CN113777327B - Antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof - Google Patents
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Abstract
The invention provides a reagent composition for leukemia/lymphoma typing, which comprises 19 antibodies. The optimized antibody combination, the fluorescence labeling combination of the corresponding antibodies and the result interpretation method only need to use 19 antibodies and one tube cell for once sample loading, can comprehensively and efficiently carry out preliminary screening on AML, ALL-B, ALL-T, MPAL, NHL-B, NHL-T, PCN, NHL-NK and chronic myeloid tumor, and achieve the purpose of definite diagnosis on AML, ALL-B, ALL-T, MPAL, NHL-B and NHL-T, PCN. The expression of tumor cell antigens in the samples was analyzed using the 19 antibody combinations of the invention to determine Leukemia Associated Immunophenotypes (LAIPs) in the antibody combinations that can be used for post-treatment Minimal Residual Disease (MRD) monitoring.
Description
Technical Field
The invention relates to the field of antibody medicines, in particular to an antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof.
Background
Leukemias and lymphomas are malignant tumors of the hematopoietic system that are hindered from differentiating at different stages to form the corresponding subtypes. The clinical classifications of leukemia and lymphoma are numerous, with the WHO classification in 2017 on hematological and lymphoid systems mainly including Acute Myeloid Leukemia (AML) and related myeloid precursor cell tumors (table 1), precursor lymphoid tumors: including B lymphoblastic leukemia/lymphoma (B-ALL/LBL), T lymphoblastic leukemia/lymphoma (T-ALL/LBL), acute series of non-clear leukemias (MPAL): including T/marrow, B/marrow mixed, etc., chronic myeloid tumors (Table 2), mature B cell tumors, mature T and NK cell tumors, Hodgkin's Lymphoma (HL), immunodeficiency-associated lymphoproliferative disorders, and histiocytic and dendritic cell tumors in total 9 major categories. Mature B and T/NK lymphomas are also known as non-Hodgkin's lymphoma (NHL). Plasma cell derived clonal tumors (PCNs) are listed within the broad class of mature B cell tumors. T, B-NHL are further divided into 20-30 subtypes according to pathological features. Therefore, the hematological lymphatic system tumor comprises nearly hundreds of different diseases, and the classification is very complex.
WHO version 1.2017 regarding Acute Myeloid Leukemia (AML) and related myeloid precursor cell tumor classification
Classification of WHO version 2.2017 on Chronic myeloid tumors
Currently, the diagnosis of hematological lymphatic system tumors is mainly based on the comprehensive diagnosis of MICM by morphology/pathology (M), immunotyping (I), genetics (C) and genes (M).
Flow Cytometry (FCM) is a detection means capable of performing quantitative analysis on a single cell, has the advantages of rapidness, high precision, multiple parameters and the like, and is one of the most advanced cell quantitative analysis methods at present. The immunophenotyping of hematological tumors such as leukemia and lymphoma by FCM is one of the important means for the diagnosis of hematological tumors. The FCM is used for comprehensively and accurately diagnosing 9 types of leukemia/lymphoma, nearly 90 antibodies (about 23 types of myeloid-related markers, about 30 types of B-related and plasma cell-related markers, about 22 types of T-related markers and about 14 types of NK-related markers) are required in total, and if all the antibodies are used at one time, comprehensive detection is carried out on 9 types of diseases, the total cost is high, and the economic burden of patients is heavy. Therefore, in the clinical examination at the present stage, a two-step method is mostly adopted, namely, in the first step, the primary screening of the AML, ALL-T, ALL-B, MPAL, NHL-B, NHL-T, NHL-NK, plasma cell diseases and chronic myelogenous diseases on the samples to be tested for 9 kinds of tumors is carried out by adopting relatively few antibodies. And selecting corresponding antibodies according to the classification result of the first step to carry out the second step of detection, wherein the second step mainly aims at disease subtype classification, trace residual disease monitoring and prognosis correlation and screening treatment targets and gene abnormality correlation marks. By two-step detection, accurate and comprehensive diagnosis of each type of leukemia and lymphoma can be realized by using relatively few antibodies. However, the principle of the two-step method is preferably applied, which antibodies are selected to be combined in each step, and which fluorescein matched antibodies are selected is a complex process, and the design of the combination schemes can be completed by highly experienced personnel.
The European Union of 2012 introduced a set of 8-colored EuroFlow antibody combinations for immunotyping acute and chronic leukemias and lymphomas in a two-step method (Van Dongen J. et al, EuroFlow antibody panels for stable n-dimensional flow cytometric immunophenotyping of normal, reactive and monoclonal leucocytes. Leukemia 2012, vol 26.1899-1907), which protocol included three screening tubes, ALOT (acute leukemia initiator tube), for a total of 8 antibodies; LST (lymphoma screening tube) 12 antibodies in total; PCD (plasma cell clonal disease) detector tube, 16 antibodies in total. The primary screening can be carried out on AML, MDS, ALL-T, ALL-B, NHL-B, NHL-T, NHL-NK, PCN and MPAL 9 by three screening tubes. In 2015, "Chinese experts consensus on four-color flow cytometry for acute leukemia immunophenotyping" was published in the Chinese journal of hematology (vol36: 265) 271, and 2 tubes of 4-color combinations were used for primary screening of AML, ALL-B, ALL-T and MAPL, not including screening of NHL-B, T, NK and plasma cell tumors. The method cannot screen all blood tumor patients through one tube. At present, the clinical routine method is a one-tube 10-color screening combination of CD7/CD33/CD117/CD19/CD10/CD34/CD38/CD 56/CD 5/CD45 for acute and chronic leukemia and lymphoma primary screening. The disadvantage is the lack of identification of the most specific series of antibodies, such as intracellular cCD3, cCD79, cCD22, cCPO, and markers for judging the clonality of B-cells and T-cells: kappa/lambda and TRBC1, a judgment error inevitably occurs, and a third step of detection is needed, so that the antibody and the time are wasted.
Therefore, the antibody combination in the prior art can not achieve the purpose of comprehensively and accurately diagnosing acute and chronic leukemia and lymphoma by a single tube, and the antibody combination which can comprehensively, accurately, economically and efficiently carry out the immunophenotyping (especially primary screening) of the hematological tumor needs to be designed at present, so that the practical clinical difficulty is solved, and the benefit is brought to patients.
Disclosure of Invention
The invention aims to provide an antibody composition for the first step of immunological typing of 9 major hematological tumors including AML, ALL-T, ALL-B, MPAL, NHL-B, NHL-T, NHL-NK, PCN and chronic myelogenous diseases, a reagent prepared from the same and a kit, so as to achieve the aim of carrying out comprehensive immunoassay detection on the hematological tumors.
In order to realize the purpose, the specific technical scheme of the invention is as follows:
the invention provides a reagent composition for leukemia/lymphoma typing, which comprises an anti-cell membrane antigen antibody and an anti-cell intracellular antigen antibody, wherein:
the anti-cell membrane (m) antigen antibody includes 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 10, anti-CD 56, anti-TRBC 1 antibodies;
the anti-intracellular (c) antigen antibodies include anti-cd 79a, anti-clamba, anti-cKappa, anti-cd 22, anti-cd 3, anti-cMPO antibodies, and anti-nTdT antibodies.
The anti-nTdT antibody is an anti-nuclear (n) antigen antibody.
Preferably, the antibodies are all monoclonal antibodies.
Preferably, the antibodies are all fluorescein dye-labeled antibodies;
in some embodiments, the fluorescein dye comprises BV785, BV750, BV711, BV650, BV605, BV421, BV510, APC-Fire750, PE-Cy5, PE-Cy7, PE-Fire700, PE-Dazle 594, Alexa Fluor 700, APC, Alexa Fluor 647, eFluor 450, FITC. Any match of the fluorescein dye to the antibody.
In specific embodiments, in the anti-cell membrane antigen antibody, the fluorescein markers of the 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 are, in order: BV785, BV750, BV711, BV650, BV605, BV421, BV510, APC-Fire750, PE-Cy5, PE-Cy7, PE-Fire700 and PE;
in the anti-intracellular antigen antibody, fluorescein labels of anti-cCD 79a, anti-cLambda, anti-cKappa, anti-cCD 22, anti-cCD 3, anti-cCPO antibody and anti-nTdT antibody are respectively PE, PE-Dazle 594, Alexa Fluor 700, APC, Alexa Fluor 647, eFluor 450 and FITC in sequence.
The cCD79a is a B cell marker and the TRBC1 is a T cell marker, both of which are not expressed simultaneously, and a fluorescent label may be used.
In a second aspect, the invention provides a kit for the immunophenotyping of acute and chronic leukemia and lymphoma, said kit comprising a first container and a second container, said first container comprising any one of said anti-cell membrane antigen antibodies described above, said second container comprising any one of said anti-cell intracellular antigen antibodies described above.
Further, the kit also comprises other containers, and the other containers are respectively filled with erythrocyte lysate and buffer solution.
In some embodiments, the acute and chronic leukemia and lymphoma diseases include Acute Myeloid Leukemia (AML), acute lymphoblastic leukemia-B line (ALL-B), acute lymphoblastic leukemia-T line (ALL-T), acute mixed-phenotype leukemia (MPAL), non-hodgkin's lymphoma-B cell type (NHL-B), non-hodgkin's lymphoma-T cell type (NHL-T), non-hodgkin's lymphoma-NK cell type (NHL-NK), plasma cell clonal tumor (PCN), chronic myeloid tumor.
In some embodiments, the chronic myeloid tumor comprises myeloproliferative neoplasm (MPN), mastocytosis, eosinophilia, and myelodysplastic syndrome (MDS)/MPN, and MDS).
The kit of the invention is used for detecting through 18-color flow cytometry based on 1 tube of 19 antibody combinations, achieves the purposes of rapid, efficient, comprehensive and accurate primary classification and diagnosis of samples to be detected, and provides necessary conditions for the second step of selecting series related antibodies to perfect immune typing detection.
The kit of the invention can specifically achieve the following aims:
1. the sample to be tested can be preliminarily screened into AML, ALL-T, ALL-B, MPAL, NHL-B, NHL-T, NHL-NK, PCN and chronic myelogenous disease 9. Class 10 is no obvious abnormality, except hematological tumors. And the diagnosis of AML, ALL-T, ALL-B, MPAL, NHL-B, NHL-T and PCN 7 large tumors is determined, namely, the 7 diseases can be judged whether to exist in one test.
2. The trace residual disease (MRD) monitoring markers can be screened, and the MRD monitoring markers of 9 main tumors including cross-series antigen expression and antigen expression abnormality, such as AML, ALL-B, ALL-T, NHL-B, NHL-T, NHL-NK, PCN, chronic myeloid tumor and MPAL, can be screened.
The appearance of the cross-series antigen expression and abnormal antigen expression is an important mark for distinguishing leukemia cells from normal cells and leukemia cells, and is called leukemia-associated immunophenotype (LAIP). The presence or absence of MRD is detected by monitoring the patient for the presence of LAIP-expressing leukemic cells after treatment.
In a third aspect, the present invention provides a system for leukemia/lymphoma typing comprising a detection section and an analysis section, wherein:
a detection part, comprising the reagent composition of any one of the above, for detecting the antigen expression of the sample to be detected by 1-tube 18-color flow cytometry;
and an analysis section for analyzing the detection result of the detection section to preliminarily determine the types of acute and chronic leukemia and lymphoma diseases.
In some embodiments, the system, when used to detect acute and chronic leukemia and lymphoma immunotypes, comprises the steps of:
processing a sample to be detected by using the reagent composition to prepare a flow cytometer sample; performing flow cytometry on the machine for detection;
wherein, when the flow cytometry is detected on the machine, the gate is arranged according to the following mode:
setting a R1 viable cell gate, removing fragments and dead cells, setting a lymphocyte gate, a granulocyte gate, a monocyte gate, a juvenile cell gate, a nucleated erythrocyte gate and an eosinophil gate by using CD45/SSC in the R1 gate; and R1 or multiple markers in each cell gate group are combined and gated to analyze the development patterns of T cells, NK cells, B cells, plasma cells, myeloid progenitor cells, granulocytes and/or monocyte gates compared with normal cells, and the tumor cells are found out.
In a third aspect, the invention provides the use of said reagent composition or said kit or said system in the manufacture of a primary screening product for leukemia/lymphoma immunotyping.
Preferably, the products include reagents, kits and systems and the like.
In a fourth aspect, the invention provides the use of the reagent composition for screening leukemia associated immunophenotypes for post-treatment Minimal Residual Disease (MRD) monitoring in patients with acute and chronic leukemia and lymphoma diseases.
In the present invention, the acute and chronic leukemia and lymphoma disease typing includes: AML, ALL-B, ALL-T, MPAL, NHL-B, NHL-T, NHL-NK, PCN and chronic myeloid disorders.
Based on the technical scheme, the invention has the following beneficial effects:
the invention discloses a method for screening the immunotyping of blood tumor by combining 1 tube of 19 antibodies, which basically covers the current common blood tumor and has the characteristics of comprehensiveness, accuracy, sensitivity, time saving and economy. Wherein the aim of definite diagnosis can be achieved for 7 types of AML, ALL-B, ALL-T, MPAL, NHL-B, NHL-T and plasma cell clonal tumors. According to the detection result and the hemogram, the primary screening can be carried out on several types of tumors of NHL-NK and chronic myeloid tumors, and the detection direction of the next step is determined. Diagnostic sensitivity and specificity to MDS is above 70% and 92%.
The factors for selecting 19 antibodies in the 1-tube channel are very complicated, and according to the judgment standard of the 2017 version WHO on the series (table 3), the antibody with the strongest series specificity is selected firstly; meanwhile, the method comprises the step of determining the clonality marks of the B cells, the plasma cells and the T cells, and the accuracy and the specificity to the blood tumor are enhanced. Secondly, the antibodies of the cell nucleus and the cell are all included in the first step of detection, and the second step of detection has no intracellular antigen detection basically, so that the repeated intracellular detection process of the second step of detection is avoided, and the overall process and time are reduced. The invention provides an effective method for comprehensive, accurate, sensitive, time-saving and economical blood tumor immunophenotyping diagnosis.
The currently widely used one-tube 8-10 color antibody combination screening method has poor specificity and accuracy because the most specific antibodies of the series of cCD3, cCD79a, cCD22 and cMCPO are not included, and moreover, lambda/Kappa and TRBC1 are not included to judge the clonality markers of B cells, plasma cells and T cells. Each tube is marked with 8-10 antibodies, so that for comprehensive screening, only 2-3 tubes can achieve the one-tube completion of the invention. The 2-3 tube screening has the defects that more cells are needed, the door antibodies need to be used repeatedly, the detection cost is increased, the operation is complicated and troublesome, and as the antibodies are dispersed in a plurality of tubes, the antibodies in different tubes are difficult or cannot be integrated together, the analysis capability is limited, the detection accuracy and sensitivity are influenced, and the comprehensive and accurate detection capability of the invention cannot be achieved. If the cost is reduced, the use amount of the antibody can only be reduced, but a lot of information is lost, so that the primary screening result is not comprehensive and accurate.
Drawings
FIG. 1 is a diagram of an example of a normal human bone marrow sample showing a 4-parameter gating assay for T-cell, B-cell, NK-cell, plasma cell, CD117+ naive myeloid cell, granulocyte, monocyte, nucleated red blood cell and ogata scores.
FIG. 2 is a graph of the detection of acute mixed phenotype leukemia-myeloid/T lines (MPAL-M/T), wherein FIG. 2a is a method using 1 tube of conventional 10-color antibody combination; FIG. 2b is a method of using the 1 tube 19 antibody combination of the present invention.
FIG. 3 is a diagram showing the detection of Acute Myeloid Leukemia (AML).
FIG. 4 is a chart of acute lymphoblastic leukemia-B line (ALL-B) detection.
FIG. 5 is a graph of acute lymphoblastic leukemia-T line (ALL-T) detection.
FIG. 6 is a map of non-Hodgkin lymphoma-B cell type (NHL-B).
FIG. 7 is a map of non-Hodgkin lymphoma-T cell type (NHL-T) detection, wherein FIG. 7a is a method using 1 tube of 19 antibody combinations of the present invention; FIG. 7b is a method using 1 tube of conventional 10 color antibody combination.
FIG. 8 is a graph showing the detection of clonal plasma cell tumor (PCN).
FIG. 9 is a graph of myelodysplastic syndrome (MDS) measurements.
FIG. 10 is a graph showing the detection of Chronic Myelogenous Leukemia (CML).
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
All materials, reagents and the like in the following examples are commercially available unless otherwise specified.
The invention adopts 1 tube 18 color flow cytometry to judge the leukemia/lymphoma cells in series and differentiation stages from samples such as marrow fluid, breast/ascites, peripheral blood, lymph nodes, tumor tissues and the like of clinical patients, thereby achieving the immunophenotype of blood tumor diseases.
The expression of the antigen in normal cells of the 19 antibodies included in the present invention is shown in Table 3.
Table 3.19 cells expressing the antigen and the effect:
the criteria for the diagnosis of ALL-B, ALL-T, AML, MPAL, chronic myeloid tumors NHL-B, NHL-T, PCN and NHL-NK using the 19 antibody combinations of the present invention are shown in tables 4 to 6:
TABLE 4 diagnostic criteria and phenotypic characteristics of different leukemias/lymphomas
Note: the cKappa/clambda ratio <0.3 or >3.0 is restriction expression and is a clonal marker.
TABLE 5.2017 WHO Standard for diagnosis of MPAL
For a specific Ogata 4 scoring system, reference is made to the Della Porta M et al, Multicenter evaluation of a reproducible flow cytometry score for the diagnosis of low-grade myeloplastic syndromes, results of an European Leukemia NET study, Haematologica.201297 (8): 1209-1217. The 4 parameters and criteria tested are shown in Table 6. And 2 or more points of diagnosis of MDS. The sensitivity and specificity of the Ogata scoring method for diagnosing MDS are 69-70% and 92-93%, respectively.
TABLE 6 ogata scoring system
EXAMPLE 1 formulation of reagents
This example uses a combination of antibodies as follows, including membrane antibodies: CD38-BV785, CD3-BV750, CD10-BV711, CD33-BV650, CD5-BV605, CD19-BV421, CD45-BV510, CD56-PE-Fire700, CD7-APC-Fire750, CD117-PE-Cy5, CD34-PE-Cy7 and TRBC 1-PE; intrabodies include: cCD22-APC, cCD3-Alexa Fluor 647, cCD79a-PE, cLambda-PE-Dazle 594, nTdT-FITC, cKappa-Alexa Fluor 700, cCPO-eFluor 450, all of which are commercially available as such, and antibodies of the present embodiments are commercially available from BD company, Biolegend, Beckman, Thermo company.
The 19 kinds of quantitative antibodies are divided into 2 parts according to membrane and intracellular antibodies, and the 2 parts are mixed and filled in 2 containers respectively for the immunophenotyping marking of samples.
The kit for detecting leukemia and lymphoma also comprises erythrocyte dissolving solution and PBS, wherein the erythrocyte dissolving solution can be self-prepared or commercially purchased (such as BD company).
Example 218 color flow cytometry analysis of leukemia/lymphoma disease immunophenotypes
1. Experiment main material and instrument
(1) Materials: 10 XPBS buffer, hemolysin for flow cytometry (BD Co.);
(2) the instrument comprises the following steps: CytekNL-3000 model full spectrum flow cytometer with three lasers of 405nm, 488nm and 635nm and 38 fluorescence detectors. A table low-speed centrifuge and a vortex mixer.
2. Method of producing a composite material
2.1 sample collection:
1-3mL of the obtained human marrow fluid is immediately placed in a heparin anticoagulation tube and quickly inverted for a plurality of times to prevent the sample from being coagulated, various cells such as hydrothorax, ascites, lavage fluid and the like, the collected human marrow fluid is sent to a laboratory as soon as possible, and the sample is placed at 4 ℃ for refrigeration and preservation. Flow Cytometry (FCM) assays must be performed within 48 hours, as described.
2.2 sample preparation procedure:
1) cell counting: when FCM detection is adopted, ensuring that cells reach a certain number, taking 10 mu l of bone marrow, adding 150 mu l of PBS, uniformly mixing, counting the number of cells per microliter by using Merrill FCM mindray, and adjusting the cell concentration to be 5-10 x10 according to a detection result ^6 Adding 50-100 μ l of cells into a test tube at a concentration of 100 μ l;
2) membrane surface antigen staining: adding corresponding fluorescein-labeled antibody premix for membrane labeling and bone marrow samples into each tube respectively, mixing well, and incubating at room temperature in dark for 15 min;
3) and (3) membrane penetration: adding 100 μ l of the solution A, and allowing to act at room temperature for 5 min.
4) Hemolysis: adding 2ml of 1 xFACS hemolysin, mixing uniformly by low-speed vortex, and incubating for 8-10 min at room temperature in a dark place; the supernatant was discarded after centrifugation at 300g for 5 min.
5) Washing: adding 1ml PBS lotion, centrifuging and washing for 5min at 300g for 2 times;
6) fixation and intracellular/nuclear antigen staining: adding 100 μ l of membrane-penetrating agent B solution, fixing cell membrane and penetrating, simultaneously adding corresponding fluorescein labeled antibody, mixing, and incubating at room temperature in dark place for 15 min.
7) Washing: the supernatant was discarded, and 1ml of a solution containing 0.1% NaN was added 3 And 1% -2% BSA PBS lotion, 300g centrifugal washing for 5 min;
8) loading on a machine for detection: after discarding the supernatant, 200. mu.l of PBS was added for detection.
2.3 determining the optimum voltage and compensation:
the voltage was set according to the conventional operating method of the spectral flow cytometer, and the unistain samples were prepared for instrument setting with reference to the fluorescent color matching of the kit.
2.4 Instrument setup, calibration and quality control:
the CytekNL-3000 is started to preheat the machine for more than 20min and is washed by deionized water, and the inner quality control products are detected to ensure that all detection values are in a control range. Calling AL-PANEL sample loading, and collecting data.
2.5, detection on a computer: according to the set instrument conditions, 5-10 ten thousand cells are obtained from each tube. If the detection can not be carried out on the machine in time, 0.5ml of 1% paraformaldehyde is added, the mixture is uniformly mixed and then is stored in a refrigerator at 4 ℃, and the detection is finished within 24 hours.
2.6 data analysis:
(1) the data were analyzed using Kaluza software, gating different types of tumors as follows:
the present invention uses a flow chart of an example of a normal bone marrow specimen (FIG. 1) to illustrate the gated analysis of each cell.
First gates A and B and R1 were set to remove debris and dead cells stepwise, with the R1 gate being a live unicellular gate (FIG. 1 a: first row). The R1 gate was selected to establish a CD45/SSC map, setting the lymphocytogate (R2 or gonorrhea), the granulocytic gate (R4 or granulocytic), the monocytic gate (R5 or monogranulocytic), the naive phylum (naive or R3), the nucleated erythrocytic gate (red or R6), the eosinophilic phylum (acid), and showing the ratio of the cells of each gate. 20-40% of lymphocytes, 2-8% of monocytes, 40-60% of granulocytes, 2-15% of nucleated erythrocytes and less than 5% of immature cells in normal bone marrow.
Each population of cells was analyzed for negative and positive expression of antigen and the proportion of positive antigen expression was calculated, < 20% defined as negative (-) or described as no expression. More than or equal to 20% is defined as positive (+), divided into 2 cases, positive cells > 60% are described as expression, positive cells are described as partial expression (part +) in 20% -60%; since each normal human is positive or negative on different cells and the intensity of antigen expression is stable and predictable, the intensity of antigen expression in normal cells is used as a standard, and when the expression of antigen in abnormal cells is stronger than that in normal cells, it is defined as strong expression (st +), otherwise it is weak expression (dim). According to the expression rule of different series of cell antigens of normal bone marrow, the existence of abnormal cell population and the antigen expression of abnormal cells are known, and finally the origin of the abnormal cells is judged according to the expression of specific series markers.
First, T and NK cells within the lymphocyte gates were analyzed (FIG. 1 a: second row): in the CD3/CD56, the ratio of gate-gated T cells is shown for CD3+ T cells, gate-gated NKT cells for CD3+ CD56+ NKT cells, and gate-gated NK cells for CD3-CD56 +. The normal T cells account for 50-70%, the NKT cells account for 20-30%, and the NK cells account for 10-40%. A CD3/TRBC1 map is established in a T cell gate, a TRBC1+ cell gate is set, the intra-gate proportion (the normal value is 15% -85%) is displayed, and if the intra-gate proportion is abnormal, the clonality T can be judged. In R1 gate, cCD3+ gate is set, which shows the expression of CD34 and nTdT cells in gate, and can judge the juvenile cells and proportion, and the normal bone marrow should not have the juvenile T cells, if abnormal, can judge ALL-T.
B cells in the lymphocyte gate were analyzed (FIG. 1 a: third row): in the CD5/CD19 plot, all CD19+ B cells were gated as mature B and CD19+ CD5+ B cells were gated as 19+5+ gates and the ratio is shown. Normal lymphocytes have B cells 10-20% and CD19+ CD5+ B cells < 5%. The 19+10+ mature B gate was set in the cCD22/CD10 map, showing the expression of cKappa, clambda in the cells within the three gates mature B, 19+5+ and 19+10+ mature B, respectively, when the ratio <0.3 or >3.0, indicating a clonal B cell. CD5+ NHL-B, CD10+ NHL-B and CD5-CD10-NHL-B were determined.
The R1 gate CD19+ ALL B cells (FIG. 1 a: fourth row) were analyzed to establish CD45/CD10, CD34/CD10, nTdT/CD34, CD10/CD38 maps, if these antigens are phenotypically abnormal, such as CD10-CD38+, CD10-CD34+, nTdT-CD34+, ALL are phenotypes of abnormal naive B cells, when > 20% of abnormal naive B cells, ALL are judged as ALL-B.
R1 gatekeeper plasma cells (FIG. 1 a: fifth row) were analyzed, CD38st + plasma cells gated, showing the expression of the intragated cells CD19/CD56, and CD56+ and CD 56-plasma cells gated, showing the expression of the three intragated cells cKappa, clambda, respectively, when the cKappa/clambda ratio <0.3 or >3.0, indicating clonal plasma cells. The result is judged to be PCN.
FIG. 1b shows a gated assay for CD34+ and CD117+ progenitor cells, granulocytes, and monocytes. The same specimen as 1 a.
For progenitor cells (FIG. 1b, first/second/third row), the detailed analysis is as follows,
analysis of Ogata 4 terms parameters (first row): a gate was set for CD34+ cells and the proportion of CD19+ B progenitors and the proportion of CD117+ myeloid progenitors in the gate were analyzed. The lymphocyte to myeloid progenitor CD45 ratio and lymphocyte to granulocyte SSC ratio were also analyzed. Scoring criteria are shown in table 6.
The proportion of CD38-, CD7+ and CD56+ cells in CD34+ cells was analyzed simultaneously, with normal values < 40%, < 20%, < 10%, respectively. In MDS, an increase in the proportion of CD38-, CD7+ and CD56+ cells may occur.
CD117+ CD34+ naive myeloid cells (as myeloid progenitors in the Ogata score) in the R1 phylum were analyzed for proportion and phenotype (FIG. 1b, third row). Normal values are between 0.1 and 1%. The proportion of CD117+ CD34+ increased > 20% in AML. In MDS the proportion of CD117+ CD34+ naive myeloid cells can be normal or increased by > 1%, but less than 20%.
A gate is arranged on CD117+ cells, non-specific cells are removed step by step, and the CD34/CD33 graph of the cells in the gate is shown, and the normal graph is a ring. The inventors have found that the CD34/CD33 pattern abnormalities (see fig. 9) in MDS are non-circular and discontinuous circles due to abnormalities (enhancement, reduction or deletion) in the expression intensity of CD34 and CD 33. By using the graph, whether the phenotype of the myeloid progenitor cell is abnormal or not can be easily judged, and the diagnosis of MDS is very helpful.
The ogata scores 2 and above for diagnosis of MDS, with <2 patients scored for ogata, if increased proportion of CD38-, CD7+ and CD56+ cells and CD34/CD33 pattern abnormalities suggest MDS, thus, increased sensitivity and specificity to MDS diagnosis.
Granulocytes (FIG. 1b, fourth), specifically analyzed as follows,
the granulocytes are graded and gated, the R4 gate mesoplasmic cells, monocytes and CD34+ cells are removed, finally the granulocytes are obtained, the relation of CD10+ (normal 40-60%), CD56+ cell proportion (normal < 10%), and CD33/CD10 is analyzed, and the early granulocytes are CD33 slightly stronger + CD 10-in normal. Decreased or abolished CD10+ cell proportion, increased CD56+ cell proportion, and abnormal CD 33-CD 10 relationship, such as slightly stronger CD33+ CD10+, may be observed with AML, MDS, or CML.
The monocytes were gated in stages (fig. 1b, fifth row), CD19+, CD3+ and CD10+ cells were removed from R5 gate, CD33st + CD10 "cells were monocytes, and the expression of CD117, CD34, CD56 cells within the monocytes was analyzed. The increase in monocyte proportion (normal value 2-8%) and CD117, CD34 proportion (normal < 1.0%) and monocyte CD56+ cell proportion (> 40%) occurred with CMML and AML-M4/5. To specifically identify CMML, MDS or AML, a comprehensive analysis is required (see table 4).
(2) MRD marker screening:
the antibody combination can be used for screening MRD monitoring markers of AML, ALL-B, ALL-T, NHL-B, NHL-T, NHL-NK, PCN, chronic myeloid tumor and MPAL 9 main tumors. Including abnormalities in the expression of the cross-series of antigens and the amount of the antigen phenotype. These aberrant phenotypes are collectively referred to as leukemia-associated immunophenotypes (LAIPs). Used for monitoring the presence of LAIP + cells in the bone marrow of each patient after treatment and determining the presence of MRD.
Detection of LAIP: comprises that
1) Expression of cross-series antigens: the expression of lymphoid antigens in myeloid leukemia or lymphoid leukemia. For samples diagnosed as AML, it appears that myeloid cells simultaneously express T-lineage markers: CD7, CD5, CD56 or B-related markers: CD19, nTdT, cpcd 79 a; ALL cells simultaneously expressed CD33, a CD117 myeloid-related marker. Plasma cells of PCN express the myeloid markers CD33, CD117 and NK marker CD 56. General antigens > 20% were determined to be positive, LAIP +.
2) Abnormal expression of antigen: the cell expressed antigens of AML, ALL-B, ALL-T, NHL-B, NHL-T, NHL-NK, PCN, chronic myeloid tumor and MPAL 9 general tumors are detected to determine whether the expression is enhanced, reduced and deleted. E.g., whether CD34, CD117, CD38, CD33 is abnormally deleted in AML samples, and if positively expressed, whether expression is increased or decreased. For PCN, whether CD19 is negative, whether CD5, CD7, CD38, CD56 expression is absent or expression intensity is abnormal in T-line tumors. Whether the expression intensity is abnormal and the expression is lost in the B-series tumor CD19, CD10, cCD22, cCD79a, CD38, CD34 and nTdT. The judgment principle of other tumors is the same, and the judgment is carried out by taking normal cells of the same series at the same differentiation stage as a standard.
2.7 results
The results of gate-setting analysis performed by the above method using one tube of 19 antibodies of the present invention to detect 70 bone marrow samples showed that 11 of the samples were determined to be ALL-B, 2 of the samples were determined to be ALL-T, 20 of the samples were determined to be AML, 3 of the samples were determined to be MPAL, 10 of the samples were determined to be NHL-B, 5 of the samples were determined to be PCN, 4 of the samples were determined to be NHL-T, and 11 of the samples were determined to be chronic myelogenous diseases, which included: supporting 3 cases of MDS, 2 cases of CMML, 2 cases of MPN, 1 case of suspicious MDS and 3 cases of preliminary judgment CML. There were 4 cases of normality.
These samples were also tested for conventional 8-10 color 4-8 tube antibody combination immunotyping.
The first step of the conventional method is to perform 1-tube 10-color screening tube including CD45, CD34, CD117, CD19, CD10, CD7, CD5, CD56, CD38, CD3310 color antibody screening using a conventional general flow meter (Canto triple laser, 10 color, BD company). A10-color screening combination allowed a preliminary determination of diagnosis for AML and ALL-B. For other tumors such as: MPAL, ALL-T, NHL-B, NHL-T, NHL-NK and plasma cell diseases can be determined only by largely determining the detection direction of the next step due to the lack of antibodies required for determining the diagnosis, and a few subsequent samples can be determined by a third step of detection, for example, for mixed phenotype leucocytes, 3 steps of detection are often required for definite diagnosis.
The 19 antibody screening tubes comprise antibodies with the strongest cell series specificity, and comprise a marker for judging the clonality of B, T cells and plasma cells. Therefore, the diagnosis of acute leukemia and NHL has higher precision and specificity.
FIG. 2 shows an example of the results of an MPAL examination. The samples were screened with one tube of conventional 10-color antibody (FIG. 2a) and expressed CD7, CD33, CD34, partially expressed CD45, CD38, CD10, CD117, CD56, and not expressed CD 5. The suggestion may be from the myeloid and T lines, but could not be confirmed, followed by the detection of 4 tubes of the 10 colored antibody combination, for a total of 40 antibodies. However, in the conventional 10-color method, due to different matching of different antibodies, fluorescein is different, so that the expression of cCD3-PE and cCPO-FITC is weak, and MPAL cannot be diagnosed.
However, when a tube of 19 antibodies of the present invention was used for screening the tube (FIG. 2b), the results show that: ALL-T can be determined at near normal T cell levels for cCD3+ CD3-CD5-CD7st + nTdT, cCD3+, with CD19-CD10part + cCD79-cCD 22-indicating no B lineage involvement. The origin of the myeloid lineage can be determined by CD33+ CD34+ CD117 fraction + CD38+ MPO +. Thus, MPAL-T/medulla is determined in one step.
The comparative experiment for examination of MPAL in the above example shows that the conventional method detects 40 antibodies in a total of 5 tubes by 10-color combination, and cannot confirm the source of T and medulla. The one-tube 19-color antibody combination diagnosis of the MPAL has higher sensitivity and accuracy, and is obviously superior to the common 8-10 color flow detection. The LAIP marker is CD34+ CD117+ CD33+ CD7st + CD10+ CD56part +.
FIG. 3 shows an example of AML examination results, showing in the CD45/SSC map a population of naive cells (R3) with low CD45 Weak + SSC, representing 37.47%, which express CD34, CD117, CD33, cMCP Weak, partially express CD7, do not express cCD3, CD5, cCD79, cCD22, CD19, CD10, CD56, with the exception of T and B lines, determined to be naive myeloid cells. The mature T, B, NK and plasma cells were not abnormal, and were judged to be Acute Myeloid Leukemia (AML). LAIP analysis: myeloid cells express the T-lineage marker CD7, LAIP is CD34+ CD117+ CD33+ CD7 +.
FIG. 4 shows an example of the results of the examination after ALL-B treatment, the first row showing: a population of naive cells accounting for 6.78% was seen in the CD45/SSC map, which also showed that T cells and mature B cells were normal. The remaining panels show expression of CD19+ cell antigen, positive: the expression of the genes cCD79, CD19, CD34, CD38, CD56, partial expression of CD117, no expression of cCD22, nTdT, CD10, cCD3, cMCPO, CD33, ckappa, clambda, and can exclude the source of naive myeloid lines and T-lines, and certainly be naive B-cells. No abnormality was observed in mature T, B, NK and plasma cells, and it was judged that acute lymphoblastic leukemia-B line (ALL-B) was not remitted after treatment. LAIP analysis: line B expresses NK and myeloid markers: CD56 and CD117, while CD38+ early B cells should express CD10, whereas in this case CD10-, is aberrant. LAIP: CD34+ CD19+ CD10-CD38+ CD56+ CD117part +.
FIG. 5 shows the results of an ALL-T assay, in which 57.15% of naive cells were observed in the CD45/SSC map, and the population of cells expressed cCD3, nTdT, CD5, CD7, CD38, and CD3, CD19, cMCPO, CD33, CD34, CD117, and CD56, which did not express membranes, were naive T cells. CD34+ CD117+ naive myeloid cells were 0.61%, with a low proportion. Can be used in addition to AML and ALL-B. Mature T, B, NK and plasma cells were not abnormal, with the exception of T, B, NK lymphoma and PCN. This example can be identified as acute lymphoblastic leukemia-T line (ALL-T). LAIP analysis T cells express the naive marker nTdT, LAIP: cCD3+ CD3-nTdT + CD34-CD5+ CD7 +.
FIG. 6 shows an example of the results of NHL-B examination, showing that 25.92% of lymphocytes were present in a small proportion in the CD45/SSC map. But of these, CD19+ CD5+ B accounted for 61.11%, with a significantly higher proportion (< 5% normal). CD19+ B cells accounted for 78.89% in R1 (viable single cell gate), with a significant increase in the proportion (< 10% normal), this group of cells expressing CD19, CD79a, CD5, CD22, ckappa, and not CD34, CD10, nTdT, clambda, cMPO, CD33, being mature and clonal B cells. No apparent CD34+ CD117+ naive myeloid cells. The exception may be AML or ALL-B, ALL-T. Mature T, NK and plasma cells and granulocytes were not abnormal, and were judged to be CD5+ CD 10-NHL-B. LAIP analysis B cells express the T line marker CD5, which is a cross-series antigen + and is a clonal B cell. LAIP: CD19+ CD5+ ckappa + clambda-.
FIG. 7a shows an example of the results of NHL-T assay, showing that CD45/SSC shows a marked increase in the percentage of 62.22% lymphocytes, 96.66% CD3+ T cells and a marked increase in the percentage of normal 50-70% CD7 and CD5 expression in this population of 6 cells, 35.97% total TRBC1 and a normal percentage (15% -85%) TRBC1 and an abnormally increased percentage of 86.16% CD5-CD7-CD3+ cells, suggesting clonal T and 9.53% T cells in this population. The remaining 5 populations of cells, TRBC1 in proportion were all within the normal range, normal T cells. No obvious immature myeloid cells, immature B cells and immature T cells were found in the sample. The exception may be AML or ALL-B, ALL-T. Mature B, NK, plasma cells and granulocytes were not abnormal, except for NHL-B, NHL-NK and PCN. Was judged to be non-Hodgkin lymphoma-T cell type (NHL-T). LAIP analysis: t cells do not express CD5 and CD7, and are absent of antigen expression. LAIP: CD3+ CD5-CD7-TRBC1+ CD 56-.
The sample is subjected to 4-tube 10-color flow detection (fig. 7b) by a conventional method according to NHL-T combination, and the CD4/CD8 ratio is found to be abnormally low, the CD8, CD45RA, CD2 and CD25 are mainly expressed, the CD57 is partially expressed, the CD56 and CD16 are not expressed, the phenotype is abnormal, but the T cells cannot be divided into 6 subgroups due to weak fluorescein of a CD7 antibody, and the clear diagnosis of NHL-T cannot be made because the cloned T cells are not found. The combination of the combined antibody and the fluorescein has better sensitivity and accuracy.
FIG. 8 shows the results of one PCN assay, in which CD38 strongly expresses plasma cells accounting for 6.59%, and the population also expresses CD56, clambda, but not CD19, CD34, CD117, nTdT, cCD79a, CD5, cCD22, ckappa, cMPO, CD33, and is clonal plasma cells, with the exception of AML, ALL-B, ALL-T. Mature T, NK and granulocytes were not abnormal, and this sample was judged to be PCN. LAIP analysis, the expression of plasma cell CD19 is deleted, CD56 is abnormally expressed, and clonality is obtained. LAIP CD38st + CD56+ CD19-clambda + ckappa-.
FIG. 9 shows an example of MDS assay results, with CD117+ CD34+ naive myeloid cells accounting for 4.40%, increased by > 1% but less than 20%, and CD34+ cells with CD19+ B progenitor cells accounting for 1.29% and less than 5%. The SSC ratio of granulocytes to lymphocytes was 1.91(<5.0), CD45 ratio of lymphocytes to myeloid progenitors was 4.1, normal. Ogata scored 3 points. 96.20% of CD34+ CD7+ is evident in > 20%, and the CD33/CD34 pattern is not circular. Naive T, B cells were not seen. Mature T, B, NK and plasma cells were not abnormal and were diagnosed as myelodysplastic syndrome (MDS). LAIP analysis: myeloid naive cells express the T-lineage marker CD7, LAIP: CD34+ CD117+ CD7 +.
FIG. 10 shows the results of a CML assay, showing from the CD45/SSC plot that R4 (granulocytes) accounts for 91.20%, the proportion is significantly increased, and lymphocytes accounts for 0.77%, the proportion is decreased. The lymphocyte has normal T, NK and B cell proportion, and ckappa/clambda and TRBC1 expression. Plasma cells account for 0.01%, with an extremely low proportion, but no clonal abnormalities. CD34+ accounted for 1.41%, and the proportion increased, with only partial expression of CD117 in CD34+ cells, indicating a decrease in expression intensity. CD33/CD34 patterns were normal (circular) in CD117+ cells. The granulocyte expresses a marrow marker CD33, MPO is weak, 10.14% of cells express CD56, the proportion is increased (> 10%), the granulocyte does not express juvenile cell markers such as CD34, CD117, CD38 and the like, and the granulocyte is an abnormal mature granulocyte. The kit has no naive T cells and naive B cells, so AML, ALL-T, ALL-B, NHL-B and clonal plasma cell diseases can be excluded, and the combined disease history is similar to MDS, NHL-T and NK cell abnormalities, and is preliminarily judged to be Chronic Myelocytic Leukemia (CML) along with WBC (white blood cell)/PLT (platelet) height, splenomegaly and the like. CML uses genes to monitor MRD, does not need FCM to detect MRD, and does not need LAIP to be judged.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A reagent composition for flow cytometric leukemia/lymphoma typing comprising an anti-cell membrane antigen antibody and an anti-cell intracellular antigen antibody, said reagent composition being used in 1 flow tube at the time of detection, wherein:
the anti-cell membrane antigen antibody consists of 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 anti-cCD 79a, anti-cLambda, anti-cKappa, anti-cCD 22, anti-cCD 3, anti-cCPO antibody and anti-nTdT antibody.
2. Reagent composition according to claim 1, wherein the antibodies are all monoclonal antibodies.
3. Reagent composition according to claim 2,
in the anti-cell membrane antigen antibody, fluorescein markers of 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 are respectively as follows in sequence: BV785, BV750, BV711, BV650, BV605, BV421, BV510, APC-Fire750, PE-Cy5, PE-Cy7, PE-Fire700 and PE;
in the anti-intracellular antigen antibody, fluorescein labels of anti-cCD 79a, anti-cLambda, anti-cKappa, anti-cCD 22, anti-cCD 3, anti-cCPO antibody and anti-nTdT antibody are respectively PE, PE-Dazle 594, Alexa Fluor 700, APC, Alexa Fluor 647, eFluor 450 and FITC in sequence.
4. A kit for the immunotyping of acute and chronic leukemias and lymphomas, comprising a first container and a second container,
the first container contains an anti-cell membrane antigen antibody in the reagent composition according to any one of claims 1 to 3, and the second container contains an anti-cell intracellular antigen antibody in the reagent composition according to any one of claims 1 to 3.
5. The kit according to claim 4, further comprising other containers, wherein the other containers are filled with red blood cell lysate and buffer solution, respectively.
6. The kit of claim 5, wherein said acute and chronic leukemia and lymphoma disease comprises acute myeloid leukemia, acute lymphoblastic leukemia-B lineage, acute lymphoblastic leukemia-T lineage, acute mixed phenotype leukemia, non-Hodgkin lymphoma-B cell type, non-Hodgkin lymphoma-T cell type, non-Hodgkin lymphoma-NK cell type, plasma cell clonal tumor, chronic myeloid tumor.
7. A system for the detection of acute and chronic leukemia and lymphoma immunotypes, comprising a detection portion and an analysis portion, wherein:
a detection part comprising the reagent composition of any one of claims 1 to 3 for detecting the antigen expression of a sample to be tested by 1-tube 18-color flow cytometry;
and an analysis section for analyzing the detection result of the detection section to preliminarily determine the types of acute and chronic leukemia and lymphoma diseases.
8. The system of claim 7, wherein the system is used for the detection of acute and chronic leukemia and lymphoma immunotyping comprising the steps of:
preparing a flow-cytometric sample after treating a sample to be tested with the reagent composition according to any one of claims 1 to 3; performing flow cytometry on the machine for detection;
wherein, when the flow cytometry is detected on the machine, the gate is arranged according to the following mode:
setting a R1 viable cell gate, removing fragments and dead cells, setting a lymphocyte gate, a granulocyte gate, a monocyte gate, a juvenile cell gate, a nucleated erythrocyte gate and an eosinophil gate by using CD45/SSC in the R1 gate; r1 or multiple markers in each population of phyla were gated to analyze the developmental patterns of T cells, NK cells, B cells, plasma cells, myeloid progenitors, granulocytes and/or monocytes relative to normal cells to identify tumor cells.
9. Use of a reagent composition according to any one of claims 1 to 3 or a kit according to any one of claims 4 to 6 or a system according to claims 7 to 8 in the preparation of a primary screening product for the immunophenotyping of leukemia/lymphoma; the leukemia/lymphoma is acute myelocytic leukemia, acute lymphatic leukemia-B line, acute lymphatic leukemia-T line, acute mixed phenotype leukemia, non-Hodgkin lymphoma-B cell type, non-Hodgkin lymphoma-T cell type, non-Hodgkin lymphoma-NK cell type, plasma cell clonal tumor and chronic myelogenous tumor.
10. Use of a reagent composition according to any one of claims 1 to 3 for the preparation of a product for screening Leukemia Associated Immunophenotype (LAIP) for post-treatment minimal residual disease monitoring markers in leukemia/lymphoma patient; the leukemia/lymphoma is acute myelocytic leukemia, acute lymphoblastic leukemia-B line, acute lymphoblastic leukemia-T line, acute mixed phenotype leukemia, non-Hodgkin lymphoma-B cell type, non-Hodgkin lymphoma-T cell type, non-Hodgkin lymphoma-NK cell type, plasma cell clonal tumor and chronic myelogenous tumor.
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