Eight-color multi-parameter flow cytometry detection Panel and application thereof in plasma cell tumor diagnosis

Abstract

The invention discloses an eight-color multi-parameter flow cytometry detection Panel and application thereof in plasma cell tumor diagnosis, the Panel comprises Tube1 and Tube2, the Tube1 is composed of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138 and CD20, and the Tube2 is composed of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117 and CD81. Two tubes are creatively designed to detect Panel based on the eight-color multi-parameter flow cytometry, by synchronously analyzing the clonality of B lymphocytes and plasma cells, the tumor cell source can be precisely discriminated, the disease subtype can be clearly distinguished, and a powerful objective basis is provided for clinical diagnosis, prognosis evaluation and risk degree stratification.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to an 8-color multi-parameter flow cytometry detection panel and its application in the diagnosis of plasma cell tumors. Background Technology

[0002] Plasma cell (PC) tumors are the second most common malignant clonal tumors of the hematologic malignancies. They are highly malignant and have a poor prognosis, with a median age of onset of approximately 65-70 years. Clonal plasma cells often produce large amounts of nonfunctional, structurally uniform monoclonal immunoglobulins (M proteins), isomorphically displacing normal hematopoietic cells and leading to a series of health problems, including monoclonal immunoglobulinemia of undetermined significance (MGUS), plasmacytomas (solitary osteoplasmacytoma and extramedullary plasmacytoma), multiple myeloma (MM), immunoglobulin deposition diseases (primary light chain amyloidosis, light chain and heavy chain deposition diseases), POEMS syndrome, etc.

[0003] Currently, the diagnosis and prognostic analysis of plasma cell tumors, both domestically and internationally, heavily rely on morphology, immunology, cytogenetics, and molecular biology. As is well known, plasma cells, also known as antibody-secreting cells, primarily differentiate from B lymphocytes. Normal B lymphocytes mature through differentiation from bone marrow hematopoietic stem cells and are activated by corresponding antigens or polyclonal stimulants to become activated B lymphocytes. Subsequently, immunoglobulin gene heavy chain class switching gradually forms a plasma cell population with specific antibody-secreting functions, while acquiring unique plasma cell antigens (CD38, CD138, CD229, etc.). Summary of the Invention

[0004] The purpose of this invention is to provide an 8-color multi-parameter flow cytometry panel and its application in the diagnosis of plasma cell tumors. By simultaneously analyzing the clonality of B lymphocytes and plasma cells, the origin of tumor cells can be accurately identified and the disease subtypes can be clearly distinguished, providing a strong objective basis for clinical diagnosis, prognostic assessment and risk stratification.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] In a first aspect, the present invention provides an 8-color multiparameter flow cytometry detection panel, comprising Tube1 and Tube2, wherein Tube1 is composed of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, and CD20, and Tube2 is composed of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, and CD81.

[0007] Secondly, the present invention provides the application of the above-mentioned Panel in the preparation of reagents for the diagnosis of plasma cell tumors.

[0008] Thirdly, the present invention provides the application of the above-mentioned Panel in the preparation of reagents for differentiating plasma cell tumor subtypes.

[0009] Fourthly, the present invention provides the application of the above-mentioned Panel in the preparation of reagents for prognostic assessment of plasma cell tumors.

[0010] Fifthly, the present invention provides the application of the above-mentioned Panel in the preparation of reagents for plasma cell tumor risk stratification.

[0011] In a sixth aspect, the present invention provides a flow cytometry kit containing antibodies labeled with different fluorescein. The antibodies are divided into two groups: the first group consists of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, and CD20; the second group consists of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, and CD81. Each antibody in each group is packaged separately.

[0012] In a seventh aspect, the present invention provides a diagnostic reagent comprising antibodies labeled with different fluorescein, the antibodies being divided into two groups. The first group consists of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, and CD20. The second group consists of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, and CD81. Each antibody in each group is packaged separately.

[0013] Eighthly, the present invention provides a diagnostic system comprising a detection part and an analysis part, wherein the detection part comprises the diagnostic reagents described above, and the analysis part is used to analyze the detection results of the detection part.

[0014] The beneficial effects of this invention are as follows: This invention innovatively designs a two-tube detection panel based on 8-color multi-parameter flow cytometry. By simultaneously analyzing the clonality of B lymphocytes and plasma cells, it can accurately identify the origin of tumor cells and clearly distinguish disease subtypes, providing strong objective evidence for clinical diagnosis, prognostic assessment and risk stratification. Attached Figure Description

[0015] Figure 1 This is a flowchart of the detection method of the present invention; Figure 2 This is a flow cytometry detection diagram (Tube 1) from one embodiment of the present invention. Figure 3 This is a flow cytometry detection diagram (Tube2) from one embodiment of the present invention. Figure 4 This is a flowchart of the diagnostic analysis process of this invention; Figure 5 This is a flow cytometry detection diagram (Tube 1) from one embodiment of the present invention. Figure 6 This is a flow cytometry detection diagram (Tube2) from one embodiment of the present invention. Detailed Implementation

[0016] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0017] This invention provides an 8-color multi-parameter flow cytometry panel and its application in the diagnosis of plasma cell tumors. By simultaneously analyzing the clonality of B lymphocytes and plasma cells, the origin of tumor cells can be accurately identified and the disease subtypes can be clearly distinguished, providing a strong objective basis for clinical diagnosis, prognostic assessment and risk stratification.

[0018] 1. Panel Design Principles This invention, based on consensus regarding flow cytometry detection and incorporating diagnostic criteria from both the WHO (2022 edition) and CSCO (2024) guidelines, proposes for the first time a flow cytometry panel for detecting plasma cell clonal activity. When developing the panel, rigorous selection of certified antibodies with proven track records and proven track records is crucial to ensure consistency and reproducibility of detection levels. Simultaneously, antibody specificity and affinity are paramount. Antibody specificity primarily ensures that the antibody binds only to the target antigen, reducing false positives caused by non-specific binding. Both excessively high and low antibody affinity are detrimental; excessively high affinity may lead to difficulty in dissociating the antigen-antibody complex, affecting results; excessively low affinity results in unstable binding and weak signals. Therefore, antibodies with appropriate affinity constants must be selected. Furthermore, the expression status of cell surface antigens (differences in expression levels and co-expression) must be considered. Using high-expression antigens, low-concentration antibodies are selected; for low-expression antigens, high-concentration or high-affinity antibodies are selected to improve detection sensitivity and avoid mutual interference. The core design concept of the antibody combination ultimately selected in this scheme is to precisely anchor the characteristic antigen expression patterns of B lymphocytes and plasma cells along their complete differentiation trajectory. The epitopes targeted by these antibodies can specifically identify each developmental stage from naïve B cells to plasmablasts and mature plasma cells, providing a clear "biological window" for us to accurately identify and delineate target cell populations. This is the molecular basis for distinguishing between normal differentiation and abnormal clonal proliferation.

[0019] 2. Panel Composition

[0020] Note: This represents the detection of intracellular proteins.

[0021] The characteristics of each antibody are as follows: CD19 is a type I transmembrane glycoprotein with a molecular weight of 95 kDa, expressed in B cells (from pre-B cells to blastoblast-like B cells, not expressed in plasma cells) and follicular dendritic cells. CD19 is involved in B cell development, activation, and differentiation. CD19 forms a complex with CD21 (CR2) and CD81 (TAPA-1) and functions as a BCR co-receptor.

[0022] CD45: It is a tyrosine phosphatase expressed on the plasma membrane of all hematopoietic cells except red blood cells and platelets.

[0023] Kappa: Each human immunoglobulin molecule antibody consists of two identical heavy chains and two identical light chains. Kappa (κ) and Lambda (λ) are two clonal molecules expressed on the membrane of mature B lymphocytes, while plasma cells often express them both intracellularly and on the cell membrane. The light chain (κ) is an important marker reflecting polyclonal activity.

[0024] Lambda: Every human immunoglobulin molecule antibody consists of two identical heavy chains and two identical light chains. Kappa (κ) and Lambda (λ) are two clonal molecules expressed on the membrane of mature B lymphocytes, while plasma cells often express them both intracellularly and on the cell membrane. The light chain (λ) is an important marker reflecting polyclonal activity.

[0025] CD38: It is an ADP-ribosyl hydrolase that exhibits diversity in intensity in hematopoietic cells and certain non-hematopoietic tissues (such as the brain, muscle, and kidneys). It is upregulated in B lymphocytes and plasma cells, but the expression patterns are inconsistent, with plasma cells often showing higher expression intensity than B lymphocytes.

[0026] CD56, also known as NCAM (neural cell adhesion molecule), is a single transmembrane glycoprotein. It is a member of the Ig superfamily. The 140kD isoform is expressed on NK and NKT cells. CD56 is also expressed in certain large granular lymphocytic (LGL) leukemias, small cell lung cancer, neuronal-derived tumors, myeloma, and myeloid leukemias.

[0027] CD138: It is expressed in normal and malignant human plasma cells, pre-B cells, epithelial cells, and endothelial cells, but not in mature circulating B lymphocytes. It is also expressed in some non-hematopoietic cells, including embryonic mesenchymal cells, vascular smooth muscle cells, endothelial cells, and nerve cells.

[0028] CD20, also known as B1 and BP35, is a four-transmembrane protein. It is expressed on mature B cells, while normal plasma cells typically do not express it.

[0029] Ki67 is a nuclear protein expressed in two isoforms with molecular weights of 395 kDa and 345 kDa, respectively. Ki67 is essential for cell proliferation, and its expression is limited to the G1, S, G2, and M phases of the cell cycle. This characteristic makes Ki67 a good marker for proliferating cells and it is often used as a prognostic factor in cancer research. It is frequently highly expressed in plasmablastic lymphomas.

[0030] CD229 is a glycoprotein with a molecular weight of 100-120 kDa, belonging to the CD2 subset of the immunoglobulin superfamily. CD229 is expressed on T cells, B cells, NK cells, plasma cells, and thymocytes, with upregulated expression on plasma cells.

[0031] CD27 is a type I membrane protein with a molecular weight of 50-55 kDa, belonging to the lymphocyte-specific member of the TNF receptor superfamily. It is expressed in myeloid thymocytes, almost all mature T cells, some B cells, and NK cells, playing a role in the co-stimulation of T cell activation and the regulation of B cell differentiation and proliferation. However, its expression intensity varies in plasma cell tumors.

[0032] CD117: It is the receptor for stem cell factor or c-kit ligand. CD117 is expressed in pluripotent hematopoietic progenitor cells (approximately 1-4% of bone marrow cells), mast cells, and acute myeloid leukemia cells (AML). Patients with CD117-positive multiple myeloma may have a relatively good prognosis.

[0033] CD81 is a 26 kDa non-glycosylated member of the tetraspanic membrane protein superfamily (TM4SF), also known as TAPA-1 (a target of antiproliferative antibodies). CD81 is expressed on T cells, B cells, NK cells, monocytes, dendritic cells, thymocytes, endothelial cells, and fibroblasts. In normal plasma cells, CD81 is typically positively expressed. However, in plasma cell tumors such as multiple myeloma, CD81 expression is significantly downregulated or even completely absent.

[0034] 3. The test results for each tube are analyzed as follows: (1) Tube1: such as Figure 2As shown, it is mainly used to determine the clonality basis of B lymphocytes and plasma cells. In normally differentiated B lymphocytes, B lymphocytes are divided into the naive stage (CD19+CD38dim+CD20-) and the mature stage (CD19+CD38-CD20+ / -) by two different markers of CD19 / CD38 / CD20. At the same time, the distribution of B lymphocytes is determined by the CD45 / SSC and CD45 / CD19 two-dimensional scatter plots. The cell size indicated by the forward scatter light (FSC) of normally differentiated naive B lymphocytes is usually similar to that of normal lymphocytes. However, in the CD45 / SSC scatter plot, due to the slightly weaker CD45 expression intensity or slightly higher intracellular complexity, it is characteristically distributed below (or lower right) the normal lymphocyte population. This unique spatial position is the key morphological indication for distinguishing it from normal cells. For the more mature B lymphocyte population, the judgment of its clonal nature depends on the detection of cytoplasmic light chains (Kappa / Lambda): normal polyclonal B cells will simultaneously express cytoplasmic Kappa and Lambda light chains, and the ratio is maintained within a specific range (1 / 4 < Kappa / Lambda < 10); while monoclonal B cells with malignant proliferation will abnormally express only one of the light chains (the κ:λ ratio is significantly imbalanced), and based on this, its clonality can be clearly distinguished. Normally differentiated plasma cells have their unique immunophenotypic characteristics, among which CD38 and CD138 are two highly specific surface markers. In flow cytometry analysis, we first use the strongly positive expression characteristics of CD38 and CD138 to accurately set gates in the CD38 vs CD138 scatter plot to initially delineate the plasma cell population. To further distinguish between benign and malignant, it is necessary to carefully evaluate the antigen expression pattern of this group of cells: abnormal plasma cells (such as multiple myeloma cells) may retain CD38 expression, but its intensity is often significantly weakened; at the same time, two-dimensional scatter plot analysis using combinations such as CD38 and CD20, CD19 and CD56 can be used to identify whether there are abnormal immunophenotypes - such as abnormal expression of CD20, deletion of CD19 or abnormal high expression of CD56, which can all be used as preliminary bases for indicating malignant populations. Finally, the gold standard for determining cell clonality still depends on cytoplasmic light chain restricted expression analysis, that is, by detecting the expression ratio of Kappa and Lambda to confirm whether there is monoclonal proliferation (the κ:λ or λ:κ ratio is significantly increased), thus providing a key confirmatory basis for the differentiation between benign and malignant plasma cell diseases.

[0035] (2)Tube2: As Figure 3As shown, normal plasma cells have a relatively stable immunophenotype, often exhibiting CD38+CD138+CD19+CD56-CD27+CD81+CD117-. However, malignant plasma cells (tumor plasma cells) undergo immunophenotype "abnormalities," manifested as the loss, gain, or alteration of expression intensity of certain markers. Different disease types (such as MGUS, MM, etc.) exhibit different flow cytometry phenotypes, and these different phenotypes are related to the different patterns and degrees of abnormality in their diseases. Based on the WHO (2022 version) and CSCO (2024 version) diagnostic criteria, the 8-color multiparameter flow cytometry panel of this invention can distinguish plasma cell tumors including: unexplained monoclonal immunoglobulinemia (MGUS), AL amyloidosis, multiple myeloma (MM), and Waldenström macroglobulinemia (WM). First, by gating CD38 and CD229 respectively, plasma cell populations are accurately identified. Furthermore, by analyzing the expression patterns of CD27, CD81, Ki67, and CD117 in this cell group (typically showing CD27 and CD81 loss / CD117 enhancement), and combining other immunophenotypic evidence from Tube1, its clonal abnormality was confirmed. Finally, a proportional count was performed on this group of confirmed clonal plasma cells. Based on the proportion of abnormal plasma cells and their unique immunophenotypic characteristics, plasma cell tumors were classified: when the proportion of abnormal cells is less than 10%, it often suggests low tumor burden diseases such as monoclonal globulinemia of undetermined significance (MGUS) or light chain amyloidosis (AL); among them, the typical immunophenotype of MGUS is CD38+CD229+CD56-CD19-CD20-CD27-Ki67-, while amyloidosis is CD38+CD229+CD56+CD19-CD20-CD27-Ki67-, suggesting that the expression of CD56 is key to distinguishing between the two. When the proportion of abnormal cells is greater than or equal to 10%, the diagnostic threshold for active multiple myeloma (MM) is met. Its classic immunophenotype is CD38+CD229+CD56+CD19-CD20-CD27-Ki67-. If the phenotype is CD38+CD229+CD56-CD19-CD20+CD27+Ki67+, it strongly suggests a more aggressive form of plasma cell leukemia (PCL). In extremely rare cases, if coexistence of B-cell and plasma cell tumors is suspected, and the abnormal phenotype of CD38+CD229+CD56-CD19+CD20+CD27+Ki67- is observed, this can provide important evidence for differentiating Waldenström macroglobulinemia from a complex tumor.

[0036] As another embodiment, such as Figure 5 and 6As shown, Tube1 analysis revealed that B lymphocytes (immature stage CD19+CD20- and mature stage CD19+CD20+) did not show monoclonal expression (Kappa, Lambda), confirming them as normal polyclonal B lymphocytes. However, plasma cells (CD38+CD138+) showed clonal expression of the cytoplasmic light chain kappa but did not express the cytoplasmic light chain lambda, suggesting possible monoclonal abnormal plasma cells. Further plasma cell subtyping revealed that these abnormal plasma cells positively expressed CD38, CD138, CD229, CD117, CD81, and CD56, and were negative for CD19, CD20, Ki67, and CD27. Furthermore, the proportion of abnormal cells was >10%, meeting the diagnostic criteria for multiple myeloma.

[0037] This invention addresses the diagnostic and subtyping needs of plasma cell tumors by designing an innovative 8-color multiparameter flow cytometry panel (including Tube 1 and Tube 2). The aim is to provide comprehensive and reliable experimental evidence for the preliminary diagnosis and precise subtyping of this type of disease. For a long time, the diagnosis of plasma cell tumors has primarily relied on CD38 and CD138 markers for plasma cell gating, followed by κ / λ light chain ratio analysis to determine cell clonality. While this method can identify abnormal plasma cells, it has significant limitations in differentiating disease subtypes and lacks multiparameter indicators that systematically reflect tumor origin and phenotypic characteristics. As the value of flow cytometry in the diagnosis of plasma cell tumors gains increasing recognition, the realization of multi-antigen, multi-dimensional detection and analysis is of great significance for clinical decision-making. Against this backdrop, this invention proposes for the first time a strategy that simultaneously integrates B-cell and plasma cell clonality analysis. By analyzing the characteristic antigen expression patterns in the differentiation trajectories of B cells and plasma cells, the origin of tumor cells can be traced, and different disease subtypes can be accurately identified, such as monoclonal globulinemia of undetermined significance (MGUS), multiple myeloma (MM), and Waldenström macroglobulinemia (WM). This panel strictly follows the WHO (2022) and CSCO (2024) guidelines, selecting high-specificity and high-affinity certified antibodies, significantly improving the consistency and reproducibility of the detection. Tube1 focuses on the basic clonality assessment of B lymphocytes and plasma cells, relying on cytoplasmic light chain restriction expression as the gold standard; Tube2 further extends to in-depth analysis of plasma cell immunophenotypes, revealing the phenotypic "abnormality" patterns specific to different disease subtypes through the combination of key markers such as CD229, CD27, CD117, CD81, and Ki67. This invention is the first to achieve clonal tracking and immunophenotypic association analysis from B cells to plasma cells at all differentiation stages, overcoming the bottlenecks of limited detection dimensions and insufficient subtype identification capabilities in previous methods. Furthermore, through ingenious marker selection (CD138 or CD229), this invention successfully constructs a CD38-independent plasma cell recognition system, directly addressing the monitoring challenges following CD38-targeted therapy and providing clinicians with continuous and precise "eyes" in the era of immunotherapy. This panel not only aids in the early diagnosis and accurate classification of plasma cell tumors but also provides objective and efficient laboratory evidence for prognostic assessment and risk stratification, possessing significant clinical application value.

[0038] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An 8-color multi-parameter flow cytometry panel for detection, characterized in that: It includes Tube1 and Tube2, wherein Tube1 is composed of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, CD20, and Tube2 is composed of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, CD81.

2. The use of the Panel of claim 1 in the preparation of reagents for the diagnosis of plasma cell tumors.

3. The use of the Panel of claim 1 in the preparation of reagents for differentiating plasma cell tumor subtypes.

4. The use of the Panel of claim 1 in the preparation of reagents for prognostic assessment of plasma cell tumors.

5. The use of the Panel of claim 1 in the preparation of a reagent for risk stratification of plasma cell tumors.

6. A flow cytometry kit, characterized in that: The kit contains antibodies labeled with different fluorescent dyes. The antibodies are divided into two groups. The first group consists of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, and CD20. The second group consists of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, and CD81. Each antibody in each group is packaged separately.

7. A diagnostic reagent, characterized in that: The diagnostic reagent includes antibodies labeled with different fluorescein. The antibodies are divided into two groups. The first group consists of the following antibodies: CD19, CD45, Kappa, Lambda, CD38, CD56, CD138, and CD20. The second group consists of the following antibodies: CD38, CD45, Ki67, CD229, CD27, CD56, CD117, and CD81. Each antibody in each group is packaged separately.

8. A diagnostic system, characterized in that: The diagnostic system includes a detection section and an analysis section. The detection section includes the diagnostic reagents of claim 7, and the analysis section is used to analyze the detection results of the detection section.

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