CN115856302A - Antibody composition for mature B cell tumor immunophenotyping, kit, application and system thereof - Google Patents

Abstract

The invention provides an antibody composition for mature B cell tumor immunophenotyping, a kit, application and a system thereof, wherein the antibody composition comprises the following group A reagents and/or group B reagents: group A: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-FMC 7, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-CD 305; group B: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-cBCL-2, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-cKi 67. The invention uses 1 tube antibody combination, reduces the demand and the operation quantity of the specimen, lightens the labor intensity and saves the operation time. Meanwhile, the repeated application of the gating antibody is reduced, and the use number of effective antibodies is increased.

Description

Antibody composition for mature B cell tumor immunophenotyping, kit, application and system thereof

Technical Field

The invention relates to the field of antibody medicines, in particular to an antibody composition for immunophenotyping of mature B cell tumors, especially mature B lymphocyte proliferative tumors, a kit and a system comprising the same, and application of the antibody composition.

Background

Mature B-cell lymphoma, also commonly referred to as B-cell non-hodgkin's lymphoma (NHL-B), is part of tumors of the blood/lymphatic system, and is classified into 29 types in the classification book of WHO blood and lymphatic system tumors, 2017. Although plasma cells are differentiated from mature B cells, their antigen expression is different from that of B cells, and the antibody combination used in the immunotyping assay is different from that of B cells, so that the immunotyping assay for a plasma cell clonal disease is not included in the present invention. The classification of NHL-B is very complex, primarily based on pathology, and is partly named after the site of disease. In addition, the onset part of lymphoma is usually in lymphoid tissues such as lymph nodes, spleen, mucosa and skin, among which chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL), B cell prolymphocytic leukemia (B-PLL), splenic B cell marginal zone lymphoma (SMZL), lymphoplasmacytic lymphoma (LPL), follicular Lymphoma (FL), hairy Cell Leukemia (HCL), mantle Cell Lymphoma (MCL), burkitt's lymphoma (Burkitti, BL) and fractional part (DLBCL) are liable to spread in blood and bone marrow or blood. Diffuse Large B Cell Lymphoma (DLBCL) has only 1/3 of the tumor mass to undergo hematogenous spread. Therefore, only a fraction of lymphomas can be typed by detecting bone marrow and peripheral blood, and the remainder can be detected as lymph nodes, spleens, or other accumulated tissues.

The diagnosis of lymphoma depends mainly on pathology, which has the advantage of allowing the simultaneous observation of the organisation and the immunological labelling of the cells by immunohistochemistry. However, the defect is that the material is invasive, and some tumors in deep part or cavity are difficult to take, so that pathological detection is difficult to realize. In addition, the pathological detection has certain subjectivity and poor repeatability. Often, a tissue section is passed through different specialists, but a different diagnosis is obtained. Moreover, it is difficult to quantify, and when the number of tumor cells is low, it is difficult to distinguish them from background cells. The detection sensitivity to certain antigens, especially the clonal markers for B cells-immunoglobulin light chain kappa and lambda-is often poorly labeled and therefore less accurate for clonal diagnosis.

Although the tissue structure cannot be observed in the immunotyping, the immunotyping has better objectivity and repeatability. The source of the cells can be easily identified, and the clonality of the cells can be determined, even if the number of tumor cells is low, the cells can be identified with the background cells. Some major NHL-B have characteristic immunophenotypes that can aid diagnosis by immunotyping. The main roles of immunotyping include: 1. b cell origin was determined. 2. The diagnosis and differential diagnosis of the lymphoma which spreads in most blood vessels can be realized by detecting blood or bone marrow. 3. Help to determine the clinical stage of lymphoma (stage 4 with hematogenous spread). 4. Lymphoma that develops hematogenous spread may be monitored for Minimal Residual Disease (MRD) by monitoring bone marrow or peripheral blood after treatment. 5. The flow-type immunity typing detection is carried out on the tissue specimen simultaneously, and the complementary effect on the pathological detection can be achieved

Currently, 8-10 color immunofluorescent markers are used internationally for immunotyping of B cell tumors with the aid of Flow Cytometry (FCM). More than 20 antibodies need to be detected, and the detection is completed by dividing into 4-5 tubes.

Problems with current detection: one is that in NHL-B, CLL/SLL is the most frequently occurring tumor, the disease condition is inert, the treatment principle is different, and the CLL/SLL needs to be identified with other B cell tumors. However, in clinical practice, it is often found that CLL/SLL cannot be distinguished from other B cell lymphomas in some cases. Although new markers are continuously discovered for the identification of CLL/SLL and NHL-B, how to apply these markers is still a problem.

Secondly, in the flow-type conventional clinical detection, the detection of about 20 antibodies can be completed by marking a plurality of tubes of cells at the same time. In order to analyze the phenotype of a certain cell in different tubes, more gating antibodies need to be repeatedly used, so that the total number of the detected antibodies is more, and the cost is high. If the cost is controlled and the number of repetitive gating antibodies is reduced, it is difficult to accurately analyze the relationship between the antibodies detected in tumor cells in different tubes, which affects the judgment of cell lines, differentiation stages, and benign and malignant cells. In addition, because several tubes of antibody combinations are needed to complete the detection, and the antibody species, clone number and fluorescein selected in each tube are different, the results of the antibody detection are seriously influenced. However, it is difficult to establish a uniform antibody combination domestically or internationally to ensure the consistency and accuracy of the detection results.

Disclosure of Invention

In order to solve the above problems, the present inventors have conducted extensive studies and have proposed an antibody composition comprising 1 tube of a combination of 19 antibodies, which has a superior differential diagnosis effect on CLL/SLL, for use in a method for B cell tumor immunotyping. B cell prolymphocytic leukemia (B-PLL), lymphoplasmacytic lymphoma (LPL), splenic B cell marginal zone lymphoma (SMZL), follicular Lymphoma (FL), hairy Cell Leukemia (HCL), mantle Cell Lymphoma (MCL), burkitt's lymphoma (Burkitti, BL) and Diffuse Large B Cell Lymphoma (DLBCL) can be determined simultaneously. The kit is also used for stage identification of lymphoma, determination of MRD monitoring markers (leukemia-related immunophenotypes, LAIP) after treatment, screening of treatment targets and MRD monitoring treatment.

In particular, the antibody composition of the present invention can perform B cell tumor immunophenotyping in one tube, and can be matched with a primary screen (see patent of applicant, 202111067074.3, "antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof", which comprises specific antibody combination, and uses of one tube to perform primary screening on AML, ALLB, ALL-T, MPAL, NHL-B, NHL-T, PCN, NHL-NK and chronic myeloid tumor), wherein a total of 2 tubes can complete immunophenotyping detection which can be completed only by 5 tubes of 8-color antibody combination, and can accurately analyze the relationship between 19 antibodies in tubes, thereby improving the analysis capability and increasing the sensitivity and specificity of detection. This is a function that cannot be achieved by the 8-color or 10-color antibody combination, and also reduces the use of repetitive antibodies. For post-treatment MRD monitoring, it is not necessary to know the pre-treatment immunotyping results, and it is applicable to all B cell tumor patients.

According to the investigation, at present, no report is available for performing comprehensive immune typing, subtype typing, stage identification and MRD (molecular resonance diagnosis) mark and treatment target screening on B cell tumors by applying a tube of 19-color primary screening antibody combination, combining a tube of 19-color fluorescein with 19 antibodies and combining 2 tubes of antibody combinations.

That is, the present invention can be regarded as a continuation of the former patent 202111067074.3, and can be used in cooperation therewith. The application is applied to the second step of detection of the specimen determined to be a mature B cell tumor in the first step.

The invention specifically provides a reagent and an antibody composition system for mature B cell tumor immunotyping, in particular for mature B cell tumor immunotyping, and a kit prepared from the same, and specifically comprises any one of two groups of antibody compositions, wherein each group comprises 19 antibodies, and the antibody compositions are respectively used for CD10 negative and CD10 positive mature B cell tumor classification diagnosis, stage identification, trace residual disease marker and treatment target screening.

That is, two sets of antibody compositions designed according to the present invention, each comprising 19 antibodies, were used for differential diagnosis of CD 10-negative and CD 10-positive mature B cell tumors, and the specific antibody types and compatible luciferins are described below, for example, in Table 1.

In a first aspect of the invention, there is provided an antibody composition for immunophenotyping of mature B cell tumors comprising the following group a and/or group B agents:

group A: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-FMC 7, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-CD 305;

group B: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-cBCL-2, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-cKi 67.

In one embodiment, the group a or group B reagents are used in 1 flow tube at the time of detection.

In one embodiment, all of the group a reagents are membrane surface markers for identifying CD10 negative (-) mature B cell tumors and the group B reagents include membrane surface and intracellular marker antibodies for identifying CD10 positive (+) mature B cell tumors.

In one embodiment, the antibodies are monoclonal antibodies.

In one embodiment, the fluorescein labels compatible with the antibodies in group a or group B are set forth in table 1 below:

TABLE 1 antibody combinations and combinations of luciferins

In one embodiment, the antibody composition is used to identify chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL), B-cell prolymphocytic leukemia (B-PLL), lymphoplasmacytic lymphoma (LPL), splenic B-cell marginal zone lymphoma (SMZL), follicular Lymphoma (FL), hairy Cell Leukemia (HCL), mantle Cell Lymphoma (MCL), burkitti Lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphomas that cannot be classified.

A second aspect of the invention provides a kit for mature B cell tumour immunophenotyping, in particular mature B cell tumour immunophenotyping, comprising a first container holding a group a antibody or group B antibody as defined above; or the group B antibodies are packaged in two containers, one container containing anti-cBCL-2 and anti-cKi 67 for intrabody antibody detection and the other container containing antibodies for membrane antibody detection other than anti-cBCL-2 and anti-cKi 67.

In one embodiment, the kit further comprises other containers comprising red blood cell lysate, hemolysin, buffer and/or a membrane permeabilizing agent.

The third aspect of the invention provides the use of the above antibody composition or the above kit for the preparation of a product for mature B cell tumor immunophenotyping, in particular a product for mature B cell tumor immunophenotyping.

In one embodiment, for ease of use, the reagents, antibody composition systems and kits of the invention may also include directly the prescreening reagents described in patent 202111067074.3, which prescreening reagents are used in prescreening reagent compositions for leukemia/lymphoma immunotyping, including anti-cell membrane antigen antibodies comprising 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-cellular intracellular antigen antibodies comprise anti-cCD 79a, anti-cLambda, anti-cKappa, anti-cCD 22, anti-cCD 3 and anti-cCPO antibodies and anti-nTdT antibodies; preferably, the prescreening reagent is used in 1 flow tube at the time of detection.

A fourth aspect of the present invention provides a system for mature B cell tumor immunophenotyping, in particular mature B cell tumor immunophenotyping, the system comprising a detection portion and an analysis portion, wherein:

a detection moiety comprising the antibody composition of claims 1-6 or the kit of any one of claims 7-8 for detecting the level of antigen expression in a test subject by flow cytometry;

and the analysis part is used for analyzing the detection result of the detection part, and is used for classifying and judging the mature B cell tumor, identifying in stages, determining the LAIP mark of MRD detection and/or screening a treatment target.

A fifth aspect of the invention provides a method for the comprehensive immunotyping of mature B-cell tumours, in particular mature B-cell tumours, the method comprising a detection step and an analysis step, wherein:

a detection step comprising detecting the level of antigen expression of a test subject by flow cytometry using the antibody composition of claims 1-6 or the kit of any one of claims 7-8;

and the analysis step is used for analyzing the detection result of the detection part, and is used for classifying and judging mature B cell tumors, identifying in stages, determining LAIP marks detected by MRD and/or screening treatment targets.

Based on the technical scheme, the invention has the following beneficial effects:

a group of 19-color antibody combinations are used for NHL-B immunotyping detection and matched with a screening tube, only 2 tubes of antibody combinations are needed for detection, and the detection effect is greatly superior to that of 4-color combinations and 8-color combinations. At present, a traditional flow cytometer adopts 9-10 color antibody combination, and 4 tubes are required to be detected for NHL-B. The invention uses 1 tube antibody combination, reduces the demand and the operation quantity of the specimen, lightens the labor intensity and saves the operation time. Meanwhile, the repeated application of the gating antibody is reduced, the using quantity of effective antibodies is increased, whether 19 antibodies are simultaneously expressed or not can be simultaneously observed, and the previous analysis can only detect whether 9-10 antibodies are simultaneously expressed or not. The invention greatly improves the analysis capability, and the strong analysis capability can increase the accuracy of the typing of NHL-B and increase the specificity and sensitivity of the detection.

Drawings

FIG. 1 shows the phenotype of normal B cells in an example of a normal bone marrow specimen. Panel A shows the results of the combined detection of the antibodies of the present invention. And B is the result of screening the tube. In panel A, lymphocyte, monocyte, granulocyte and nucleated red blood cell ratios are shown in the CD45/SSC plot. CD19/SSC and CD45/CD19 vs CD19 ⁺ The cells are gated. A CD45/CD81 map was created showing 19+ portal cells, which were divided into a, B, c, d4 populations of cells according to cell distribution, representing the B cell differentiation process from early to mature stages. From the kappa/lambda plots, a few b-gated cells and all c-and d-gated cells expressed polyclonal kappa and lambda, with a kappa-to-lambda ratio =1.34 normal. A CD43/FSC map was constructed showing the cells in the gonorrhea, in which CD43, the cells in the d-gate, was seen ^- FSC is the same as T cells and is the standard for minicells. The rest two-dimensional dot diagrams are all established to display the R1 portal cells, and the expression condition of the antigens detected by the B cells in different phases can be seen. Mature B cell phenotype is CD81 ⁺ CD43 ^- CD23 ^part+ CD200 ^dim+ CD79b ⁺ FMC7 ^part+ CD62 ⁺ CD27 ^part+ CD123 ^dim+ CD103 ^- CD25 ^- CD11c ^- CD305 ⁺ . CD19 in B drawing ⁺ The gating pattern of B cells was the same as that of Panel A. Mature B cell phenotype is CD34 ^- CD10 ^- TdT ^- CD38 ^- CD79a ⁺ Ckappa and clambda are polyclonal expression.

FIG. 2 shows an example of CLL-B assay results. Panel A shows the results of the combined detection of the antibodies of the present invention. Panel B is the result of screening tube, panel B demonstrates CD5 ⁺ CD10 ^- clambda ⁺ NHL-B。AIn the figure, it is shown in the CD45/SSC diagram that the proportion of lymphocytes (R2) is 79.07%, which is significantly increased. CD19/CD20 mapping for CD19 ⁺ CD20 ⁺ The proportion of B cells designated as 19+20+ is 77.08%, and the proportion is obviously increased. The CD43/FSC plot shows that B-cell volume (FSC) is smaller than T-cells, and is small. A kappa/lambda map was constructed showing cells within the 19+20+ portal cell, and B cells were seen to predominantly express lambda, 98.3% of which are clonal B cells. Kappa ⁺ 0.08% of the total antigen was retained in normal B cells, and the cells in the gate were shown in bold and served as a control for antigen expression in normal B cells. The rest of the two-dimensional dot plots are all established to show that the R1 gate cells and the clonal B cells express CD19 and CD20 compared with the normal B cells ^dim 、CD43、CD200、CD22 ^dim 、CD81 ^dim CD62L, partially expressing CD23, and not expressing CD79b, FMC7, CD305, CD103, CD25, CD123, CD11c. Meets the CLL-B phenotype characteristics. LAIP CD19 ⁺ CD20dim ⁺ CD5 ⁺ lambda ⁺ 。

FIG. 3 shows an example of the results of MCL detection. Panel A shows the results of the combined detection of the antibodies of the present invention. Panel B is the result of screening tube, panel B demonstrates CD5 ⁺ CD10 ^- ckappa ⁺ NHL-B. The CD45/SSC plot in Panel A shows that the proportion of lymphocytes (R2) is 89.37%, which is significantly higher. CD19/CD20 mapping for CD19 ⁺ CD20 ⁺ The B cell is designated as 19+20+, the ratio of the B cell to the B cell is obviously increased, and the B cell accounts for 86.92%. The volume of B cells (FSC) is shown by CD43/FSC plots to be similar to T cells, being small cells. A kappa/lambda map was constructed showing the 19+20+ portal cell, and B cells were seen to express kappa predominantly, 99.3%, as clonal B cells. lambad + accounted for 0.27% as residual normal B cells, and the gate inner cells were shown bold and served as normal B cell controls. The remaining two-dimensional dot plots were all set up to show that R1 gated cells and clonal B cells expressed CD19, CD20, CD22, CD27, CD79B, CD81, and did not express CD23, CD200, CD43, FMC7, CD305, CD103, CD25, CD123, CD11c, CD62L. Compared with normal B cells, the antigen expression is not obviously weakened or enhanced. It is in accordance with MCL phenotype characteristics. LAIP CD19 ⁺ CD20 ⁺ CD5 ⁺ lambda ⁺ 。

FIG. 4 shows an example of NHL-B-10 ⁺ FL detection junctionAnd (5) fruit. Panel A shows the results of the combined detection of the antibodies of the present invention. Panel B shows the results of screening tubes, and panel B shows CD5 ^- CD10 ⁺ clambda ⁺ NHL-B. The CD45/SSC plot in Panel A shows that 84.58% of lymphocytes (R2) are present, with a significant increase in the proportion. CD19/CD20 mapping for CD19 ⁺ CD20 ⁺ The B cell is designated as 19+20+, the ratio of the B cell to the B cell is obviously increased, and the ratio of the B cell to the B cell is 72.86%. The volume of B cells (FSC) is shown by CD43/FSC plots to be similar to T cells, being small cells. A CD45/CD43 and kappa/lambda map was established showing cells within the 19+20+ door, and seen to express lambda predominantly, 94.34%, as clonal B cells. Kappa ⁺ The percentage of 1.5% was residual normal B cells, and the gate inner cells were shown in bold and served as normal B cell controls. The remaining two-dimensional dot plots were all set up to show R1 gated cells, and clonal B cells expressed CD20, CD81, BLC-2, CD79B, and partially CD19, compared to the population of cells ^dim And does not express CD43, CD200, CD22, CD23, CD103, CD25, CD123, CD11c, CD27, CD62L, KI-67. Combined with B tube CD10 ⁺ And accords with FL phenotype characteristics. LAIP CD19 ^dim+ CD20 ⁺ CD10 ⁺ lambda ⁺ 。

FIG. 5 shows an example of the result of HCL detection. Panel A shows the results of the detection of the antibody combinations of the present invention. Panel B shows the results of screening tubes, demonstrating CD5 ^- CD10 ^- ckappa ⁺ clambda ^- NHL-B. In the A diagram, lymphocyte (R2) is shown to account for 8.34% in the CD45/SSC diagram, and the ratio is not high. CD19/CD20 mapping for CD19 ⁺ CD20 ⁺ The B cell is designated as 19+20+, and the ratio of the B cell to the B cell is not high, wherein the B cell accounts for 4.48%. The CD43/FSC plot shows that B cell volume (FSC) is significantly greater than T cells, and is large. A kappa/lambda map was constructed showing cells within the CD19+ CD20+ gate, which were seen to predominantly express kappa, 90.73%, as clonal B cells. lambda (lambda) ⁺ The percentage of 1.79% was residual normal B cells, and the cells in the gate were shown in bold and served as normal B cell controls. The remaining two-dimensional dot plots were all set up to show R1 gated cells, and clonal B cells expressed CD20 compared to this population of cells ^st 、CD19 ^st 、CD22 ^st 、CD81 ^st 、FMC7、CD103、CD11c、CD305 ^st Partially expressing CD79b, CD23, non-expressing CD43,CD200, CD25, CD123, CD27, CD62L. It is in accordance with the phenotype characteristics of HCL. LAIP CD19 ^st+ CD20 ^st+ CD5 ^- CD103 ⁺ CD305 ⁺ kappa ⁺ 。

FIG. 6 shows an example of DLBCL. Panel A shows the results of the combined detection of the antibodies of the present invention. The result of the screening tube is shown in panel B, which shows CD5 ⁺ CD10 ^- ckappa ^- clambda ⁺ NHL-B. The CD45/SSC plot in Panel A shows that lymphocytes (R2) account for 47.17%, with an increased proportion. CD19 mapping using CD19/CD20 ⁺ CD20 ⁺ The B cell is designated as 19+20+ and the ratio of the B cell to the B cell is increased, wherein the B cell accounts for 38.55%. The CD43/FSC plot shows that B cells are similar in volume (FSC) to T cells and are slightly larger in volume. A kappa/lambda map was created showing cells within the gate 19+20+, which were seen to be predominantly lambda-kappa-. However, panel B shows the expression of cLambda ⁺ ckappa ⁺ And is a clonal B cell. All the other two-dimensional dot diagrams are established to show R1 phylum cells and cloned B cells express CD20 and CD19 ^dim 、CD81 ^st CD200, CD79b, partially expressing CD43, CD305, CD62L, not expressing CD22st, CD23, FMC7, CD25, CD103, CD11c, CD123, CD27. Is CD5 ^- CD10 ^- NHL-B, clinically diagnosed as DLBCL recurrence. LAIP CD19 ^dim+ CD20 ⁺ CD22 ^- CD5 ⁺ CD200 ⁺ clambda ⁺ 。

FIG. 7 shows an example of NHL-B-DLBCLL assay results. Panel A shows the results of the combined detection of the antibodies of the present invention. The result of the screening tube is shown in panel B, which shows CD5 ⁺ CD10 ^- ckappa ^- clambda ⁺ NHL-B. The CD45/SSC plot in Panel A shows that lymphocytes (R2) account for 47.17%, with an increased proportion. CD19 mapping using CD19/CD20 ⁺ CD20 ⁺ The B cell is designated as 19+20+ and the ratio of the B cell to the B cell is increased, wherein the B cell accounts for 38.55%. The CD43/FSC plot shows that B-cell volume (FSC) is similar to that of T-cells and slightly larger. A kappa/lambda map was constructed showing cells within the gate 19+20+ cells, seen to be predominantly lambda-kappa-. However, panel B shows the expression of cLambda ⁺ ckappa ⁺ And is a clonal B cell. The rest of the two-dimensional dot plots are all established to show the R1 gate cells, and the clonal B cells express CD20 and CD19 ^dim 、CD81 ^st CD200, CD79b, partially expressing CD43, CD305, CD62L, not expressing CD22st, CD23, FMC7, CD25, CD103, CD11c, CD123, CD27. Is CD5 ^- CD10 ^- NHL-B, clinically diagnosed as DLBCL recurrence. LAIP CD19 ^dim+ CD20 ⁺ CD22 ^- CD5 ⁺ CD200 ⁺ clambda ⁺ 。

Detailed Description

The following examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention. 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 flow cytometry to carry out immunophenotyping analysis on samples of marrow fluid, hydrothorax, ascites, peripheral blood and the like of clinical patients, and carries out second-step comprehensive immunophenotyping detection on samples which are determined to be possible AML and chronic myeloid tumors by primary screening.

EXAMPLE 1 formulation of reagents

The antibody combination and compatible fluorescein are shown in table 1, and the membrane labeled antibodies are 19 types including: anti-CD 11c-cFluor420, anti-CD 23-BV421, anti-CD 20-eFluor450, anti-CD 43-BV510, anti-CD 45-cFluorV547, anti-CD 62L-BV605, anti-CD 25-BV650, anti-CD 103-BV 711, anti-CD 123-BV785, anti-FMC 7-FITC, anti-CD 200-PE, anti-lambda-PE-Dazle 594, anti-CD 22-PE-Cy5, anti-CD 19-BYG710, anti-CD 81-PE-Cy7, anti-Kappa-eFluR 720, CD79b-APC-Fire750, anti-CD 27-APC-Fire810, anti-CD 305-Alexa Fluor 647. The intracellular marker antibody 2 comprises: anti-BCL-2-FITC and anti-KI 67-APC. These antibodies were commercially available as they are, and the antibodies of the examples of the present invention were purchased from BD, biolegend, cytek, beckman, thermo.

The present invention provides two combinations, which can be selectively configured as desired, the first combination being suitable for CD 10-negative NHL-B, 19 antibodies in total, and not comprising intracellular antibody detection, manipulationThe steps are few, and the time is short. A second group suitable for CD 10-positive NHL-B, consists of 19 antibodies, including 2 intrabodies: cBCL-2 and cKi67, are useful for identifying CD10 ⁺ Follicular lymphoma and Burkitti lymphoma, but require intracellular staining procedures. Because both lymphomas have a low incidence and express CD10, CD10 can be screened through the first tube ⁺ NHL-B,2 antibody combinations were designed to avoid intracellular antibody labeling of all NHL-B specimens tested (see Table 1).

Configuring CD10 ^- Combining sample antibodies: mixing the above 19 membrane antibodies, and packaging in 1 container for CD10 according to the dosage determined by pre-experiment ^- Immunotyping of the samples.

Configuring CD10 ⁺ Combining sample antibodies: mixing the above 17 kinds of membrane antibody and 2 kinds of intracellular marker antibody (cBCL-2 and cKi 67) according to each dosage determined by pre-experiment, and packaging in 2 containers for CD10 ⁺ Immunotyping of the samples.

The kit for detecting NHL-B immunophenotyping further comprises erythrocyte dissolving solution and PBS, wherein the erythrocyte dissolving solution can be prepared by self or purchased commercially (such as BD company).

Example 2 Combined flow cytometry of Chromatous antibodies NHL-B immunophenotypes

1. Experiment main material and instrument

1. Materials: 10 × PBS buffer, hemolysin (BD corporation) dedicated to flow cytometry;

2. the instrument comprises the following steps: cytekNL-3000 model full spectrum flow cytometer with 405nm,488nm and 635nm lasers and 38 fluorescence detectors. A table low-speed centrifuge and a vortex mixer.

2. Method of producing a composite material

1. Collecting samples:

1-3mL of the obtained human bone marrow fluid is immediately placed in a heparin anticoagulation tube and quickly inverted for several times to prevent the specimen from coagulating. The cells such as hydrothorax, ascites and lavage fluid should be sent to the laboratory as soon as possible after collection, and the specimen is stored at 4 ℃ for refrigeration. The assay must be completed within 48 hours for Flow Cytometry (FCM) assays to be performed as described.

2. The sample preparation process comprises the following steps:

(1) Cell counting: adding 150 μ PBS into 10 μ l of bone marrow, mixing, counting cells per microliter with Mirey FCM (model mindray), and adjusting cell concentration to 5-10x10 according to detection result ^{^6} Adding 50ul-100 ul cells into the test tube

(2) Membrane surface antigen staining of CD 10-specimens:

a) Adding corresponding fluorescein-labeled antibody premix for membrane labeling and bone marrow specimen into each tube respectively, mixing well, incubating at room temperature in dark place for 15 min;

b) Hemolysis: adding 2ml of 1 XFACS hemolysin, mixing evenly by low speed vortex, standing for 8-10 min at room temperature in dark place. The supernatant was discarded after centrifugation at 300g for 5min.

c) Washing: 1ml of PBS wash containing 0.1% NaN3 and 1% -2% BSA was added, and centrifuged at 300g for 5min, and the supernatant was discarded. Adding 200ul PBS suspension cells, and waiting for detection on a computer.

(3) Staining of CD10+ specimen membranes and intracellular antigens:

a) Adding corresponding fluorescein-labeled antibody premixed solution for membrane labeling and bone marrow specimen into each tube respectively, mixing uniformly, and incubating at room temperature in a dark place for 15 min;

b) Membrane permeation: adding 100ul of the penetrating agent A, and acting at room temperature for 5min.

c) Hemolysis: adding 2ml of 1 XFACS hemolysin, mixing evenly by low speed vortex, standing for 8-10 min at room temperature in dark place. The supernatant was discarded after centrifugation at 300g for 5min.

d) Washing: 1ml of PBS wash was added, and the mixture was centrifuged at 300g for 5min to remove the supernatant.

e) Fixing: adding 100ul of membrane-penetrating agent B liquid, fixing and penetrating cell membrane, simultaneously adding corresponding fluorescein labeled antibody, mixing, and incubating at room temperature in dark place for 15min.

f) Washing: adding 1ml PBS washing solution containing 0.1% NaN3 and 1% -2% BSA, centrifuging and washing for 5min at 300g, and discarding the supernatant. Adding 200ul PBS suspension cells, and waiting for detection on a computer.

(4) And (3) computer detection:

a) Determining the optimum voltage and compensating: the voltage was set according to the conventional operating method of a spectral flow cytometer, and a single-stained sample was prepared for instrument setting with reference to the fluorescent color matching of the kit.

b) 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. And calling AL-PANAL sample loading and collecting data.

c) And (3) computer detection: according to the set instrument conditions, 5-10 ten thousand cells are obtained from each tube. If the detection cannot 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.

3. And (4) analyzing results: data were analyzed using Kluzaa software:

(one) case typing

(1) FSC/SSC was used to remove debris, adherent cells and dead cells, and live single cells were gated with R1.

(2) Displaying R1 gate cells, establishing a CD45/SSC diagram, gating lymphocyte, monocyte, granulocyte, nucleated erythrocyte and naive cells according to different cell distributions, and setting different colors. And observing whether the proportion of the cells of each group is normal. Whether the proportion of naive cells is increased, etc. In normal bone marrow, each population of cells has a normal range of ratios: 20-40% of gonorrhea cell, 2-8% of monocyte, 40-60% of granulocyte, 2-15% of nucleated erythrocyte and less than 5% of naive cell.

(3) A series of two-dimensional dot plots of 2 antibodies were created, consisting essentially of CD43/FSC, CD20/CD19, lambda/kappa, CD23/CD200, CD22/CD20, CD81/CD43, FMC7/CD79b, CD62L/CD27, CD103/CD123, CD25/CD11c, CD200/CD305. Wherein the CD43/FSC map shows lymphocytes, the lambda/kappa map shows CD19+ CD20+ portal cells, and the remaining maps show R1 portal cells. The expression of these antigens was analyzed. The antigen expression profile is shown in table 2.

(4) Analysis of B cells for FSC, cell volume: the size of B cells was analyzed using a lymphocyte CD43/FSC map. Normal T and B lymphocytes are similar in volume and both belong to minicells. CD43 is strongly expressed in T cells and can be distinguished from B cells. And CD43 ^st+ The size of the B cells can be determined in comparison to T cells. Volume ofSimilar to T lymphocytes are small cells, larger in volume than T lymphocytes, indicating large cells.

(5) Analysis of B cell clonality: using the CD20/CD19 map, for CD19 ⁺ CD20 ⁺ B cells were gated at 19+20+, and 19+20+ gated cells were shown in the lambda/kappa map, and expression of lambda and kappa was analyzed. Normal CD19 ⁺ CD20 ⁺ B cells with a lambda/kappa ratio of between 0.3 and 3.0,>3.0 and<0.3 is all restriction expression, is a marker of clonal B cells.

(6) Combining the results of the primary screen CD5 and CD10 and the test results of the primary screen, NHL-B was diagnosed according to the criteria in tables 3 and 4. A differential diagnostic scoring criterion was proposed internationally based on the phenotypic specificity of classical CLL-B, which is generally between score 4 and score 5, and the remaining NHL-B, which is generally between score 0 and score 2 (see Table 3). From table 4 it is shown that NHL-B diagnosis combines cellular morphology/pathology, genetic, chromosomal results. For cases with atypical phenotypes, the clinician is more required to synthesize various pieces of information for final diagnosis.

TABLE 2 Effect of antigens and expression on Normal cells

TABLE 3 Scoring criteria for identifying CLL and NHL-B

Note: CLL 4-5 points, NHL-B0-2 points

TABLE 4 NHL-B typing and major phenotypic characteristics and other diagnostic criteria

Note: b, strong expression; i: moderate expression; d: weak expression

(II) judging the LAIP mark, wherein the LAIP for NHL-B mainly comprises the following steps: 1. cross-line antigen expression or cross-line antigen expression, whether expressing myeloid and T-line related antigens: CD33, CD13, CD5, etc. 2. Abnormal antigen expression intensity, such as CD20, CD22, CD19, CD79b, FMC7, etc. Clonal abnormality, either kappa or lambda restricted expression.

And (III) screening a mark related to a treatment target, wherein the targeted drug aiming at NHL-B mainly comprises CD19, CD20 and CD22. Thus, the assay can provide clinical expression of CD19, CD20, CD22 antigens, including: whether the expression is enhanced or reduced or not provides basis for the use of the targeting drugs.

4. As a result:

by using the antibody combination of the present invention, 101 samples were tested, wherein 3 samples of normal bone marrow were tested, and the remaining 98 samples were B-series abnormal samples, wherein the first 51 samples were used as experimental group, and the last 47 samples were used as verification group. Of the experimental groups, 32 men and 20 women had a median age of 64.5 years (range 46-81 years). In the validation group, 31 men and 16 women were aged 65 years (range 34-91 years). CD10 result for 4 cases of primary screen ⁺ CD5 ^- Using CD10 ⁺ Combined detection, the rest is CD10 ^- And (4) detecting in combination. 98 samples are detected by adopting conventional 10-color immunotyping at the same time, and the results are the same.

As a result:

experimental group one:

the experimental groups were integrated according to the criteria of table 3 and the results are shown in table 5. Of the 51 specimens, 34 specimens were tested for pathology, gene and chromosome at the same time, and a comprehensive diagnosis was performed based on these results. The flow integration results of these 34 cases were compared with the comprehensive diagnosis results, and the total of 16 cases were integrated by 4 to 5, and the comprehensive diagnosis was all CLL-B, and the results of both cases were identical. And 18 cases are diagnosed as non-CLL-B through comprehensive diagnosis, wherein 12 cases have the same integral 0-2 as the comprehensive diagnosis, 5 cases have the integral 3 and cannot determine whether CLL or NHL-B, and 1 case has the integral 4, which is inconsistent with the comprehensive diagnosis. The remaining 17 specimens had clinical impressions, flow scores of 0-2, 7, 4-5, 10, and no intermediate specimens. The stream result has clear boundary, and no further detection of pathology, chromosome and the like is carried out clinically. In 3 cases of normal samples, the integration results for mature B cells were all 0 or 1. This result indicates that the patients with score 4 have false positives in 1 case, while the specimens with score 3 cannot give a clinically definite diagnosis. There are drawbacks.

TABLE 5 results of the integration method of Experimental group 5

From the results of this study, we found that in CLL-B patients, CD20 and CD81 are expressed weakly and CD43 and CD62L are positive, and the score of 1 is met and the score of 0 is not met by using <90%, <80% and >20% as the criteria, respectively. The experimental groups were grouped according to the 5-point method and the 4 parameter integrals for each group of patients were analyzed, the results are shown in table 6. 0-2 patients were integrated by 5 points method, and 4 parameters were all below 2 points. The 5 point score 4 parameters for the 5 patients were all 3 or4 points. The 2 sets of points have the same distribution trend. We combined the results of the 5-point method and the 4-parameter method to form a 9-point method integration system, and the results are shown in Table 7. The patients with CLL-B were diagnosed synthetically by 9-parameter score analysis, with a total score of more than 6, while the patients with NHL-B were diagnosed with a total score of less than 5. The distribution is the same in the clinical impression group.

TABLE 6 results of parameter integration in Experimental group 4

TABLE 7 integration results of experiment group 9 component method

Verification group:

a total of 47 cases and 37 cases in the verification group were simultaneously subjected to pathological and/or genetic and chromosomal tests, and comprehensive diagnosis was carried out based on these results. The results of integration according to 5min, 4 min and 9 min are shown in tables 8-10, respectively. The results of the 5-point method showed a total of 6 specimens with 3 points, and the diagnosis was unclear. And 4 samples are integrated in 1 case, and misjudgment occurs in the flow type. While CLL-B and NHL-B were completely separated by 9-min. All of the 7 specimens with unknown or misjudged flow diagnosis were classified as correct diagnosis. The same applies to the clinical impression group results.

TABLE 8 results of the validation set 5 integration method

TABLE 9 verification of group 4 parameter integration results

TABLE 10 verification of the 9-component integration results

Through the verification group, 4 parameters are added, integration is carried out according to a defined standard determined by a user, and the accuracy of flow type CLL-B and non-CLL-B judgment can be greatly improved. The method is good for clinical service.

Third, non-CLL-B analysis results:

experiment group (one): in total 25 non-CLL-B, 4 CD10 s ⁺ The detection is carried out by adopting the CD10+ antibody combination, all positive BCL2 cells are positive, and all positive cells are>98 percent, while KI-67 is negative (the cell ratio is between 0.26 percent and 3.87 percent). The diagnosis was FL, and 3 cases were examined for the presence of t (14. 11 cases expressed CD5 and judged CD5 ⁺ NHL-B or MCL, wherein 1 case of pathological diagnosis MCL, 2 cases of pathological diagnosis mucosa-associated NHL,1 case of DLBCL treatment, 5 cases of comprehensive diagnosis NHL-B and 2 cases of lack of comprehensive detection results. The remaining 10 specimens did not express CD5 and CD10, of which 2 expressed CD103 or CD305 strong, CD11c, diagnostic for HCL. In 2 cases, a small number of clonal plasma cells were present at the same time, and LPL was diagnosed. Remaining 6 cases of preliminary diagnosis of CD5 ^- CD10 ^- NHL-B,1 case SMZL, and the remaining 5 cases were undetected for pathology and no subtype was determined.

(II) verification group: in total 21 cases were non-CLL-B, 12 cases were CD5 ⁺ NHL-B,4 general diagnoses supporting MCL, 1 pathological examination was DLBCL,5 pathological examinations were NHL-B,2 no pathological examination. The other 9 cases are CD5 ^- CD10 ^- NHL-B,8 cases were diagnosed synthetically: 1 aggressive NHL-B,1 LargeB cell NHL,1 case MZL,5 cases NHL-B. No comprehensive test results were obtained in 1 case.

In addition, exemplary results of normal bone marrow normal B cells and different typing of antibody combination test and screening tube tests are shown in fig. 1-7.

The antibody combination detection of the invention can carry out fine analysis on the cloned B cell tumor, and CLL-B and non-CLL-B can be accurately identified after the flow 9-point method integral analysis.

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. An antibody composition for immunophenotyping of mature B cell tumors comprising the following group a agents and/or group B agents:

group A: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-FMC 7, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-CD 305;

group B: anti-CD 11C, anti-CD 23, anti-CD 20, anti-CD 43, anti-CD 45, anti-CD 62L, anti-CD 25, anti-CD 103, anti-CD 123, anti-cBCL-2, anti-CD 200, anti-lambda, anti-CD 22, anti-CD 19, anti-CD 81, anti-Kappa, anti-CD 79b, anti-CD 27, and anti-cKi 67.

2. The antibody composition of claim 1, wherein said group a or group B reagents are used in 1 flow tube at the time of detection.

3. The antibody composition of claim 1, wherein said group a reagents are used to identify CD10 negative mature B cell tumors and said group B reagents are used to identify CD10 positive mature B cell tumors.

4. The antibody composition of claim 1, wherein said antibodies are monoclonal antibodies.

5. The antibody composition of claim 3, wherein the fluorescein label compatible with the antibodies in group A or group B is as follows:

。

6. the antibody composition of claim 1, wherein said antibody composition is used to identify chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL), B-cell prolymphocytic leukemia (B-PLL), lymphoplasmacytic lymphoma (LPL), splenic B-cell marginal zone lymphoma (SMZL), follicular Lymphoma (FL), hairy Cell Leukemia (HCL), mantle Cell Lymphoma (MCL), burkitt's Lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphomas.

7. A kit for immunotyping a mature B cell tumor comprising a first container comprising a group a antibody or a group B antibody of the antibody composition of any one of claims 1-6; or

The group B antibodies are packaged in two containers, one container containing anti-cBCL-2 and anti-cKi 67 for intrabody antibody detection and the other container containing antibodies for membrane antibody detection other than anti-cBCL-2 and anti-cKi 67.

8. The kit of claim 7, further comprising additional containers comprising red blood cell lysate, hemolysin, buffer, and/or a membrane permeant.

9. Use of an antibody composition according to any one of claims 1 to 6 or a kit according to any one of claims 7 to 8 in the manufacture of a product for immunotyping of mature B cell tumours.

10. A system for immunophenotyping a mature B cell tumor, comprising a detection portion and an analysis portion, wherein:

a detection moiety comprising the antibody composition of any one of claims 1-6 or the kit of any one of claims 7-8 for detecting the level of antigen expression in a test subject by flow cytometry;

and the analysis part is used for analyzing the detection result of the detection part, and is used for classifying and judging the mature B cell tumor, identifying in stages, determining the LAIP mark of MRD detection and/or screening a treatment target.

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