CN103675277B - Flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe and kit - Google Patents
Flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe and kit Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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Abstract
The invention discloses a kind of flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe and kit, described fluorescence probe comprises four kinds of fluorescent-labeled antibody, described four kinds of fluorescent-labeled antibody are all with fluorescently-labeled GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody, wherein, different antibody is with different fluorescence labelings.Described kit comprises probe solution; Hemolytic agent; Phosphate buffer and positive criteria product.Fluorescence probe of the present invention respectively can with the rhabdomyosarcoma in marrow and neuroblastoma cell and acute leukemia initial cell specific binding, can by tumour cell common in flow cytometry quick and precisely antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and this three class of leukaemia children marrow, guiding clinical treatment, has good application prospect.
Description
Technical field
The present invention relates to a kind of medical treatment and detect reagent, particularly relate to a kind of flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe and kit.
Background technology
Clinically, occur tumour cell commonly leukaemia, rhabdomyosarcoma and neuroblastoma in children's marrow, the treatment of correct discriminating three to patient has important directive significance.Rhabdomyosarcoma and neuroblastoma are the common malignant entity tumors of children, have serious threat and harm to children's life.Some patients can obtain rehabilitation by operative treatment.But this disease easily bone marrow neoplasms occurs, often jeopardize infant life, chemotherapy, the radiotherapy even remedy measures such as autologous stem cell transplantation need be taked in time.Therefore be the key improving cure rate, reduce mortality ratio to the early detection of bone marrow neoplasms, early diagnosis and early treatment.
Diagnose rhabdomyosarcoma and neuroblastoma bone marrow neoplasms mainly through Bone Marrow Cell Morphology inspection both at home and abroad at present, but cytomorphology is insensitive for the diagnosis of the patient of early stage transfer, easy generation false negative result, and both cytomorphologies are similar with myeloblast to lymphoblast, very easily be misdiagnosed as leukaemia, simultaneously rhabdomyosarcoma and more difficult discriminating also similar to the cytomorphology of neuroblastoma in marrow.
Also cannot meet the requirement of clinician just because of the current various inspection methods to diagnosis and differential diagnosis rhabdomyosarcoma and neuroblastoma bone marrow neoplasms, seeking a kind of fast and convenient and accuracy and the high method of susceptibility, to come diagnosis and differential diagnosis rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic method very important.Flow cytometry (Flow Cytometry, FCM) is the high science and technology that development in recent years is got up, and it integrates computer technology, laser technology, fluid mechanics, cytochemistry, cellular immunology, has analysis and sorting cells function simultaneously.It not only can measure cell size, the proterties of internal particle, also can detect cell surface and cytoplasmic antigen, DNA in cell, rna content etc., multi parameter analysis can be carried out on individual cell level to colony's cell, namely on same cell, carry out the analysis of the co expression of 2-3 kind or plurality of antigens, detect at short notice and analyze a large amount of cell, and collect, store and process data, carry out multiparameter quantitative test, there is susceptibility and the high feature of accuracy, the diagnosis applying to leukaemia and lymphadenomatous marrow specimen clinically at present more, and the rare report of diagnosis and differential diagnosis to rhabdomyosarcoma and neuroblastoma.Because both cells do not have single specific antigen markers at present, flow cytometry is the combination of Multiple Antibodies to antidiastole rhabdomyosarcoma and knurl neuroblastoma bone marrow neoplasms and leukaemia key.
Summary of the invention
The invention provides a kind of flow cytometry antidiastole diagnosis rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe, utilize this fluorescence probe can obtain diagnostic result fast and accurately.
A kind of flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe, comprise four kinds of fluorescent-labeled antibody, described four kinds of fluorescent-labeled antibody are all with fluorescently-labeled GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody, wherein, different antibody is with different fluorescence labelings.
Rhabdomyosarcoma is the modal soft tissue neoplasms of children, and neuroblastoma belongs to neuroendocrine, and CD56 is the membrane glycoprotein N-CAM in an immunoglobulin like protein sample region, at the cell surface expression of neuroectodermal origin.CD90 and CD56 as adhesion molecule expression in human rhabdomyosarcoma cells and neuroblastoma, NK cell mainly also expresses the CD56 isomeride of this 140kDa, and CD45 is the restrictive membrane protein molecule of haemocyte series, express and comprising candidate stem cell except exo-erythrocytic film on the surface to mature blood cell (comprising NK cell), therefore CD90+CD56+/CD45-phenotype can avoid mistaken diagnosis to be NK cell tumour and leukaemia, and can as accusing of of judging that neuroendocrine tumor and rhabdomyosarcoma exist.
Neuroblastoma cell derives from the gangliocyte of differentiation difference, there is the expression of neuropeptide Y nerves CD56 antigen and gangliosides GD2 on its surface, and do not express leukocyte common antigen (LCA) CD45, rhabdomyosarcoma originates from striated muscle cell or the mesenchymal a kind of malignant tumour to striated muscle cell differentiation, do not express gangliosides GD2.Same leukaemia does not express GD2 but it expresses leukocyte common antigen (LCA) CD45 and initial cell mark CD90, therefore can be expressed identifying tumour cell common in this three class of rhabdomyosarcoma, neuroblastoma and leukaemia initial cell children marrow specimen by the difference of these four kinds of antigens of polychrome Flow Cytometry Assay.Three has different immunophenotypes, that is:
Human rhabdomyosarcoma cells: GD2
-cD90
+cD56
+cD45
-;
Neuroblastoma cell: GD2
+cD90
+cD56
+cD45
-;
Acute leukemia initial cell: GD2
-cD90
+cD56
-cD45
+.
The present invention combines GD2, CD90, CD56 and CD45 tetra-kinds of fluorescent-labeled antibody as probe, use the immunophenotype (tube method) of the common polychrome flow cytomery cell of Hospitals at Present can antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukaemia, specificity can reach 100%.
Concrete, described four kinds of fluorescent-labeled antibody are GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody.
For ensureing the specificity detected, four kinds of fluorescent-labeled antibody are monoclonal antibody.
Present invention also offers a kind of Flow Cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic kit, comprising:
(1) probe solution, includes four kinds of fluorescent-labeled antibody, and wherein, four kinds of fluorescent-labeled antibody are that different antibody is with different fluorescence labelings all with fluorescently-labeled GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody;
(2) hemolytic agent;
(3) phosphate buffer;
(4) positive criteria product.
Described probe solution take phosphate buffer as solvent.
Four kinds of described fluorescent-labeled antibody are specially GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody.Wherein, CD90-PE can purchased from American BD company, article No.: 555596, CD45-PerCP can purchased from American BD company, article No.: 347464, CD56-APC can purchased from American BD company, article No.: 341025, GD2-FITC preparation method can refer to CANCER RESEARCH, 46 phase in 1986 2988 pages, Highly selective recognition of human neuroblastoma cells by mouse monoclonal antibody to a cytoplasmic antigen.
The concentration ratio of described four kinds of fluorescent-labeled antibody is 1:1:1:1.
Consisting of of described hemolytic agent: 8.3% ammonium chloride and 15% paraformaldehyde.
Described probe solution comprises antiseptic, and described antiseptic is Sodium azide.
Described phosphate buffer is also containing containing antiseptic, and described antiseptic is Sodium azide.
The addition of described Sodium azide is 0.05 ~ 0.1%, is preferably 0.1%.
The concentration of the phosphate buffer used in kit is 0.8 ~ 1M, is preferably 1M, the pH=7.4 of this phosphate buffer.
It is pointed out that because children are without chronic lymphocytic leukemia, chronic myelocytic leukemia is few, and if no special instructions, leukaemia mentioned in the application all refers to acute leukemia.
Compared with prior art, beneficial effect of the present invention is:
Fluorescence probe of the present invention can be combined by the tumor cell specific in marrow, no cross reaction, can accurately quickly through Flow cytometry rhabdomyosarcoma and neuroblastoma whether bone marrow neoplasms, there is very high specificity (can reach 100%) and sensitivity, acute leukemia initial cell can be distinguished simultaneously, accurately simple and quick, there is good potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is the Wright's staining result figure of the present invention to embodiment 1 Bone Marrow of Patients sample; Wherein, A is rhabdomyosarcoma bone marrow neoplasms, and B is neuroblastoma bone marrow neoplasms.
Fig. 2 is the Flow cytometry result figure of the present invention to embodiment 1 rhabdomyosarcoma Bone Marrow of Patients sample; Wherein, A:CD45-and CD56+ cell establishes R1 door, B:GD2-and CD90+ cell establishes R2 door; C:FSC and SSC cell establishes R3 door; R2 derives from R1, and R3 derives from R2.
Fig. 3 is the Flow cytometry result figure of the present invention to embodiment 1 neuroblastoma Bone Marrow of Patients sample; Wherein, A:CD45-and CD56+ cell establishes R1 door, B:GD2+ and CD90+ cell establishes R2 door; C:FSC and SSC cell establishes R3 door; R2 derives from R1, and R3 derives from R2.
Fig. 4 is the Wright's staining result figure of embodiment 1 acute leukemia initial cell.
Fig. 5 is the Flow cytometry result figure of the present invention to embodiment 1 acute leukemic patient marrow specimen; Wherein, A:CD45+ and CD90+ cell establishes R1 door, B: cell display GD2-and CD45+ deriving from R1; C: cell display GD56-and CD45+ deriving from R1.
Fig. 6 a is the flow cytometry sensitivity Detection result figure of the present invention to embodiment 1 rhabdomyosarcoma Bone Marrow of Patients sample (dilution ratio is 1:100).
Fig. 6 b is the flow cytometry sensitivity Detection result figure of the present invention to embodiment 1 rhabdomyosarcoma Bone Marrow of Patients sample (dilution ratio is 1:1000).
Fig. 6 c is the flow cytometry sensitivity Detection result figure of the present invention to embodiment 1 rhabdomyosarcoma Bone Marrow of Patients sample (dilution ratio is 1:10000).
Fig. 7 is the Wright's staining result figure of the present invention to embodiment 2 rhabdomyosarcoma Cerebrospinal Fluid in Patients sample.
Fig. 8 is the Flow cytometry result figure of the present invention to embodiment 2 rhabdomyosarcoma Cerebrospinal Fluid in Patients sample, and wherein, A:CD45-and CD56+ cell establishes R1 door, B:GD2-and CD90+ cell establishes R2 door; C:FSC and SSC cell establishes R3 door; R2 derives from R1, and R3 derives from R2.
Embodiment
The present invention is explained further below in conjunction with embodiment.
Embodiment 1
GD2-FITC(provides for oneself, with neuroblastoma cell line LAN-1 immunity BALB/c mouse, get mouse bone-marrow-derived lymphocyte and murine myeloma cell strain NS1 to be fused into hybridoma and to prepare monoclonal antibody GD2 and flag F ITC fluorescein, concrete technical step with reference to magazine CANCER RESEARCH, 46 phase in 1986 2988 pages; Highly selective recognition of human neuroblastoma cells by mouse monoclonal antibody to a cytoplasmic antigen), CD90-PE(U.S. company BD, 555596), CD45-PerCP(U.S. company BD article No.:, 347464), CD56-APC(U.S. company BD article No.:, article No.: 341025).
One, kit composition:
1) probe solution (2mL × 1 bottle):
GD2-FITC:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of marked by fluorescein isothiocyanate;
CD90-PE:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of phycoerythrin mark;
CD45-PerCP:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of paramecium phyllochlorin mark;
CD56-APC:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of allophycocyanin mark;
Composition antibody concentration ratio GD2-FITC:CD90-PE:CD45-PerCP:CD56-APC is 1:1:1:1.
2) 10 × hemolytic agent (100mL × 1 bottle): 8.3% ammonium chloride, containing 15% paraformaldehyde;
3) 10 × phosphate buffer (PBS, pH=7.4,1M, containing 1% Sodium azide) (100mL × 1 bottle);
4) positive criteria product (2mL × 1 bottle): fix the cell line (5 × 10 with conserving liquid process
7individual cell/mL).
Two, the application of kit
Research object: the marrow specimen of 15 routine non-leukaemia and tumour children, 21 routine rhabdomyosarcomas, 25 routine neuroblastomas and 22 routine acute myeloblastic leukemia patients, marrow specimen is all from The Children's Hospital, Zhejiang University School of Medicine's outpatient service and inpatient.
Operation steps: 100 μ L bone marrow fluids, add each 20 μ L of monoclonal fluorescent-labeled antibody (GD2-FITC, CD90-PE, CD45-PerCP and CD56-APC) respectively, 4 DEG C of lucifuges react 30 minutes, add hemolytic agent 1mL, leave standstill 5 minutes, add PBS5mL, 500G centrifugation 5 minutes, abandon supernatant, sediment adds PBS300 μ L, and mixing, flow cytometer detects 50000 cells.
Streamed image analysis strategy:
Rhabdomyosarcoma: 1, CD45-and CD56+ establishes R1 door; 2, GD2-and CD90+ cell establishes R2 door; 3, FSC and SSC cell establishes the analysis of R3 door; 4, the positive cell number in R3 door is calculated.(R2 derives from R1; R3 derives from R2).
Neuroblastoma: 1, CD45-and CD56+ establishes R1 door; 2, GD2+ and CD90+ cell establishes R2 door; 3, FSC and SSC cell establishes the analysis of R3 door; 4, the positive cell number in R3 door is calculated.(R2 derives from R1; R3 derives from R2).
Acute leukemia initial cell: 1, CD45+ and CD90+ establishes R1 door; 2, CD45+ and GD2-cell is analyzed; 3, CD45+ and CD56-cell is analyzed.
Set up negative control simultaneously.
With immunophenotype, positive findings judges that cell type is as follows:
Phenotype is GD2
-cD90
+cD56
+cD45
-for human rhabdomyosarcoma cells;
Phenotype is GD2
+cD90
+cD56
+cD45
-for neuroblastoma cell;
Phenotype is GD2
-cD90
+cD56
-cD45
+for acute leukemia initial cell.
Detection sensitivity measures: respectively human rhabdomyosarcoma cells normal marrow cell is diluted to 1:100, the ratio of 1:1000,1:10000, detects equally in order to upper method.
Statistical method: utilize software SPSS11.5 (SPSS Inc., Chicago, IL, USA), the Sensitivity and Specificity of assay diagnostic method.
The testing result of all types of sample as shown in Figure 1, Figure 2, shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6 a, Fig. 6 b, Fig. 6 c.
Result shows: 15 routine non-leukaemia and tumour children all do not detect above three para-immunity phenotype cells; In 21 routine human rhabdomyosarcoma cells patients, 5 examples detect GD2
-cD90
+cD56
+cD45
-immunophenotype cell, clinical this 5 example of finally making a definite diagnosis is rhabdomyosarcoma bone marrow neoplasms; In 25 routine neuroblastoma patients, 11 examples detect GD2
+cD90
+cD56
+cD45
-immunophenotype cell, clinical this 11 example of finally making a definite diagnosis is neuroblastoma bone marrow neoplasms; 22 routine acute leukemia cellses are GD2
-cD90
+cD56
-cD45
+immunophenotyping; The specificity of flow cytometry to three class tumour cell diagnosis in marrow is 100%, and susceptibility is 1/10
-4(namely 1 tumour cell can be detected in 10000 normal cells).
Embodiment 2
One, kit composition:
1) probe solution (2mL × 1 bottle):
GD2-FITC:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of marked by fluorescein isothiocyanate;
CD90-PE:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of phycoerythrin mark;
CD45-PerCP:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of paramecium phyllochlorin mark;
CD56-APC:2mL PBS(is containing 0.1% Sodium azide) containing the 100 μ g antibody proteins (50 μ g/mL) of allophycocyanin mark;
Composition antibody concentration ratio GD2-FITC:CD90-PE:CD45-PerCP:CD56-APC is 1:1:1:1.
2) 10 × hemolytic agent (100mL × 1 bottle): 8.3% ammonium chloride, containing 15% paraformaldehyde;
3) 10 × phosphate buffer (PBS, pH=7.4,1M, containing 1% Sodium azide) (100mL × 1 bottle);
4) positive criteria product (2mL × 1 bottle): fix the cell line (5 × 10 with conserving liquid process
7individual cell/mL).
Two, the application of kit
Research object: 2 examples are suspected to be that rhabdomyosarcoma cerebrospinal fluid transfer patient punctures samples of CSF.
Operation steps, analysis strategy and statistical method are with embodiment 1.
Result shows: 2 routine rhabdomyosarcoma clinical signs of suspected are that cerebrospinal fluid transfer patient carries out puncture cerebrospinal fluid through this Antibody Combination flow cytomery, find GD2
-cD90
+cD56
+cD45
-immunophenotype cell (Fig. 7, Fig. 8), this two example of final clinical diagnosis is the transfer of rhabdomyosarcoma cerebrospinal fluid, and carries out intrathecal chemotherapy and craniospinal radiation therapy.Show that fluorescence probe of the present invention can monitor the transfer of rhabdomyosarcoma cerebrospinal fluid, instruct clinical rational drug use.
Claims (3)
1. fluorescence probe is preparing the application in flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic kit, described fluorescence probe comprises four kinds of fluorescent-labeled antibody, described four kinds of fluorescent-labeled antibody are all with fluorescently-labeled GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody, wherein, different antibody is with different fluorescence labelings.
2. apply as claimed in claim 1, it is characterized in that, described four kinds of fluorescent-labeled antibody are GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody.
3. apply as claimed in claim 1 or 2, it is characterized in that, four kinds of fluorescent-labeled antibody are monoclonal antibody.
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| CN112391263B (en) * | 2020-11-18 | 2022-05-27 | 深圳市儿童医院 | Neuroblastoma circulating tumor cell capturing chip and manufacturing method thereof |
| CN115389766B (en) * | 2022-08-29 | 2023-09-22 | 深圳市瑞格生物科技有限公司 | Marker for diagnosing whether neuroblastoma is subjected to bone marrow infiltration and application thereof |
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