CN108982839A - Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer - Google Patents
Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer Download PDFInfo
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- CN108982839A CN108982839A CN201810600371.1A CN201810600371A CN108982839A CN 108982839 A CN108982839 A CN 108982839A CN 201810600371 A CN201810600371 A CN 201810600371A CN 108982839 A CN108982839 A CN 108982839A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The circulating tumor cell detection method based on immunomagnetic beads and flow cytometer that the present invention provides a kind of, it is the following steps are included: human peripheral blood carries out puncture blood drawing, then blood is subjected to resuspension haemocyte, removes most of red blood cell, added CD326 magnetic bead and separated；By first time selected by flow cytometry apoptosis, remove most of lymphocyte, then carry out second of selected by flow cytometry apoptosis, remove whole lymphocytes, third time selected by flow cytometry apoptosis finally is carried out to remaining cell, that is, detects whether that there are circulating tumor cells.Method of the invention is simple to operation, and detection sensitivity is high.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of circulating tumor based on immunomagnetic beads and flow cytometer
Cell detection method.
Background technique
Circulating tumor cell (Circulating tumor cell, CTC) is that all kinds of tumours that are present in peripheral blood are thin
The general designation of born of the same parents.A large number of studies show that CTC is present in peripheral blood with different shape, existing free single CTC also has aggregation
Pockets of cell mass (CTM, CirculatingTumorMicroemboli).Tumour cell is in the process for entering Peripheral Circulation
In can occur epithelial-mesenchymal transformation (EMT, EpithelialMesenchymalTransition), therefore CTC is there are different type,
Phenotype is mixed with interstitial cell including epithelial cell phenotype, interstitial cell phenotype and epithelial cell.CTM and interstitial cell phenotype
CTC has stronger metastatic potential.CTC in early detection blood controls patient's Index for diagnosis, therapeutic evaluation and individuation
Treatment suffers from important directive function.CTC detection by capturing CTC existing for trace in detection peripheral blood, monitoring CTC type and
The trend of quantity variation realizes real-time individual treatment so as to real-time monitoring tumour dynamic, assessment therapeutic effect.
Currently, that clinically applies obtains unique detection circulating tumor cell of U.S. FDA and Chinese Bureau of Drugs Supervision's approval
The method of (Circulating tumor cell, CTC) is the instrument (trade name CellSearch) of Johson & Johnson, this is a kind of
Use epidermal cell specific proteins as tumor markers, while being enriched with circulating tumor cell using the method for immunomagnetic beads
And finally carry out a kind of automanual circulating tumor cell method of counting of immunofluorescence microscopy observation detection.This method exists
Detect the lower problem of positive coincidence rate.Simultaneously because its system costly, individually detect sample higher cost, it is time-consuming compared with
Long, application is more difficult.Therefore, high, at low cost and easy to operate circulation that clinically there is an urgent need to a kind of specificity sensitivities
Tumour cell detection technique.Other are being applied or granted patent, and there are also use first cell separation nucleic acid hybridization and fluorescence again
Microscope detection, also all heavy dependence operation and labor intensive, it is more troublesome.
Summary of the invention
It is high, at low cost the purpose of the present invention is aiming at the above technical problems to be solved, providing a kind of specificity sensitivity
And circulating tumor cell detection method easy to operate.
In order to realize the above goal of the invention, the present invention provides following technical schemes:
A kind of circulating tumor cell detection method based on immunomagnetic beads and flow cytometer comprising following steps:
(1) human peripheral blood carries out puncture blood drawing, and blood is then carried out resuspension haemocyte, removes most of red blood cell, then
CD326 magnetic bead is added to be separated；
(2) pass through first time selected by flow cytometry apoptosis, remove most of lymphocyte, then carry out second of flow cytometer
Sorting, removes whole lymphocytes, finally carries out third time selected by flow cytometry apoptosis to remaining cell, that is, detects whether exist
Circulating tumor cell.
Preferably, the concrete operations of step (1) are as follows:
1) peripheral blood 7.5ml is adopted with the ACD pipe containing sodium citrate；
2) blood is transferred in 15ml centrifuge tube, about 5ml HBSS is added, after haemocyte is resuspended, be added slowly to containing 7.5ml
On the 50ml centrifuge tube liquid level of lymphocyte separation medium, 1500rpm room temperature is centrifuged 18min, and intermediate misty mononuclearcell is sucked out
Layer；
3) match erythrocyte cracked liquid working solution during being centrifuged；
4) 5ml HBSS is added, 1500rpm is centrifuged 5min；Supernatant is removed, erythrocyte cracked liquid working solution weight described in 3ml is added
Outstanding cell, room temperature are protected from light 10min, and 1500rpm is centrifuged 5min, removes supernatant；
5) 5ml HBSS is added, cell is resuspended, 1500rpm centrifugation 5min removes supernatant, it is thin that MACS buffer5ml resuspension is added
Born of the same parents, 1500rpm are centrifuged 5min, remove supernatant；
6) CD326 magnetic bead (20ul/10 is added7A cell), 4 DEG C of shadings are incubated for 30min, are washed carefully with MACS Buffer
Born of the same parents, 1500rpm are centrifuged 5min, remove supernatant；
7) LS Beads enrichment pipe is taken to be installed on magnetic sheet, with 3ml MACS buffer rinse splitter, with 500 μ l MACS
Splitter is added after cell is resuspended in buffer, and liquid is waited all to flow out；
8) splitter is added after rinsing tube wall with 3ml MACS buffer；
9) 5ml MACS buffer is added, and then taking-up splitter is put on streaming pipe rapidly, and the liquid of splitter is pushed away
Enter in the streaming pipe, 1500rpm is centrifuged 5min, removes supernatant；
10) cell is resuspended after 1ml HBSS is added, 1500rpm is centrifuged 5min, removes supernatant；
11) three kinds of antibody CK, CD326 and CD45 are added, every kind of 10ul is mixed well, and room temperature, which is protected from light, is incubated for 12min, then
5ml PBS is added, 1500rpm centrifugation 5min removes supernatant, 500 μ l PBS flow cytometers are added.
Result of practical application shows that method of the invention is based on immunomagnetic beads and flow cytometer, can be effectively detected
Circulating tumor cell, specificity sensitivity are high, at low cost and easy to operate.
Detailed description of the invention
Fig. 1 is the process mode schematic diagram with CTC detection method of the invention.
Fig. 2 is the result of the flow cytometry analysis first step.
Fig. 3 is the result of flow cytometry analysis second step.
Fig. 4 is the result of flow cytometry analysis third step.
Fig. 5 is the data summarization of three step of flow cytometry analysis.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention, range and is not intended to limit the present invention.
One, pretreatment and immunomagnetic beads processing
As shown in Figure 1, being the process mode schematic diagram using CTC detection method of the invention.Firstly, human peripheral blood carries out
Blood drawing is punctured, blood is then subjected to resuspension haemocyte, removes most of red blood cell, CD326 magnetic bead is added and is separated, and
By first time selected by flow cytometry apoptosis, remove most of lymphocyte, then carry out second of selected by flow cytometry apoptosis, removes complete
Portion's lymphocyte finally carries out third time selected by flow cytometry apoptosis to remaining cell, that is, it is thin to detect whether that there are circulating tumors
Born of the same parents.
Concrete operations are as follows:
1) peripheral blood 7.5ml is adopted with the ACD pipe containing sodium citrate；
2) blood is transferred in 15ml centrifuge tube, is added about 5ml HBSS (Hank's balanced salt solution), haemocyte is resuspended
Afterwards, it is added slowly on the 50ml centrifuge tube liquid level of the lymphocyte separation medium containing 7.5ml, 1500rpm room temperature is centrifuged 18min, is sucked out
Intermediate mist mononuclearcell layer；
3) it works during being centrifuged with erythrocyte cracked liquid (U.S. company BD, trade name Pharm Lyse, article No. 555899)
Liquid (concentrate: sterilizing pure water volume ratio is 1:9)
4) 5ml HBSS (Hank's balanced salt solution) is added, 1500rpm is centrifuged 5min；Supernatant is removed, it is above-mentioned red that 3ml is added
Cell is resuspended in cell pyrolysis liquid working solution, and room temperature is protected from light 10min, and 1500rpm is centrifuged 5min, removes supernatant；
5) 5ml HBSS is added, cell is resuspended, 1500rpm centrifugation 5min goes supernatant (printing dry extraction raffinate with paper), MACS is added
Cell is resuspended in buffer 5ml, and 1500rpm is centrifuged 5min, removes supernatant；
6) CD326 magnetic bead (20ul/10 is added7A cell) (magnetic bead separation kit of German Mei Tian Ni company, product are compiled
Number 130-061-101), 4 DEG C of shadings are incubated for 30min, with MACS Buffer (107/ 1-2ml) washing cell, 1500rpm centrifugation
5min removes supernatant；
7) LS Beads enrichment pipe is taken to be installed on magnetic sheet, with 3ml MACS buffer rinse splitter, with 500 μ l MACS
buffer(≤50×106) be resuspended cell after splitter is added, wait liquid all flow out；
8) splitter (2 times) are added after rinsing tube wall with 3ml MACS buffer；
9) 5ml MACS buffer is added, and then taking-up splitter is put on streaming pipe rapidly, takes pusher by separating pipe
Liquid push-in streaming pipe in, 1500rpm be centrifuged 5min, remove supernatant；
10) cell is resuspended after 1ml HBSS is added, 1500rpm is centrifuged 5min, removes supernatant；
11) three kinds of antibody CK, CD326 and CD45 are added, every kind of 10ul is mixed well, and room temperature, which is protected from light, is incubated for 12min, then
5ml PBS is added, 1500rpm centrifugation 5min removes supernatant, 500 μ l PBS flow cytometers are added.
Two, flow cytometry analysis
The workflow of flow cytometry analysis is referring to figs. 2 to shown in Fig. 5.
Fig. 2 is the flow cytometry analysis first step, and machine, each flow cytometer on the good cell of antibody incubation are harvested altogether
200000 cells are analyzed, and forward angle light scatter (FSC) registration is less than 200 and agglomerating existing cell is with the complete of nucleus
Cell.Circle takes complete cell (region P1,19.75 ten thousand) to carry out next step detection on the screen, abandons cell fragment
Fig. 3 is flow cytometry analysis second step, the result figure obtained after intact cell is taken for circle, from intact cell centre circle
Taking CD45- cell, (CD45-PE registration is less than 100 and agglomerating existing comprising including CTC, i.e., the region P2, machine reading are
3.98 ten thousand)
Fig. 4 is flow cytometry analysis third step, the result figure obtained after CD45- cell is taken for circle, from CD45- cell
CK and CD326 double positive cells are detected, (region Q2 is that CK and CD326 registration is both greater than 100 and agglomerating presence to as CTC in figure
Region, as double positive cells, machine reading is 4)
Fig. 5 is the data summarization of three step of flow cytometry analysis, is that flow cytometer exists the data summarization of above-mentioned three step
In one frame.
Through 50 non-tumour Healthy Peoples and 500 Advanced cancers blood samples of patients Samples detections, Healthy People is examined except only a few
Surveying result tumor cell number is outside 1, is all 0；Tumor patient is according to state of an illness difference numerical value from 1-40 etc..Actually detected result table
Bright, method of the invention is easy quickly (half a day can go out result) for detecting circulating tumor cell, more acurrate using machine counting,
Accuracy is high.
Claims (2)
1. a kind of circulating tumor cell detection method based on immunomagnetic beads and flow cytometer, it is characterised in that including following step
It is rapid:
(1) human peripheral blood carries out puncture blood drawing, and blood is then carried out resuspension haemocyte, removes most of red blood cell, adds
CD326 magnetic bead is separated；
(2) pass through first time selected by flow cytometry apoptosis, remove most of lymphocyte, then carry out second of flow cytometer point
Choosing, removes whole lymphocytes, finally carries out third time selected by flow cytometry apoptosis to remaining cell, that is, detects whether to exist and follow
Ring tumour cell.
2. the method according to claim 1, wherein the concrete operations of the step (1) are as follows:
1) peripheral blood 7.5ml is adopted with the ACD pipe containing sodium citrate；
2) blood is transferred in 15ml centrifuge tube, about 5ml HBSS is added, after haemocyte is resuspended, be added slowly to lymph containing 7.5ml
On the 50ml centrifuge tube liquid level of cell separating liquid, 1500rpm room temperature is centrifuged 18min, and intermediate misty mononuclearcell layer is sucked out；
3) match erythrocyte cracked liquid working solution during being centrifuged；
4) 5ml HBSS is added, 1500rpm is centrifuged 5min；Supernatant is removed, erythrocyte cracked liquid working solution described in 3ml is added and is resuspended carefully
Born of the same parents, room temperature are protected from light 10min, and 1500rpm is centrifuged 5min, removes supernatant；
5) 5ml HBSS being added, cell is resuspended, 1500rpm centrifugation 5min removes supernatant, MACS buffer5ml is added, cell is resuspended,
1500rpm is centrifuged 5min, removes supernatant；
6) CD326 magnetic bead (20ul/10 is added7A cell), 4 DEG C of shadings are incubated for 30min, cell is washed with MACS Buffer,
1500rpm is centrifuged 5min, removes supernatant；
7) LS Beads enrichment pipe is taken to be installed on magnetic sheet, with 3ml MACS buffer rinse splitter, with 500 μ l MACS
Splitter is added after cell is resuspended in buffer, and liquid is waited all to flow out；
8) splitter is added after rinsing tube wall with 3ml MACS buffer；
9) 5ml MACS buffer is added, and then taking-up splitter is put on streaming pipe rapidly, and the liquid of splitter is pushed into institute
It states in streaming pipe, 1500rpm is centrifuged 5min, removes supernatant；
10) cell is resuspended after 1ml HBSS is added, 1500rpm is centrifuged 5min, removes supernatant；
11) three kinds of antibody CK, CD326 and CD45 are added, every kind of 10ul is mixed well, and room temperature, which is protected from light, is incubated for 12min, is added
5ml PBS, 1500rpm centrifugation 5min removes supernatant, and 500 μ l PBS flow cytometers are added.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113432959A (en) * | 2021-05-21 | 2021-09-24 | 赛雷纳（中国）医疗科技有限公司 | Preparation method of quality control product for sperm DNA fragmentation detection |
| CN115197914A (en) * | 2022-05-25 | 2022-10-18 | 广州复大医疗有限公司 | Method for separating and detecting circulating tumor cells |
| CN115992096A (en) * | 2023-02-22 | 2023-04-21 | 赛尔医学科技(山东)有限公司 | Tumor cell detection method |
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| CN103026228A (en) * | 2010-03-31 | 2013-04-03 | 维亚塔有限责任公司 | Methods, systems and devices for separating tumor cells |
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| CN103026228A (en) * | 2010-03-31 | 2013-04-03 | 维亚塔有限责任公司 | Methods, systems and devices for separating tumor cells |
| CN102527353A (en) * | 2011-12-21 | 2012-07-04 | 深圳市老年医学研究所 | Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113432959A (en) * | 2021-05-21 | 2021-09-24 | 赛雷纳（中国）医疗科技有限公司 | Preparation method of quality control product for sperm DNA fragmentation detection |
| CN115197914A (en) * | 2022-05-25 | 2022-10-18 | 广州复大医疗有限公司 | Method for separating and detecting circulating tumor cells |
| CN115992096A (en) * | 2023-02-22 | 2023-04-21 | 赛尔医学科技(山东)有限公司 | Tumor cell detection method |
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