CN106596938A - Rapid detection kit for circulating tumor cells - Google Patents
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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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
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Abstract
The invention discloses a rapid detection kit for circulating tumor cells. The rapid detection kit comprises a cell density gradient separating medium, a porous diaphragm separator tube, a centrifuge tube with a volume of 5 mL, epidermal tumor marker antibodies and mesenchymal tumor marker antibodies. The kit employs a density gradient centrifugation method to remove background interference of most erythrocytes and leucocytes in blood and enrich circulating tumor cells in the blood; then epidermal and mesenchymal tumor marker antibodies, altogether four fluorescent tumor marker antibodies, are used for fluorescence labeling of the circulating tumor cells; and a general flow cytometer is used for detecting and counting the circulating tumor cells. The kit provided by the invention takes the epidermal-mesenchymal transformation process of the tumor cells into consideration, increases captured mesenchymal circulating tumor cells, improves the positive coincident rate of detection, and is high in accuracy, rapid, convenient, and low in cost and time consumption; and detection counting is automatically accomplished by the flow cytometer, so good repeatability is realized.
Description
Technical field
The present invention relates to tumor prognosis detection technique field, and in particular to a kind of circulating tumor cell quick detection reagent
Box.
Background technology
In China, the sickness rate of malignant tumor has become now and has a strong impact on health of people in ascendant trend year by year
Public health problem.Due to the biological characteristicses to tumor not yet completely clearly, except traditional iconography and pathology method
Outward, side that is simple, quick, being evaluated the operation of tumor or the prognosis of chemicotherapy is clinically lacked at present noninvasive
Method, and the effective means that the recurrence to patient and disease progression are tracked.
Circulating tumor cell (CTCs) is malignant tumor primary tumor or metastasis shed into Peripheral Circulation tumor it is thin
Born of the same parents, it has the biological characteristicses such as tumor specific antigen, gene expression characteristicses, and can be in peripheral blood long-term survival, and they are
The important channel of tumor tissues far-end transfer and cell derived.The detection and analysis of circulating tumor cell is in recent years tumor
One hot issue of area research, has had at present substantial amounts of document to demonstrate the circulating tumor cell in tumour patient blood
The number of quantity and the Overall survival of patient's malignancy, disease progression and prognosis have clear and definite dependency, and
Confirm the circulating tumor cell and tumor primary lesion tumor tissues in blood in biological characteristicses sides such as drug resistance, mutational sites
There is higher concordance in face.Therefore clinically the detection of circulating tumor cell is treated to clear and definite transfer process, auxiliary diagnosis, monitoring
Imitate, instruct the aspects such as individualized treatment, judging prognosis that there is important clinical value.While the method for this in-vitro diagnosis
It is simple and quick and there is no wound to patient, can effectively solve the problem that the difficult problem of ground tumour patient prognostic evaluation.
Circulating tumor cell is very rare in blood, there is about 1,000,000,000 erythrocyte (RBCs) peace treaties in 5mL whole bloods
5000000 leukocyte (WBCs), only several to tens circulating tumor cells.So that obtaining pure under so big background
Circulating tumor cell there is greatly challenge.
Most of circulating tumor cell detection technique is related to remove the whole blood pre-treatment of RBCs, such as RBCs lysates are processed
Comprehend and cause part circulating tumor cell loss Deng, such place, and often have that analysis process is complicated, efficiency is low, special
Property and the low defect of sensitivity.At present, that what is clinically applied obtains unique detection of U.S. FDA and Chinese Bureau of Drugs Supervision's approval
The method of CTC is the CellSearch methods of Johson ＆ Johnson, and this is that one kind uses epidermis cell specific proteinses as tumor mark
Will thing, while being enriched with and finally carrying out immunofluorescence microscopy observation detection by circulating tumor cell using the method for immunomagnetic beadses
A kind of automanual circulating tumor cell method of counting.This method there is a problem of detecting that positive coincidence rate is relatively low.While by
In its system costly, single detection sample it is relatively costly, time-consuming longer, application is more difficult.Therefore, clinically compel to be essential
Want a kind of high specificity sensitivity, low cost and circulating tumor cell detection technique simple to operate.
The content of the invention
In order to solve the defect of existing circulating tumor cell detection technique, the invention provides one kind from peripheral blood simultaneously
Extract circulating tumor cell detection kit.
Technical scheme is specific as follows：
A kind of circulating tumor cell quick detection kit, composition includes：
Cell density gradient separations liquid；
Porous septum separating pipe；
The centrifuge tube of 5mL volumes；
Epidermis class tumor markerses antibody；
Interstitial class tumor markerses antibody.
It is dry that this test kit eliminates most of erythrocyte and leukocyte background in blood using density－gradient centrifuga－tion method
Disturb, while being enriched with to the circulating tumor cell in blood.Use afterwards with epidermis class and the big class of interstitial class two totally 4 kinds of tumors
The fluorescent antibody of mark carries out fluorescent labeling to circulating tumor cell, finally completes in general flow cytometer to circulation
Tumor cell is detected and counted, and the test kit of the present invention take into account the epidermis interstitial transformation process of tumor cell, increased and catches
The circulating tumor cell of interstitial class is obtained, detection positive coincidence rate is increased, degree of accuracy is high.
Preferably, above-mentioned circulating tumor cell quick detection kit, the cell density gradient separations liquid is Germany
The product Oncoquick TCD gradient separations liquid of Geriner companies.
Preferably, above-mentioned circulating tumor cell quick detection kit, the epidermis class tumor markerses are EpCAM, CK7
And CK8.
Preferably, above-mentioned circulating tumor cell quick detection kit, in the epidermis class tumor markerses antibody CK7 and
CK8 antibody is the product Alexa of U.S. company BD 488MouseAnti-CK7/CK8。MouseAnti-CK7/CK8
It is an antibody, both antigens can be recognized.
Preferably, above-mentioned circulating tumor cell quick detection kit, EpCAM in the epidermis class tumor markerses antibody
Antibody is the product BB515MouseAnti-Human CD326 antibody of U.S. company BD.
Preferably, above-mentioned circulating tumor cell quick detection kit, the interstitial class tumor markerses are N-
Cadherin。
Preferably, above-mentioned circulating tumor cell quick detection kit, the interstitial class tumor markerses antibody is the U.S.
Product Anti-Human CD325 (N-Cadherin) PE of eBioscience companies.
Preferably, above-mentioned circulating tumor cell quick detection kit, the porous septum separating pipe is Wuhan Hai Jili
The product of bio tech ltd：Porous septum separating pipe, volume is 15ml.When this centrifuge tube avoids sample-adding sample and from
The premixing of heart medium, reduces the pollution caused because centrifuge is unstable, and artificial disturbance causes test when eliminating sampling
As a result disturb, improve success of the test rate and cell recoveries.
Preferably, above-mentioned circulating tumor cell quick detection kit, the porous septum separating pipe quantity is 10.
Compared with prior art, the invention has the advantages that：
1. compare with existing CTC detection methods, this test kit need sample size it is few (this method only needs 5ml peripheral bloods,
And Cell Search methods need 7.5ml)；It is simple to operate, quickly, it is easy to clinical staff grasp (this test kit only need it is simple from
The heart, incubation, single is taken less than 90 minutes altogether, and other technologies in society are typically taken at 2 and a half hours or so), cell
Counting is automatically performed by fluidic cell, and convenient operation, result are objective.
2. this test kit single testing cost is low, and can be adapted to the flow cytometer of most of models on the market, makes
User reduces the burden of patient without the need for individually buying instrument.
3. the overwhelming majority on the market will be fixed so as to lose the work of cell for the product of CTC to cell
Property, and the CTC cells detected by the enrichment of this test kit maintain preferable cytoactive, under can be used to carry out further
Trip analysis, such as：Unicellular sequencing, the analysis of cell surface specific antigen, drug sensitive test of tumor cell analysis etc..
4. this test kit sensitivity specificity is higher, can preferably realize multiple to tumorigenic early screening, tumor
The various clinical purposes such as early warning and oncotherapy curative effect evaluation is sent out, for the reference that doctor provides medication and treatment.
5. this test kit using method fast and easy, cost is relatively low and time-consuming shorter, detection count by flow cytometer from
Dynamic to complete, degree of accuracy is high, repeatability is strong.A kind of preferable circulating tumor cell detection method of operability is provided for doctor,
The blank of current like product is filled up.
Description of the drawings
Fig. 1, the test kit detection process flow chart of embodiment 1；
The state change of sample before and after Fig. 2, the centrifugation of embodiment 2；
Fig. 3, the Healthy People of embodiment 3 and tumour patient peripheral blood sample data composition；
Fig. 4, the normal population of embodiment 3 and tumour patient crowd monitoring results contrast.
Specific embodiment
With reference to specific embodiment, the invention will be further described, so that those skilled in the art can be more preferable
Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1：Kit forms and using method.
(1) test kit component mainly includes：
1. Oncoquick TCDs gradient separations liquid 4ml (Geriner, Germany)；
2. the porous septum separating pipe of 15mL volumes 10 (Wuhan Hygiea Bioscience Co., Ltd.)；
3. the centrifuge tube of 5mL volumes 10 (Eppendorf, Germany)；
④488Mouse Anti-CK7/CK8 (BD, USA)；
5. BB515MouseAnti-Human CD326 antibody (BD, USA)；
6. Anti-Human CD325 (N-Cadherin) PE (eBioscience, USA).
(2) concrete detection process is following (referring to Fig. 1, test kit detection process flow chart)：
1. taking a blood sample, (4 degree preserve and pre-cooling for 5mL whole blood EDTA anticoagulants；Detect in 24 hours)
2. taking 5mL blood of cancer patients and adding has in the centrifuge tube of separating liquid, and on 1200g acceleration of gravitys 25 are centrifuged
Minute.Peripheral blood is divided into plasma layer and red blood cell layer after centrifugation, and on separating liquid, red blood cell layer is under separating liquid for plasma layer
Face, tumor cell takes out about 80% volume plasma and individually preserves (attention on blood plasma with separating liquid bed boundary：Should not be drawn onto
CTC cellular layers, in order to avoid cause the loss of cell).
3. take out separating pipe porous septum on aim cell layer and separating liquid, be added in 5ml centrifuge tubes with
2mlPBS buffer is mixed.
4. the centrifugation of 200g acceleration of gravitys, removes supernatant, and cell precipitation is resuspended with 300 μ LPBS.
5. mixing cell adds mixtures of antibodies, lucifuge to be incubated 30 minutes.
6. FCM analysis are counted, detection time about 10 minutes.
Embodiment 2：Healthy human peripheral blood mixes the detection and analysis of the analog sample of tumor cell line
The present embodiment is the MCF-7 cells for using healthy human peripheral blood to mix gradient dilution, and as analog sample pair
It is tested and analyzed, the performance such as the tumor cell enrichment efficiency for carrying out kits for evaluation with this and the response rate of test kit, tool
Details are as follows for body：
1st, prepared by sample
By the MCF-7 cell dissociations in culture dish and single cell suspension is made, counted using red blood cell count(RBC) plate, made
Suitable concn is diluted to PBS.Add the MCF-7 cells of variable concentrations in the 5mL healthy human peripheral bloods of EDTA anticoagulants,
Its concentration be respectively 800/pipe, 400/pipe, 100/pipe, 20/pipe, 5/pipe, 0/pipe.Each concentration is respectively provided with 5
Group repeats.
2nd, pattern detection
(1) simulation blood sample gentle inversion is mixed for several times.
(2) the porous septum separating pipe for having pre-installed Oncoquick separating liquids is taken out, using front by sharp separation pipe 200g,
Centrifugation 1min, to ensure that centrifugal liquid is completely in the lower floor of porous septum.
(3) PBS solution of 500 μ L is added in the blood sample of 5mL, is mixed, be transferred to the barrier film upper strata of separating pipe, 4
DEG C, 1600g is centrifuged 20min (acceleration maximum, the deceleration for arranging centrifuge is minimum).
(4) after the completion of being centrifuged, four layers should be from top to bottom divided in separating pipe.Ground floor is plasma layer, the second layer be containing
The centrifugation liquid layer of aim cell, third layer is leukocytic cream, and the 4th layer is red blood cell layer (see accompanying drawing 2).Action is soft, removes
The flaxen plasma layers of 70%-80%, never pipette tips touching aim cell layer is (due to the ratio shared by blood plasma in different human bloods
Difference, can suitably adjust the Plasma volumes for removing according to practical situation), the remaining whole liquid in barrier film upper strata is taken out, it is placed in
It is stand-by in 5mL centrifuge tubes.
(5) add the PBS solution of 1.5mL, cleaning to separate tube wall and barrier film upper strata to separating pipe, take out PBS liquid with
Solution containing aim cell mixing, 200g is centrifuged 10min, removes supernatant, and the PBS solution for adding 300 μ L is resuspended thin
Born of the same parents.
(6) 10 μ L mixtures of antibodies (Kit components in embodiment 1 are 4., 5. and 6.) are added in cell suspension, gently
It is light to mix, room temperature lucifuge incubation 30min.
(7) machine testing on flow cytometer, carries out cell counting.
Its testing result such as table 1 below：
The average recovery rate of table 1. and the response rate are linear
Equation of linear regression is：Y=0.918x-0.875 (R2=0.992), average recovery rate is：91.8%.
The present embodiment result shows, this test kit has to tumor cell and preferably specifically reclaims ability, is being enriched with
The ambient interferences of hemocyte can be effectively removed in journey, it is good in low cellular density conditions lower linear.
Embodiment 3：Circulating tumor cell DNA independent sample extraction and analysis
1st, prepared by sample
Choose 100 healthy human peripheral bloods, 79 breast cancer disease human peripherals, 80 lung cancer patient peripheral bloods, 30 other
Primary position tumour patient peripheral blood is test sample (accompanying drawing 3).
2nd, pattern detection：
Experimentation such as embodiment 2.
3rd, testing result：Referring to accompanying drawing 4.
Normal population detects that median is：1.8；
Tumour patient crowd monitoring median is：7.9；
There is significant difference (P between two groups<0.0001).
The tumour patient crowd monitoring positive coincidence rate table of table 2.
Select 5 for cutoff values when, drawn by ROC curve model：Sensitivity 73.8%, specificity is 91%.I phases phase II
Detection specificity 53%；III phases phase IV detect specificity 81%, and total specificity is 73.8%.
Experimental result by more than can be seen that this test kit can effectively subregion tumour patient crowd and normal population,
Product sensitivity is high, high specificity, and has preferable Detection results to different types of tumor.
Embodiment described above is only the preferred embodiment lifted to absolutely prove the present invention, the protection model of the present invention
Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (9)
1. a kind of circulating tumor cell quick detection kit, it is characterised in that composition includes：
Cell density gradient separations liquid；
Porous septum separating pipe；
The centrifuge tube of 5mL volumes；
Epidermis class tumor markerses antibody；
Interstitial class tumor markerses antibody.
2. circulating tumor cell quick detection kit according to claim 1, it is characterised in that the cell density ladder
Degree separating liquid is the product Oncoquick TCD gradient separations liquid of German Geriner companies.
3. circulating tumor cell quick detection kit according to claim 1, it is characterised in that the epidermis class tumor
Mark is EpCAM, CK7 and CK8.
4. circulating tumor cell quick detection kit according to claim 3, it is characterised in that the epidermis class tumor
CK7 and CK8 antibody is the product Alexa of U.S. company BD in mark antibody Mouse Anti-CK7/
CK8。
5. circulating tumor cell quick detection kit according to claim 3, it is characterised in that the epidermis class tumor
EpCAM antibody is the product BB515Mouse Anti-Human CD326 antibody of U.S. company BD in mark antibody.
6. circulating tumor cell quick detection kit according to claim 1, it is characterised in that the interstitial class tumor
Mark is N-Cadherin.
7. circulating tumor cell quick detection kit according to claim 6, it is characterised in that the interstitial class tumor
Mark antibody is product Anti-Human CD325 (N-Cadherin) PE of eBioscience companies of the U.S..
8. circulating tumor cell quick detection kit according to claim 1, it is characterised in that the porous septum point
From the product that pipe is Wuhan Hygiea Bioscience Co., Ltd.：Porous septum separating pipe, volume is 15ml.
9. circulating tumor cell quick detection kit according to claim 8, it is characterised in that the porous septum point
It it is 10 from pipe quantity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107589060A (en) * | 2017-09-30 | 2018-01-16 | 北京大学第三医院 | Digestive system tumor recovery marker evaluation system and method |
| CN108548920A (en) * | 2018-02-28 | 2018-09-18 | 江苏医诺万细胞诊疗有限公司 | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry |
| CN109030322A (en) * | 2018-08-17 | 2018-12-18 | 成都赋智健康科技有限公司 | A kind of flow cytometry assays of save the cost |
| CN110029090A (en) * | 2019-05-05 | 2019-07-19 | 江苏康为世纪生物科技有限公司 | It is a kind of for separating the preparation method of the separating pipe of tumour cell |
| CN115558644A (en) * | 2022-10-20 | 2023-01-03 | 长沙普方德医疗器械有限公司 | Method for rapidly extracting circulating tumor cells from body fluid |
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