WO2021213290A1 - 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 - Google Patents
一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 Download PDFInfo
- Publication number
- WO2021213290A1 WO2021213290A1 PCT/CN2021/087978 CN2021087978W WO2021213290A1 WO 2021213290 A1 WO2021213290 A1 WO 2021213290A1 CN 2021087978 W CN2021087978 W CN 2021087978W WO 2021213290 A1 WO2021213290 A1 WO 2021213290A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peripheral blood
- solution
- add
- filter
- pancreatic cancer
- Prior art date
Links
- 210000005259 peripheral blood Anatomy 0.000 title claims abstract description 35
- 239000011886 peripheral blood Substances 0.000 title claims abstract description 35
- 206010061902 Pancreatic neoplasm Diseases 0.000 title claims abstract description 33
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 title claims abstract description 33
- 201000002528 pancreatic cancer Diseases 0.000 title claims abstract description 33
- 208000008443 pancreatic carcinoma Diseases 0.000 title claims abstract description 33
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 19
- 238000012360 testing method Methods 0.000 title abstract description 6
- 239000012192 staining solution Substances 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 22
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 19
- 239000003085 diluting agent Substances 0.000 claims abstract description 11
- 230000000306 recurrent effect Effects 0.000 claims abstract description 10
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- 241000283707 Capra Species 0.000 claims abstract description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 7
- 229920004890 Triton X-100 Polymers 0.000 claims abstract description 6
- 239000013504 Triton X-100 Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 210000005266 circulating tumour cell Anatomy 0.000 claims description 32
- 210000004369 blood Anatomy 0.000 claims description 15
- 239000008280 blood Substances 0.000 claims description 15
- 238000005374 membrane filtration Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical group C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 210000004881 tumor cell Anatomy 0.000 claims description 9
- 238000011161 development Methods 0.000 claims description 8
- 239000011521 glass Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical group [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000004042 decolorization Methods 0.000 claims description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical group CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 4
- GKJMOBYTOXEHKJ-UHFFFAOYSA-N dihydroxymethylurea Chemical compound NC(=O)NC(O)O GKJMOBYTOXEHKJ-UHFFFAOYSA-N 0.000 claims description 4
- 229940089468 hydroxyethylpiperazine ethane sulfonic acid Drugs 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- 239000008096 xylene Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229940009662 edetate Drugs 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 230000005484 gravity Effects 0.000 claims description 3
- 238000003364 immunohistochemistry Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- QMFYDAZWMAZXKO-UHFFFAOYSA-N sodium;pyridine Chemical compound [Na+].C1=CC=NC=C1 QMFYDAZWMAZXKO-UHFFFAOYSA-N 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- REHUGJYJIZPQAV-UHFFFAOYSA-N formaldehyde;methanol Chemical compound OC.O=C REHUGJYJIZPQAV-UHFFFAOYSA-N 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000002055 immunohistochemical effect Effects 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 16
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 239000002699 waste material Substances 0.000 description 6
- 229910052742 iron Inorganic materials 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- -1 GPC-3 Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000011897 real-time detection Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- 101100243447 Arabidopsis thaliana PER53 gene Proteins 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 description 1
- 102100033587 DNA topoisomerase 2-alpha Human genes 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 101000960235 Dictyostelium discoideum Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 description 1
- 102100027100 Echinoderm microtubule-associated protein-like 4 Human genes 0.000 description 1
- 102100038595 Estrogen receptor Human genes 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 description 1
- 101001057929 Homo sapiens Echinoderm microtubule-associated protein-like 4 Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000006809 Pancreatic Fistula Diseases 0.000 description 1
- 102100021768 Phosphoserine aminotransferase Human genes 0.000 description 1
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 1
- 101150080074 TP53 gene Proteins 0.000 description 1
- 108010046308 Type II DNA Topoisomerases Proteins 0.000 description 1
- 102100035071 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000002978 thoracic duct Anatomy 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- 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
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Definitions
- the invention relates to a kit and a detection method for detecting the expression of CA199 in peripheral blood circulating tumor cells of patients with pancreatic cancer, and belongs to the technical field of molecular biology.
- Pancreatic cancer is a common malignant tumor of the digestive system and one of the worst-prognostic malignancies. Among deaths caused by malignant tumors, pancreatic cancer ranks fourth. 90% of patients die within one year after diagnosis, with a 5-year survival rate. Less than 5%. Because the early symptoms of pancreatic cancer are insidious, not specific, aggressive, early metastasis, most of them are in the middle and late stages at the time of diagnosis, the surgical resection rate is low, only 15% at the time of diagnosis, so the selection of treatment methods for patients with pancreatic cancer is particularly important , Can make some patients avoid unnecessary surgical trauma and risks.
- CAl99 (carbohydrate antigenl99), also known as tumor-associated 199 antigen, gastrointestinal cancer-associated antigen or carbohydrate antigen, is a mucin-like tumor marker, which is a glycolipid on the cell membrane and exists in the form of mucin in the blood.
- CAl99 is an antigenic substance highly related to adenocarcinoma. It is produced by adenocarcinoma cells and drained into the blood through the thoracic duct.
- the normal reference value of serum CAl99 is 39kU/L.
- the concentration of CAl99 in serum of malignant tumors increases significantly, especially in patients with pancreatic cancer and colon cancer. It is used for the early diagnosis of pancreatic cancer.
- the specimens detected by CA199 in pancreatic cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, which is difficult to achieve multiple or real-time detection.
- Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
- This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
- the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
- the present invention provides a kit and a detection method for detecting the expression of CA199 in peripheral blood circulating tumor cells in patients with pancreatic cancer, using membrane filtration
- the device separates and obtains CTC in the peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens, and further uses immunohistochemical technology to detect the expression of CTC CA199.
- a kit for detecting the expression of CA199 in circulating tumor cells in the peripheral blood of patients with pancreatic cancer including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, goat anti-CA199 primary antibody 100 ⁇ L, rabbit anti-goat IgG/HRP 100 ⁇ L , 0.1% Triton X-100 100 ⁇ L, 0.3% H 2 O 2 100 ⁇ L, Reagent A 15ml, Reagent B 50ml, 6 ⁇ PBS buffer 60mL.
- the diluent is potassium chloride 19g/L, dipotassium edetate 4.8g/L, sodium pyridinium 2.6g/L, potassium dihydrogen phosphate 17g/L, sodium hydroxide 9g/L, hydroxyethyl Piperazine ethanesulfonic acid 0.2g/L, dihydroxymethyl urea 2g/L, the balance is purified water.
- the decolorizing liquid is composed of 0.5% hydrochloric acid-70% ethanol solution.
- the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.
- the reagent A is formaldehyde-methanol in a volume ratio of 2:1; the reagent B is xylene.
- the method for detecting the expression of CA199 in circulating tumor cells in the peripheral blood of patients with pancreatic cancer for non-diagnostic purposes of the above kit includes the following steps:
- Peripheral blood sample pretreatment Dilute the collected peripheral blood sample with 45ml of diluent, add paraformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, and fix the final concentration to 0.25%;
- the specific method for detecting the CA199 expression of CTC in the step (5) is as follows:
- the membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid cylinder, and an iron stand.
- the iron stand is provided with a base, a stand and a bracket.
- the blood sample container is set on the upper part of the iron stand through the bracket.
- a filter which is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
- the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
- the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
- the filter membrane is made of a hydrophobic material, and the filter holes with a diameter of 8 micrometers are evenly spread on it.
- the detection method provided by the present invention can detect the expression of CA199 in patients with advanced or recurrent pancreatic cancer without puncture biopsy to obtain tissue samples. This technology is minimally invasive and can be detected in real time.
- the method provided by the present invention can avoid false positive results caused by edge effects that may occur during the staining process, has good stability, reduces cell loss, and improves detection accuracy.
- Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention.
- FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
- FIG. 3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
- Figure 4 is an image of circulating tumor cells obtained from the peripheral blood of a patient with pancreatic cancer.
- Component content 6 ⁇ PBS buffer 60mL Diluent 50mL Decolorizing liquid 1mL Staining solution A 0.5mL Staining Solution B 1mL Goat anti-CA199 primary antibody 100 ⁇ L Rabbit anti-goat IgG/HRP 100 ⁇ L 0.1% Triton X-100 100 ⁇ L 0.3% H 2 O 2 100 ⁇ L Reagent A 15mL Reagent B 50mL
- the configuration of the diluent potassium chloride 19g, dipotassium edetate 4.8g, sodium pyridinium 2.6g, potassium dihydrogen phosphate 17g, sodium hydroxide 9g, hydroxyethylpiperazine ethanesulfonic acid 0.2g, Dihydroxymethyl urea 2g, purified water 945.4g.
- the membrane filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
- the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter. The filter hole 10 is trapped on the filter membrane 7.
- Figure 4 is an image of circulating tumor cells isolated from the peripheral blood of a patient with pancreatic cancer. It can be seen from the figure that the nucleus is atypia, the nucleus is larger, the shape of the nucleus is irregular, and the cell diameter (long end) is greater than 15um; nuclear chromatin Side shift, huge nucleoli, abnormal mitosis.
- the prepared blood sample has poor stability, and some blood samples will also form stratification, and blood cells are prone to aggregation and adhesion, which affects the final The detection effect.
- the decolorizing solution prepared from a 0.5% hydrochloric acid-70% ethanol solution has a better decolorizing effect than a decolorizing solution composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- the accuracy of the detection result is 85%.
- the cells of the present invention are concentrated, with clear morphology, good dispersion uniformity, and the accuracy rate can reach 99%.
- the detected circulating tumor cells were used to confirm the expression of CA199 by immunohistochemistry and compared with the results of CA199 expression in pancreatic cancer surgery or puncture tissue specimens to observe their differences, verifying the potential of CTCs as a non-invasive biopsy for real-time evaluation of pancreatic cancer CA199 expression. Provide an important basis for evaluating the prognosis of pancreatic cancer chemotherapy combined with immunotherapy.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hospice & Palliative Care (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
组分 | 含量 |
6×PBS缓冲液 | 60mL |
稀释液 | 50mL |
脱色液 | 1mL |
染色液A | 0.5mL |
染色液B | 1mL |
羊抗CA199一抗 | 100μL |
兔抗羊IgG/HRP | 100μL |
0.1%Triton X-100 | 100μL |
0.3%H 2O 2 | 100μL |
试剂A | 15mL |
试剂B | 50mL |
Claims (7)
- 一种检测胰腺癌患者外周血循环肿瘤细胞CA199表达的试剂盒,其特征在于,包括稀释液45mL、脱色液1mL、染色液A 0.5mL、染色液B 1mL、羊抗CA199一抗100μL、兔抗羊IgG/HRP 100μL、0.1%Triton X-100 100μL、0.3%H 2O 2 100μL、试剂A 15ml、试剂B 50ml、6×PBS缓冲液60mL。
- 根据权利要求1所述的试剂盒,其特征在于,所述稀释液为氯化钾19g/L,乙二胺四乙酸二钾4.8g/L,吡啶鎓钠2.6g/L,磷酸二氢钾17g/L,氢氧化钠9g/L,羟乙基哌嗪乙硫磺酸0.2g/L,二羟基甲基脲2g/L,余量为纯化水。
- 根据权利要求1所述的试剂盒,其特征在于,所述脱色液是由0.5%盐酸-70%乙醇溶液组成。
- 根据权利要求1所述的试剂盒,其特征在于,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
- 根据权利要求1所述的试剂盒,其特征在于,所述试剂A为甲醛-甲醇按照体积比2:1组成;所述试剂B为二甲苯。
- 一种利用权利要求1-5任一项所述的试剂盒非诊断目的检测胰腺癌患者外周血循环肿瘤细胞CA199表达的方法,其特征在于,包括以下步骤:(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发胰腺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发胰腺癌患者外周血:肘正中静脉外周血5ml;(2)外周血样预处理:将采集的外周血样采用稀释液45ml进行稀释,稀释后加多聚甲醛固定外周血样10分钟,固定终浓度为0.25%;(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;(5)运用免疫组化技术检测CTC的CA199表达情况。
- 根据权利要求6所述的检测方法,其特征在于,步骤(5)所述检测CTC的CA199表达的具体方法如下:S1脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液;S2滴加100μl 0.1%Triton X-100,室温孵育15min,DI水洗2min×3次;S3滴加100μl 0.3%H 2O 2,室温孵育10min,PBS洗2min×3次;滴加100μl羊抗CA199一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;S4滴加100μl兔抗羊IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;S5滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;S6显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;S7盐酸酒精分化8秒,自来水返蓝5min;S8将返蓝后的CTC采用75%乙醇,95%乙醇,100%乙醇梯度乙醇脱水各10min,然后加入15mL试剂A,振荡均匀后过滤;加入试剂B,脱色30min,干燥,中性树脂封固;S9光学显微镜下镜检。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010312529.2A CN111562377A (zh) | 2020-04-20 | 2020-04-20 | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 |
CN202010312529.2 | 2020-04-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021213290A1 true WO2021213290A1 (zh) | 2021-10-28 |
Family
ID=72071888
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/087978 WO2021213290A1 (zh) | 2020-04-20 | 2021-04-19 | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN111562377A (zh) |
WO (1) | WO2021213290A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111562377A (zh) * | 2020-04-20 | 2020-08-21 | 山东第一医科大学(山东省医学科学院) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 |
CN111521789A (zh) * | 2020-04-20 | 2020-08-11 | 山东第一医科大学(山东省医学科学院) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的免疫荧光试剂盒及检测方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105588943A (zh) * | 2016-01-28 | 2016-05-18 | 山东省肿瘤防治研究院 | 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法 |
CN108020666A (zh) * | 2018-02-07 | 2018-05-11 | 福州大学 | 一种可同时定量检测血液中cea和ca19-9的磁性免疫层析试纸条及制备方法 |
CN110412282A (zh) * | 2019-07-26 | 2019-11-05 | 北京健平金星生物科技有限公司 | Vegf多肿瘤标记物的荧光免疫层析联合检测试剂盒 |
CN111562377A (zh) * | 2020-04-20 | 2020-08-21 | 山东第一医科大学(山东省医学科学院) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 |
-
2020
- 2020-04-20 CN CN202010312529.2A patent/CN111562377A/zh not_active Withdrawn
-
2021
- 2021-04-19 WO PCT/CN2021/087978 patent/WO2021213290A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105588943A (zh) * | 2016-01-28 | 2016-05-18 | 山东省肿瘤防治研究院 | 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法 |
CN108020666A (zh) * | 2018-02-07 | 2018-05-11 | 福州大学 | 一种可同时定量检测血液中cea和ca19-9的磁性免疫层析试纸条及制备方法 |
CN110412282A (zh) * | 2019-07-26 | 2019-11-05 | 北京健平金星生物科技有限公司 | Vegf多肿瘤标记物的荧光免疫层析联合检测试剂盒 |
CN111562377A (zh) * | 2020-04-20 | 2020-08-21 | 山东第一医科大学(山东省医学科学院) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 |
Non-Patent Citations (1)
Title |
---|
MENG FANBIN, GUO KEJIAN GE CHUNLIN SONG SHAOWEI: "The Value of Pancreatic CT Perfusion on Biological Behavior Assessment in Pancreatic Cancer", ZHONGGUO AI ZHENG ZA ZHI, vol. 25, no. 5, 30 May 2015 (2015-05-30), pages 387 - 391, XP055859765, ISSN: 1007-3639, DOI: 10.3969/j.issn.1007-3969.2015.05.012 * |
Also Published As
Publication number | Publication date |
---|---|
CN111562377A (zh) | 2020-08-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2021213316A1 (zh) | 一种检测肾癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 | |
WO2022001824A1 (zh) | 一种检测小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 | |
WO2021213323A1 (zh) | 一种通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因突变的非诊断目的方法 | |
WO2021213290A1 (zh) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的试剂盒及检测方法 | |
WO2021213262A1 (zh) | 一种检测胃癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法 | |
WO2021213322A1 (zh) | 一种检测肾癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法 | |
WO2021213295A1 (zh) | 检测小细胞肺癌患者外周血循环肿瘤细胞nse基因突变的免疫荧光试剂盒及检测方法 | |
WO2021213304A1 (zh) | 一种检测小细胞肺癌患者外周血循环肿瘤细胞nse基因突变的试剂盒及检测方法 | |
WO2021213306A1 (zh) | 一种检测非小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 | |
WO2021213302A1 (zh) | 检测非小细胞肺癌患者外周血循环肿瘤细胞cea基因突变的免疫荧光试剂盒及检测方法 | |
WO2021213297A1 (zh) | 检测非小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的免疫荧光试剂盒及方法 | |
WO2021213310A1 (zh) | 一种通过外周血循环肿瘤细胞检测食道鳞癌患者pd-l1基因表达的免疫荧光试剂盒 | |
WO2021213315A1 (zh) | 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变表达的试剂盒 | |
CN111638357A (zh) | 非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin突变的免疫荧光试剂盒及方法 | |
WO2021213292A1 (zh) | 一种检测前列腺癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法 | |
WO2021213298A1 (zh) | 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的免疫荧光试剂盒及检测方法 | |
WO2022001825A1 (zh) | 一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法 | |
WO2022001826A1 (zh) | 一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的免疫荧光试剂盒 | |
WO2021213261A1 (zh) | 一种检测胃癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 | |
WO2021213318A1 (zh) | 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变的非诊断目的方法 | |
WO2022001823A1 (zh) | 检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及方法 | |
WO2021213299A1 (zh) | 一种检测前列腺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 | |
WO2021213311A1 (zh) | 通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因表达的免疫荧光试剂盒 | |
WO2021233037A1 (zh) | 一种检测胆管癌患者外周血循环肿瘤细胞fgfr基因突变的试剂盒及检测方法 | |
CN111534586B (zh) | 一种检测非小细胞肺癌患者外周血循环肿瘤细胞cea基因突变的试剂盒及检测方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21793527 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21793527 Country of ref document: EP Kind code of ref document: A1 |
|
32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21793527 Country of ref document: EP Kind code of ref document: A1 |