CN105911269A - Retroperitoneal children's neuroblastoma CTC detection kit and detection method thereof - Google Patents
Retroperitoneal children's neuroblastoma CTC detection kit and detection method thereof Download PDFInfo
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Abstract
The invention provides a retroperitoneal children's neuroblastoma CTC detection kit and a detection method thereof. The invention provides the circular tumor cell detection kit. The kit contains i, an erythrocyte-leucocyte combined antibody, ii, a leucocyte surface common antigen-antibody complex, iii, a cell nucleus fluorescent dye and iv, a chromosome FISH probe. The detection kit and method realize accurate and fast detection of circulating tumor cells under the condition of less blood collection amount of 2ml or less so that diagnosis of a retroperitoneal children's neuroblastoma prevalence risk is realized.
Description
Technical field
The present invention relates to cell detection field；In particular it relates to a kind of circulating tumor cell detection kit and
Its detection method.
Background technology
Tumors in children is the major issue threatening children's health, has the most become and has caused child after wound
Dead second largest reason.Wherein, neuroblastoma is the modal abdominal malignant tumor of children's, accounts for institute
Have the 8～10% of pediatric tumor, account for the 28% of the total cancer morbidity of baby, the highest for infant stage sickness rate
Malignant tumor, occupies and treats up to now first of the common malignant entity tumor of the most thorny children's.
Neuroblastoma (neuroblastoma, NB) be derived from joint after sympathetic nervous system Embryo dislike
Property tumor, be originating primarily from adrenal gland, but also can originate from the sympathetic god such as the outer neck of adrenal gland, vertical diaphragm, vertebra be other
Through chain distribution any position, as after peritoneum, chest, cervical region etc., its sickness rate be only below leukemia and in
Pivot nervous system tumor.Clinically, neuroblastoma often holds important feature internal organs, complete resection
Rate is only 70%, although with comprehensive measures such as operation, chemotherapy, radiotherapy and Biotherapeutics, the most not
Can be eradicated it and be colonizated in internal minimal residual stove, thus cause the recurrence after treatment stopping and again shifting.
The data of American-European countries show, the neuroblastoma of high-risk group (tumor holds important feature internal organs, it is impossible to
Complete resection, has metastasis, N-myc gene amplification and the knubble biological that histological type is prognosis mala type
Learn type) 5 years overall survival be only 30-40%, and be only 20-30% at home.Situation residing for it it
Sternness makes the diagnosis to this tumor and Therapy study be always focus and the difficult point of medical circle research.
Neuroblastoma has that grade malignancy is high, happening part is wide, progression of disease rapidly, easily occurs in early days
The feature of the other organs transfers such as bone marrow, bone regulating liver-QI, brain, lung, and disease is the most without specificity whole body disease
Shape, during when the most most of infants (more than 50%) are made a definite diagnosis, disease advances to, late period, prognosis is poor, long
Phase survival rate is low.It addition, the concealment of this tumor onset, with Ewing sarcoma, primary nervous ectoderm tumor
Similar Deng small round cell neoplasm origin, pathomorphism is similar, it is difficult to differentiate.In addition conformability when children's haves a medical check-up
Poor, and symptom etc. can not be described also bring difficulty to making a definite diagnosis of disease in complete and accurate ground, result in certain journey
Degree mistaken diagnosis, fail to pinpoint a disease in diagnosis.
Existing result of study shows, is starting infant with abdominal mass, if early finding disease, early dividing
Phase, even if the most poor infant does not the most occur bone marrow neoplasms, therapeutic effect may be well.As can be seen here, existing
On the basis of some treatment levels, finding in early days or the diagnostic method of extreme early is the most important, this has undoubtedly
Great clinic and social meaning.
At present, making a definite diagnosis of neuroblastoma, mainly by operation obtains tumor tissues, carry out pathology
Checking, this method typically requires and carries out under general anesthesia, anaesthetizes and performs the operation and has huge risk, severe patient
Cause infant postoperative death in art；And, open surgery destroys the integrity of tumor capsule, can enter again
One step increases aggressivity and the transfer of tumor, and result loses more than gain often.
Enter the tumor cell of peripheral blood time CTC (circulating tumor cell) refers to spontaneous or operation of diagnosis and treatment.Tool
The CTC having height vigor and height metastatic potential can be survived in blood circulation, and in suitable environment
Propagation, leads oncogenic recurrence and transfer.Monitoring to CTC can be sentenced for the clinical progress of tumor, curative effect
Disconnected and prognosis evaluation etc. provides valuable scientific basis.
Existing CTC detection method is generally by being enriched with and identify that two parts form, and wherein enrichment is CTC detection
Difficult point, be also CTC characterization of molecules detection and CTC counting key.Conventional detection method such as Filtration,
The sample collection amount that magnetic activated cell seperation, CTC chip etc. need is bigger, and operating process is complicated, detection
Expense is the highest.
Additionally, current standard or conventional method need to gather about 7.5ml or more blood, and child is outstanding
It is infant blood sampling relatively difficulty, also should not take a blood sample in a large number, and this is undoubtedly infant and family's warp thereof
Ji situation is proposed huge challenge.
Therefore, this area is in the urgent need to a kind of Wicresoft of exploitation, and blood sampling volume is few, detects simple and quick and can realize
Examine the detection kit of circulating tumor cell (such as the neuroblastoma of origin after peritoneum) in early days.
Summary of the invention
It is an object of the invention to provide a kind of Wicresoft, blood sampling volume is few, detects simple and quick and can realize examining in early days
The detection kit of circulating tumor cell (such as the neuroblastoma of origin after peritoneum).
First aspect present invention provides a kind of circulating tumor cell detection kit, described test kit contain as
Lower component:
I () erythrocyte combines antibody with leukocyte；
(ii) LCA antibody；
(iii) nucleus fluorescent dye；With
(iv) chromosome FISH probe.
In another preference, each component lays respectively in different vessels.
In another preference, described LCA antibody includes CD45 antibody.
In another preference, described nucleus fluorescent dye includes DAPI dyestuff.
In another preference, described chromosome FISH probe includes visiting for the FISH of chromosome centromere
Pin.
In another preference, described chromosome FISH probe includes the FISH probe for No. 8 chromosomes.
In another preference, described chromosome FISH probe includes CEP8.
In another preference, described LCA antibody includes with detectable label or not
Erythrocyte with detection labelling combines antibody with leukocyte.
In another preference, described chromosome FISH probe includes with detectable label or without can
The chromosome FISH probe of detection labelling.
In another preference, described detectable label is selected from lower group: chromophore, chemiluminescent groups, glimmering
Light blob, isotope or enzyme.
In another preference, described test kit also includes the sampling container for gathering blood sample, sampling volume
≤ 4ml, it is preferred that≤3ml, more preferably ,≤2.5ml.
In another preference, described sampling volume >=1.
In another preference, described test kit also includes a kind of or plants extra detection related reagent, described
Detection related reagent is selected from lower group: separating medium, CTC Cell Wash Buffer, erythrocyte cracked liquid, thin
Born of the same parents' fixative, penetrating inorganic agent, lavation buffer solution mother solution (SSC), NP-40, antibody diluent or its group
Close.
In another preference, described detection related reagent can be conc forms, stock solution form or through dilution
The form that can be used directly.
In another preference, described penetrating inorganic agent includes pepsin and/or formaldehyde.
In another preference, described antibody diluent includes PBS.
In another preference, described antibody diluent includes the PBS containing FBS, the content of described FBS
For 0.5-5%, it is preferred that 1-4%, more preferably, 1-3%, with total restatement of antibody diluent.
In another preference, described test kit also includes label or description, described label or description note
The blood sample amount of bright described test kit collection is 0.5-3.5ml, it is preferred that 1-3ml, more preferably, 1.5-2.5ml.
In another preference, swelling of the neuroblastoma that described circulating tumor cell originates from after including peritoneum
Oncocyte.
A second aspect of the present invention provides the purposes of detection kit described in a kind of first aspect present invention, uses
Product in preparation diagnosis circulating tumor cell.
In another preference, the neuroblastoma that described circulating tumor cell originates from after including peritoneum.
In another preference, described detection kit can be used for supporting detection equipment automatization operation.
Third aspect present invention provides the method for the detection circulating tumor cell of a kind of nondiagnostic, including step
Rapid:
A () provides a blood sample to be measured, described blood sample volume is 1-4ml；
B () removes blood plasma after, process by the detection kit described in first aspect present invention, obtain treated
Blood sample；With
C treated blood sample is detected by () by fluorescence in situ hybridization technique and immunofluorescence dyeing technology,
See whether to there is described circulating tumor cell.
In another preference, described process also includes processing with described coherent detection reagent, obtain through
The blood sample processed.
In another preference, described mammal includes people.
In another preference, the age of described people is 0.5-15 year, it is preferred that 1-12 year, more preferably,
1-10 year.
In another preference, described is detected as detection by quantitative or qualitative detection.
In another preference, described method includes step (d), and treated blood sample is carried out imaging analysis,
According to imaging results, it is determined whether there is described circulating tumor cell, and observe described circulating tumor cell
Quantity.
In another preference, when the total cellular score (i.e. CTC sum) >=3 of DAPI+/CD45-/CEP8,
It is preferred that 4-9, more preferably, during 4-7, then can determine whether that this cell is circulating tumor cell (CTC cell).
In another preference, swelling of the neuroblastoma that described circulating tumor cell originates from after including peritoneum
Oncocyte.
In another preference, when the total cellular score (i.e. CTC sum) >=3 of DAPI+/CD45-/CEP8,
It is preferred that 4-9, more preferably, during 4-7, then can determine whether the neuroblastoma that object originates from after suffering from peritoneum.
Fourth aspect present invention provides a kind of agent combination, and described agent combination includes:
I () erythrocyte combines antibody with leukocyte；
(ii) LCA antibody；
(iii) nucleus fluorescent dye；With
(iv) chromosome FISH probe.
Fifth aspect present invention provides the purposes of agent combination described in a kind of fourth aspect present invention, is used for making
The test kit of the neuroblastoma of origin after standby diagnosis peritoneum.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (as implemented
Example) in can be combined with each other between each technical characteristic of specifically describing, thus constitute new or preferred skill
Art scheme.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 shows CTC cell cytological map in each fluorescence channel.
Fig. 2 shows CTC cell cytological map in each fluorescence channel.
Fig. 3 shows operation consent, CTC cell cytological map in each fluorescence channel.
Fig. 4 shows Post operation, CTC cell cytological map in each fluorescence channel.
In accompanying drawing, merge represents merging or superposition, and WBC represents leukocyte；CTC represents circulating tumor cell.
Detailed description of the invention
The present inventor is through long-term extensively in-depth study, by a large amount of screenings and test, the most unexpectedly
Find, combine antibody with containing (i) erythrocyte with leukocyte；(ii) LCA antibody (as
CD45 antibody)；(iii) nucleus fluorescent dye (such as DAPI)；(iv) chromosome FISH probe (such as CEP8)
Test kit detection circulating tumor cell (such as the neuroblastoma of origin after peritoneum), it is only necessary to gather few
Amount blood (such as 2ml or less) just can quickly, be accurately detected circulating tumor cell, be particularly suitable for
The neuroblastoma (the especially Neuroblastoma of origin after peritoneum) of origin after diagnosis peritoneum.?
On the basis of this, the present inventor completes the present invention.
Detection kit
The present invention provide a kind of quickly, neuroblastoma CTC of origin after low cost, effective detection peritoneum
Detection kit.
In the present invention, the test kit of the present invention comprises following components:
I () erythrocyte combines antibody with leukocyte；
(ii) LCA antibody；
(iii) nucleus fluorescent dye；With
(iv) chromosome FISH probe.
In the present invention, the ratio between each component in the test kit of the present invention, and each component
Content is unrestricted.
Each component in the reagent of the present invention is available commercially, or prepares by conventional method.
The test kit of the present invention can accurately detect circulating tumor cell (neuroblastoma of origin after peritoneum),
And only need to gather little need (0.5-3.5ml, it is preferred that 1-3ml, more preferably, 1.5-2.5ml).
Detection method
Present invention also offers a kind of detection circulating tumor cell (such as the neuroblastoma of origin after peritoneum)
Method.
In a preferred embodiment, detection method of the present invention includes step:
I peripheral blood in patients 2mL is in BD anticoagulant tube in () collection, try by the present invention in 48 hours (as far as possible in 24h)
Agent box detects.
(ii) circulating tumor cell in whole blood is enriched with, after the cell of enrichment is fixing, is coated onto microscope slide
On；
(iii) cell on above-mentioned (2) described microscope slide is used fluorescence in situ hybridization technique and immunofluorescence
Staining technique is identified；
(iv) above-mentioned (3) gained Cell sheet glass being placed in interpretation under fluorescence microscope, under Taking Pictures recording, CTC is thin
Born of the same parents scheme, and add up CTC total cellular score.
(v) interpretation of result.If the purpose of detection is to differentiate tumor type, as CTC cell number >=15/2mL
Blood, then can determine whether that this patient tumor type is that the probability of the neuroblastoma of origin is the biggest after peritoneum；As
Really testing goal is the real-time monitoring of tumour progression, then may compare this testing result and testing result last time
(interval time of usual monitor and detection is one month), by CTC detection change in value indication tumour progression feelings
Condition, and then determine the need for combining other tumor interference methods or change therapeutic scheme.
Specifically, when the neuroblastoma CTC detection kit of origin after the detection peritoneum using the present invention
When detecting, typically can include following detecting step and follow-up CTC cell interpretation process:
(1) (reagent of employing is Human CD45Depletion Cocktail to circulating tumor cell enriching section
Test kit, Primary Reference original reagent box description)
1.1 2mL whole blood 800g are centrifuged 8 minutes, remove upper plasma, add and the isopyknic CTC of blood plasma
Cell Wash Buffer, reverse mixing.
1.2 in above-mentioned (1.1) system add 100 μ L leukocyte and combine antibody with erythrocyte, the most mixed
Even, incubated at room 20 minutes.
1.3 take 3mL separating medium in clean 15mL centrifuge tube.In above-mentioned (1.2), system adds 2mL
After CTC Cell Wash Buffer, fully mixing, complete soln system is added on 3mL separating medium gently,
Within centrifugal 20 minutes, (test kit description is 1200g to 963g (2200rpm), and the parameter after we adjust is centrifuged effect
Fruit is more preferably).
1.4 centrifugal rear visible delamination.Transfer upper strata cellular layer, in new 15mL centrifuge tube, adds CTC
Cell Wash Buffer is supplied volume and is centrifuged 5 minutes to 15ml, 2000rpm, abandons upper solution to 1ml
Place.(washing step after transfer is that we revise)
Precipitation is observed with or without significantly not removing clean erythrocyte, if do not had after 1.5 (optional steps) are centrifugal
Having or seldom can directly carry out following 1.6 steps, if had, needing to carry out erythrocyte process.Concrete operations are:
Erythrocyte cracked liquid 3ml after above-mentioned 1.4 step gained solution systems add 27 DEG C of preheatings, shakes up liquid
Body, stands 3 minutes, shook up a liquid every 1 minute.
1.6 are shaken gently for centrifuge tube supplies body with cell precipitation of scattering, addition CTC Cell Wash Buffer
Amass to 15ml, 2000rpm and be centrifuged 5 minutes.
1.7 take an anticreep microscope slide, go out the square dispensing area of 1cm × 1cm with SABC stroke.
1.8 abandon upper solution at 100 μ L, dispel cell precipitation gently, add 100 μ L cells solid
Determining liquid, fully mix, gained cell suspension is all applied to the cell dispensing area on microscope slide.
1.9 slides stand dry or 31～32 DEG C dry overnight.
(2) fluorescence in situ hybridization technique combined immunization fluorescence dye identifies circulating tumor cell part jointly
(CEP8 probe is commercially available, such as purchased from Vysis company)
Before 2.1 experiments, reagent prepares:
A. by 1% formaldehyde that measures of 180 μ L/ sheets, 37 DEG C preheat 20 minutes.
The most in advance the 10 times of dilutions of 20X SSC (PH=5.3) mother solution sterile deionized water are prepared as work molten
Liquid, this solution can be prepared more, at most can room temperature place 6 months.
Preparing lavation buffer solution the most in advance, can prepare more, lavation buffer solution can room temperature be placed 6 months.
2.2 by 1% formaldehyde that is fully warmed-up with pepsin (0.05mg/mL) by 180 μ L/ sheets: 20 μ L/
The ratio of sheet mixes rapidly, is added drop-wise to the cell dispensing area of above-mentioned 1.8 gained slides immediately, it is ensured that solution
Fully cover whole specimen district, incubated at room 10 minutes.
2.3 inhale and abandon the liquid on slide, with 2X SSC 200 μ L/ time. and sheet washs the specimen area on slide,
Washing 3 times altogether.During front twice washing, solution is inhaled immediately after adding and is abandoned, after the dropping of last solution,
Inhale after standing 1min and abandon.
Specimen is put in the color jar containing 70%, 85%, 100% dehydrated alcohol and to be stood respectively by 2.4
Within 1 minute, 1 minute, 2 minutes, carry out CTC cell dehydration process.Slide after processed is erected at dustless
Crack between the teeth gently on paper, to blot the debris that slide flows down, and with miniature hair-dryer, slide is dried up.Completely
The slide being dried carries out next-step operation immediately.
2.5 No. 8 chromosome FISH probe of dropping.By the amount of 5 μ L/ sheets, probe is dropped in specimen district
Centre, covered, with mounting glue mounting.This operation and following operation are both needed under the conditions of lucifuge carry out.
2.6 fluorescence in situ hybridization.Hybridization instrument put by above-mentioned slide, arranges hybridization procedures and is: 73 DEG C, and 10
Minute；37 DEG C, 6 hours.
2.7 hybridization terminate first 10 minutes, carry out the preparation of antibody mixed liquor.LCA
Antibody (CD45 antibody) is diluted by 1:100 with antibody diluent, and antibody mixed liquor is by 200 μ L/ sheets
Amount prepares.
2.8 specimen immunofluorescence pre-treatments.
A., after hybridization terminates, use tweezers to tear mounting glue off, slide is placed in the color jar containing 2X SSC
In, rock and remove coverslip.Carry out following cleaning step immediately.
B.2X SSC 200 μ L/ time. the specimen area on sheet washing slide, one is washed 2 times, the most quiet
Put 1min.
C. lavation buffer solution 200 μ L/ time. the specimen area on sheet washing slide, washing 3 times altogether.Front two
During secondary washing, solution is inhaled immediately after adding and is abandoned, and after the dropping of last solution, inhales and abandon after standing 1min.
Slide is placed in wet box by 2.9,200 μ L/ sheets dropping antibody mixed liquors to slide sample district, 30 DEG C
Lucifuge hatches 1 hour.
The antibody mixed liquor on slide is abandoned in 2.10 suctions, thin with the CTC after lavation buffer solution washing antibody incubation
Born of the same parents.Washing step ibid 2.8 (c).
2.11 is central in specimen district by 5 μ L/ sheet dropping DAPI Mounting, covered.The completeest
Become the test kit detection part of CTC.
(3) interpretation under CTC mirror.Cell sheet glass obtained as above is placed in interpretation under fluorescence microscope, takes pictures simultaneously
Record all CTC cells cytological map in each fluorescence channel.When reading Cell sheet glass, need first from regarding
The wild upper left corner starts, and does not have the omission in the visual field during mobile microscope slide.Under 40 times of object lens, first
In red channel, first find suspicious cells, i.e. there is no the bare nucleus cell of red outer ring (CD45 does not expresses), so
After confirm the most respectively this suspicious cells whether have in blue channel blue-fluorescence signal and its at orange passage
In the fluorescent orange signaling point number seen, and then judge whether this cell is CTC cell.The while of every
Possess the thin of DAPI+/CD45-/CEP8 (that is: CD45 expresses feminine gender, and CEP8 is accredited as heteroploid) condition
Born of the same parents can be determined as CTC cell.
Generally, when quantity >=3 of DAPI+/CD45-/CEP8 cell, then can determine that in detected sample
Suffer from a fairly large number of CTC cell (such as the tumor cell of the neuroblastoma of origin after peritoneum), and then
Judge that detected object suffers from neuroblastoma (the especially neuroblastoma of origin after peritoneum).
Circulating tumor cell can be detected fast, accurately by the detection method of the present invention, thus diagnose exactly
The neuroblastoma that (or auxiliary diagnosis) object originates from after whether suffering from peritoneum.
Main advantages of the present invention include:
(1) present invention only needs 2mL peripheral blood in patients, Wicresoft and amount of blood collected few, and sample acquisition compares appearance
Easily, the injury of the child of one full year of life especially not enough to infant is the most greatly alleviated.
(2) detection kit of the present invention and detection method, simple to operate, can quickly, accurately assist diagnosis
Whether infant tumor type is origin neuroblastoma after peritoneum, reduces misdiagnosis rate, also achieves tumor
Early find, the most by stages, early treatment, improve infant therapeutic efficiency and survival rate.
(3) compared to existing CTC detection method, detection kit of the present invention and detection method greatly reduce
Testing cost, significantly reduces patient and household economy burden thereof, to the trouble that need to carry out continuous monitor and detection
Person is especially suitable.
(4) test kit of the present invention can detect infant Peripheral Circulation tumor cell situation, auxiliary diagnosis infant
Whether tumor type is the neuroblastoma of origin after peritoneum, or origin is neural female thin after monitoring peritoneum in real time
The tumour progression situation of born of the same parents' tumor infant, to realize accomplishing such disease early to find early treatment or timely the most by stages
Adjust therapeutic scheme purpose, this to after peritoneum origin neuroblastoma diagnosis and treatment has and
Important meaning.
(5) present invention is detected by regular peripheral blood CTC, also to having been carried out operation or can carry out other treatment
Infant carry out the monitoring of progression of disease, in order to adjust therapeutic scheme in time, improve therapeutic efficiency.
(6) test kit of the present invention can coordinate the equipment of automatization so that the detection of circulating tumor cell can
Automatically complete.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise
Percentage ratio and number are percentage by weight and parts by weight.
Material used by the present invention if no special instructions, is commercially available prod.
Embodiment 1
After peritoneum, preparation and the Detection results of the diagnostic kit of the neuroblastoma of origin are verified
The neuroblastoma CTC detection kit of origin after preparation detection peritoneum, described test kit includes:
Leukocyte combine with erythrocyte antibody, separating medium, CTC Cell Wash Buffer (2%FBS+PBS), red carefully
Cellular lysate liquid, cell fixative, pepsin (0.05mg/mL), 1% formaldehyde, 20 × SSC, No. 8 dyeing
Body FISH probe, NP-40, antibody diluent (1%FBS+PBS), LCA antibody (CD45
Antibody), nucleus fluorescent dye (DAPI).
Test kit uses forward part working solution need to carry out following preparation in advance:
The preparation of (1) 2 × SSC: take 50mL 20 × SSC (PH=5.3) mother solution, add 450mL aseptic go from
Sub-water, fully mixing can obtain 500mL 20X SSC (PH=5.3) solution.
(2) preparation of 2 × SSC solution (PH=7.0～7.5) of the i.e. 0.1%NP-40 of lavation buffer solution: take
State 20 × SSC (PH=5.3) 100mL of (1) gained, separately take 1mL NP-40 and 849mL sterile deionized water
Mixing, adjusts PH to 7.0～7.5, is finally settled to 1000mL.
(3) preparation of antibody diluent (1%FBS+PBS): take 1mL FBS and add 99mL PBS, the most mixed
Even.
Embodiment 2
By neuroblastoma CTC detection kit of originating after the peritoneum of embodiment 1 preparation, carry out detecting also
Verify, specifically comprise the following steps that
(1) numerical example is gathered:
First batch is tested: originate after the peritoneum made a definite diagnosis neuroblastoma infant peripheral blood 2mL, or right
According to individual peripheral blood 2ml (the peripheral blood C1 that other tumor is individual, or the peripheral blood of 10 normal individuals
C2)；
Second batch experiment: originate after the peritoneum made a definite diagnosis neuroblastoma infant peripheral blood 2mL, and
The peripheral blood C3 to C14 that other tumor is individual, and the peripheral blood C15 of 20 normal individuals
(2) enrichment of CTC and the preparation of Cell sheet glass are carried out:
A.2mL peripheral blood 800g is centrifuged 8 minutes, removes blood plasma, adds corresponding isopyknic CTC cell and wash
Wash buffer, add 100 μ L leukocyte after mixing and combine antibody, incubated at room 20 minutes with erythrocyte.
Adding 2mLCTC Cell Wash Buffer, fully after mixing, whole systems are added to 3mL separating medium
On, centrifugal 20 minutes of 963g (2200rpm), in collection upper strata cellular layer to new 15mL centrifuge tube, the most in fact
Show the enrichment of CTC；
B. to the above cell 15mL Cell Wash Buffer collected, 2000rpm is centrifuged 5 minutes and carries out carefully
Born of the same parents wash, and repeat above washed once.It is eventually fabricated 100 μ L cell suspension, adds 100 μ L cells solid
Determine liquid, fully mix, the cell dispensing area that all cells suspension is all applied on microscope slide, make
Cell sheet glass.Cell sheet glass 32 DEG C is dried overnight, identifies for CTC and prepares；
(3) fluorescence in situ hybridization and immunofluorescence dyeing identify CTC jointly:
A. the fluorescence in situ hybridization detection of Cell sheet glass.20 μ L pepsin (0.05mg/mL) and 180 μ L
1% formaldehyde being preheated to 37 DEG C in advance is prepared by mixing into covering immediately extremely whole specimen district, room after working solution
Temperature is hatched 10 minutes and is processed cell membrane.Specimen area is washed by 2X SSC.With 70%, 85%,
100% dehydrated alcohol carries out processed to cell successively.Miniature hair-dryer drips after being dried up completely by slide immediately
Add No. 8 chromosome FISH probe of 10 μ L, with mounting glue mounting, hybridization instrument carries out fluorescence in situ hybridization.
Hybridization procedures is: 73 DEG C, 10 minutes；37 DEG C, 6 hours；
B. the Immunofluorescence test of Cell sheet glass.Hybridization terminates first 10 minutes, carries out CD45 antibody mixing
The preparation of liquid.After hybridization terminates, tear mounting glue off with tweezers, slide is placed in the dyeing containing 2X SSC
In cylinder, rock and remove coverslip.Wash respectively to the specimen district on slide with 2X SSC and lavation buffer solution
Territory is washed.Slide after washing is placed in wet box, drips 200 μ L antibody mixed liquors to slide mark
Local area, 30 DEG C of lucifuges hatch 1 hour.Antibody incubation inhales the antibody mixed liquor abandoning on slide, with washing after terminating
Wash buffer and carry out cell washing；
C. nuclear targeting.Dripping 5 μ L DAPI Mounting central in specimen district, covered makes
DAPI Mounting opens in whole specimen district profit, inhales and abandons the surplus liquid around slide, i.e. completes CTC
Identification and detection part；
(4) interpretation under the mirror of CTC:
According to CTC interpretation standard DAPI+/CD45-/CEP8+ (triploid or above polyploid) to more than
Gained Cell sheet glass is placed in interpretation under fluorescence microscope, and under Taking Pictures recording, all CTC cells are glimmering at each simultaneously
Cytological map in optical channel.
Testing result is as shown in table 1.Wherein, >=3 CTC cells for 2ml blood sample, detected, can
It is judged to suffer from source neuroblastoma after peritoneum.
Table 1CTC testing result (peripheral blood 2ml)
The CTC testing result (peripheral blood 2ml) of other tumor cases of table 2
| Sample sequence number | Age | Clinical diagnosis or remarks | CTC sum |
| 4 | 20 months | Neuroblastoma (adrenal area after the peritoneum of right side) | 20 |
| 5 | 5 years old | Ganglioneuroblastoma (posterior peritoneum) after peritoneum | 40 |
| C3 | 5 years old | Between fluidity mucus pernicious cellule type tumor | 1 |
| C4 | JIUYUE | Adrenal Neuroblastoma (left side adrenal gland) | 0 |
| C5 | 4 years old | Adult form cystic teratoma | 1 |
| C6 | 3 years old | Mixed germ cell tumor | 0 |
| C7 | 13 months | Right adrenal gland neuroblastoma | 1 |
| C8 | 5 years old | Left side adrenal gland is inclined to neuroblastoma | 1 |
| C9 | 3 years old | Ganglioneuroma | 0 |
| C10 | 15 months | Nephroblastoma | 1 |
| C11 | 2 years old | Ganglion cell's property neuroblastoma | 0 |
| C12 | 11 years old | Pernicious minicell tumor | 1 |
| C13 | 7 years old | Ganglioneuroma | 0 |
| C14 | 2 years old | Adrenal gland originates ganglion cell's property neuroblastoma | 0 |
| C15 | 2-15 year | Normal individual (n=20) | 0 |
The above results shows, after peritoneum, the neuroblastoma of origin and the tumor in other sources are in peripheral blood
There is the difference of highly significant in the quantity of CTC, in the former peripheral blood, CTC quantity is the most, the most about
About 50/2mL, be the most tumor of CTC content.Therefore, detection kit or the side of the present invention are used
Method, only with the blood sample of 0.5-2 milliliter, that originates from after being just enough to relatively accurately judge peritoneum is neural female
Glucagonoma.
As a example by 2 the CTC cells detected in a detected individuality, result is as depicted in figs. 1 and 2.
Result shows, the detection method of the present invention can be accurately detected source neuroblastoma after peritoneum.
Embodiment 3
The feasibility checking of the neuroblastoma CTC detection kit of origin after peritoneum
The detection kit of Application Example 1 preparation carries out feasibility checking, and concrete condition is as follows:
(1) there is FUO and anemia phenomenon in certain 5 years old infant, suffers from suspected of neuroblastoma
Person, gathers its peripheral blood；
(2) apply retroperitoneal neuroblastoma CTC detection kit of the present invention that the peripheral blood gathered is carried out
The enrichment of circulating tumor cell and detection, detecting step is with (2) in embodiment 1～(4).
(3) testing result shows, the CTC sum that this patient's 2mL peripheral blood detects is 16, Qi Zhongsan
Times body CTC 7, tetraploid CTC 4, pentaploid and above CTC 5, CTC cytological map such as Fig. 3
Shown in.CTC sum is more than 10, and this patient be that the probability of the neuroblastoma patient originated from after peritoneum is non-
Chang great；
(4) this patient has been carried out checking targetedly by doctor according to this testing result, at second time abdominal part
Find suspicious mass time ultrasonic, and it is implemented in time operation and associated treatment；
(5) 3 months after operation, then this patient has been carried out the detection of Peripheral Circulation tumor cell, detect process
With (2)；
(6) after testing, neuroblastoma peripheral blood in patients CTC sum of originating after this peritoneum is 2, its
Middle triploid CTC 1, tetraploid CTC 1, CTC cytological map is as shown in Figure 4.Peripheral blood in patients CTC
Quantity drastically reduces, and other Clinical detection indexs display that patient's recovery is splendid, it is seen that therapeutic effect is relatively
Good, original therapeutic scheme can be maintained to proceed treatment；
(7) aftertreatment is after 6 months, gathers its peripheral blood, carries out Peripheral Circulation tumor cell detection,
Detection process is with (2) in embodiment 1～(4)；
(8) neuroblastoma peripheral blood in patients CTC sum of after testing, originating after this peritoneum reduces to 0, with it
He is consistent at Clinical detection result, all shows that patient outcomes is splendid.
Result shows, after the detection method of the present invention and test kit can be accurately detected peritoneum, Derived Nerve is female thin
Born of the same parents' tumor also reflects therapeutic effect.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document coverlet
Solely it is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, this area
The present invention can be made various changes or modifications by technical staff, and these equivalent form of values fall within right appended by the application equally
Claim limited range.
Claims (10)
1. a circulating tumor cell detection kit, it is characterised in that described test kit contains following group
Point:
I () erythrocyte combines antibody with leukocyte；
(ii) LCA antibody；
(iii) nucleus fluorescent dye；With
(iv) chromosome FISH probe.
2. detection kit as claimed in claim 1, it is characterised in that described leukocyte surface is common
Antigen-antibody includes CD45 antibody.
3. detection kit as claimed in claim 1, it is characterised in that described nucleus fluorescent dye
Including DAPI dyestuff.
4. detection kit as claimed in claim 1, it is characterised in that described chromosome FISH probe
Including the FISH probe for chromosome centromere.
5. detection kit as claimed in claim 1, it is characterised in that described test kit also includes
Planting or plant extra detection related reagent, described detection related reagent is selected from lower group: separating medium, CTC are thin
Born of the same parents' lavation buffer solution, erythrocyte cracked liquid, cell fixative, penetrating inorganic agent, lavation buffer solution mother solution
(SSC), NP-40, antibody diluent or a combination thereof.
6. detection kit as claimed in claim 1, it is characterised in that described circulating tumor cell bag
The tumor cell of the neuroblastoma originated from after including peritoneum.
7. the purposes of detection kit described in a claim 1, it is characterised in that be used for preparing diagnosis
The product of circulating tumor cell.
8. the method for the detection circulating tumor cell of a nondiagnostic, it is characterised in that include step:
A () provides a blood sample to be measured, described blood sample volume is 1-4ml；
B () removes blood plasma after, process by the detection kit described in claim 1, obtain treated blood
Sample；
C treated blood sample is detected by () by fluorescence in situ hybridization technique and immunofluorescence dyeing technology,
See whether to there is described circulating tumor cell.
9. an agent combination, it is characterised in that described agent combination includes:
I () erythrocyte combines antibody with leukocyte；
(ii) LCA antibody；
(iii) nucleus fluorescent dye；With
(iv) chromosome FISH probe.
10. the purposes of agent combination as claimed in claim 9, it is characterised in that be used for preparing diagnosis peritoneum
The test kit of the neuroblastoma of rear origin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970224A (en) * | 2017-03-16 | 2017-07-21 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application |
| RU2797690C1 (en) * | 2022-07-06 | 2023-06-07 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии имени Дмитрия Рогачева" Министерства здравоохранения Российской Федерации | Method of increasing the radicality of surgical treatment of children with locally advanced form of neuroblastoma |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101587043A (en) * | 2008-05-20 | 2009-11-25 | 林平 | Integrated method for enriching and detecting rare cell in biological fluid sample |
| CN103031276A (en) * | 2012-09-17 | 2013-04-10 | 孙小娟 | Method for obtaining circulating tumor single-cell |
| CN104007257A (en) * | 2013-02-24 | 2014-08-27 | 北京莱尔生物医药科技有限公司 | Method for detecting non-humoral rare karyotes, and kit thereof |
| CN204086278U (en) * | 2014-07-31 | 2015-01-07 | 北京海思特临床检验所有限公司 | For detecting the kit of Peripheral Circulation tumour cell |
-
2016
- 2016-03-03 CN CN201610121071.6A patent/CN105911269A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101587043A (en) * | 2008-05-20 | 2009-11-25 | 林平 | Integrated method for enriching and detecting rare cell in biological fluid sample |
| CN103031276A (en) * | 2012-09-17 | 2013-04-10 | 孙小娟 | Method for obtaining circulating tumor single-cell |
| CN104007257A (en) * | 2013-02-24 | 2014-08-27 | 北京莱尔生物医药科技有限公司 | Method for detecting non-humoral rare karyotes, and kit thereof |
| CN204086278U (en) * | 2014-07-31 | 2015-01-07 | 北京海思特临床检验所有限公司 | For detecting the kit of Peripheral Circulation tumour cell |
Non-Patent Citations (2)
| Title |
|---|
| WEI GUO等: "Clinical Significance of EpCAM mRNA-Positive Circulating Tumor Cells in Hepatocellular Carcinoma by an Optimized Negative Enrichment and qRT-PCR-Based Platform", 《CLINICAL CANCER RESEARCH》 * |
| 汤宏峰 等: "外周血和骨髓微量神经母细胞瘤细胞检测的临床意义", 《中华小儿外科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970224A (en) * | 2017-03-16 | 2017-07-21 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application |
| CN106970224B (en) * | 2017-03-16 | 2018-06-26 | 武汉康录生物技术股份有限公司 | A kind of kit and its application using CD45 immunofluorescences joint CEP probe identification circulating tumor cells |
| RU2797690C1 (en) * | 2022-07-06 | 2023-06-07 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии имени Дмитрия Рогачева" Министерства здравоохранения Российской Федерации | Method of increasing the radicality of surgical treatment of children with locally advanced form of neuroblastoma |
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