CN109439732B - Kit for three-dimensional noninvasive tumor early screening - Google Patents
Kit for three-dimensional noninvasive tumor early screening Download PDFInfo
- Publication number
- CN109439732B CN109439732B CN201811587184.0A CN201811587184A CN109439732B CN 109439732 B CN109439732 B CN 109439732B CN 201811587184 A CN201811587184 A CN 201811587184A CN 109439732 B CN109439732 B CN 109439732B
- Authority
- CN
- China
- Prior art keywords
- blood
- detection module
- sample
- liquid
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 63
- 238000012216 screening Methods 0.000 title claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 171
- 238000001514 detection method Methods 0.000 claims abstract description 103
- 210000004027 cell Anatomy 0.000 claims abstract description 75
- 210000004369 blood Anatomy 0.000 claims abstract description 64
- 239000008280 blood Substances 0.000 claims abstract description 64
- 239000007788 liquid Substances 0.000 claims abstract description 53
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 49
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 43
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 38
- 239000007853 buffer solution Substances 0.000 claims abstract description 29
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 29
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 29
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 29
- 238000004140 cleaning Methods 0.000 claims abstract description 24
- 210000000601 blood cell Anatomy 0.000 claims abstract description 21
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 claims abstract description 16
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 claims abstract description 16
- 238000004458 analytical method Methods 0.000 claims abstract description 16
- 238000002509 fluorescent in situ hybridization Methods 0.000 claims abstract description 16
- 238000002331 protein detection Methods 0.000 claims abstract description 16
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims abstract description 15
- 239000000427 antigen Substances 0.000 claims abstract description 15
- 102000036639 antigens Human genes 0.000 claims abstract description 15
- 108091007433 antigens Proteins 0.000 claims abstract description 15
- 239000004005 microsphere Substances 0.000 claims abstract description 12
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims abstract description 11
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 210000000265 leukocyte Anatomy 0.000 claims abstract description 11
- 239000003550 marker Substances 0.000 claims abstract description 11
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 8
- 239000000725 suspension Substances 0.000 claims abstract description 7
- 239000011521 glass Substances 0.000 claims abstract description 5
- 239000012089 stop solution Substances 0.000 claims abstract description 5
- 238000011529 RT qPCR Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 210000005259 peripheral blood Anatomy 0.000 claims description 33
- 239000011886 peripheral blood Substances 0.000 claims description 33
- 238000012360 testing method Methods 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 21
- 238000011144 upstream manufacturing Methods 0.000 claims description 15
- 208000032839 leukemia Diseases 0.000 claims description 10
- 238000007789 sealing Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 239000013049 sediment Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 210000000349 chromosome Anatomy 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 210000001519 tissue Anatomy 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 239000012916 chromogenic reagent Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003146 anticoagulant agent Substances 0.000 claims description 3
- 229940127219 anticoagulant drug Drugs 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 239000008119 colloidal silica Substances 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 230000005484 gravity Effects 0.000 claims description 3
- 210000003917 human chromosome Anatomy 0.000 claims description 3
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 3
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 3
- 239000011159 matrix material Substances 0.000 claims description 3
- 230000003204 osmotic effect Effects 0.000 claims description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000011701 zinc Substances 0.000 claims description 3
- 229910052725 zinc Inorganic materials 0.000 claims description 3
- 108010004729 Phycoerythrin Proteins 0.000 claims description 2
- 238000003018 immunoassay Methods 0.000 claims description 2
- 210000005087 mononuclear cell Anatomy 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 239000000872 buffer Substances 0.000 abstract description 4
- 238000010790 dilution Methods 0.000 abstract description 4
- 239000012895 dilution Substances 0.000 abstract description 4
- 230000003902 lesion Effects 0.000 abstract description 4
- 239000012898 sample dilution Substances 0.000 abstract description 2
- 210000005266 circulating tumour cell Anatomy 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 14
- 239000012192 staining solution Substances 0.000 description 10
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 239000000439 tumor marker Substances 0.000 description 7
- 101150084041 WT1 gene Proteins 0.000 description 6
- 239000006059 cover glass Substances 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 101500016899 Arabidopsis thaliana C-terminally encoded peptide 8 Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000009401 metastasis Effects 0.000 description 4
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000010191 image analysis Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 206010048612 Hydrothorax Diseases 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 208000007660 Residual Neoplasm Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004880 lymph fluid Anatomy 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000006853 reporter group Chemical group 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 101150084967 EPCAM gene Proteins 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000018732 detection of tumor cell Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 102000050607 human TSGF Human genes 0.000 description 1
- 108700002006 human TSGF Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit for three-dimensional noninvasive tumor early screening, and relates to the technical field of diagnostic reagents. The blood sample collection module in the kit comprises at least three blood collection tubes and blood collection tube jacks; the cell detection module comprises a sample density separation liquid, a first buffer liquid, a first cleaning liquid, an antigen retrieval buffer liquid, a tissue fixing liquid, a sample dilution liquid, a second buffer liquid, a dilution liquid for blood cell analysis, a FISH probe, a second cleaning liquid and a fluorescence labeling antibody, wherein the fluorescence labeling antibody is a fluorescence labeling anti-human leukocyte surface marker antibody CD31, CD45 and CD54; the nucleic acid detection module comprises an oligonucleotide double-fluorescence labeling Taqman probe, a specific primer and a fluorescent quantitative qPCR reaction system reagent; the protein detection module comprises a 96-well reaction plate, a third buffer solution, a labeled mixed antibody solution, microsphere suspension, microsphere solution, stop solution, a blood collection tube and a glass slide. The kit can detect the tumor cells with abundant types and high detection precision, and can detect free cancerous components at the stage of precancerous lesions.
Description
Technical Field
The invention relates to the technical field of diagnostic reagents, in particular to a kit for three-dimensional noninvasive tumor early screening.
Background
Malignant tumors are one of the important diseases that endanger human health. The world health organization cancer research Institute (IARC) data shows that: the global cases of malignant tumor are over 1400 ten thousand in 2012, and the number of malignant tumors is about 800 ten thousand. Statistics of the national cancer center show that in China, new cancer cases reach 429 ten thousand annually, and account for 22% of the global new cases, and mortality accounts for 27% of the global mortality. In china, 80% of cancers have entered advanced stages of cancer when first diagnosed, thus losing optimal treatment opportunities.
Circulating tumor cells (circulating tumor cells, CTCs) are key to tumor metastasis and recurrence, and they shed from primary foci, invade the blood circulation, become CTCs, evade body immunity, colonize distant organs or primary organs, and form metastasis and recurrence lesions. It has been found that detecting the number of CTCs helps to detect cancer early, to determine prognosis of cancer patients, and to evaluate the effect of chemotherapy on cancer patients; when CTCs exhibit epithelial-to-mesenchymal transition (EMT), over-expressed epithelial cell adhesion molecule (epithelial cellular adhesion molecule, epCAM), it is often suggested that tumor patients have poor prognosis, so that detection of CTCs is visually referred to as "liquid biopsy", and has important clinical research and application value.
The WT1 gene is highly expressed in most Acute Myelogenous Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). This makes WT1 mRNA a tumor marker for leukemia blasts, and one leukemia cell out of 10 ten thousand normal Peripheral Blood Mononuclear Cells (PBMCs) can be detected by quantifying WT1 mRNA (WT 1 detection). In Chronic Myelogenous Leukemia (CML) and myelodysplastic syndrome (MDS), WT1 mRNA expression levels increase with disease progression. WT1 detection is currently considered an important test for controlling acute leukemia and MDS by detecting Minimal Residual Disease (MRD) of leukemia. The WT1 gene is expressed at high levels in almost all types of solid tumors, with its expression level acting as a significant prognostic factor.
At present, the tumor early screening technology in the world is in the starting stage, and the means of tumor early screening are very limited. The latest early detection method of malignant tumor at present comprises a multi-tumor marker protein chip detection system (C12 chip), a tumor gene mutation detection kit, a six-tumor marker determination kit and a human malignant Tumor Specific Growth Factor (TSGF) enzyme-linked immunosorbent assay (ELISA) analysis kit. However, the detection false positive of the multi-tumor marker protein chip detection system (C12 chip) is high, and the detection efficiency is low. The tumor gene mutation detection reagent depends on the complex operation of Polymerase Chain Reaction (PCR), is easy to generate false positive, can only be used for susceptibility risk assessment, and can not detect leukemia in a plurality of kits at present.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a kit for three-dimensional noninvasive tumor early screening.
According to an aspect of an embodiment of the present invention, there is provided a kit for three-dimensional noninvasive tumor early screening, characterized in that the kit includes a blood sample collection module, a cell detection module, a nucleic acid detection module, a protein detection module, and at least one test tube receptacle;
the blood sample collection module comprises at least three blood collection tubes and blood collection tube jacks;
the cell detection module comprises a sample density separating liquid, a first buffer solution, a first cleaning solution, an antigen retrieval buffer solution, a tissue fixing liquid, a sample diluting liquid, a second buffer solution, a blood cell analysis diluting liquid, a FISH probe, a second cleaning solution and a fluorescence labeling antibody, wherein the fluorescence labeling antibody is a fluorescence labeling anti-human leucocyte surface marker antibody CD31, CD45 and CD54, and the FISH probe is a fluorescence labeling probe capable of specifically recognizing human chromosome 8;
the nucleic acid detection module comprises an oligonucleotide double-fluorescence labeling Taqman probe, a specific primer and a fluorescent quantitative qPCR reaction system reagent;
the protein detection module comprises a 96-hole reaction plate, a third buffer solution, a marked mixed antibody solution, microsphere suspension, microsphere solution, a stop solution, a blood collection tube with an anticoagulant and a glass slide, wherein the marked mixed antibody solution comprises at least one of antibodies AFP, CEA, NSE, SCC, CA, CA125, CA153 and CA 125;
the test tube jack is arranged on the periphery of the blood sample collection module, the cell detection module, the nucleic acid detection module and the protein detection module and is used for inserting a test tube for testing.
In a preferred embodiment, the 5 'end of the oligonucleotide double fluorescent label Taqman probe in the nucleic acid detection module is a fluorescent reporter group, the 3' end is a fluorescent quenching group, and the specific primer comprises 7-10 exon-encoded zinc finger structural regions.
In a preferred embodiment, the nucleic acid detection module comprises at least one set of oligonucleotide double fluorescent label Taqman probes and specific primers shown in combination one, combination two, combination three, wherein:
in combination one, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-CCTTCAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-GGCATCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GAGAGTCAG ACTTGAAAGCACT-3';
in the second combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-AAGCTAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-TTACTCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GCATGTCAG ACTTGAAAGTCTC-3';
in combination three, the probe sequence of the oligonucleotide double fluorescent label Taqman probe is 5'-TTCAAGCTTACACTTACATCGCTTA-3', the upstream primer base sequence of the specific primer is 5'-CCCATGCTACGGAGGCTAGCATTT-3', and the downstream primer base sequence of the specific primer is 5'-GTACTAAGCGTTAGCATTACTGCATTA-3'.
In a preferred embodiment, the sample density separation fluid comprises ferric sulfate, sucrose, polyvinylpyrrolidone coated colloidal silica, and has a specific gravity of 1.07125 to 1.08158 g/ml at 20 ℃; the first buffer solution contains anti-leukocyte antibody coupled immunomagnetic beads, the osmotic pressure is 260-300 mOsm/kgH2O, and the pH is 6.8-7.8.
In a preferred embodiment, the cell detection module, the nucleic acid detection module and the protein detection module are located at respective predetermined positions corresponding to a predetermined number of test tube receptacles.
In a preferred embodiment, the kit detects tumor cells in peripheral blood.
Compared with the prior art, the kit for three-dimensional noninvasive tumor early screening has the following advantages:
the CTC capturing technology method for the kit for three-dimensional noninvasive tumor early screening is simple, convenient, quick and efficient, and is suitable for blood, lymph fluid, bone marrow, hydrothorax, ascites, urine, cerebrospinal fluid and the like in any volume; WT1-mRNA detection overcomes the defect that leukemia cannot be detected by the existing CTC, improves the sensitivity of a cancer screening method to the greatest extent, and can detect free cancerous components at a precancerous lesion stage, so that a wider intervention space is obtained for preventing and treating tumors; in addition, the invention adopts a plurality of groups of oligonucleotide double fluorescent markers Taqman probes and specific primers to detect the nucleic acid of the cells, so that the technical problem of lower detection result precision caused by incomplete gene complementation progress of a single group of Taqman probes and specific primers in the detection process can be avoided, and the accurate detection of the cancerous cells can be realized from the nucleic acid level; on the basis of distinguishing blood-borne and non-blood-borne cells by immunofluorescence staining, tumor marker staining and FISH detection are synchronously carried out on the enriched non-blood-borne tumor epithelial cells, so that cells with abnormal numbers of non-blood-borne chromosomes and different tumor surface markers are effectively identified, and CTC subclasses with different clinical meanings such as drug resistance, tumor metastasis and recurrence are identified. Proved by a large number of clinic tests. Therefore, the kit for three-dimensional noninvasive tumor early screening provided by the invention has extremely high accuracy and precision in the aspects of potential detection and diagnosis of cancers, and can shorten the detection interval of the same cell sample through the rapid comprehensive detection of a cell layer, a nucleic acid layer and a protein layer, so that the problem of error of a detection result caused by continuous division of the cell sample in the processes of placement and pretreatment is avoided.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
Fig. 1 is a schematic block diagram of a kit for three-dimensional noninvasive tumor early screening according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a kit for three-dimensional noninvasive tumor early screening according to an embodiment of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the following specific embodiments (but not limited to the illustrated embodiments) and the accompanying drawings, and the specific methods of the present embodiments are only for illustrating the present invention, and the scope of the present invention is not limited to the embodiments, and the present invention can be modified and changed in various forms and structures in application, which are also within the scope of protection of the claims of the present application based on the equivalents of the present invention.
Referring to fig. 1, a schematic block diagram of a kit 100 for three-dimensional noninvasive tumor early screening according to an embodiment of the present invention is shown, and referring to fig. 1, the kit 100 includes a blood sample collection module 110, a cell detection module 120, a nucleic acid detection module 130, a protein detection module 140, and at least one test tube jack 150.
The blood sample collection module 110 includes at least three blood collection tubes and blood collection tube receptacles.
The cell detection module 120 includes a sample density separating solution, a first buffer solution and a first cleaning solution, an antigen retrieval buffer solution, a tissue fixing solution, a sample dilution solution, a second buffer solution, a dilution solution for blood cell analysis, a FISH probe, a second cleaning solution and a fluorescence labeling antibody, wherein the fluorescence labeling antibody is a fluorescence labeling anti-human leukocyte surface marker antibody CD31, CD45 and CD54, and the FISH probe is a fluorescence labeling probe capable of specifically recognizing human chromosome 8.
The nucleic acid detection module 130 comprises an oligonucleotide double-fluorescence labeling Taqman probe, a specific primer and a fluorescent quantitative qPCR reaction system reagent.
The protein detection module 140 comprises a 96-well reaction plate, a third buffer solution, a labeled mixed antibody solution, a microsphere suspension, a microsphere solution, a stop solution, a blood collection tube with an anticoagulant, and a glass slide, wherein the labeled mixed antibody solution comprises at least one of antibodies AFP, CEA, NSE, SCC, CA, CA125, CA153 and CA 125.
The test tube jack 150 is disposed on the peripheral sides of the blood sample collection module 110, the cell detection module 120, the nucleic acid detection module 130, and the protein detection module 140, and is used for inserting test tubes for testing.
The invention comprehensively uses fluorescent labeled antibodies as fluorescent labeled anti-human leukocyte surface marker antibodies CD31, CD45 and CD54 in the cell detection module 120 to jointly carry out tumor labeling, wherein the CD54 belongs to a member of immunoglobulin superfamily (IGSF) in adhesion molecules, and is an important adhesion molecule for mediating adhesion reaction. CD54 is expressed on resting vascular endothelial cells at a low level, and exerts biological activity through binding with specific receptors on the surface of the vascular endothelial cells, and as protein of leucocytes and endothelial cells transmembrane, CD54 plays an important role in stabilizing intercellular interaction and promoting migration of leucocytes and endothelial cells, so that the kit provided by the embodiment of the invention can identify blood cell samples to be detected at a cell level by adopting CD31, CD45 and CD54 together for tumor marking at a cell detection module, thereby accurately determining whether target users corresponding to the peripheral blood samples suffer from tumors.
It should be noted that, after enriching blood cells, the existing detection method for tumor cells only carries out single-aspect detection on the same blood cells, and the detection method can only carry out single detection results of cancer or non-cancer on the diseased condition of a blood cell provider, but can not determine whether the blood cells provide a potential possibility of cancer.
In a preferred embodiment, the 5 'end of the oligonucleotide double fluorescent label Taqman probe in the nucleic acid detection module 130 is a fluorescent reporter group, the 3' end is a fluorescent quenching group, and the specific primer comprises 7-10 exon-encoded zinc finger structural regions.
In a preferred embodiment, the nucleic acid detection module 130 comprises at least one set of oligonucleotide double fluorescent label Taqman probes and specific primers shown in combination one, combination two, combination three, wherein:
in combination one, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-CCTTCAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-GGCATCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GAGAGTCAG ACTTGAAAGCACT-3';
in the second combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-AAGCTAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-TTACTCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GCATGTCAG ACTTGAAAGTCTC-3';
in combination three, the probe sequence of the oligonucleotide double fluorescent label Taqman probe is 5'-TTCAAGCTTACACTTACATCGCTTA-3', the upstream primer base sequence of the specific primer is 5'-CCCATGCTACGGAGGCTAGCATTT-3', and the downstream primer base sequence of the specific primer is 5'-GTACTAAGCGTTAGCATTACTGCATTA-3'.
For example, the kit provided by the embodiment of the invention can adopt any one group of oligonucleotide double fluorescent labeling Taqman probes and specific primer combinations shown in the combination I, the combination II and the combination III; or, adopting any two groups of oligonucleotide double fluorescent markers Taqman probes and specific primers shown in the combination I, the combination II and the combination III; or, the combination of the Taqman probe and the specific primer with the double fluorescent markers of the three groups of oligonucleotides of the combination one, the combination two and the combination three is adopted.
It should be noted that, the combination of the gene sequences of the oligonucleotide double-fluorescence-labeled Taqman probe and the specific primer adopted by the invention is not limited to any one of the three groups of sequence pairs, and the number of the combinations adopted by the kit is not limited to three, and any invention based on the combination of the gene sequences of the oligonucleotide double-fluorescence-labeled Taqman probe and the specific primer belongs to the technical teaching provided by the invention.
The kit provided by the embodiment of the invention adopts a plurality of groups of oligonucleotide double-fluorescence labeling Taqman probes and specific primers to detect the nucleic acid of cells, so that the technical problem of lower detection result precision caused by incomplete gene complementation progress of a single group of Taqman probes and specific primers in the detection process can be avoided, and the accurate detection of cancerous cells can be realized from the nucleic acid level.
In a preferred embodiment, the sample density separation fluid comprises ferric sulfate, sucrose, polyvinylpyrrolidone coated colloidal silica, and has a specific gravity of 1.07125 to 1.08158 g/ml at 20 ℃; the first buffer solution contains anti-leukocyte antibody coupled immunomagnetic beads, the osmotic pressure is 260-300 mOsm/kgH2O, and the pH is 6.8-7.8.
In a preferred embodiment, the cell detection module 120, the nucleic acid detection module 130, and the protein detection module 140 are located at respective predetermined positions corresponding to a predetermined number of test tube receptacles 150.
Fig. 2 shows a schematic diagram of a kit for three-dimensional noninvasive tumor early screening according to an embodiment of the present invention.
Through in the kit cell detection module nucleic acid detection module each predetermine the position that the protein detection module is located respectively correspond and set up the test tube jack, can make each detection module wait to examine or the reagent of preparation arrange in the module area that corresponds, can accelerate inspector's detection efficiency simultaneously, prevent that inspector from taking the reagent by mistake, the inspector of being convenient for detects.
In a preferred embodiment, the kit 100 detects tumor cells in peripheral blood.
It should be noted that, the kit provided by the invention can complete the accurate detection of tumor cells only by collecting 18mL of peripheral blood at least, and the tumor cells comprise circulating solid tumor cells with epithelial sources or tumor cells without epithelial sources and leukemia cells.
The CTC capturing technology method for the kit for three-dimensional noninvasive tumor early screening is simple, convenient, quick and efficient, and is suitable for blood, lymph fluid, bone marrow, hydrothorax, ascites, urine, cerebrospinal fluid and the like in any volume; WT1-mRNA detection overcomes the defect that leukemia cannot be detected by the existing CTC, improves the sensitivity of a cancer screening method to the greatest extent, and can detect free cancerous components at a precancerous lesion stage, so that a wider intervention space is obtained for preventing and treating tumors; in addition, the invention adopts a plurality of groups of oligonucleotide double fluorescent markers Taqman probes and specific primers to detect the nucleic acid of the cells, so that the technical problem of lower detection result precision caused by incomplete gene complementation progress of a single group of Taqman probes and specific primers in the detection process can be avoided, and the accurate detection of the cancerous cells can be realized from the nucleic acid level; on the basis of distinguishing blood-borne and non-blood-borne cells by immunofluorescence staining, tumor marker staining and FISH detection are synchronously carried out on the enriched non-blood-borne tumor epithelial cells, so that cells with abnormal numbers of non-blood-borne chromosomes and different tumor surface markers are effectively identified, and CTC subclasses with different clinical meanings such as drug resistance, tumor metastasis and recurrence are identified. Proved by a large number of clinic tests. Therefore, the kit for three-dimensional noninvasive tumor early screening provided by the invention has extremely high accuracy and precision in the aspects of potential detection and diagnosis of cancers, and can shorten the detection interval of the same cell sample through the rapid comprehensive detection of a cell layer, a nucleic acid layer and a protein layer, so that the problem of error of a detection result caused by continuous division of the cell sample in the processes of placement and pretreatment is avoided.
In order to better illustrate the kit 100 for three-dimensional noninvasive tumor early screening provided by the embodiment of the invention, a corresponding use method of the kit 100 is also shown, and the use method comprises the following steps:
(1) Peripheral blood sample collection: at least three peripheral blood samples are collected from a target user using a blood collection tube and subjected to homogenization and centrifugation.
(2) Enrichment of non-blood-borne nucleated rare cells: and (3) adding a first cleaning solution into the collected peripheral blood sample, uniformly mixing, adding the mixture into a sample density separating solution, centrifuging, collecting peripheral blood sample cells with the upper layer removed of red blood cells, adding a first buffer solution into the mixture, shaking, placing the mixture on a magnetic rack, sucking and collecting liquid which is not adsorbed at the center of the mixture, adding the first cleaning solution into the mixture, centrifuging, and removing the supernatant to 100ul to obtain the enriched cell sediment of non-blood-origin nucleated rare cells.
(3) Identification of non-blood-borne nucleated rare cells: adding fluorescent labeled antibodies, namely fluorescent labeled anti-human leukocyte surface marker antibodies CD31, CD45 and CD54, into the enriched cell sediment, incubating for a preset period of time, washing to remove unbound fluorescent labeled anti-human leukocyte surface marker antibodies, fixing and drying to prepare a chromosome fluorescent in-situ hybridization sample, adding an immune chromogenic reagent FISH probe, hybridizing the sealing sheet, placing the sealing sheet under a fluorescent microscope for shooting and storing cell images, and identifying non-blood-induced nucleated rare cells according to the cell images, thereby determining whether a target user corresponding to the peripheral blood sample has tumors.
(4) Detection of WT 1-mRNA: extracting total RNA of mononuclear cells in peripheral blood, carrying out reverse transcription to obtain cDNA, quantitatively detecting copy numbers of target genes and reference genes ABL in a sample by using an AB7500 type fluorescent quantitative PCR instrument and a TaqMan probe method, and determining whether a target user corresponding to the peripheral blood sample has potential leukemia risk according to the copy numbers of the ABL, wherein a gene sequence combination of an oligonucleotide double-fluorescence labeling Taqman probe and a specific primer used by the TaqMan probe method comprises at least one group of oligonucleotide double-fluorescence labeling Taqman probes and specific primers shown in a combination one, a combination two and a combination three.
The gene sequence combinations of the oligonucleotide double fluorescent labeling Taqman probes and the specific primers in each combination are as follows:
in combination one, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-CCTTCAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-GGCATCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GAGAGTCAG ACTTGAAAGCACT-3';
in the second combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-AAGCTAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-TTACTCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GCATGTCAG ACTTGAAAGTCTC-3';
in combination three, the probe sequence of the oligonucleotide double fluorescent label Taqman probe is 5'-TTCAAGCTTACACTTACATCGCTTA-3', the upstream primer base sequence of the specific primer is 5'-CCCATGCTACGGAGGCTAGCATTT-3', and the downstream primer base sequence of the specific primer is 5'-GTACTAAGCGTTAGCATTACTGCATTA-3'.
(5) Detection of saccharide antigen: the method comprises the steps of adopting a double-antibody sandwich immunoassay method, adding mixed liquid of phycoerythrin PE marked detection antibodies into suspension of fluorescent coding microspheres respectively crosslinked with anti-preset type tumor antigen capture monoclonal antibodies after reacting with tumor antigens in serum of a peripheral blood sample, finally forming microsphere-capture antibody-detection antibody-PE complex, detecting on a Luminex multifunctional flow dot matrix instrument, calculating to obtain concentration values of all tumor markers according to detection data, and determining whether a target user corresponding to the peripheral blood sample has potential tumor risks according to the concentration values of all tumor markers, wherein the detection of the carbohydrate antigens comprises detection of at least one of antigens AFP, CEA, NSE, SCC, CA, CA125, CA153 and CA 125.
It should be noted that, specifically, the operation steps of the method for using the kit for three-dimensional noninvasive tumor early screening are as follows:
1. preparation work
Step 101, respectively collecting 6.0ml peripheral blood samples from veins by using at least three blood collection tubes, and uniformly mixing the blood collection tubes upside down after the collection is finished; for each blood collection tube, after the blood collection tube with the collected peripheral blood sample is subjected to centrifugal treatment for 15 minutes at room temperature, the supernatant of the peripheral blood sample inside and outside the tube is discarded to the position 5 mm above the brownish red sediment, and then the blood collection tube is inserted into a blood collection tube jack of a blood collection module for standby.
Step 102, placing 110ml of the first cleaning fluid and 3ml of the sample density separating fluid into a water bath kettle with the temperature of 27 ℃ for preheating and uniformly mixing.
Step 103, adding a proper amount of first buffer solution into a 2ml centrifuge tube, standing for 20 seconds, then absorbing and discarding the supernatant, adding 1ml of first cleaning solution for washing, uniformly mixing and standing for 20 seconds, then absorbing and discarding the supernatant of the first buffer solution again, and continuously adding a proper amount of first buffer solution into the 2ml centrifuge tube to reach the original volume.
Step 104, preparing a second buffer solution: after accurate quantification of all second buffers, 10-fold dilution was performed with deionized water.
Step 105, 2 staining jars, respectively designated as jar a and jar B, were prepared, and 40ml of the corresponding liquid was added to each jar in the following order:
1) Cylinder a: the second buffer solution is preheated to 37 ℃ in a constant-temperature water bath kettle before use;
2) Cylinder B: absolute ethyl alcohol and preserving at room temperature.
And 106, taking out 16m of the first cleaning solution before each detection, sub-packaging the sample diluent according to the quantity of 80 mu l of each detection sample, preheating to 37 ℃ in a constant-temperature water bath for later use, sub-packaging the diluent for blood cell analysis according to the quantity of 80 mu l of each detection sample, sub-packaging the second cleaning solution according to the quantity of 600 mu l of each detection sample, and preheating the liquids to 27 ℃ for later use.
Step 107, after melting the immune chromogenic reagent FISH probe stored at the temperature of minus 20 ℃ at room temperature, performing high-speed centrifugation on the needle tube of the FISH probe on an oscillator for 5 seconds after head and tail oscillation, and performing head and tail oscillation again and centrifugation.
2. Cell-level detection
Step 201, taking out a blood collection tube with a peripheral blood sample from a blood collection tube jack of a blood collection module, adding a first cleaning solution to the blood collection tube to a preset position, sealing, mixing the blood collection tube with the first cleaning solution upside down, and then placing the blood collection tube into a test tube jack corresponding to a cell detection module for testing.
Step 202, adding 3ml of sample density separating liquid into 50ml of centrifuge tube a, slowly adding blood cells in a blood collection tube to the top layer of the sample density separating liquid along the liquid surface of the tube wall of the centrifuge tube a, and centrifuging the mixed liquid in the centrifuge tube a at room temperature for 6 minutes.
The mixed liquid in the centrifugal tube a after centrifugal treatment is divided into three layers of liquid, wherein the upper layer of liquid is yellow, the middle layer of liquid is transparent or light red, and the bottom layer of liquid is dark brown red sedimentary red blood cells.
Step 203, transferring the upper layer liquid and the middle layer liquid in the centrifuge tube a into a centrifuge tube b with the volume of 50ml, and uniformly mixing the mixed liquid in the centrifuge tube b.
Step 204, slowly adding the first buffer solution into the centrifuge tube b according to the amount of 300 mu l of each tube, then obliquely fixing the centrifuge tube b on a shaking table, and shaking for 20 minutes at the room temperature according to the rotation speed of 125rpm, wherein the maximum shaking of the liquid level is between 30 ml and 35 ml.
Step 205, fixing the centrifuge tube b on a magnetic rack, standing for 2 minutes, then extending into the center of the centrifuge tube b by using a 5ml gun head, sucking the cell liquid to be tested and transferring the cell liquid to a 50ml centrifuge tube c.
And 206, adding the first cleaning solution to 45ml into the centrifuge tube c, balancing, mixing the mixed solution in the centrifuge tube c uniformly, centrifuging at room temperature for 5 minutes, discarding the supernatant to 100 mu l, gently oscillating the mixed cells in a vortex mixer, adding the first cleaning solution to 45ml, mixing uniformly to obtain enriched cell sediment of non-blood-induced nucleated rare cells, centrifuging at room temperature for 5 minutes, and then testing.
Step 207, adding 2 mu l of antigen retrieval buffer solution into the enriched cell sediment of the non-blood-related nucleated rare cells in the centrifuge tube c, gently shaking and uniformly mixing the precipitated cells by using a shaking mixer, and then standing for 10 minutes at room temperature.
Step 208, 200. Mu.l of a blood cell analysis dilution, 1. Mu.l of a blood cell analysis staining solution (CD 45), 1. Mu.l of a blood cell analysis staining solution (CD 31), and 1. Mu.l of a blood cell analysis staining solution (CD 54) were mixed to prepare a blood cell analysis staining solution mixture, and the blood cell analysis staining solution mixture was gently blown 10 times with a sample applicator to thoroughly mix the antibodies, and then the prepared blood cell analysis staining solution mixture was left at room temperature in a dark place.
Step 209, adding 200. Mu.l of the staining solution mixture for blood cell analysis into the centrifuge tube c under the condition of shading, gently shaking and uniformly mixing the precipitated cells by using a shaking mixer, and incubating for 20 minutes at room temperature under the condition of shading.
Step 210, adding 5ml of first cleaning liquid into a 50ml centrifuge tube c, gently blowing and mixing the mixture by using a 5ml pipette, transferring the mixture into a new 15ml centrifuge tube, adding the first cleaning liquid to 14ml, balancing, reversing the mixture, mixing the mixture uniformly, centrifuging the mixture at room temperature for 5 minutes, and discarding the supernatant to 100 mu l.
Step 211, lightly blowing and mixing the mixed liquid in the centrifuge tube c, adding 100 mu l of tissue fixing liquid, lightly blowing and mixing by using a reserved gun head of a sample adder, respectively coating the mixed liquid in the centrifuge tube c in a glass slide specimen frame, and drying to obtain a specimen to be tested.
And 212, placing the sample to be tested in a drying oven at 30-32 ℃ for drying and ventilation for 10-12 hours, closing the temperature of the drying oven, then continuously standing the sample to be tested in the drying oven for 30 minutes, cooling to room temperature, and immediately performing subsequent detection.
Step 213, performing chromosome fluorescence in situ hybridization, wherein the steps are as follows:
(a) Mixing 20 μl of tissue fixing solution and 180 μl of sample diluent to obtain mixed solution, slightly dripping 200 μl of mixed solution along the inner side corner of a specimen frame of a specimen to be detected to cover the specimen frame, standing at room temperature in dark for 10 min, and sucking and discarding redundant mixed solution of the specimen to be detected;
(b) 200 mu l of second buffer solution is gently dripped along the inner side corner of a specimen frame of a specimen to be detected, and then the excessive second buffer solution is immediately sucked and removed, and the process is repeated for 2 times.
(c) 200 mu l of absolute ethyl alcohol is gently dripped along the inner side corners of a specimen frame of a specimen to be detected, and then the excessive absolute ethyl alcohol is immediately absorbed and removed, and the process is repeated for 2 times.
(d) Inserting a sample to be tested into a jar B (absolute ethyl alcohol) for standing for 2 minutes, taking out the sample to be tested, standing on filter paper to suck residual liquid, using a miniature blower to gently blow the sample to be tested to be completely dried, taking out a split charging immune chromogenic reagent FISH probe (CEP 8 probe), dropwise adding 10 mu l of probe into the center of a sample frame of the sample to be tested, immediately using tweezers to lay a cover glass on the probe liquid, and spreading the liquid to the periphery of the whole sample frame; cutting off the tip of a 1ml sample feeder, sealing the edges of four sides of the cover glass by using a sealing glue, and directly placing the cover glass into a hybridization instrument for hybridization, wherein the parameters of the hybridization process comprise: denaturation at 76 ℃,10 min; the hybridization temperature was 37℃and the hybridization time was 3 hours.
(e) And taking out the sample to be detected after hybridization, tearing off the sealing glue of the sample to be detected by using tweezers, placing the sample to be detected in a cylinder A (second buffer solution) preheated to 37 ℃, and after standing for 1 minute, gently shaking the cylinder A until the cover glass falls off.
(f) After the cover glass is fallen, the sample to be detected is kept in a jar A for 5 minutes, and is shaken for 5 seconds to be taken out, residual liquid is sucked by using filter paper, peripheral liquid of the sample frame is wiped off, 200 mu l of second cleaning liquid is gently dripped along the inner side edge of the sample frame, the solution is immediately sucked and discarded, the total is repeated for 2 times, 200 mu l of second cleaning liquid is gently dripped along the inner side edge of the sample frame, the sample is kept at room temperature for 2 minutes, the solution is sucked and discarded, and a blower is used for blowing the slide sample frame.
(g) And (3) carrying out instantaneous high-speed centrifugation on a staining solution DAPI split charging tube for blood cell analysis, taking 10 mu l of the staining solution DAPI for blood cell analysis, dripping the staining solution DAPI into the center of a specimen frame of a specimen to be detected, placing a cover glass, extruding and drying overflowed superfluous liquid by using a vacuum pump suction head or filter paper, and sealing the specimen to be detected, wherein no air bubble can appear.
(h) Detecting a sample to be detected under a fluorescence microscope, storing a cell picture of the sample to be detected, and identifying non-blood-related nucleated rare cells according to the cell picture so as to determine whether a target user corresponding to the sample to be detected has tumor.
3. Detection of nucleic acid layer:
WT-mRNA detection was performed on the enriched cell pellet, the detection steps including:
step 301, taking out a blood collection tube with a peripheral blood sample from a blood collection tube jack of the blood collection module, and then placing the blood collection tube into a test tube jack corresponding to the nucleic acid detection module for testing.
Step 302, separating single non-blood-related nucleated rare cells in the blood sample enrichment cell sediment at the periphery of the blood collection tube, extracting total RNA of the single nucleated cells of the non-blood-related nucleated rare cells, and performing reverse transcription to obtain cDNA.
And 303, quantitatively detecting copy numbers of target genes and reference genes ABL in non-blood-related nucleated rare cells by using an AB7500 type fluorescent quantitative PCR instrument and a TaqMan probe method to obtain a quantitative detection result.
The quantitative detection result in the detection step is expressed by the ratio (percentage) of copy numbers of the target gene and the reference gene, and the sensitivity to the target gene and the reference gene ABL is 5 copies/reaction system.
In this embodiment, if the ABL copy number is detected to be greater than 10e+04, it is determined that the target user corresponding to the peripheral blood sample has a potential leukemia risk.
The gene sequence combination of the oligonucleotide double fluorescent labeling Taqman probe and the specific primer used in the Taqman probe method comprises at least one group of oligonucleotide double fluorescent labeling Taqman probes and the specific primer shown in a combination one, a combination two and a combination three. The gene sequence combinations of the oligonucleotide double fluorescent labeling Taqman probes and the specific primers in each combination are as follows:
in combination one, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-CCTTCAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-GGCATCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GAGAGTCAG ACTTGAAAGCACT-3';
in the second combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-AAGCTAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-TTACTCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GCATGTCAG ACTTGAAAGTCTC-3';
in combination three, the probe sequence of the oligonucleotide double fluorescent label Taqman probe is 5'-TTCAAGCTTACACTTACATCGCTTA-3', the upstream primer base sequence of the specific primer is 5'-CCCATGCTACGGAGGCTAGCATTT-3', and the downstream primer base sequence of the specific primer is 5'-GTACTAAGCGTTAGCATTACTGCATTA-3'.
Preferably, combinations two and three are used together as a gene sequence combination of oligonucleotide double fluorescent labeled Taqman probes and specific primers in a nucleic acid detection module.
4. Detection of protein layer:
the detection steps of the protein layer surface comprise:
step 401, taking out the blood collection tube with the peripheral blood sample from the blood collection tube jack of the blood collection module, and then placing the blood collection tube into the test tube jack corresponding to the protein detection module for testing.
And step 402, balancing each reagent in the protein detection module at room temperature for 30min, and fully and uniformly mixing each reagent before sample addition.
Step 403, redissolving the calibrator with purified water according to the marked amount, standing for 2 minutes, and fully mixing the calibrator for later use.
Step 404, diluting the microsphere solution with microsphere suspension according to the usage amount by 10 times.
Step 405, taking out the blood collection tube with the peripheral blood sample from the blood collection tube jack of the blood collection module, sequentially adding a third buffer solution (25 ul/hole) on the 96-hole reaction plate, marking the peripheral blood sample (10 ul/hole) in the blood collection tube, and an antibody mixed solution (25 ul/hole), covering a sealing paper, fully and uniformly mixing on an oscillator, and placing a 37 ℃ incubator for light-shielding reaction for 5min, wherein the antibody mixed solution comprises at least one of antibodies AFP, CEA, NSE, SCC, CA199, CA125, CA153 and CA 125.
Step 406, after the reaction is finished, adding a stop solution (100 ul/hole) into the mixed liquid in the 96-hole reaction plate, and uniformly mixing;
and step 407, reading on a Luminex multifunctional flow dot matrix instrument, and finally, importing the result to calculate the concentration of the corresponding tumor marker in the peripheral blood sample.
The identification criteria for CTCs were:
1. non-blood borne CTC judgment criteria: DAPI+/CD45-/CD31-/CD54+ or-/CEP8+ (monomer, or multimer)
CD45: staining was negative, and sometimes whole cells were seen as pale red under the red channel. The most obvious difference from white blood cells is the vivid red light ring that does not surround the nucleus
CEP8: monomers or multimers. The signal is clear in either the red or orange channel
CD31: the green signal was negative. Because CD31 is combined with blood platelets, clustered spot dyeing is carried out under green light, and the background is different from person to person
CD54: pink signal positive or negative. Because the excitation light is invisible to the naked eye, a ZeissMetafer-iFISH full-automatic CTC scanning and image analysis system is needed.
DAPI: blue, round or oval
2. Circulating vascular endothelial (CEC) judgment criteria: DAPI+/CD45-/CD31+/CD54+ or-/CEP 8.
CD45: staining was negative, and sometimes whole cells were seen as pale red under the red channel. The most obvious difference from white blood cells is the bright red annulus that does not surround the nucleus.
CEP8: there are various numbers of chromosomes. The signal is clear in either the red or orange channel.
CD31: the green signal was positive. The signal brightness varies depending on the CD31 expression level. CD31 binds to platelets and therefore has clustered spot staining under green light, background level varies from person to person.
CD54: pink signal was negative and a few were positive. Because the excitation light is invisible to the naked eye, a ZeissMetafer-iFISH full-automatic CTC scanning and image analysis system is needed.
3. Blood-borne White Blood Cell (WBC) criteria: dapi+/cd45+/cd31+ or-/cd54+ or-/cep8+ (most diploids).
CD45: positive staining, visible in the red channel was a red halo CEP8 surrounding the blue nucleus: most chromosomal diploids. The signal is clear in either the red or orange channel.
CD31: green signal is negative and a few are positive.
CD54: pink signal positive or negative. Because the excitation light is invisible to the naked eye, a ZeissMetafer-iFISH full-automatic CTC scanning and image analysis system is needed.
DAPI: blue, round or oval.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is to be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings, which have been described above, and that various modifications and changes may be effected without departing from the scope thereof.
Claims (4)
1. The kit for the three-dimensional noninvasive tumor early screening is characterized by comprising a blood sample collection module, a cell detection module, a nucleic acid detection module, a protein detection module and at least one test tube jack;
the blood sample collection module comprises at least three blood collection tubes and blood collection tube jacks;
the cell detection module comprises a sample density separating liquid, a first buffer solution, a first cleaning solution, an antigen retrieval buffer solution, a tissue fixing liquid, a sample diluting liquid, a second buffer solution, a blood cell analysis diluting liquid, a FISH probe, a second cleaning solution and a fluorescence labeling antibody, wherein the fluorescence labeling antibody is a fluorescence labeling anti-human leucocyte surface marker antibody CD31, CD45 and CD54, and the FISH probe is a fluorescence labeling probe capable of specifically recognizing human chromosome 8;
the nucleic acid detection module comprises an oligonucleotide double-fluorescence labeling Taqman probe, a specific primer and a fluorescent quantitative qPCR reaction system reagent;
the protein detection module comprises a 96-hole reaction plate, a third buffer solution, a marked mixed antibody solution, microsphere suspension, microsphere solution, a stop solution, a blood collection tube with an anticoagulant and a glass slide, wherein the marked mixed antibody solution comprises at least one of antibodies AFP, CEA, NSE, SCC, CA, CA125, CA153 and CA 125;
the test tube jack is arranged on the periphery of the blood sample collection module, the cell detection module, the nucleic acid detection module and the protein detection module and is used for inserting a test tube for testing;
the using method of the kit comprises the following steps:
(1) Peripheral blood sample collection: collecting at least three peripheral blood samples from a target user by using a blood collection tube, and uniformly mixing and centrifuging the blood samples;
(2) Enrichment of non-blood-borne nucleated rare cells: adding a first cleaning solution into the collected peripheral blood sample, mixing uniformly, adding the mixture into a sample density separating solution, centrifuging, collecting peripheral blood sample cells with the upper layer removed of red blood cells, adding a first buffer solution into the mixture, shaking the mixture, placing the mixture on a magnetic rack, sucking and collecting liquid which is not adsorbed at the center of the mixture, adding the first cleaning solution into the mixture, centrifuging the mixture, and removing the supernatant to 100ul to obtain enriched cell sediment of non-blood-origin nucleated rare cells;
(3) Identification of non-blood-borne nucleated rare cells: adding fluorescent labeled antibodies, namely fluorescent labeled anti-human leukocyte surface marker antibodies CD31, CD45 and CD54, into the enriched cell sediment, incubating for a preset period of time, washing to remove unbound fluorescent labeled anti-human leukocyte surface marker antibodies, fixing and drying to prepare a chromosome fluorescent in-situ hybridization sample, adding an immune chromogenic reagent FISH probe, hybridizing a sealing sheet, placing under a fluorescent microscope, shooting and storing cell images, and identifying non-blood-induced nucleated rare cells according to the cell images, thereby determining whether a target user corresponding to the peripheral blood sample has tumors;
(4) Detection of WT 1-mRNA: extracting total RNA of mononuclear cells in the peripheral blood sample, reversely transcribing the total RNA into cDNA, quantitatively detecting copy numbers of target genes and reference genes ABL in the sample by using an AB7500 type fluorescent quantitative PCR instrument and a TaqMan probe method, and determining whether a target user corresponding to the peripheral blood sample has potential leukemia risk according to the copy numbers of the ABL, wherein a gene sequence combination of an oligonucleotide double fluorescent marker Taqman probe and a specific primer used by the TaqMan probe method comprises at least one group of oligonucleotide double fluorescent marker Taqman probe and a specific primer shown in a combination one, a combination two and a combination three; the 5 'end of the oligonucleotide double-fluorescence labeling Taqman probe is a fluorescence report group, the 3' end of the oligonucleotide double-fluorescence labeling Taqman probe is a fluorescence quenching group, and the specific primer comprises 7-10 exon-encoded zinc finger structural regions; the gene sequence combinations of the oligonucleotide double fluorescent labeling Taqman probes and the specific primers in each combination are as follows:
in combination one, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-CCTTCAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-GGCATCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GAGAGTCAG ACTTGAAAGCACT-3';
in the second combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-AAGCTAATGTGTGCTTACCCAGG-3', the upstream primer base sequence of the specific primer is 5'-TTACTCTGAGACCAGTGAGAA-3', and the downstream primer base sequence of the specific primer is 5'-GCATGTCAG ACTTGAAAGTCTC-3';
in the third combination, the probe sequence of the oligonucleotide double-fluorescence labeling Taqman probe is 5'-TTCAAGCTTACACTTACATCGCTTA-3', the upstream primer base sequence of the specific primer is 5'-CCCATGCTACGGAGGCTAGCATTT-3', and the downstream primer base sequence of the specific primer is 5'-GTACTAAGCGTTAGCATTACTGCATTA-3';
(5) Detection of saccharide antigen: the method comprises the steps of adopting a double-antibody sandwich immunoassay method, adding mixed liquid of phycoerythrin PE marked detection antibodies into suspension of fluorescent coding microspheres respectively crosslinked with anti-preset type tumor antigen capture monoclonal antibodies after reacting with tumor antigens in serum of a peripheral blood sample, finally forming microsphere-capture antibody-detection antibody-PE complex, detecting on a Luminex multifunctional flow dot matrix instrument, calculating to obtain concentration values of all tumor markers according to detection data, and determining whether a target user corresponding to the peripheral blood sample has potential tumor risks according to the concentration values of all tumor markers, wherein the detection of the carbohydrate antigens comprises detection of at least one of antigens AFP, CEA, NSE, SCC, CA, CA125, CA153 and CA 125.
2. The kit according to claim 1, wherein the sample density separation liquid comprises ferric sulfate, sucrose, polyvinylpyrrolidone-coated colloidal silica, and has a specific gravity of 1.07125 to 1.08158 g/ml at 20 ℃; the first buffer solution contains anti-leukocyte antibody coupled immunomagnetic beads, the osmotic pressure is 260-300 mOsm/kgH2O, and the pH is 6.8-7.8.
3. The kit of claim 1, wherein each of the predetermined locations of the cell detection module, the nucleic acid detection module, and the protein detection module corresponds to a predetermined number of test tube receptacles, respectively.
4. The kit of claim 1, wherein the kit detects tumor cells in peripheral blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811587184.0A CN109439732B (en) | 2018-12-25 | 2018-12-25 | Kit for three-dimensional noninvasive tumor early screening |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811587184.0A CN109439732B (en) | 2018-12-25 | 2018-12-25 | Kit for three-dimensional noninvasive tumor early screening |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109439732A CN109439732A (en) | 2019-03-08 |
| CN109439732B true CN109439732B (en) | 2024-04-12 |
Family
ID=65537944
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811587184.0A Active CN109439732B (en) | 2018-12-25 | 2018-12-25 | Kit for three-dimensional noninvasive tumor early screening |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439732B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462065A (en) * | 2020-11-16 | 2021-03-09 | 浙江博真生物科技有限公司 | Antibody composition, kit and detection method for detecting solid tumor |
| CN112946270B (en) * | 2021-01-28 | 2023-03-17 | 北京大学人民医院 | Method and kit for diagnosing malignant solid tumors in vivo |
| CN216847366U (en) * | 2021-02-03 | 2022-06-28 | 深圳市帝迈生物技术有限公司 | Kit and detection cup assembly of POCT blood cell analyzer |
| CN113189071B (en) * | 2021-04-29 | 2022-11-22 | 上海交通大学 | Kit and imaging method for accurate imaging of three-dimensional network of blood vessel of complete organ |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013002631A1 (en) * | 2011-06-28 | 2013-01-03 | Erasmus University Medical Center Rotterdam | New flow cytometric method for detection of circulating endothelial cells |
| CA2934344A1 (en) * | 2013-12-20 | 2015-06-25 | David T. TING | Methods and assays relating to circulating tumor cells |
| CN106771185A (en) * | 2016-12-05 | 2017-05-31 | 湖北省肿瘤医院 | A kind of nasopharyngeal carcinoma circulating tumor cell detection kit |
| CN108531585A (en) * | 2017-03-05 | 2018-09-14 | 南京科维思生物科技股份有限公司 | A kind of method of counting of the circulating tumor cell of based on PCR |
| CN209555255U (en) * | 2018-12-25 | 2019-10-29 | 迈迪速能医学技术（天津）有限公司 | A kind of kit early sieved for three-dimensional noninvasive tumour |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL2972359T3 (en) * | 2013-03-15 | 2021-03-08 | Menarini Silicon Biosystems S.P.A. | Enrichment of circulating tumor cells by depleting white blood cells |
| WO2017053887A1 (en) * | 2015-09-23 | 2017-03-30 | The Regents Of The University Of California | Methods for detection and eradication of myeloid leukemia stem cells |
-
2018
- 2018-12-25 CN CN201811587184.0A patent/CN109439732B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013002631A1 (en) * | 2011-06-28 | 2013-01-03 | Erasmus University Medical Center Rotterdam | New flow cytometric method for detection of circulating endothelial cells |
| CA2934344A1 (en) * | 2013-12-20 | 2015-06-25 | David T. TING | Methods and assays relating to circulating tumor cells |
| CN106771185A (en) * | 2016-12-05 | 2017-05-31 | 湖北省肿瘤医院 | A kind of nasopharyngeal carcinoma circulating tumor cell detection kit |
| CN108531585A (en) * | 2017-03-05 | 2018-09-14 | 南京科维思生物科技股份有限公司 | A kind of method of counting of the circulating tumor cell of based on PCR |
| CN209555255U (en) * | 2018-12-25 | 2019-10-29 | 迈迪速能医学技术（天津）有限公司 | A kind of kit early sieved for three-dimensional noninvasive tumour |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109439732A (en) | 2019-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109439732B (en) | Kit for three-dimensional noninvasive tumor early screening | |
| Gogoi et al. | Development of an automated and sensitive microfluidic device for capturing and characterizing circulating tumor cells (CTCs) from clinical blood samples | |
| CN104007257B (en) | Method for detecting non-humoral rare karyotes, and kit thereof | |
| Fehm et al. | Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells | |
| CN106635995B (en) | Negative enrichment method for circulating tumor cells | |
| CN105954246B (en) | Method and kit for detecting free rare tumor cells in human biological fluid sample | |
| CN108179134B (en) | EpCAM/PSMA-based double-antibody functionalized microfluidic chip and preparation method and application thereof | |
| US20140308669A1 (en) | Methods for obtaining single cells and applications of single cell omics | |
| JP6485759B2 (en) | Method for detecting malignancy of peripheral circulating tumor cell unit and kit thereof | |
| CN1234117A (en) | Use of anti-embryonic hemoglobin antibodies to identify fetal cells | |
| KR20100012847A (en) | A high sensitivity multiparameter method for rare event analysis in a biological sample | |
| CN107449713B (en) | Method for mixed antibody dependent sorting and enrichment of circulating tumor cells | |
| Lei et al. | Combined detection of aneuploid circulating tumor‐derived endothelial cells and circulating tumor cells may improve diagnosis of early stage non‐small‐cell lung cancer | |
| CN105087493A (en) | Joint application of three types of monoclonal antibody-coupled immunomagnetic beads to sorting tumor cells | |
| EP2450457A1 (en) | Method of analyzing genetically abnormals cells | |
| TWI790399B (en) | Information evaluating and analysis methods of circulating tumor cells thereof | |
| CN112067589A (en) | Malignant tumor recurrence and metastasis risk assessment system based on circulating tumor cell detection | |
| Jee et al. | Midkine concentrations in fine‐needle aspiration of benign and malignant thyroid nodules | |
| CN111812071A (en) | Novel circulating tumor cell identification technology | |
| CN209555255U (en) | A kind of kit early sieved for three-dimensional noninvasive tumour | |
| CN105925719A (en) | Gene related to liver cancer differentiation and application of gene | |
| CN108531585A (en) | A kind of method of counting of the circulating tumor cell of based on PCR | |
| CN108603233A (en) | The unicellular Genome Atlas of circulating tumor cell (CTC) is analyzed to characterize disease heterogeneity in metastatic disease | |
| CN110628721A (en) | Isolated culture method and kit for circulating tumor cells | |
| Ao et al. | Fiber-Optic Array Scanning Technology (FAST) for detection and molecular characterization of circulating tumor cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |