A multi-cell subset detection kit and its application

Abstract

The present invention discloses a kit for detecting multiple cell subsets, which kit comprises two sets of small molecule antibodies conjugated with different fluorescent groups, a reaction buffer and a red blood cell lysate. The antibodies include CD3 molecule labeled with APC / CY7, CD4 molecule labeled with BV605, CD8 molecule labeled with Alexa700, HLA-DR molecule labeled with Percp / Cy5.5, CD45RA molecule labeled with APC, CD19 molecule labeled with FITC, CD197 molecule labeled with BB700, CD16 molecule labeled with APC and CD56 molecule labeled with PE. Among them, the combination of CD3, CD19, CD56, CD16 and HLA-DR fluorescent antibodies is set B, and the combination of CD3, CD4, CD8, CD45RA and CD197 fluorescent antibodies is set T. The present invention also provides the use of the kit for detecting multiple cell subsets.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically, to a multi-cell subset detection kit and its application. Background Art

[0002] Multiple myeloma (MM) is a malignant hematological disease of plasma cells, characterized by abnormal proliferation of clonal plasma cells in the bone marrow, accompanied by abnormal accumulation of monoclonal immunoglobulins, followed by destructive bone damage, kidney damage, anemia, and hypercalcemia. MM is the second most common hematological malignancy in developed countries and is highly heterogeneous in terms of clinical manifestations, prognosis, and treatment response. Despite the continuous development of treatment methods, a significant number of MM patients still experience multiple disease relapses and ultimately die from the disease itself or treatment-related complications. Therefore, early identification of the disease and finding effective prognostic indicators to guide accurate risk stratification of patients and formulate individualized treatment plans have always been the focus of clinical attention.

[0003] The pathogenic mechanism of MM is complex and has not been clearly explored yet. It is currently believed that its onset is mainly affected by two interacting mechanisms, namely the intramedullary evolution of malignant plasma cells and the progressive dysfunction of the immune microenvironment. The abnormal immune microenvironment of MM usually shows that the functions and numbers of T cells, B cells, and NK cells are inhibited, the production of immunosuppressive cytokines increases, and the expression of inhibitory immune checkpoints in immune cells increases, etc. The interaction between myeloma cells and the immunosuppressive microenvironment has been found to be related to disease progression, and the abnormality of the immune microenvironment indicates poor prognosis of patients. The currently latest and widely recognized MM staging system is the Revised-International Staging System (R-ISS) system formulated by the International Myeloma Working Group in 2014, including serum albumin, serum β2-microglobulin, and detection of high-risk factors in cytogenetics. However, the current staging criteria still cannot accurately predict the treatment response of patients. In recent years, some studies have found new MM prognostic immune markers, such as the proportion of Th22 cells, the level of plasma IL-17, and the relative proportions of neutrophils and monocytes, etc., which have guiding effects on the prognosis of the disease. It can be seen that the changes in the immune subsets of MM patients are crucial for the prognosis of patients, and the immune subsets have important prognostic value for MM patients.

[0004] Chinese Patent Document CN: 202110317284.7 discloses an antibody composition, its kit and application for detecting the therapeutic effect of multiple myeloma. The antibody composition includes: the first group of antibodies consisting of antibody CD138, antibody CD38, antibody CD45, antibody CD269 and antibody CD27; the second group of antibodies consisting of antibody CD138, antibody CD38, antibody CD45, antibody CD56, antibody CD19, antibody ckappa and antibody clamda. The antibody combination provided by the present invention includes a flow antibody combination that has never been clinically used for detecting tumor plasma cells in peripheral blood, and specifies the number of cells to be detected, the standardized operation steps, and the degree of sensitivity that needs to be achieved. Summary of the Invention

[0005] In the first aspect, the present invention provides a kit for detecting multiple cell subsets, including two groups of small molecule antibodies conjugated with different fluorescent groups, wherein: the first group consists of antibody CD3, antibody CD19, antibody CD56, antibody CD16 and antibody HLA-DR molecule to form Set B; the second group of antibodies consists of antibody CD3, antibody CD4, antibody CD8, antibody CD45RA and antibody CD197 to form Set T.

[0006] As a preferred example of the present invention, the kit further includes a reaction buffer and a red blood cell lysate.

[0007] In the second aspect, the present invention provides the application of the kit in the preparation of a diagnostic reagent for detecting the therapeutic effect of multiple myeloma.

[0008] In the third aspect, the present invention provides the application of the kit in the preparation of an immunohistochemical reagent for prognosis monitoring of multiple myeloma.

[0009] The advantages of the present invention are as follows:

[0010] (1) At present, there is a lack of a systematic immune assessment method for the prognosis of MM, and most methods involve bone marrow sampling, which is difficult to sample, causes greater damage to patients, and has a long detection time. The present invention combines multiple immune cell subsets, and according to the changes in peripheral blood immune subsets, can quickly complete the prediction of therapeutic effects with less damage to patients.

[0011] (2) It can predict the therapeutic effect in the early stage and provide a reference basis for subsequent treatment. Brief Description of the Drawings

[0012] Figure 1 It is a flow cytometry result of Set B and a gating method diagram of each flow cytometry cell subset in the result;

[0013] Figure 2 It is a flow cytometry result of Set T and a gating method diagram of each flow cytometry cell subset in the result;

[0014] Figure 3 To quantify the PFS probability graphs of MM patients at one month, three months, and five months;

[0015] Figure 4 A is a graph of the AUC values for the PFS probabilities at one month, three months, and five months in the training set; Figure 4 B is a graph showing the clinical benefit of the prognostic model in the 1-month, 3-month, and 5-month predictions; Figure 4 C is a graph of the nomogram calibration curve showing that this model has good accuracy; Figure 4 D is a graph of the Kaplan-Meier survival curve showing that the prognosis of the high-risk group is significantly worse compared to the low-risk group;

[0016] Figure 5 A is a graph of the ROC curve in the validation set showing the AUC values for the PFS probabilities at one month, three months, and five months; Figure 5 B is a graph showing the clinical benefit of the prognostic model in the 1-month, 3-month, and 5-month predictions; Figure 5 C is a graph of the nomogram calibration curve showing that this model has good accuracy; Figure 5 D is a graph of the Kaplan-Meier survival curve showing that the prognosis of the high-risk group is significantly worse compared to the low-risk group. Detailed implementation manner

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0018] Example 1

[0019] The present invention uses flow cytometry to detect the expression levels of CD3, CD4, CD8, HLA-DR, CD45RA, CD19, CD197, CD16, CD183, CD56, CD196, CD45RO, CD25, and CD127 on the surface of peripheral blood cells of multiple myeloma patients, where neutrophils, CD16 + dendritic cells (DCs), CD56 + , CD16 dimNatural killer cells (NKs) and naïve cytotoxic T cells (Tc) have predictive value for the prognosis of multiple myeloma patients. The detection kit includes: ① Two sets of small molecule antibodies conjugated with different fluorescent groups, which can specifically bind to cell surface molecules and emit specific fluorescence when excited by different wavelengths of excitation light and are detected by a flow cytometer; ② Reaction buffer, including 1% bovine serum albumin (BSA) and phosphate buffer saline (PBS); ③ Red blood cell lysate.

[0020] 1. Experimental protocol

[0021] The antibodies and fluorescein labels used in this invention are as follows: Nine different flow cytometry fluorescent antibodies are selected to label the corresponding cell surface molecules, including CD3 molecule labeled with APC / CY7, CD4 molecule labeled with BV605, CD8 molecule labeled with Alexa 700, HLA-DR molecule labeled with Percp / Cy5.5, CD45RA molecule labeled with APC, CD19 molecule labeled with FITC, CD197 molecule labeled with BB700, CD16 molecule labeled with APC, and CD56 molecule labeled with PE. Among them, the combination of CD3, CD19, CD56, CD16 and HLA-DR fluorescent antibodies is set B, and the combination of CD3, CD4, CD8, CD45RA and CD197 fluorescent antibodies is set T.

[0022] 2. Experimental operation

[0023] Take 200 μl of fresh peripheral blood from the patient and centrifuge at 1500 rpm for 5 minutes. After centrifugation, discard the supernatant plasma, take 100 μl of the lower layer of blood cells, add 300 μl of lysing solution, gently pipette to mix evenly, place on a shaker, and slowly shake for 5 minutes to lyse red blood cells. After the lysis is completed, add 1 ml of PBS buffer, centrifuge at 1500 rpm for 5 minutes in a centrifuge. After centrifugation, discard the upper layer of liquid, and resuspend the cells with 100 μl of reaction buffer. Take two flow cytometry tubes, labeled as tube T and tube B. Pipette 50 μl of the cells resuspended with the reaction buffer into each tube, and add the flow cytometry antibodies of set T and set B respectively, 1 μl of each flow cytometry antibody per tube. Pipette the antibodies and cells in the flow cytometry tubes to mix evenly, and incubate at room temperature in the dark for 30 minutes for staining. After the staining is completed, add 500 μl of reaction buffer to each tube, centrifuge at 1500 rpm for 5 minutes, discard the upper layer of liquid, and resuspend the lower layer of cells with 500 μl of reaction buffer. After mixing by oscillation, perform on-machine detection with a flow cytometer to detect the differences in various immune cell subtypes in the patient's peripheral blood, and use Flowjo software to analyze the flow cytometry results. The flow cytometry results of set B and the gating method for each flow cytometry cell subset in the results (seeFigure 1 ) Flow cytometry results of the T suit and the gating method for each flow cytometry cell subset in the results (see Figure 2 ).

[0024] Example 2

[0025] Inclusion criteria for MM patients: According to the 2014 International Myeloma Working Group criteria: Inpatients diagnosed with multiple myeloma between October 2020 and January 2022.

[0026] Exclusion criteria:

[0027] (1) Age < 16 years old;

[0028] (2) Patients receiving other immunosuppressive or immunomodulatory drugs for the treatment of other diseases;

[0029] (3) Patients who disagreed to participate.

[0030] Thirty-five MM patients were recruited from Jinshan Hospital, Fudan University, Shanghai, China. At the same time, 60 healthy controls from the outpatient clinic were included. Among them, 22 patients had disease progression during the treatment, and the average follow-up time was 6 months. This study was approved by the Institutional Review Board of Jinshan Hospital, Fudan University, and complied with the Helsinki Declaration and relevant laws. According to international clinical practice guidelines, all MM patients received standard-dose chemotherapy and follow-up. The induction regimen was a dual-drug or triple-drug combination, and at least included a proteasome inhibitor (such as bortezomib) or an immunomodulatory drug (such as thalidomide) and dexamethasone. Patients received routine clinical and follow-up evaluations 1 - 3 days before each chemotherapy. The treatment response was evaluated according to the guidelines specified by the International Myeloma Working Group. Disease progression was defined as including progressive disease (PD) and recurrence. The study endpoint was disease progression, and the progression-free survival time of the patients was statistically followed up.

[0031] First, the MM patients and healthy controls were mixed and sorted, and all participants were randomly divided into a training set and a validation set according to a ratio of 2:1. In the training set, immune cell subtypes with P < 0.05 in the log-rank test were included in the LASSO regression algorithm to determine the cell types with the greatest impact on MM prognosis, and 9 cell subsets were screened out. According to the Youden index, the cut-off value of the variable was set, and the variable was divided into 0 or 1, as shown in Table 1.

[0032] Table 1 Cell subsets and cut-off values after LASSO screening

[0033]

[0034]

[0035] The transformed data was incorporated into a multivariate Cox regression model, and immune cell subsets with the best prognostic value for MM were determined through forward selection. The results of the multivariate Cox regression showed that CD16 + DCs (HR = 3.50, P = 0.003), CD56 + CD16 dim NKs (HR = 2.78, P = 0.010), naive cytotoxic T cells (HR = 1.69, P = 0.092), and neutrophils (HR = 1.67, P = 0.148) were prognostic factors for MM. A nomogram based on these predictors was established to quantify the PFS probabilities of MM patients at one month, three months, and five months ( Figure 3 ), and based on this, a prognostic detection kit was designed.

[0036] In the training set, the ROC curve showed that for the PFS probabilities at one month, three months, and five months, the AUC values were 0.707, 0.725, and 0.848 respectively ( Figure 4 A), and the decision curve analysis (DCA) demonstrated that this prognostic model had clinical benefits in the predictions at 1 month, 3 months, and 5 months ( Figure 4 B). The nomogram calibration curve showed that this model had good accuracy ( Figure 4 C). According to the prognostic scores, patients were divided into high-risk and low-risk groups, and the cut-off point selected by the Youden index was 2.292. The Kaplan-Meier survival curve showed that compared with the low-risk group, the prognosis of the high-risk group was significantly poor (P = 0.0041) ( Figure 4 D).

[0037] In the validation set, the ROC curve showed that for the PFS probabilities at one month, three months, and five months, the AUC values were 0.724, 0.672, and 0.716 respectively ( Figure 5 A), and the decision curve analysis (DCA) demonstrated that this prognostic model had clinical benefits in the predictions at 1 month, 3 months, and 5 months ( Figure 5 B). The nomogram calibration curve showed that this model had good accuracy ( Figure 5 C). According to the prognostic scores, patients were divided into high-risk and low-risk groups, and the cut-off point selected by the Youden index was 2.292. The Kaplan-Meier survival curve showed that compared with the low-risk group, the prognosis of the high-risk group was significantly poor (P = 0.0074) ( Figure 5 D).

[0038] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a multi-cell subset detection kit in the preparation of an immunohistochemical reagent for prognosis monitoring of multiple myeloma, characterized in that, It includes small molecule antibodies conjugated with two different fluorescent groups, where: the first group consists of antibody CD3, antibody CD19, antibody CD56, antibody CD16 and antibody HLA-DR molecule to form Set B; the second group consists of antibody CD3, antibody CD4, antibody CD8, antibody CD45RA and antibody CD197 to form Set T.

2. The application according to claim 1, wherein It also includes reaction buffer and red blood cell lysate.

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