CN115856302B - Antibody composition for mature B cell tumor immunophenotyping and application thereof - Google Patents

Abstract

The invention provides an antibody composition, a kit and application and a system thereof for mature B cell tumor immunophenotyping, 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 the 1-tube antibody combination, reduces the amount of the required specimens and the operation quantity, lightens the labor intensity and saves the operation time. Meanwhile, repeated application of the door setting antibody is reduced, and the use quantity of the effective antibody is increased.

Description

Antibody composition for mature B cell tumor immunophenotyping and application thereof

Technical Field

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

Background

Mature B cell lymphomas, also commonly referred to as B cell non-hodgkin's lymphoma (NHL-B), are part of the blood/lymphatic system tumors, classified as 29 in the WHO blood and lymphatic system tumor classification book, 2017. Although the plasma cells are produced by differentiation of mature B cells, the antigen expression is different from that of B cells, and the antibody combination used in the immunophenotyping test is different from that of B cells, so that the immunophenotyping test of a plasma cell-cloned disease is not included in the present invention. The classification of NHL-B is very complex, mainly based on pathology, partly named pathogenesis. In addition, the sites of onset of lymphomas are generally in lymphoid tissues such as lymph nodes, spleen, mucosa, skin, etc., where 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 lymphoma (Burkitti, BL), and part (DLBCL) are prone to spreading in blood and bone marrow or blood. Whereas diffuse large B-cell lymphoma (DLBCL) only 1/3 occurs with blood spreading. Therefore, only a portion of lymphomas can be typed by examining bone marrow and peripheral blood, and the rest can examine lymph nodes, spleen, or other accumulated tissue.

The diagnosis of lymphoma mainly depends on pathology, and the advantage of pathological detection is that tissue mechanisms can be observed simultaneously, and cells are subjected to immune labeling by means of immunohistochemistry. However, the defect is that the materials are invasive, and some deep or intracavity tumors are difficult to obtain, so that pathological detection is difficult to realize. In addition, the pathological detection has certain subjectivity and poor repeatability. Often a tissue section is subjected to different specialists, but is diagnosed differently. And is difficult to quantify, and is not easily distinguishable from background cells when the number of tumor cells is low. The sensitivity of detection to cross-talk of certain antigens, especially the cloning markers for B cells-immunoglobulin light chain kappa and lambda, tends to be unsatisfactory, and thus the accuracy of the cloning diagnosis is poor.

Although the tissue structure can not be observed in the immune typing, the immune typing has better objectivity and repeatability. The cell source is easy to identify, and the clonality of the cells is determined, so that the cells can be identified from the background cells even if the number of tumor cells is low. Some of the major NHL-B have a characteristic immunophenotype that can aid in diagnosis by immunophenotyping. The main effects of immunophenotyping include: 1. b cell sources were determined. 2. The lymphoma spread in most blood can be diagnosed and identified by detecting blood or bone marrow. 3. Helping to determine the clinical stage of lymphoma (stage 4 with bleeding). 4. Lymphomas exhibiting bleeding can be monitored for Minimal Residual Disease (MRD) by detecting bone marrow or peripheral blood after treatment. 5. The flow type immunity parting detection is carried out on the tissue specimen at the same time, which can supplement the pathological detection

Currently, immunotyping of B cell tumors by means of Flow Cytometry (FCM) using 8-10 color immunofluorescent markers is widely used internationally. More than 20 antibodies need to be detected, and the detection is completed in 4-5 tubes.

Problems of current detection: in NHL-B, CLL/SLL is the tumor with highest incidence, the disease condition is relatively inert, and the treatment principle is different, so that the identification of the CLL/SLL and other B cell tumors is needed. However, in clinical practice, it is often found that some cases fail to identify CLL/SLL from other B-cell lymphomas. Although new markers are continually being discovered for the identification of CLL/SLL and NHL-B, how to apply these markers remains a problem.

Secondly, in the conventional clinical detection of the flow type, several tubes of cells need to be marked simultaneously to finish detection of about 20 antibodies. In order to facilitate analysis of the phenotype of a cell in a different tube, more gating antibodies need to be reused, so that the total number of detected antibodies is more and the cost is high. If the cost is controlled, the number of repeated gating antibodies is reduced, it is difficult to accurately analyze the relationship between the detected antibodies in tumor cells in different tubes, and the judgment of cell line, differentiation stage and benign and malignant cells is affected. In addition, since several tubes of antibody combinations are required to complete the assay, the antibody species, clone number and fluorescein selected for each tube are different, which can severely impact the results of the antibody assay. However, it is difficult to establish a uniform antibody combination in China and even internationally so as to ensure the consistency and accuracy of detection results.

Disclosure of Invention

In order to solve the above problems, the present inventors have made intensive studies and have proposed an antibody composition comprising 1 tube of 19 antibody combinations for use in a method of B cell tumor immunophenotyping, which has a good differential diagnosis effect on CLL/SLL. Meanwhile, B cell prolymphocytic leukemia (B-PLL), lymphoplasmacytoma (LPL), splenic B cell marginal zone lymphoma (SMZL), follicular Lymphoma (FL), hairy Cell Leukemia (HCL), mantle Cell Lymphoma (MCL), burkitt lymphoma (Burkitti, BL), and Diffuse Large B Cell Lymphoma (DLBCL) can be determined. The method is also used for stage identification of lymphoma, determination of a marker (leukemia-related immunophenotype, LAIP) of MRD monitoring after treatment and screening of treatment targets, and can also be used for MRD monitoring.

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

Through examination, no report of comprehensive immune typing, subtype typing, stage identification and screening of MRD markers and treatment targets on B cell tumors by using a tube 19-color primary screening antibody combination and combining a tube 19-color fluorescein 19 antibodies and a total 2-tube antibody combination is available at present.

That is, the present invention can be regarded as a continuation of the former patent 202111067074.3 and can be used in combination therewith. The second step of detection was performed using the present application on specimens determined to be mature B cell tumors in the first step.

Specifically, the invention provides a reagent for mature B cell tumor immunophenotyping, in particular to a reagent for mature B cell tumor immunophenotyping, an antibody composition system and a kit containing the preparation of the reagent, wherein any one of two groups of antibody compositions specifically comprises 19 antibodies which are respectively used for CD10 negative and CD10 positive mature B cell tumor classification diagnosis, stage identification and trace residual disease sign and treatment target screening.

That is, two sets of antibody compositions designed according to the present invention each comprise 19 antibodies for CD10 negative and CD10 positive mature B cell tumor classification diagnosis, respectively, the specific antibody types and compatible luciferins are described below, for example, see table 1.

In a first aspect of the invention, there is provided an antibody composition for use in mature B cell tumor immunophenotyping comprising the following group a 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.

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 shown in table 1 below:

table 1 antibody combinations and compatible 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), burkitt's lymphoma (Burkitti, BL), and Diffuse Large B Cell Lymphoma (DLBCL), and other non-sortable B cell lymphomas.

In a second aspect the present invention provides a kit for immunophenotyping of mature B cell tumors, in particular for immunophenotyping of mature B cell tumors, the kit comprising a first container containing a group a or a group B antibody as described above; or the B-group antibody is dispensed into two containers, one container containing anti-cBCL-2 and anti-cKi 67 for intracellular antibody detection and the other container containing other antibodies for membrane antibody detection than anti-cBCL-2 and anti-cKi.

In one embodiment, the kit further comprises additional containers containing red blood cell lysate, hemolysin, buffer and/or permeabilizing agent.

In a third aspect the invention provides the use of an antibody composition as described above or a kit as described above for the preparation of a product for immunophenotyping of mature B cell tumours, in particular for immunophenotyping of mature B cell tumours.

In one embodiment, for ease of use, the reagents, antibody composition systems and kits of the invention may also directly comprise the prescreening reagent described in patent 202111067074.3 for use in a prescreening reagent composition for leukemia/lymphoma immunophenotyping comprising an anti-cell membrane antigen antibody 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-intracellular antigen antibodies comprise anti-cd 79a, anti-clapda, anti-cKappa, anti-cd 22, anti-cd 3 and anti-cMPO antibodies and anti-nTdT antibodies; preferably, the primary screening reagent is used in 1 flow tube at the time of detection.

In a fourth aspect the invention provides a system for immunophenotyping of mature B-cell tumors, in particular mature B-cell tumors, the system comprising a detection part and an analysis part, wherein:

a detection section comprising the antibody composition of claims 1-6 or the kit of any one of claims 7-8 for detecting the antigen expression level of an individual to be tested by flow cytometry;

the analysis part is used for analyzing the detection result of the detection part, classifying and judging mature B cell tumors, stage identifying, determining the LAIP mark of the MRD detection and/or screening treatment targets.

In a fifth aspect the present invention provides a method for comprehensive immunophenotyping of mature B cell tumors, in particular mature B cell tumors, the method comprising a detection step and an analysis step, wherein:

a detection step comprising detecting the level of antigen expression of an individual to be tested by flow cytometry using the antibody composition of claims 1-6 or the kit of any one of claims 7-8;

and an analysis step, which is used for analyzing the detection result of the detection part, classifying and judging mature B cell tumor, stage identification, determining LAIP mark of MRD detection and/or screening treatment target points.

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

a group of 19-color antibody combinations are used for detecting NHL-B immunophenotyping, and are matched with a screening tube for use, and only detection of 2-tube antibody combinations is needed, so that the detection effect is greatly superior to that of 4-color combinations and 8-color combinations. At present, a traditional flow cytometer adopts a 9-10 color antibody combination, and 4 tubes are required to be detected for NHL-B. The invention uses the 1-tube antibody combination, reduces the amount of the required specimens and the operation quantity, lightens the labor intensity and saves the operation time. Meanwhile, repeated application of the gated antibodies is reduced, the number of effective antibodies is increased, whether 19 antibodies are expressed simultaneously can be observed simultaneously, and in the past, only 9-10 antibodies can be detected to be expressed simultaneously. The invention greatly improves the analysis capability, and the powerful analysis capability can increase the accuracy of NHL-B typing and the specificity and sensitivity of detection.

Drawings

FIG. 1 shows the phenotype of a normal B cell of a normal bone marrow specimen. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube. The proportion of lymphocytes, monocytes, granulocytes and nucleated erythrocytes is shown in the CD45/SSC plot in panel A. CD19 using CD19/SSC and CD45/CD19 pair ⁺ The cells were gated. CD45/CD81 map was constructed showing cells in the 19+ gate, based on cellsDistribution, which is divided into a, B, c, d4 populations of cells, represents the process of B cell differentiation from early to mature stage. From the kappa/lambda plot, a few b-gate cells and all c and d-gate cells expressed polyclonal kappa and lambda with a kappa and lambda ratio = 1.34 normal. Establishing a CD43/FSC diagram, showing cells in the strangles, wherein d-door cells CD43 can be seen in the diagram ^- FSC is the same as T cells and is the standard for minicells. And establishing other two-dimensional point diagrams to show the R1 portal cells, and the expression condition of antigens detected by B cells in different periods can be seen. Mature B cell phenotype CD81 ⁺ CD43 ^- CD23 ^part+ CD200 ^dim+ CD79b ⁺ FMC7 ^part+ CD62 ⁺ CD27 ^part+ CD123 ^dim+ CD103 ^- CD25 ^- CD11c ^- CD305 ⁺ . CD19 in Panel B ⁺ B cells were gated in the same manner as in panel A. Mature B cell phenotype is CD34 ^- CD10 ^- TdT ^- CD38 ^- CD79a ⁺ Ckappa and clambda are polyclonal expressions.

FIG. 2 shows an example of the results of the CLL-B test. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, and panel B shows CD5 ⁺ CD10 ^- clambda ⁺ NHL-B. In panel A, the ratio of lymphocytes (R2) was 79.07% in the CD45/SSC, and the ratio was significantly increased. CD19 using CD19/CD20 map ⁺ CD20 ⁺ The B cells were designated as 19+20+, the ratio was 77.08%, and the ratio was significantly increased. By the CD43/FSC plot, B cell volumes (FSCs) are shown to be smaller than T cells, being minicells. A kappa/lambda map was established showing 19+20+ in-gate cells, and B cells were seen to express mainly lambda, accounting for 98.3%, as clonal B cells. Kappa (Kappa) ⁺ 0.08% of the total cells were residual normal B cells, and the cells in this gate were shown by thickening, as a normal B cell antigen expression control. The other two-dimensional point diagrams are established to show that R1 portal cells, compared with normal B cells, the cloned B cells express CD19 and CD20 ^dim 、CD43、CD200、CD22 ^dim 、CD81 ^dim CD62L, partially expressed CD23, not CD79b, FMC7, CD305, CD103, CD25, CD123, CD11c. Meets the phenotype characteristics of CLL-B. LAIP CD19 ⁺ CD20dim ⁺ CD5 ⁺ lambda ⁺ 。

FIG. 3 shows an example of the MCL detection results. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, and panel B shows CD5 ⁺ CD10 ^- ckappa ⁺ NHL-B. In panel A, in the CD45/SSC, 89.37% of lymphocytes (R2) are shown, and the proportion is markedly increased. CD19 using CD19/CD20 map ⁺ CD20 ⁺ The B cells were designated as 19+20+, and the proportion of the B cells was 86.92% and the ratio was significantly increased. B cell volumes (FSCs), similar to T cells, are shown as minicells by CD43/FSC plots. A kappa/lambda plot was established showing 19+20+ in-gate cells, and B cells were seen to express predominantly kappa, 99.3% of which were clonal B cells. lambad+ was 0.27% and was residual normal B cells, and this in-gate cell was shown bolded as a normal B cell control. The remaining two-dimensional plots were all constructed to show R1 portal cells, with clonal B cells expressing CD19, CD20, CD22, CD27, CD79B, CD81, and not expressing CD23, CD200, CD43, FMC7, CD305, CD103, CD25, CD123, CD11c, CD62L. There was no significant decrease or increase in antigen expression compared to normal B cells. Meets the phenotype characteristics of MCL. LAIP CD19 ⁺ CD20 ⁺ CD5 ⁺ lambda ⁺ 。

FIG. 4 shows an example of NHL-B-10 ⁺ FL detection results. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, and panel B shows CD5 ^- CD10 ⁺ clambda ⁺ NHL-B. In panel A, the ratio of lymphocytes (R2) was 84.58% and significantly increased in the CD 45/SSC. CD19 using CD19/CD20 map ⁺ CD20 ⁺ The B cells were designated as 19+20+, and the proportion of the B cells was 72.86% and was significantly increased. B cell volumes (FSCs), similar to T cells, are shown as minicells by CD43/FSC plots. CD45/CD43 and kappa/lambda plots were established showing 19+20+ in-gate cells, which were seen to express mainly lambda, 94.34%, as clonal B cells. Kappa (Kappa) ⁺ 1.5% of the total cells were residual normal B cells, and the cells in this gate were shown by thickening, as a normal B cell control. The other two-dimensional point diagrams are all established to show that R1 portal cells, compared with the population of cells, the cloned B cells express CD20, CD81, BLC-2, CD79B and partially express CD19 ^dim Non-expression of CD43, CD200, CD22, CD23, CD103, CD25, CD123, CD11c, CD27, CD62L, KI-67. CD10 combined with B tube ⁺ Meets the FL phenotype characteristics. LAIP CD19 ^dim+ CD20 ⁺ CD10 ⁺ lambda ⁺ 。

FIG. 5 shows an example of HCL detection results. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, panel B shows CD5 ^- CD10 ^- ckappa ⁺ clambda ^- NHL-B. In panel A, the ratio of lymphocytes (R2) was 8.34% in the CD45/SSC panel, but the ratio was not high. CD19 using CD19/CD20 map ⁺ CD20 ⁺ B cells were designated as 19+20+, and the proportion of these cells was not high, but 4.48%. By the CD43/FSC plot, B cell volume (FSC) is shown to be significantly increased over T cells, being large cells. A kappa/lambda plot was established showing CD19+CD20+ cells within the gate, which were seen to express predominantly kappa, 90.73% of which were clonal B cells. lambda type ⁺ 1.79% of the total cells were residual normal B cells, and the cells in this gate were shown in bold as normal B cell controls. The remaining two-dimensional plots were constructed to show R1 portal cells, compared to this population, the clonal B cells expressed CD20 ^st 、CD19 ^st 、CD22 ^st 、CD81 ^st 、FMC7、CD103、CD11c、CD305 ^st Partially express CD79b, CD23, not express CD43, CD200, CD25, CD123, CD27, CD62L. Meets the phenotype characteristics of HCL. LAIP CD19 ^st+ CD20 ^st+ CD5 ^- CD103 ⁺ CD305 ⁺ kappa ⁺ 。

Fig. 6 shows an example DLBCL. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, panel B shows CD5 ⁺ CD10 ^- ckappa ^- clambda ⁺ NHL-B. In panel A, the ratio of lymphocytes (R2) was 47.17% in the CD45/SSC, and the ratio was increased. CD19 using CD19/CD20 map ⁺ CD20 ⁺ The B cells were designated as 19+20+, and the proportion of these cells was increased by 38.55%. By the CD43/FSC plot, it was shown that B cell volumes (FSCs) were similar to T cells, with slightly larger volumes. A kappa/lambda plot was established showing 19+20+ in-gate cells, which were seen to be predominantly lambda-kappa-. But Panel B shows expression of cLambda ⁺ ckappa ⁺ Is a clonal B cell. Establishing other two-dimensional point diagrams all show R1 portal cells, and cloning B cells express CD20 and CD19 ^dim 、CD81 ^st CD200, CD79b, partially expressed CD43, CD305, CD62L, not CD22st, CD23, FMC7, CD25, CD103, CD11c, CD123, CD27. Is CD5 ^- CD10 ^- NHL-B, which is clinically diagnosed as a recurrence of DLBCL. LAIP CD19 ^dim+ CD20 ⁺ CD22 ^- CD5 ⁺ CD200 ⁺ clambda ⁺ 。

FIG. 7 shows one example of NHL-B-DLBCLL assay results. Panel A shows the results of the antibody combination assay of the present invention. Panel B shows the results of the screening tube, panel B shows CD5 ⁺ CD10 ^- ckappa ^- clambda ⁺ NHL-B. In panel A, the ratio of lymphocytes (R2) was 47.17% in the CD45/SSC, and the ratio was increased. CD19 using CD19/CD20 map ⁺ CD20 ⁺ The B cells were designated as 19+20+, and the proportion of these cells was increased by 38.55%. By the CD43/FSC plot, it was shown that B cell volumes (FSCs) were similar to T cells, with slightly larger volumes. A kappa/lambda plot was established showing 19+20+ in-gate cells, which were seen to be predominantly lambda-kappa-. But Panel B shows expression of cLambda ⁺ ckappa ⁺ Is a clonal B cell. The other two-dimensional point diagrams are established to show that R1 portal cells and cloned B cells express CD20 and CD19 ^dim 、CD81 ^st CD200, CD79b, partially expressed CD43, CD305, CD62L, not CD22st, CD23, FMC7, CD25, CD103, CD11c, CD123, CD27. Is CD5 ^- CD10 ^- NHL-B, which is clinically diagnosed as a recurrence of DLBCL. LAIP CD19 ^dim+ CD20 ⁺ CD22 ^- CD5 ⁺ CD200 ⁺ clambda ⁺ 。

Detailed Description

The following examples are given to illustrate the invention but are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

The experimental methods used in the examples below are conventional methods unless otherwise specified.

All materials, reagents, etc. in the examples described below are commercially available unless otherwise specified.

The invention adopts flow cytometry to carry out immunophenotype analysis on samples such as bone marrow fluid, hydrothorax and 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 by primary screening as AML and chronic marrow tumor.

Example 1 formulation of reagents

In this example, 19 kinds of membrane labeled antibodies are shown in Table 1, including: anti-CD 11c-cFluor420, anti-CD 23-BV421, anti-CD 20-eFuor 450, 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-eFuor 720, CD79b-APC-Fire750, anti-CD 27-APC-Fire810, anti-CD 305-Alexa Fluor 647. An intracellular marker antibody 2, comprising: anti-BCL-2-FITC and anti-KI 67-APC. These antibodies are commercially available as they are, and the antibodies of the examples of the present invention are purchased from BD company, biolegend, cytek company, beckman, thermo company.

The invention provides two combinations, which can be selected and configured according to the requirement, wherein the first combination is suitable for CD10 negative NHL-B, and totally 19 antibodies, which do not comprise intracellular antibody detection, and has the advantages of fewer operation steps and short time. The second combination was applicable to CD10 positive NHL-B for a total of 19 antibodies, including 2 intracellular antibodies: cBCL-2 and cKi67 are used to identify CD10 ⁺ Follicular lymphoma and Burkitti lymphoma, but requires an intracellular staining procedure. Because of the low incidence of both lymphomas, CD10 is expressed and can be screened through the first tube ⁺ NHL-B in order to avoid the labeling of intracellular antibodies on all NHL-B samples, 2 antibody combinations were designed (see Table 1).

Configuration CD10 ^- Sample antibody combination: mixing the above 19 membrane antibodies in 1 container according to the predetermined dosage for CD10 ^- Immunotyping markers of samples.

Configuration CD10 ⁺ Sample antibody combination: taking 17 kinds of the membrane antibodies and 2 kinds of intracellular marker antibodies (cBCL-2 and cKi 67) respectively, mixing and filling the membrane antibodies and the intracellular marker antibodies into 2 containers according to each dosage determined by a pre-experiment,for CD10 ⁺ Immunotyping markers of samples.

The kit for detecting NHL-B immunotyping also comprises erythrocyte lysis solution and PBS, wherein the erythrocyte lysis solution can be self-configured or can be purchased commercially (such as BD company).

EXAMPLE 2 immunophenotype analysis of NHL-B by flow cytometry with color 19 antibodies

1. Experimental main material and instrument

1. Materials: 10 XPBS buffer, flow cytometer specific hemolysin (BD Co.);

2. instrument: cytekNL-3000 model full spectrum flow cytometer is equipped with 405nm,488nm, 635nm three lasers, 38 fluorescence detectors. A desk type low-speed centrifuge and a vortex mixer.

2. Method of

1. Sample collection:

1-3mL of the obtained human bone marrow fluid is immediately placed in a heparin anticoagulation tube and rapidly inverted for several times to prevent the sample from coagulating. Various cells such as hydrothorax and ascites, lavage fluid and the like are required to be sent to a laboratory as soon as possible after collection, and specimens are placed at 4 ℃ for cold storage. Flow Cytometry (FCM) detection must be completed within 48 hours, operating in accordance with instructions.

2. Sample preparation process:

(1) Cell count: taking 10 mu l of bone marrow, adding 150 mu PBS, uniformly mixing, counting each microliter of cells by using Michaelis-FCM (model mindray), and adjusting the cell concentration to 5-10 x10 according to the detection result ^{^6} 100 μl, taking 50ul-100 μl cells and adding 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, mixing, and incubating at room temperature in dark place for 15 min;

b) Hemolysis: 2ml of 1 XFACS hemolysin is added, the mixture is stirred evenly at a low speed, and the mixture is kept stand for 8 to 10 minutes at room temperature in a dark place. Centrifuge wash at 300g for 5min and discard supernatant.

c) Washing: 1ml of PBS wash containing 0.1% NaN3 and 1% -2% BSA was added, and the supernatant was discarded by washing with 300g centrifugation for 5min. 200ul of PBS was added to the suspension cells to be detected on-line.

(3) Cd10+ specimen membrane and intracellular antigen staining:

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

b) Film permeation: adding 100ul of film penetrating agent A solution, and allowing the mixture to act for 5min at room temperature.

c) Hemolysis: 2ml of 1 XFACS hemolysin is added, the mixture is stirred evenly at a low speed, and the mixture is kept stand for 8 to 10 minutes at room temperature in a dark place. Centrifuge wash at 300g for 5min and discard supernatant.

d) Washing: 1ml PBS wash was added, and the supernatant was discarded by centrifugation at 300g for 5min.

e) Fixing: adding 100ul of membrane permeable agent B solution, fixing cell membrane and making it permeable, adding corresponding fluorescein labeled antibody, mixing, and incubating at room temperature in dark place for 15min.

f) Washing: 1ml of PBS wash containing 0.1% NaN3 and 1% -2% BSA was added, and the supernatant was discarded by washing with 300g centrifugation for 5min. 200ul of PBS was added to the suspension cells to be detected on-line.

(4) And (3) detecting:

a) Determining an optimum voltage and compensation: setting voltage according to the conventional operation method of the spectrum flow cytometer, preparing a single-dye sample by referring to fluorescent color matching of the kit, and setting the single-dye sample by using the single-dye sample.

b) Instrument setup, calibration and quality control: and starting the preheating machine for more than 20min by CytekNL-3000, washing by deionized water, detecting an internal quality control product, and ensuring that each detection value is within a control range. And (5) calling AL-PANAL for loading and collecting data.

c) And (3) detecting: according to the set instrument conditions, 5-10 ten thousand cells are obtained per tube. If the detection can not be carried out on the machine in time, 0.5ml of 1% paraformaldehyde is added, and the mixture is uniformly mixed and then is stored in a refrigerator at 4 ℃ for 24 hours to finish the detection.

3. Analysis of results: data were analyzed using Kluzaa software:

case typing

(1) The FSC/SSC was used to remove debris, adherent cells and dead cells, and the viable single cells were gated on R1.

(2) R1 portal cells are displayed, a CD45/SSC map is established, and lymphocytes, monocytes, granulocytes, nucleated erythrocytes and naive cells are gated and set with different colors according to the distribution of the cells. The cell proportion of each group was observed to be normal. Whether the proportion of naive cells is increased, etc. In normal bone marrow, each population of cells has a normal range of proportions: 20-40% of gonococcal cells, 2-8% of monocytes, 40-60% of granulocytes, 2-15% of nucleated erythrocytes and less than 5% of naive cells.

(3) A series of 2 antibody two-dimensional plots were constructed, 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 plot shows lymphocytes, the lambda/kappa plot shows CD19+CD20+ intragate cells, and the remaining plots all show R1-gate cells. The expression of these antigens was analyzed. The antigen expression profile is shown in Table 2.

(4) FSC of B cells was analyzed, i.e. cell volume: the B cell volumes were analyzed using a gonocyte CD43/FSC plot. Normal T and B lymphocytes are similar in volume and belong to the minicells. CD43 is strongly expressed in T cells and distinguishable from B cells. With CD43 ^st+ Compared with T cells, the volume size of B cells can be determined. The volume is similar to T lymphocytes as small cells, and the volume is larger than T lymphocytes, indicating large cells.

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

(6) The NHL-B was diagnosed according to the criteria of tables 3 and 4 by combining the results of the primary screen CD5, CD10 and the results of the present tube test. A differential diagnostic score criterion (see Table 3) was proposed internationally based on the phenotypic specificity of a typical CLL-B, which is typically 4-5 points, and the remaining NHL-B, which is typically 0-2 points. From Table 4, it is shown that NHL-B diagnosis is to integrate cell morphology/pathology, genes, chromosomal results. For cases with atypical phenotypes, it is more desirable for the clinician to make a final diagnosis by integrating the information of all aspects.

TABLE 2 action of antigen and expression on Normal cells

TABLE 3 scoring criteria for identifying CLL and NHL-B

Note that: CLL 4-5 min, NHL-B0-2 min

TABLE 4 NHL-B typing and major phenotypic characterization and other diagnostic grounds

Note that: b, strong expression; i: moderately expressing; d: weak expression

(II) judging the LAIP mark, wherein the LAIP for NHL-B mainly comprises: 1. cross-series antigen expression or cross-line antigen expression, note whether myeloid and T-line related antigens are expressed: CD33, CD13, CD5, etc. 2. Antigen expression intensity abnormalities such as CD20, CD22, CD19, CD79b, FMC7, etc. Third, cloning abnormalities, either kappa or lambda restricted expression.

(III) screening therapeutic target related markers, and targeting drugs for NHL-B mainly comprise CD19, CD20 and CD22. Thus, the present assay can provide clinically the expression of CD19, CD20, CD22 antigens, including: whether the expression is enhanced or reduced provides a basis for the use of these targeting agents.

4. Results:

using the antibody combination of the present invention, a total of 101 samples were examined, of which 3 of normal bone marrow and the remaining 98 were B-series abnormal specimens, of which the first 51 were used as experimental groups and the last 47 were used as validation groups. In the experimental group, 32 men and 20 women, the median age was 64.5 years (range 46-81 years). In the authentication group, men31, 16 women, 65 years of age (range 34-91 years). 4 primary screen results are CD10 ⁺ CD5 ^- By CD10 ⁺ Combination detection, all the others adopt CD10 ^- The combination is tested. The 98 samples were all tested simultaneously using conventional 10-color immunophenotyping, with the same results.

Results:

experimental group:

the experimental groups were integrated according to the criteria of table 3 and the results are shown in table 5. Of the 51 samples, 34 were examined for pathology, gene and chromosome at the same time, and comprehensive diagnosis was performed based on these results. Comparing the 34 cases of the flow integration results with the comprehensive diagnosis results, integrating 4-5 cases, wherein the comprehensive diagnosis is all CLL-B, and the results are consistent. And 18 cases are comprehensively diagnosed to be non-CLL-B, wherein 12 cases are integrated by 0-2, which is the same as the comprehensive diagnosis, 5 cases are integrated by 3 points, which cannot be determined to be CLL or NHL-B, and 1 case is integrated by 4 points, which is inconsistent with the comprehensive diagnosis. The rest 17 specimens only have clinical impressions, the flow integral is divided into 0-2 for 7, 4-5 for 10, and no intermediate specimens exist. The flow result has clear demarcation, and further pathological and chromosome detection is not carried out clinically. The results of the integration of mature B cells in 3 normal samples were either 0 or 1. This result indicates that the patient with score 4 had a misjudgment in 1 case, whereas the patient with score 3 had a clinically clear diagnosis. There are drawbacks.

TABLE 5 Experimental group 5 integration results

From the results of this study, we found that in CLL-B patients, CD20 and CD81 were expressed weakly, CD43 and CD62L were positive, and that <90%, <80% and >20%, >20% were used as criteria, respectively, for the integral 1 and the integral 0. The experimental groups were grouped according to the 5-way method and the 4-parameter integral of each group of patients was analyzed, and the results are shown in table 6. The score of 5 is integrated for 0-2 patients, and the parameters of 4 are all below 2 minutes. Patient 4 parameters for score 5 integration 5 were either 3 or 4. The 2 sets of integrals have the same distribution trend. We combine the 5-way and 4-parameter results to form a 9-way integration system, the results are shown in table 7. By 9-parameter integral analysis, the comprehensive diagnosis is that the patient with CLL-B has integral more than 6 minutes, and the patient with NHL-B has integral less than 5 minutes. And the same is true of the distribution in the clinical impression group.

TABLE 6 Experimental group 4 parameter integration results

TABLE 7 integration results of experimental component 9 method

Second, verify group:

the verification group has 47 cases, 37 cases simultaneously detect pathology and/or genes and chromosomes, and comprehensive diagnosis is carried out according to the results. The integration results of the 4-parameter and 9-component methods according to the 5-component method are shown in tables 8-10 respectively. From the results of the 5-component method, the total of 6 samples were integrated 3, and the diagnosis was not clear. And 1 case integrates 4 samples, and erroneous judgment occurs in the flow type. Whereas CLL-B and NHL-B were completely separated by a 9-way. All of the 7 samples with unknown or erroneous flow diagnosis were classified as correct diagnosis. The same is true of the clinical impression group results.

TABLE 8 validation of group 5 integration results

TABLE 9 validation of group 4 parameter integration results

TABLE 10 verification of the group 9 integration results

By verifying the group, the addition of 4 parameters, integrating according to our defined criteria, can greatly increase the accuracy of the stream-based determination of CLL-B versus non-CLL-B. And the method is good for clinical service.

non-CLL-B analysis results:

experimental group (a): totally 25 cases of non-CLL-B, 4 cases of CD10 ⁺ All BCL2 positive cells and positive cells are detected by adopting CD10+ antibody combination>98% while KI-67 was negative (cell ratio between 0.26% and 3.87%). FL was diagnosed, and the presence of t (14; 18) ectopic sites in 3 cases was confirmed by chromosome inspection. 11 cases expressed CD5, judged as CD5 ⁺ NHL-B or MCL, wherein 1 case of pathological diagnosis MCL, 2 cases of pathological diagnosis mucosa-associated NHL, 5 cases of comprehensive diagnosis NHL-B after 1 case of DLBCL treatment, and 2 cases of lack of comprehensive detection results. None of the remaining 10 samples expressed CD5 and CD10, with 2 expressing CD103 or CD305 strong, CD11c, diagnosing HCL. In 2 cases, there were small numbers of clonal plasma cells at the same time, and LPL was diagnosed. Preliminary diagnosis of CD5 in the remaining 6 cases ^- CD10 ^- NHL-B,1 SMZL, and the remaining 5 were not pathologically detected, and subtypes could not be determined.

(II) verification group: a total of 21 cases were non-CLL-B, 12 cases CD5 ⁺ NHL-B,4 cases of comprehensive diagnosis support MCL, 1 case of pathology detection is DLBCL,5 cases of pathology detection is NHL-B, and 2 cases of no pathology detection. The other 9 cases were CD5 ^- CD10 ^- NHL-B,8 cases were comprehensively diagnosed: 1 case aggressive NHL-B,1 case large B cell NHL,1 case MZL,5 case NHL-B.1 case has no comprehensive detection result.

In addition, exemplary results of the combined detection and screening tube detection of normal bone marrow normal B cells and antibodies of different types are shown in fig. 1-7.

The invention shows that the antibody combination detection can be used for carrying out fine analysis on the cloned B cell tumor, and the CLL-B and the non-CLL-B can be accurately identified after the flow 9-component integral analysis.

While the invention has been described in detail in the general context and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (8)

1. An antibody composition for use in the immunophenotyping of mature B cell tumors, comprising the following group a 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 group a or group B reagents were used in 1 flow tube at the time of detection,

the antibody composition is used for identifying 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 lymphoma BL and diffuse large B cell lymphoma DLBCL and other B cell lymphomas.

2. The antibody composition of claim 1, wherein the group a reagent is used to identify CD10 negative mature B cell tumors and the group B reagent is used to identify CD10 positive mature B cell tumors.

3. The antibody composition of claim 1, wherein the antibodies are monoclonal antibodies.

4. An antibody composition according to claim 3, wherein the fluorescein label compatible with the antibodies in group a or group B is as follows:

。

5. a kit for immunophenotyping of mature B-cell tumors, comprising a first container containing an antibody of group a or an antibody of group B of the antibody composition of any one of claims 1-4; or alternatively

The B-group antibody was dispensed into two containers, one container containing anti-cBCL-2 and anti-cKi 67 for intracellular antibody detection and the other container containing other antibodies for membrane antibody detection than anti-cBCL-2 and anti-cKi.

6. The kit of claim 5, further comprising additional containers containing red blood cell lysate, hemolysin, buffer and/or permeabilizing agent.

7. Use of the antibody composition of any one of claims 1-4 or the kit of any one of claims 5-6 for the preparation of a product for mature B cell tumor immunotyping.

8. A system for immunophenotyping mature B cell tumors, comprising a detection section and an analysis section, wherein:

a detection section comprising the antibody composition of any one of claims 1 to 4 or the kit of any one of claims 5 to 6 for detecting the antigen expression level of an individual to be tested by flow cytometry;

the analysis part is used for analyzing the detection result of the detection part, classifying and judging mature B cell tumors, stage identifying, determining the LAIP mark of the MRD detection and/or screening treatment targets.

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