CN-121114422-B - High-flux 24-color flow detection kit for acute lymphoblastic leukemia

Abstract

The invention relates to a high-flux flow type detection kit for acute leukemia (ALL), which belongs to the field of leukemia detection, and adds 16 ALL cell antigens on the basis of original 8-color flow type, so that 24 antigens of the same cell can be detected simultaneously, the detection precision of a tiny residual focus (MRD) is greatly improved, and the ALL cells can be divided into 12 development stages to more accurately identify the immunophenotyping at the initial diagnosis and the immunophenotyping after bone marrow transplantation and CAR-T cell treatment, so that the kit has very important guiding significance for evaluating the treatment effect of patients and adjusting the treatment scheme.

Inventors

• JING DUOHUI
• MI JIANQING
• WANG JIN
• XU WENQIAN
• TIAN FENG
• Weng Xiangqin
• JIANG MIAO

Assignees

• 上海交通大学医学院附属瑞金医院

Dates

Publication Date

20260512

Application Date

20250806

Priority Date

20240812

Claims (5)

1. 1. The high-throughput 24-color flow detection kit for acute lymphoblastic leukemia is characterized by comprising a cell suspension treatment solution, a cell membrane surface antigen antibody premix solution, a solidification treatment solution and an intracellular antigen antibody premix solution, wherein the cell membrane surface antigen antibody premix solution comprises an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 73 antibody, an anti-CD 58 antibody, an anti-IgHs antibody, an anti-CD 45RB antibody, an anti-CD 95 antibody, an anti-CD 24 antibody, an anti-CD 33 antibody, an anti-CD 22 antibody, an anti-CD 38 antibody, an anti-CD 45 antibody, an anti-CD 127 antibody, an anti-CD 20 antibody, an anti-CD 34 antibody and an anti-FVS 620 antibody, and the intracellular antigen antibody premix solution comprises an anti-p 4EBP1 antibody, an anti-ZAP 70 antibody, an anti-IgHi antibody, an anti-CD 179a antibody, an anti-CD 179b antibody, an anti-TdT 6 antibody and an anti-pCreb antibody.
2. 2. The kit of claim 1, wherein the cell suspension treatment fluid comprises Stain Buffer and Brilliant Stain Buffer.
3. 3. The kit according to claim 1, wherein the curing treatment liquid comprises Fix/Perm and Perm/Wash.
4. 4. The kit according to claim 1, wherein 24 streaming antibodies are packaged in one antibody combination tube to achieve parallel detection of 24 surface antigens and intracellular proteins in the same cell in the same tube in leukemia.
5. 5. Use of the high-throughput 24-color flow assay kit for acute lymphoblastic leukemia according to any one of claims 1-4 for preparing an assay product for acute lymphoblastic leukemia immunophenotyping and prognosis risk stratification.

Description

High-flux 24-color flow detection kit for acute lymphoblastic leukemia Technical Field The invention belongs to the field of leukemia detection, and particularly relates to a high-throughput 24-color flow detection kit for acute lymphoblastic leukemia. Background Leukemia is a malignant clonal disease of hematopoietic stem and progenitor cells, which stagnates at different stages of cellular development due to increased self-renewal, uncontrolled proliferation, impaired differentiation, and blocked apoptosis of leukemia cells. Leukemia cells accumulate in massive proliferation, inhibiting normal hematopoiesis and infiltrating other organs and tissues. Cell differentiation arrest in acute leukemia is early stage, and is mostly primitive and early naive, and the disease is rapid. In china, leukemia is the first ten new cases of tumor and the number of cases of tumor death, with Acute Lymphoblastic Leukemia (ALL) being the most frequently occurring malignancy in people under 35 years of age. Although its cure rate has been significantly improved (up to 80% or more), it remains one of the important causes of death in adolescents due to disease, and the prognosis for adult ALL patients remains poor—only about 40% of patients achieve long-term complete remission. Thus, there is an urgent need to increase the diagnostic and therapeutic levels of ALL to improve patient survival. While ALL patients who respond poorly to standard chemotherapy regimens, despite being assigned to high risk groups, a high dose of chemotherapy regimen, may remain with small numbers of leukemia cells (MRD) in the body. Among ALL, the prognostic value and clinical significance of MRD were suggested in the early 1990 s at several centers in europe and the united states, and related studies during this period have shown that residual leukemia cell levels have a strong predictive effect on disease recurrence, and that risk of recurrence increases significantly when MRD levels remain at positive levels at the end of induction therapy. MRD cells often exhibit a strong tolerance to chemotherapeutic agents and are rare in number, difficult to detect after long-term concealment in patients, often proliferate again in large amounts after chemotherapy, causing disease recurrence. Whereas survival in relapsed ALL patients is generally only less than 30%. Internationally, typically bounded by one ten-thousandth, MRD exceeding one ten-thousandth generally means a higher cancer recurrence rate. Although there is diversity in the detection methods for MRD, due to their limited sensitivity, true MRD negativity is practically difficult to achieve, and small numbers of leukemia cells often remain in the patient's bone marrow, these leukemia residual cells (MRD cells) often become the root cause of leukemia recurrence, so that by monitoring MRD levels, disease recurrence can be predicted earlier and more accurately while assessing efficacy, and intervention therapy is suggested to be taken when there is no recurrence or at an early stage of recurrence, to increase the patient's survival probability, and to improve the patient's prognosis. At present, the clinical MRD monitoring in China is mainly 8-12 flow detection, and the accuracy can reach 10 -3～-5 by detecting the expression of cell surface antigens and monitoring the MRD level in the disease progress. As the gold standard of the current clinical MRD detection, the 8-color flow detection technology has the advantages of wide application range, high speed and the like, is clinically used for evaluating early treatment response and prognosis risk evaluation of ALL patients, and the detection indexes mainly comprise surface proteins related to development stages such as CD10, CD19, CD20, CD33, CD34, CD38 and the like, and the 8-color flow detection results (shown in figure 1) of five clinical bone marrow samples are selected for illustration. CD10 is a surface protein present in B precursor cells and mature neutrophils, CD38 is a multifunctional transmembrane glycoprotein which is itself a membrane receptor capable of affecting B cell differentiation and proliferation and expression in B precursor cells, however, CD38 expression is reduced in most B-ALL cells. The above detection indexes have undergone long-term laboratory and clinical trial exploration and optimization, and the combinations used by different research institutes are still not exactly the same at present. In addition, ALL cells often express other important and complex surface antigens in addition to the common detection antigens, and ALL cell phenotypes often change during treatment, and patients after bone marrow transplantation often have little circulating leukocytes until hematopoietic engraftment and reconstitution. Current flow assays are difficult to accurately assess immunophenotype variability and accurately detect MRD levels in patients following bone marrow transplantation, and we refer to "Leukemia associated immunophenotype" (