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CN-121994683-A - Flow detection method and system for circulating plasma cells of multiple myeloma

CN121994683ACN 121994683 ACN121994683 ACN 121994683ACN-121994683-A

Abstract

The invention relates to the technical field of biomedical detection, in particular to a flow type detection method and a flow type detection system for circulating plasma cells of multiple myeloma, which are characterized in that a peripheral blood sample is used for preparing single cell suspension, the single cell suspension is extracted to prepare a tube to be detected (TEST tube), a nonspecific site is sealed on the TEST tube, a multi-fluorescent antibody panel containing nano antibodies for BCMA and GPRC5D is added for performing cell staining operation, the TEST tube is subjected to flow type cell detection analysis to obtain flow type cell detection data, and the flow type cell detection data is subjected to loop gate analysis according to a preset detection analysis strategy to identify and count the number and the phenotype characteristics of Circulating Plasma Cell (CPC) groups so as to generate circulating plasma cell detection information.

Inventors

  • ZHAO ZHANRAN
  • LIU SIYING
  • FANG XU
  • HU YAN

Assignees

  • 广州金墁利医药科技有限公司

Dates

Publication Date
20260508
Application Date
20260306

Claims (6)

  1. 1. A method for flow-through detection of circulating plasma cells of multiple myeloma, comprising: preparing a single cell suspension using the peripheral blood sample, and extracting the single cell suspension to prepare a TEST tube; Performing nonspecific site blocking on the TEST tube, and adding a multiplex fluorescent antibody panel comprising fluorescent labeled antibodies against CD138, CD38, CD45, CD19, CD56, CD27, CD81, CD117, CD20, CD28, BCMA (CD 269) and GPRC5D for cell staining operations; carrying out flow cytometry detection analysis on the TEST tube to obtain flow cytometry detection data; performing loop gate analysis on the flow cytometry detection data according to a preset detection analysis strategy to identify and count the number and the phenotype characteristics of circulating plasma cell populations, thereby generating circulating plasma cell detection information.
  2. 2. The method of flow cytometry in circulating plasma cells of multiple myeloma according to claim 1, wherein the step of preparing a single cell suspension using a peripheral blood sample and extracting said single cell suspension to prepare a TEST tube comprises: Collecting an anticoagulated peripheral blood sample of a detection object, taking 300 mu L-3mL of the peripheral blood sample, and placing the peripheral blood sample in a centrifuge tube; Adding 5-10 times volume of erythrocyte lysate, and lysing for 10-15min at room temperature under dark condition to remove erythrocyte interference; centrifuging at room temperature for 5min at 300-500g, and discarding supernatant; adding PBS buffer solution to resuspend cell sediment, washing, centrifuging again, discarding supernatant, repeating the washing step for 1-2 times to remove residual lysate and plasma proteins; adding PBS buffer solution or staining buffer solution to resuspend the cells to prepare single cell suspension; And (3) taking part of the single cell suspension to carry out cell counting, regulating the cell density according to the concentration of the white blood cells, and taking the suspension of 1X 10-6-2X 10-6 cells to a flow tube to serve as a TEST tube.
  3. 3. The method of flow cytometry in circulating plasma cells of multiple myeloma according to claim 1, wherein the step of non-specific site blocking the TEST tube and adding multiple fluorescent antibody panels for cell staining procedures comprises: Adding an Fc receptor blocker to the TEST tube, and incubating at room temperature or 4 ℃ for 10-15min to block nonspecific binding of the cell surface Fc receptor; Adding all antibodies in the multiple fluorescent antibody panel into a TEST tube, and fully and uniformly vortex; incubating for 20-30min at room temperature or 4 ℃ in dark condition to realize the specific binding of antigen and antibody; Adding 2mL of PBS buffer solution for washing, centrifuging 300-500g for 5 minutes at room temperature, and discarding the supernatant; The cells were resuspended by adding 300-500. Mu.L PBS or flow staining buffer and detected on-line.
  4. 4. The method of claim 1, wherein the step of performing a loop gate analysis on the flow cytometry detection data according to a predetermined detection analysis strategy comprises: The ALL EVENTS gates are arranged on the FSC-A/SSC-A diagram to exclude obvious cell fragments; Setting SINGLE CELLS gates on the FSC-A/FSC-H diagram, and selecting single cell signals at diagonal positions to remove adherents; On the CD45/SSC-A or CD38/SSC-A panels, A population of cells that is strongly positive for CD38 and either strongly or weakly positive for CD45 is circled, initially defined as plasmA cells; on the CD138/CD38 plot, the double positive cells of CD138+/CD38++ are further circled, identified as the total plasma cell population; in the total plasma cell population, normal plasma cells are distinguished from abnormal circulating plasma cells by using the expression patterns of CD19, CD56, CD27, CD81, CD117, CD20 and CD 28; the expression intensity of BCMA and GPRC5D in the abnormal circulating plasma cell population was evaluated to determine the abundance of the drug target.
  5. 5. The method of claim 1, wherein the generating of the circulating plasma cell detection information comprises: Counting the percentage of abnormal circulating plasma cells in total nucleated cells or total white blood cells; determining the immunophenotype characteristics of the abnormal circulating plasma cells according to the phenotypes of CD56, CD19, CD27, CD81, CD117 and the like; Reporting expression levels of BCMA and GPRC5D to aid in immunotherapy decisions; if counting microspheres are added to the sample or a volumetric flow cytometer is used, the absolute count of abnormal circulating plasma cells per microliter of blood is further calculated.
  6. 6. A circulating plasma cell flow assay system for multiple myeloma, which is used for realizing the circulating plasma cell flow assay method for multiple myeloma according to any one of claims 1 to 5.

Description

Flow detection method and system for circulating plasma cells of multiple myeloma Technical Field The invention relates to the technical field of biomedical detection, in particular to a circulating plasma cell flow type detection method and system for multiple myeloma. Background Multiple myeloma (Multiple Myeloma, MM) is a malignant hematological tumor derived from terminally differentiated B lymphocytes, plasma cells, characterized by abnormal proliferation of cloned plasma cells in the bone marrow and excessive secretion of monoclonal immunoglobulins. Although the use of proteasome inhibitors (e.g., bortezomib), immunomodulators (e.g., lenalidomide), and anti-CD 38 monoclonal antibodies has significantly improved patient prognosis in recent years, MM is still considered an incurable disease, and relapse and resistance are almost unavoidable. As the disease progresses, malignant plasma cells gradually get rid of the dependence on the bone marrow microenvironment, escape bone marrow into the peripheral blood circulation, and form Circulating plasma cells (Circulating PLASMA CELLS, CPC). Studies have demonstrated that CPC is an important biomarker reflecting tumor burden and disease biological aggressiveness in MM patients. The detection of high levels of CPC (e.g., >5% or > 2%) in peripheral blood not only suggests a very poor prognosis, but also an important window for judging early recurrence, extramedullary invasion and Minimal Residual Disease (MRD). Therefore, high-sensitivity detection of peripheral blood CPC by "liquid biopsy" has become an urgent need for clinical accurate diagnosis and treatment. Currently, flow cytometry is the gold standard method for detecting CPC. However, existing clinical routine detection protocols rely primarily on basic markers such as CD38, CD138, CD45, CD19 and CD 56. These approaches present significant challenges in addressing modern precision medical needs, firstly, inadequate sensitivity. CPC is extremely rare (typically < 0.01%) in peripheral blood and down-regulation of CD38 or CD138 expression often occurs after drug treatment (e.g., CD38 detection is disturbed after Daratumumab use), resulting in missed detection. Second, the authentication capability is limited. It is difficult to distinguish all types of malignant plasma cells by only using CD19 and CD56, and especially for atypical clones with weak positive or negative CD19, the lack of comprehensive study of auxiliary markers such as CD27, CD81, CD117, CD28, etc. is liable to cause false negative or false positive. Finally, target monitoring is lacking. Most importantly, existing panels do not contain detection of novel immunotherapeutic targets (BCMA and GPRC 5D), and it is not possible to assess whether patients are suitable for CAR-T therapy or bispecific antibody therapy nor to monitor antigen escape after treatment. B cell maturation antigen (B-cell Maturation Antigen, BCMA), also known as CD269 or TNFRSF17, belongs to a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily. BCMA is expressed mainly on the surface of terminally differentiated plasma cells and partially mature B cells, but hardly in hematopoietic stem cells and non-hematopoietic tissues, and this high expression and strong specificity make it the preferred target for MM immunotherapy (e.g. idecel, cilta-cel, teclistamab). Biologically, BCMA, after binding to its ligands BAFF (B cell activating factor) and APRIL (proliferation-inducing ligand), activates downstream NF- κb (nuclear factor κb), JNK and p38 MAPK signaling pathways by recruiting intracellular TRAF (TNFR-related factor) family proteins (mainly TRAF1, TRAF2, TRAF 3). Among them, activation of NF-. Kappa.B pathway is critical for up-regulating anti-apoptotic proteins such as Bcl-xL, bcl-2, mcl-1, etc., thereby maintaining survival and proliferation of long-lived plasma cells. However, clinical data indicate that some patients relapse after receiving BCMA targeted therapy, and their drug resistance mechanism is mainly involved in antigen escape. This includes a double allele deletion or mutation of the BCMA gene (TNFRSF 17) on chromosome 16, resulting in no longer expression of cell surface BCMA (anti-body Loss), and cleavage of membrane surface BCMA by Gamma-secretase (Gamma-secretase), releasing soluble BCMA (sBCMA) into the blood, sBCMA as a "bait" to neutralize CAR-T cells or antibody drugs, reducing efficacy (ANTIGEN SHEDDING). G protein coupled receptor group C, member D (GPRC 5D) is an orphan receptor, its endogenous ligand and specific signaling mechanisms have not been fully elucidated, but its expression profile has extremely high tissue specificity. GPRC5D is mainly limited to malignant plasma cells, which are hardly detected in normal hematopoietic cells, and which are expressed only in small amounts in keratinized tissues such as hair follicles, except for plasma cells. This unique expression profile makes it an ideal target for a variety of novel immunoth