioned at the upper left position (about 300-330 ℃), cCD3 is positioned at the lower left position (about 210-240 ℃), CD16 and CD56 are positioned at the upper right position (about 40-70 ℃), and TdT is positioned at the lower right position (about 125-155 ℃), so that mature T cells can be positioned at the upper left, NK cells are positioned at the upper right, and the rest areas are vacant positions, namely areas where MRD is easy to appear.
In some embodiments of the invention, the invention may also be used for simultaneous immunological evaluation: first flow tube analysis of CD3 in-door CD4, CD8 each sub-population of T cells, CD4/CD8 ratio; second flow tube analysis of NK cell door, CD16, CD56 each subgroup NK cells. When the ratio of CD4 positive/CD 8 positive is severely inverted (especially below 0.1-0.2), it is suggested that immunosuppression is too severe, the risk of infection is high, and the use of chemotherapeutic drugs and immunosuppressive agents needs to be reduced or stopped.
In some embodiments of the present invention, the analyzing unit compares the displayed distribution pattern of each subpopulation of T cells with the normal cells in the multi-marker combination setting to find out tumor cells when analyzing the results of the detecting unit, 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; if the expression intensity or composition pattern of several antigens in the specimen to be tested is changed compared with that of Normal differential expression (Di ff energy-from Normal, DFN), i.e., the distribution pattern of several Normal expression antigens in the development or subpopulation, the expression intensity or composition pattern is changed to DFN. The invention adopts a mode of combining the two. Normal T cell development processes are as follows: pan T markers include cCD3, CD7, CD2, and CD 5. Of these, CD7 and CD3 are true pan T markers, with CD7 being strongly expressed in the promyelocytic stage, least strongly expressed in the cortical stage, and moderately intense in the mature stage. CD2 and CD5 appeared in the second stage, with no major change in CD2 throughout, with increased expression of CD5 with cell maturation, but with weak expression or even negative in the TCR γ δ subpopulation. The primitive stage markers include TdT, CD34, CD10, and CD99 strong expression (bright, bri). T cells at the thymic cortex stage express CD1a, CD4 and CD8 are simultaneously expressed (i.e., double positive), TCR is weakly expressed (dim). Mature T lymphocytes are classified into TCR α β cells (accounting for more than 90% of T cells) and TCR γ δ cells (accounting for 3-10% of T cells, which may be elevated in special cases) according to the difference in TCR expression. The difference of TCR expression and CD5 expression is mainly the difference of CD4 and CD8, the TCR alpha beta cell is divided into two sub-groups of CD4 and CD8 single positive, the ratio is about 0.5-2.5, and the ratio is changed in special immune state; TCR γ δ subset, did not express CD4, CD8 was expressed with a weak positive to negative continuation.
In conclusion, the invention provides a reagent composition and application thereof in detecting acute T lymphocyte leukemia after targeted therapy. The invention has the following outstanding characteristics and beneficial technical effects: (1) the diagnosis missing caused by early marker loss (for example, the positive rate of TdT in T-ALL is 70-95%, the positive rate of CD34 in T-ALL is about 30%, even if the positive rate of CD99 strong expression in T-ALL is more than 95%, the inventor finds out through experience that the strong expression of CD99 and the positive rate of TdT are gradually weakened to loss along with treatment, the positive rates of the two early markers with high positive rates in MRD are only about 30%), the CD7 expression is weakened or lost after the treatment of CD7-CAR-T, and the like can be avoided; (2) help to find potential CAR-T targets of tumor cells, CD7, CD2, CD5 and even CD4, CD34+ CD1a expression conditions, and guide further treatment; (3) the method has the advantages that T cell gates are set in a multi-parameter and multi-dimension mode, expression of each subgroup of T cells is analyzed, complexity is reduced, dozens of complex pictures are concentrated on a plurality of pictures, efficiency is improved, missed diagnosis rate is reduced, the method is suitable for current manual analysis, and a prototype is provided for development of FCM software artificial intelligence; (4) the clinical practicability of the invention is ensured to the maximum extent, the machine model with the highest clinical coverage rate is a 10-color flow cytometer at present, and in the combined scheme for detecting T-ALL MRD, although T cells have the characteristics of phenotypic diversity and large antigen change after treatment, the MRD can be observed by adopting CD99/CD34+ CD1a/CD3/CD4/CD8/CD45/CD5, 90-95% or even higher cases can be covered, the MRD can be further verified and diagnosed by adopting a second flow tube TdT/cCD3/CD3/CD56/CD16, 85-95% of cases can be covered, and the sensitivity and specificity can be improved to more than 98%; even under the condition of extremely low proportion, the two tubes can mutually verify, so that false positives and false negatives can be conveniently eliminated; (6) can be used for detection during initial treatment, can also be used for follow-up after treatment, and especially can be used for detection after targeted treatment such as CAR-T. (7) Can simultaneously carry out immune evaluation, prompt immunosuppression in time and prevent serious infection. The invention will have profound effects on clinical efficacy and treatment cost saving.
Drawings
FIGS. 1 and 2 show flow cytometry gated analyses of the same bone marrow specimen with complete remission after treatment, according to one embodiment of the present invention. Wherein:
FIG. 1 is a sample of bone marrow with complete remission after treatment, for nail tube analysis. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using FSC/SSC to set a living cell gate P2 in P2, using CD5, CD2, CD7 and SSC four-dimensional four-parameter setting T NK1 cell gate, using CD3/CD45 to set a CD3 negative CD45 strong positive (bri) NK cell gate, using CD3 positive CD45 strong positive CD3 gate, and using CD3 gate to set a CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis graphs of CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD8 are respectively shown in a T NK1 cytogate, an NK cell, a CD4CD8DN cytogate and a T cytogate. The proportion of each subset of T cells, CD4, CD8, was analyzed in CD3 gate.
FIG. 2: bone marrow specimens were completely relieved after the same treatment as in FIG. 1 and analyzed by tube B. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; and carrying out six-dimensional six-parameter analysis on the T NK 2 phylum, the T phylum and the NK phylum by using CD45/TdT/cCD3/CD3/CD56/CD 16. NK cell in vivo analysis CD16, CD56 each subgroup NK proportion.
FIG. 3 and FIG. 4 show bone marrow specimens of T-ALL relapse after the same treatment. Wherein:
FIG. 3: T-ALL bone marrow specimens that recur after treatment, were subjected to nail tube analysis. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using CD5, CD2, CD7 and SSC to set a T NK1 cell gate in a four-dimensional four-parameter setting in a P2 gate; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8.
FIG. 4: T-ALL bone marrow samples, which had relapsed after the same treatment as in FIG. 3, were analyzed by tube B. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD16/CD56, respectively.
FIGS. 5-10 show bone marrow samples from the same T-ALL patient, and FIGS. 5 and 6 show MRD positive samples from the patient before treatment with CD7-CAR-T, both normal T cells and tumor T cells being CD7 positive; FIG. 7, FIG. 8 are samples taken 1 month after CD7-CAR-T treatment in this patient, MRD negative, but with loss of normal T cell CD 7; FIG. 9, FIG. 10 are samples of the patient 2 months after CD7-CAR-T treatment, MRD negative, but partially restored normal T cell CD 7. Wherein:
FIG. 5: bone marrow specimens from CD7-CAR-T pre-treatment MRD positive T-ALL patients, for analysis in the nail canal. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using CD5, CD7 and SSC to set a T NK1 cell gate in three-dimensional three parameters (CD 2 is not done at the time) in P2; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8.
FIG. 6: the same specimen was analyzed in the same tube as in FIG. 5. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD16/CD56, respectively.
FIG. 7: complete remission bone marrow specimens of CD7 loss 1 month after CD7 CAR-T treatment in the same case as in fig. 5, 6, were analyzed by nail tube. FSC-A/H sets a single cell gate P1, a P1 sets a living cell gate P2 by using FSC/SSC, a P2 gate is internally provided, CD5, CD2, CD7 and SSC four-dimensional four-parameter sets a T NK1 cell gate, although CD7 is lost, the T cell gate can still be set because CD5 and CD2 exist; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8.
FIG. 8: complete remission bone marrow specimens of CD7 loss 1 month after CD7 CAR-T treatment identical to figure 7, were analyzed by tube b. FSC-A/H sets a single cell gate P1, FSC/SSC is used in P1 to set a living cell gate P2, P2 is internally set, three-dimensional parameters of cCD3, CD7 and SSC are used for setting a T cell gate T NK 2, although CD7 is lost, the setting of the gate is not influenced, and three-dimensional graphs of CD3, CD16 and CD56 are used for setting the NK cell gate in the T NK 2; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD7/CD56, respectively.
FIG. 9: complete remission bone marrow specimens, nail tube analysis, of CD7 recovery 2 months after CD7 CAR-T treatment in the same case as in fig. 5-8. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using CD5, CD2, CD7 and SSC to set a T NK1 cell gate in a four-dimensional four-parameter setting in a P2 gate; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8.
FIG. 10: complete remission bone marrow specimens of CD7 recovery 2 months after CD7 CAR-T treatment identical to figure 9, were analyzed by tube b. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD7/CD56, respectively.
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 antibody combinations used in this example were a first set of antibodies, a second set of antibodies, and a third set of antibodies:
in the first group of antibodies, the fluorescein markers of the anti-CD 99 antibody, the anti-CD 1a antibody, the anti-CD 34 antibody, the anti-CD 3 antibody, the anti-CD 4 antibody, the anti-CD 5 antibody, the anti-CD 8 antibody, the anti-CD 7 antibody, the anti-CD 45 antibody and the anti-CD 2 antibody are respectively: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, BV421, V500, BV 605; taking the ten monoclonal antibody reagents according to the volume ratio of 5: 5: 5: 5: 3: 2: 3: 3: 3: 3, mixing and filling in a first container.
In the second group of antibodies, the fluorescein labels of the anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody and anti-CD 45 antibody are, in order: PerCP-Cy5.5, APC-Cy7, BV421, V500; taking the five monoclonal antibody reagents according to the volume ratio of 5: 2: 3: 3: 3, mixing and filling in a second container.
In the third group of antibodies, the fluorescein of the anti-TdT antibody is labeled as FITC, and the fluorescein of the anti-cytoplasmic CD3 antibody is labeled as PE; taking the two monoclonal antibody reagents according to the volume ratio of 2: 5, mixing and filling in a third container.
Each of the antibodies in this example is commercially available, with TdT-FITC and cCD3-PE being Beckman Coulter, USA, and the remaining fluorescein-labeled antibodies being Becton Dickinson, USA.
And (3) optionally preparing hemolysin and placing the hemolysin into a fourth container, placing the membrane breaking agent A solution into a fifth container, placing the membrane breaking agent B solution into a sixth container, placing the PBS buffer solution into a seventh container, wherein the hemolysin, the membrane breaking agent and the PBS buffer solution are all commercially available, wherein the hemolysin and the membrane breaking agent are both products of Becton Dickinson company of America, and the PBS buffer solution is a product of Beckman Coulter company of America.
EXAMPLE 2 treatment of specimens
According to the cell count result, heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added into the first flow tube (tube A) to ensure that the added cell amount is about 2X 106Adding 37 mu l of ten cell membrane monoclonal antibody reagents marked by different fluorescein into a flow tube according to the table 1, fully mixing the cell membrane monoclonal antibody reagents with cell suspension, incubating the mixture for 15 minutes at normal temperature in the dark, adding 3ml of 1 Xhemolysin, incubating the mixture for 10 minutes in the dark to crack red blood cells, centrifuging the mixture at 1500rpm for 5 minutes, removing supernatant, adding 3ml of PBS buffer solution for washing, centrifuging the mixture at 1500rpm for 5 minutes, removing supernatant, and resuspending the cells by using 0.5ml of PBS buffer solution to obtain a processed sample which can be used for machine-on detection.
According to the cell counting result, heparin or EDTA anticoagulated bone marrow or peripheral blood sample is added into the second flow tube (tube B) to ensure that the added cell amount is about 2 x 106Adding 16 mu l of five cell membrane monoclonal antibody reagents marked by different fluorescein into a flow tube according to the table 1, fully and uniformly mixing the five cell membrane monoclonal antibody reagents with cell suspension, incubating for 15 minutes at normal temperature in the dark, adding 100 mu l of a membrane breaking agent A solution, incubating for 5 minutes at room temperature in the dark, adding 3ml of 1 x hemolysin, incubating for 10 minutes in the dark to lyse red blood cells, centrifuging at 1500rpm for 5 minutes to remove supernatant, adding 50 mu l of a membrane breaking agent B solution, 2 mu l of a cytoplasmic monoclonal antibody reagent TdT-FITC and 5 mu l of a cytoplasmic monoclonal antibody reagent cCD3-PE, incubating for 15 minutes at room temperature in the dark, then adding 3ml of PBS buffer solution for washing, centrifuging at 1500rpm for 5 minutes, removing supernatant, and resuspending cells by using 0.5ml of PBS buffer solution to obtain a processed sample which can be used for machine detection.
EXAMPLE 3 detection of specimens
The specimens processed according to the method of example 2 were tested on a 3-laser 10-color FACS Canto Plus or similar type flow cytometer from Becton Dickinson, USA, preferably taking 100 million cells per tube (at least 30 million suggested), and then the data was analyzed using diva 2.8 software and other software such as kaluza.
Wherein, when the flow cytometry is detected on the machine, the gate analysis is set according to the following modes:
firstly, a door is fixedly arranged: the FSC-A/H setting was used in order to remove the adhesion gate P1 and the FSC/SSC setting was used to set the live cell gate P2. CD45/SSC set the major blood cell gate, within the P2 gate; setting a T cell gate by multidimensional parameters: the first tube uses a CD7, CD2 and CD5 combined SSC to set a four-parameter four-dimensional T NK1 cell gate, and the second tube uses a CD7 and cCD3 combined SSC to set a three-parameter three-dimensional T NK 2 cell gate; (iii) other related phyla: in TNK1 cell gates, a tube A uses CD3/CD45 to set a CD3 negative CD45 strong positive NK cell gate, a CD3 positive CD45 strong positive CD3 gate and a CD4CD8DN cell gate in the CD3 gate, and T NK1 is divided into an NK and CD4CD8 double negative gate (an NK cell and CD4CD8DN cell gate) and a T cell gate; the tube B sets an NK cell gate in a three-dimensional mode by using three parameters including CD3, CD16 and CD56, and splits a T NK 2 cell gate into an NK cell gate and a T cell gate; can be observed more accurately, and eliminate interference of NK cells and CD4CD8DN cells (mainly TCR gamma delta cells); fourthly, multidimensional analysis: in the phylum of T NK1 cell, NK cell, CD4CD8DN cell and T cell, each subgroup of T and NK cells is observed by using a seven-parameter seven-dimensional map set by CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8; in a T NK 2 cell gate, an NK cell gate and a T cell gate of the vascular system, a six-parameter six-dimensional graph is set by using CD45/TdT/cCD3/CD3/CD16/CD56 to observe the distribution of each subgroup of T and NK cells; fifthly, immunity evaluation: within the nail canal CD3 gate, CD4 positive, CD8 positive, CD4CD8DNT, CD4CD8DPT were analyzed; in the angio NK cell gate, CD56briCD16negNK, CD56dimCD16posNK, CD56negCD16pos NK cells were analyzed; sixthly, further target screening: if an abnormal cell population is present in addition to the normal cell population, the MRD cell population is considered excluding other factors, and two-dimensional dot plots of CD4/CD34+ CD1a, CD5/CD2 and CD5/CD7 of the MRD cell population are displayed to evaluate the expression.
The marker combination can accurately lock target cells, and the multi-dimensional multi-parameter MRD can be detected simply, quickly and sensitively, so that a prototype can be provided for artificial intelligence; moreover, the immune evaluation can be carried out simultaneously, the severe immunosuppression state can be found in time, and the severe infection can be prevented; further target screening can be carried out, and guidance is provided for further treatment.
In this example, 20 samples of other disease-free complete remission bone marrow were selected for testing, and all of the complete remission bone marrow T cells had different ratios and different ratios of T and NK cells in each subset, but had regular expression profiles of antigen appearance time, expression intensity and combinations of two of them (fig. 1 and fig. 2). And meanwhile, multidimensional software analysis is carried out, so that a superposition effect can be achieved, the sensitivity is enhanced, and seven-parameter or six-parameter pictures of normal T and NK cells are displayed in one picture. Further setting gates to distinguish NK cell gate and CD4CD8DN cell gate and T cell gate of the first tube and NK cell gate and T cell gate of the second tube, respectively displaying seven-dimensional and six-dimensional pictures of seven parameters in each gate, and being capable of eliminating interference of NK cells and CD4CD8DN cells, thereby, tumor cells are more or less different from normal cells on various antigens and antigen combination expression modes, MRD positive can be judged according to the difference, and the proportion of malignant cells exceeds that of nucleated cells by 5%, and relapse is considered. If MRD cells are found, the nail tube may be further analyzed for expression of CD34+ CD1a, CD4, CD5, CD2, CD7 for selection of further treatment regimens. Simultaneously, the immune evaluation is carried out: the sub-populations of CD4 and CD8 cells in the vascular CD3, CD4/CD8 less than 0.1-0.2, are severe immunosuppression, and even if complete remission is achieved, immunosuppression should be reduced or stopped to prevent severe infection due to excessive immunosuppression; each subset of NK cells was analyzed in tube b.
This example provides as an example a case of complete remission after treatment of other unrelated diseases, relapse after treatment of T-ALL, a case of MRD positive CD7 positive before CD7 CAR-T, a case of CD7 loss but complete remission 1 month after CD7-CAR-T treatment, and a case of complete remission recovery of CD7 2 months after treatment.
In particular, FIG. 1 shows complete remission of bone marrow specimens after treatment of other unrelated diseases, for analysis in the nail canal. After the conventional setting of the detachment gate P1 and the live cell gate P2, the CD45/SSC in P2 sets each blood cell gate, and the CD7, CD2 and CD5 in P2 set the four-parameter four-dimensional radar chart to set the T NK1 cell gate. In TNK1, CD3/CD45 was used to set the CD 3-negative CD 45-strong-positive NK cell gate, CD 3-positive CD 45-strong-positive CD3 gate, and CD4CD8DN cell gate in CD3 gate, so that T NK1 was divided into NK cells and CD4CD8DN and T cell gates. In a T NK1 cell gate, an NK cell gate, a CD4CD8DN cell gate and a T cell gate, a seven-parameter seven-dimensional graph is set by using CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD8 to observe each subgroup of T cells and NK cells, coordinate positions are adjusted to enable normal groups of T cells and NK cells to be in scattered positions, and positions where MRD frequently appears on the right side and the lower side are vacated. And (3) immune evaluation: within the CD3 gate, CD4 positive, CD8 positive, CD4CD8DNT, CD4CD8DPT were analyzed. Although the patient was normal bone marrow, CD4/CD8=0.11, and the use of immunosuppressive agents was reduced for a severe immunosuppressive state.
Specifically, FIG. 2 shows the same normal bone marrow specimen as in FIG. 1, analyzed by tube B: conventional settings for the debonding gate P1, Living cell gate P2: CD45/SSC in P2 set each blood cell gate; the combination of CD7 and cCD3 SSC in the P2 gate sets a three-parameter three-dimensional radar chart to set a T NK 2 cell gate. Setting an NK cell gate in a three-dimensional manner by using three parameters of CD3, CD16 and CD56 in the T NK 2 cell gate, and splitting the T NK 2 cell gate into the NK cell gate and the T cell gate; and in a T NK 2 cell gate, an NK cell gate and a T cell gate, a TdT/cCD3/CD3/CD16/CD56/CD45 six-parameter six-dimensional radar chart is used for observing the distribution process of normal various subgroups of T and NK cells. And adjusting the angle to ensure that the normal T cells and NK cells of each subgroup are the T cells and the NK cells from top to bottom on the left side, and the right side is a region with MRD high frequency. And (3) immune evaluation: in NK cell lines, CD56briCD16negNK, CD56dimCD16posNK, CD56negCD16pos NK cells were analyzed.
It can be clearly seen that the traditional streaming requires dozens of complex two-dimensional dot pictures, and the respective relationships are clearly shown in fig. 1 and 2 by only 4 pictures. And can be performed separately for the immunological evaluation of T and NK cell subsets.
Specifically, FIG. 3 is a bone marrow sample from a T-ALL patient that relapsed after treatment, and is analyzed by nail tube analysis. The detadhesive adhesion cell gate P1 is set conventionally, FSC/SSC in P1 sets the live cell gate P2, CD7, CD2, CD5 in P2 set the four-parameter four-dimensional radar chart sets the T NK1 cell gate. In TNK1, CD3/CD45 was used to set the CD 3-negative CD 45-strong-positive NK cell gate, CD 3-positive CD 45-strong-positive CD3 gate, and CD4CD8DN cell gate in CD3 gate, so that T NK1 was divided into NK cells and CD4CD8DN and T cell gates. In the T NK1 cell gate, NK cell and CD4CD8DN cell gate, T cell gate, each subpopulation of T cells and NK cells was observed using a seven-parameter seven-dimensional map set with CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8. In FIG. 3, the tumor cell population evident on the right side is shown as MRD 1. And (3) immune evaluation: within the CD3 gate, CD4 positive, CD8 positive, CD4CD8DNT, CD4CD8DPT were analyzed. In this case, CD4/CD8=0.33, and treatment can be continued despite the existence of an immunosuppressive state.
Specifically, FIG. 4 shows bone marrow samples from the same example as FIG. 3, in a T-ALL patient, after treatment, and analyzed by tube B. The FSC-A/H setting is used for setting a de-adhesion cell gate P1, the FSC/SSC setting is used for setting a living cell gate P2 in P1, and the CD7 and the cCD3 in P2 gate are combined with SSC setting for setting a three-parameter three-dimensional radar chart for setting a T NK 2 cell gate. Setting an NK cell gate in a three-dimensional manner by using three parameters of CD3, CD16 and CD56 in the T NK 2 cell gate, and splitting the T NK 2 cell gate into the NK cell gate and the T cell gate; in T NK 2 cell gate, NK cell gate and T cell gate, TdT/cCD3/CD3/CD16/CD56/CD45 six-parameter six-dimensional radar map is used to observe the distribution of T and NK cells of each subgroup, in FIG. 4, the upper right significant tumor cell group can be seen, and the value is set as MRD 2. The proportion of each NK cell subset was analyzed in NK cells. CD16+/CD56+ NK account for NK cells 80.87%.
Specifically, FIGS. 5-10 are the same T-ALL patient CD7-CAR-T pre-and post-treatment assays. FIG. 5 and FIG. 6 show the CD7 positive period of MRD positive tumor cells before treatment; FIGS. 7, 8 are 1 month post CD7-CAR-T treatment, with complete remission but loss of T cells CD 7; FIG. 9, FIG. 10 is the complete remission phase of CD7 recovery 2 months after CD7-CAR-T treatment.
In particular, FIG. 5 shows bone marrow specimens from CD7-CAR-T pre-treatment MRD positive T-ALL patients, for nail tube analysis. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using CD5, CD7 and SSC to set a T NK1 cell gate in three-dimensional three parameters (CD 2 is not done at the time) in P2; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8. A distinct MRD1 positive cell population was seen in the center of the picture. CD4/CD8=2.12 in CD3 positive T cells.
Specifically, FIG. 6 shows the same sample as in FIG. 5, analyzed by the same tube B. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 cell gate is divided into an NK cell gate and a T cell gate; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD16/CD56, respectively. A distinct MRD2 positive cell population is seen below the T cells on the left side of the panel. The NK cells are all CD16-CD56+ NK.
Specifically, fig. 7 shows a complete remission bone marrow specimen, nail tube analysis, of CD7 loss 1 month after CD7 CAR-T treatment in the same case as fig. 5, 6. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, CD5, CD2, CD7 and SSC are used for setting a T NK1 cell gate in a four-dimensional four-parameter mode in P2, although CD7 is lost, the T cell gate can still be set by using CD5 and CD 2; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8. No tumor cells were observed. Normal T cells lost CD7, and within the T cell population, CD4/CD8= 0.93.
In particular, figure 8 shows a complete remission bone marrow specimen, in vitro analysis, of CD7 loss 1 month after the same CD7 CAR-T treatment as figure 7. FSC-A/H sets up single cell gate P1, use FSC/SSC to set up living cell gate P2 in P1, P2 in the door, cCD3, CD7, SSC three-dimensional three-parameter set T cell gate T NK 2, although CD7 is lost, do not influence and set up T cell gate similarly, in T NK 2, CD3, CD16, CD56 three-dimensional map set up NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD7/CD56, respectively. No tumor cell population was observed. The NK cells are all CD16-CD56+ NK.
In particular, fig. 9 shows a complete remission stage bone marrow specimen, nail tube analysis, of CD7 recovery 2 months after CD7 CAR-T treatment in the same case as fig. 5-8. FSC-A/H setting a single cell gate P1, using FSC/SSC to set a living cell gate P2 in P1, using CD5, CD2, CD7 and SSC to set a T NK1 cell gate in a four-dimensional four-parameter setting in a P2 gate; in T NK1 cell gate, CD3/CD45 is used for setting CD3 negative CD45briNK cell gate, CD3 positive CD45 strong positive CD3 gate and CD3 gate is used for setting CD4CD8DN cell gate; the T NK1 phylum is divided into NK cells, a CD4CD8DN phylum and a T cell phylum; seven-dimensional seven-parameter analysis of T NK1 phylum, NK cells, CD4CD8DN phylum, and CD45/CD99/CD34+ CD1a/CD3/CD4/CD5/CD 8. No tumor cells were observed. Most of the CD7 in normal T cells was recovered, and within the T cell population, CD4/CD8= 0.93.
Specifically, fig. 10 shows a complete remission stage bone marrow specimen, in vitro analysis, of CD7 recovery 2 months after the same CD7 CAR-T treatment as in fig. 9. FSC-A/H sets a single cell gate P1, FSC/SSC is used for setting a living cell gate P2 in P1, a P2 gate is used, three-dimensional three-parameter setting of cCD3, CD7 and SSC is performed on a T cell gate T NK 2, three-dimensional parameter setting of T NK 2 is performed on a CD3, CD16 and CD56 three-dimensional graph is performed on an NK cell gate; the T NK 2 phylum is divided into an NK phylum and a T cell phylum; six-dimensional six-parameter analysis was performed on T NK 2, T and NK phyla using CD45/TdT/cCD3/CD3/CD7/CD56, respectively. No tumor cell population was observed. CD16+ CD56+ NK accounted for 92.15% in NK cells.
Clinical validation was performed using the method of this example: MRD detection of T-ALL (the first tube lacks CD2, the second tube lacks CD 16) is carried out by adopting the antibody composition prototype scheme of the invention at the beginning of 2018 in Hebei Yanda Ludao-Doe-culture hospital, more than 2000 times of detection are carried out so far, and the rapid detection is tried to be carried out by adopting the multidimensional software analysis method of the invention at the beginning of 2021 in the basis of a classical two-dimensional dot diagram, and the bone marrow MRD detection is carried out for 128 times, wherein 18 positive cases and 110 negative cases,the simultaneous verification of the methods of morphology, genetics, clinical manifestations, follow-up and the like shows that the sensitivity of the method for detecting MRD is 10-4Sensitivity 94.74%, specificity 99.1%. The positive predictive value is 94.74 percent, and the negative predictive value is 99.1 percent. In addition, the multidimensional analysis method is simple and easy to implement, and can improve the analysis efficiency by 30-40%. 68 persons started to test MRD of bone marrow at 8 months of 2021, the test was positive for 8 cases and negative for 60 cases, and the sensitivity for testing MRD was 10-4Sensitivity and specificity reach 100%, and NK cell subset immunity evaluation and CD2 target measurement can be carried out. CD7-CAR-T treatment of T-ALL was performed starting at 12 months 2020, and 30 cases were completed to date with a 90% remission rate, and MRD could be effectively detected using this protocol.
Claims (12)
1. A reagent composition for flow cytometry detection of acute T lymphocyte leukemia after targeted therapy, wherein the reagent composition comprises a first set of antibodies, a second set of antibodies and a third set of antibodies, and a two-tube parallel protocol is used for flow cytometry detection, wherein:
the first group of antibodies comprises: anti-CD 99 antibody, anti-CD 1a antibody, anti-CD 34 antibody, anti-CD 3 antibody, anti-CD 4 antibody, anti-CD 5 antibody, anti-CD 8 antibody, anti-CD 7 antibody, anti-CD 45 antibody, anti-CD 2 antibody; each antibody in the first group of antibodies is used for being added into a first flow tube of which the sample to be detected is in a single cell suspension state;
the second group of antibodies includes: anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody, anti-CD 45 antibody; each antibody in the second group of antibodies is used for being added into a second flow tube of which the sample to be detected is in a single cell suspension state;
the third group of antibodies includes: anti-nuclear TdT antibody, anti-cytoplasmic CD3 antibody; each antibody in the third set of antibodies is used to add to the second flow tube after the second set of antibodies has been added and subjected to membrane rupture treatment.
2. Reagent composition according to claim 1, characterized in that each antibody is a monoclonal antibody.
3. The reagent composition of claim 1, wherein each antibody is a fluorescein-labeled antibody;
in the first group of antibodies, the fluorescein markers of the anti-CD 99 antibody, the anti-CD 1a antibody, the anti-CD 34 antibody, the anti-CD 3 antibody, the anti-CD 4 antibody, the anti-CD 5 antibody, the anti-CD 8 antibody, the anti-CD 7 antibody, the anti-CD 45 antibody and the anti-CD 2 antibody are respectively: FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, BV421, V500, BV 605;
in the second group of antibodies, the fluorescein labels of the anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody and anti-CD 45 antibody are, in order: PerCP-Cy5.5, APC-Cy7, BV421, V500;
in the third group of antibodies, the fluorescein label for the anti-TdT antibody was FITC and the fluorescein label for the anti-cytoplasmic CD3 antibody was PE or APC.
4. Reagent composition according to claim 1, characterized in that:
the first set of antibodies is anti-CD 99 antibody, anti-CD 1a antibody, anti-CD 34 antibody, anti-CD 3 antibody, anti-CD 4 antibody, anti-CD 5 antibody, anti-CD 8 antibody, anti-CD 7 antibody, anti-CD 45 antibody, anti-CD 2 antibody according to 5: 5: 5: 5: 3: 2: 3: 3: 3: 3 in a volume ratio;
the second group of antibodies is anti-CD 3 antibody, anti-CD 7 antibody, anti-CD 16 antibody, anti-CD 56 antibody, anti-CD 45 antibody according to 5: 2: 3: 3: 3 in a volume ratio;
the third group of antibodies was anti-TdT and anti-cytoplasmic CD3 antibodies according to 2: 5 volume ratio of the mixture.
5. A kit for detecting acute T-lymphocyte leukemia minimal residual disease and lymphocyte subpopulations after targeted therapy, comprising a first container, a second container and a third container, each container containing a first set of antibodies, a second set of antibodies and a third set of antibodies, respectively, of a reagent composition according to 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 a reagent composition according to any one of claims 1 to 4 for the preparation of a flow cytometric sample for the detection of acute T-lymphocyte leukemia following targeted therapy.
8. The use according to claim 7, wherein the process of preparing a flow cytometric sample for the detection of acute T-lymphocyte leukemia after targeted therapy comprises the steps of:
(1) respectively adding the sample to be detected into the first and second flow tubes 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 reagent composition of any one of claims 1-4 into the sample obtained by the step (1) and a second group of antibodies in the reagent composition of any one of claims 1-4 into a first flow tube, uniformly mixing, and incubating at room temperature in a dark place;
(3) adding the membrane breaking agent A solution into the second flow tube incubated in the step (2), and incubating at room temperature in a dark place;
(4) adding 1 Xhemolysin into the first flow tube incubated in the step (2) and the second 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) and removing supernatant;
(6) then adding the solution B of the membrane breaking agent and the third group of antibodies in the reagent composition of any one of claims 1 to 4 into the second flow tube after the supernatant is removed in the step (5), and incubating at room temperature in a dark place;
(7) and (4) adding a PBS buffer solution into the first flow tube after the supernatant is removed in the step (5) and the second flow tube after the room-temperature light-shielding incubation in the step (6) for washing, removing the supernatant after centrifugation, and resuspending the cells by using the PBS buffer solution to obtain the flow cell on-machine sample.
9. A device for detecting acute T-lymphocyte leukemia after targeted therapy, characterized in that the device comprises 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 a reagent composition of any one of claims 1-4;
the analysis unit is used for analyzing the detection result of the detection unit.
10. The device of claim 9, when used for detecting acute T-lymphocyte leukemia following targeted therapy, 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 a reagent composition according to any one of claims 1 to 4;
performing flow cytometry on the machine for detection; wherein, when the flow cytometry is detected on the machine, the gate analysis is set according to the following modes:
a first flow tube: setting a debonding cell gate P1 and a living cell gate P2 in a P1 gate to obtain a single living cell; setting each blood cell gate within P2 using CD45/SSC antibody; performing four-parameter four-dimensional gating by using CD7, CD2, CD5 and SSC in a P2 gate, and circling a T NK1 cell gate which is CD7 positive, CD2 positive and/or CD5 positive; setting an NK cell gate by using CD45 strong positive/CD 3 negative in the T NK1 cell gate; a CD3 cytogate is arranged in a T NK1 cytogate by using CD45 strong positive/CD 3 positive, and a CD4CD8 double-negative cytogate is arranged in a CD3 cytogate; dividing T NK1 cells into NK and CD4CD8 double negative cells and the T cell gate; and/or
A second flow tube: sequentially arranging a cell gate P1 for removing adhesion and a cell gate P2 for living cells; setting each blood cell gate within P2 using CD45/SSC antibody; in a P2 gate, three-parameter three-dimensional gating is carried out by using CD7, cytoplasmic CD3 and SSC, and any cell T NK 2 gate with positive mark is circled; in the T NK 2 gate, CD3/CD16/CD56 three-parameter three-dimensional setting is used for setting CD16+ CD56 positive/CD 3 negative NK cell gates, and the T NK 2 gate is divided into an NK cell gate and a T cell gate.
11. The apparatus of claim 10, wherein:
the gated analysis of the first flow tube further comprises:
the proportion of each subgroup of T cells of CD4 and CD8 is analyzed in a CD3 cell gate to carry out immune evaluation; and/or
After dividing the T NK1 cells into an NK and CD4CD8 double negative cell gate and a T cell gate, performing seven-parameter seven-dimensional gating on the T NK1 cell gate, the T cell gate, the NK and CD4CD8 double negative cell gates by adopting CD45/CD99/CD34+ CD1a/CD3/CD4/CD8/CD5 respectively, so that normal T cells and NK cells are distributed in different regions according to subgroups;
the gated analysis of the second flow tube further comprises:
in NK cell gate, analyzing NK cell subgroup of CD56briCD16neg, CD56dimCD16pos and CD56negCD16pos, and carrying out immune evaluation; and/or
After dividing the T NK 2 gate into an NK cell gate and a T cell gate, adopting CD 45/cytoplasmic CD 3/nuclear TdT/CD3/CD16/CD56 to respectively carry out six-parameter six-dimensional gating in the T NK 2 cell gate, the NK cell gate and the T cell gate, so that normal T cells and NK cells are distributed in different regions according to subgroups.
12. The apparatus of claim 11, wherein:
the gated analysis of the first flow tube further comprises: if the seven-parameter seven-dimension gate shows a cell group except normal cells, the seven-dimension gate is defined as MRD1 gate; performing two-dimensional dot diagram analysis of CD4/CD34+ CD1a and CD5/CD7 in MRD1 gate; and/or
The gated analysis of the second flow tube further comprises: if the six-parameter six-dimension gate shows a cell group except normal cells, the cell group is defined as MRD2 gate, and whether MRD is positive is further judged.
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| CN114778857A (en) * | 2022-06-17 | 2022-07-22 | 信纳克(北京)生化标志物检测医学研究有限责任公司 | Reagent composition for detecting nonhematopoietic system tumor and application thereof |
| CN116593699A (en) * | 2023-07-13 | 2023-08-15 | 天津市肿瘤医院空港医院 | Antibody composition for detecting CD39 molecules on surface of T cells by flow cytometry |
| CN117384289A (en) * | 2023-11-09 | 2024-01-12 | 济南金域医学检验中心有限公司 | Antibody combination and kit for detecting T cell large granule lymphocyte and application of antibody combination and kit |
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