CN105087779A - Method and related kit for detecting EGFR/CEP7 gene status based on rare cells - Google Patents
Method and related kit for detecting EGFR/CEP7 gene status based on rare cells Download PDFInfo
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- CN105087779A CN105087779A CN201510441614.8A CN201510441614A CN105087779A CN 105087779 A CN105087779 A CN 105087779A CN 201510441614 A CN201510441614 A CN 201510441614A CN 105087779 A CN105087779 A CN 105087779A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention relates to a method and a related kit for detecting EGFR/CEP7 gene status based on rare cells, wherein the method comprises the following steps: 1. acquiring a blood sample or a body fluid sample, wherein the sample includes a mixed cell population of circulating tumor cells or other rare cells and white blood cells; 2. diluting the sample by virtue of a buffer solution, removing plasma and recovering CTC or other rare cells; 3. cracking by virtue of a red blood cell lysis buffer, removing red blood cells and recovering CTC or other rare cells; 4. uniformly mixing and breeding magnetic particles coated with related anti-human leucocyte antigens and antibodies with the recovered cells, and combining so as to form a magnetic particle-white blood cell mixture; 5. isolating the magnetic particle-white blood cell mixture from other cells so as to obtain a mixed cell population containing CTC or other rare cells and a few of white blood cells; and 6. by virtue of a method combining immunofluorescence cytochemistry and fluorescence in-situ hybridization, determining the status of EGFR/CEP7, and simultaneously identifying CTC or other rare cells.
Description
Technical field
The present invention relates to medical diagnostics, particularly the detection of tumour cell.More particularly, the present invention relates to the diagnostic method of the treatment plan detecting cancer and assess patient.
Background technology
Circulating tumor cell (CirculatingTumorCell, CTC) refers to the tumour cell entering into blood circulation, and it can come off into blood by primary tumor or metastasis, also may advancing in blood in formation solid tumor focus.Tumour cell invades the recycle system, major part due to the Immune discrimination of body, machinery kill and wound and self apoptosis dead in a short time, only have only a few survive, and plant at far-end or former tissue and organ, being further development of metastasis, is the most direct factor causing metastases and recurrence.
CTC as a kind of " liquid biopsy " sample that can represent primary tumo(u)r, peripheral blood sample easily obtains, traumatic little, can repeatedly gather, be clinical detection more preferably Specimen origin, real-time comprehensive monitoring can be carried out to oncotherapy.Can effectively realize tumor recurrence early warning, curative effect is evaluated and tumor individual therapy medication guide in time, current CTC detect be widely used in clinical tumor patient curative effect Real-Time Evaluation, state of an illness Real-Time Monitoring, resistance factorial, in Index for diagnosis.
Fluorescence in situ hybridization (fluorescentinsituhybridization, FISH) is a kind of Cytogenetic techniques, can be used for carrying out detection and positioning to nucleic acid.FISH technology at present based on the rare cell that comes off is generally qualitative detection, is difficult to accomplish precisely quantitative and ensure its effect.Original position of searching to the bottom is to be applicable to the FISH detection method of tissue sample and common exfoliative cytology FISH detection method at present, when after the cell smear of the order of magnitude between 0-100, and effectively cannot keep obtaining in cell balancing and keeping higher stability preferably at coloring effect, more be difficult to form the moulding product effectively stablizing detection by quantitative.Though have corresponding patent to set forth a kind of effective ways (US6 at present, 524,798), but need the special inspecting equipment of extremely sky high cost, cannot Routine Test Lab complete this based on and the FISH testing of a small amount of rare cell or even unicellular aspect.
Describe the authentication method of the tumour cell of biological sample, rare cells or other biological entity before this (as Chinese Patent Application No.: 200810097889.4,201310057307.0).This two-step approach requires that effective enrichment is to guarantee to obtain target cell before analysis, removes a large amount of fragment and other interfering substances simultaneously, makes to carry out cellular assay by imaging technique.With the negative enrichment strategy of uniqueness by immune magnetic particle and multi-density centrifugation, fluorescence in situ hybridization and Immunofluorescence analysis bank altogether, accurate quantification ground detects in nearly all variety of solid tumor types and drops to containing the rare cell (containing CTC) in the multiple biological fluid samples such as blood, chest/seroperitoneum, urine, cerebrospinal fluid this method.This combined method is used for the chromosome amplification type tumour cell in enrichment and counting blood sample, because herein is provided a kind of instrument measuring cancer.
EGFR (EpidermalGrowthFactorReceptor) is the acceptor of epidermal growth factor (EGF) cell proliferation and intracellular signaling.Research shows in many noumenal tumours, there is EGFR high expression level or unconventionality expression.The propagation of EGFR and tumour cell, vasculogenesis, tumor invasion, transfer and apoptotic suppression are relevant.
Iressa is for previously accepting the evident in efficacy of chemotherapeutical Locally Advanced or Metastatic Nsclc patient.Multinomial research display, the patient of EGFR gene amplification, its targeted drug is efficient reaches 35%, and disease control rate reaches 70%, and does not have the efficient of gene amplification and be only 9.8%.
But during clinical detection, due to such as many situations that effectively cannot obtain tumor tissues such as excision, the not accomplice under duress of patient's subjective desire, and following limitation can be there is:
1, as having wound property and being limited to the histology of lesions position, there is specimen sampling and the limitation such as not easily maybe cannot to draw materials, and postoperatively can not repeatedly to draw materials, cause tissue sampling cannot provide the monitoring information of real-time.
2 and subject impact etc., the molecular information of tumour cell likely occurrence dynamics changes.As: studies have reported that, accept the patient with breast cancer of new adjuvant chemotherapy, before chemotherapy, accept biopsy, and result contrasted with postoperative pathological, before and after finding to there is treatment, EGFR expresses inconsistent situation.
3, the present situation of just local message reflection that causes of the existence of tumor tissues heterogeneity and single-point biopsy, makes traditional histological detect the evaluation lacking comprehensive globality, as the comprehensive evaluation etc. of primary tumor, multiple metastasis information.
The invention reside in while providing a kind of CTC of detection or other rare cells the method that quantity and the ratio of EGFR/CEP7 detect.To obtain more comprehensively information.The present invention is being separated on the basis obtaining CTC (or other rare cells) in blood, carry out unicellular analysis of molecules, detect EGFR expression, come from individual cell level with this, immediately find the molecular information of EGFR, to carry out Real Time Observation to the state of patient.
Summary of the invention
To the method that quantity and the ratio of EGFR/CEP7 detect while the invention provides a kind of CTC of detection or other rare cells.Direct help clinicist understands the real-time gene appearance of tumour patient, drafts next step treatment plan with auxiliary prediction Survival.Method of the present invention comprise following all and part steps: step 1, obtains blood sample from patient or other contain the body fluid sample of rare cell, comprises CTC or other rare cells and leukocytic mixed cellularity group in sample;
Step 2, uses damping fluid diluted sample, centrifugal segregation blood plasma, and reclaims CTC or other rare cells;
Step 3, uses erythrocyte cracked liquid cracking, and centrifugal segregation red corpuscle also reclaims CTC or other rare cells;
Step 4, with the recovery cell blending incubation of magnetic particle with step 3 gained being coated with anti-human white corpuscle associated antigen, fully to combine with target white corpuscle to make magnetic particle and forms magnetic particle-white corpuscle mixture;
Step 5, centrifugal by magnetic particle-white corpuscle mixture and other cellular segregation, obtain containing CTC or other rare cells and a small amount of leukocytic mixed cellularity group;
Step 6, uses Immunofluorescence and the method combined of fluorescence in situ hybridization, measures the state of EGFR/CEP7, carry out the discriminating of CTC or other rare cells simultaneously.
Method of the present invention, wherein, damping fluid described in step 2 is a kind of damping fluid close with density of blood, containing materials such as BSA, PBS, between pH7-8, while can ensureing to remove blood plasma, protects various karyocyte.
Method of the present invention, wherein, erythrocyte cracked liquid described in step 3 is a kind of erythrocyte cracked liquid, utilizes isotonic anisotonic principle, while splitting erythrocyte, can not damage other karyocytes.
Lysate as following formula:
NH4Cl82.9g
KHCO310g
EDTA0.37g
Add H2O to 1000ml (autoclaving, 4 DEG C of preservations)
Or
Erythrocyte cracked liquid (Tris-NH4Cl): take 3.735g ammonium chloride, Tutofusin tris (Tris) 1.3g is dissolved in water and is diluted to 500ml.0.22 μm of membrane filtration is degerming, 4 DEG C of preservations.
Method of the present invention, wherein, the magnetic particle being coated with anti-human white corpuscle associated antigen described in step 4 is that a kind of bag is by the streptavidin magnesphere of anti-leukocyte common antigen (LCA) antibody.Can maximum ground, securely in conjunction with white corpuscle, be beneficial to follow-up sorting process.
Method of the present invention, wherein centrifugation medium described in step 5 is the density gradient centrifugation liquid of a kind of density between 1.070-1.080, wherein optimum with 1.072, removing in conjunction with magnetic bead leukocytic while, other single karyocytes can be preserved and do not lose.
Method of the present invention, wherein, uses the method for Immunofluorescence and fluorescence in situ hybridization described in step 6, carry out CTC discriminating to CTC or other rare cells, Simultaneously test EGFR/CEP7, method is as follows:
(1) EGFR amplification type CTC: observe under sample being placed in fluorescent microscope respective channel, if find multiple EGFR signaling point distribution (>=3), and the signal that number is greater than CEP7 is counted, and without CD45 antigen presentation, then count a CTC positive cell, and be directly EGFR positive cell depending on this cell;
(2) chromosome amplification type CTC: count single karyocyte CEP7 signal number, if be more than or equal to 3 signaling points and without CD45 antigen presentation, then count a CTC positive cell;
(3) EGFR/CEP7-CTC overall number: record all EGFR amplification types and chromosome amplification type CTC number, summation is considered as EGFR/CEP7-CTC overall number;
(4) EGFR gene amplification state recording: if there is obvious and typical EGFR to increase in all specimen area, be then designated as EGFR amplification; If nothing, then by the result of EGFR and CEP7 signal ratio in CEP7 number of signals, average each nucleus in EGFR number of signals, average each nucleus in each nucleus average in all CTC cells.
A kind of test kit one for aforesaid method, comprises following composition:
Wherein, 10 ×, 20 × indicated concentration, namely this solution is the concentration of 10 times/20 times.
Test kit preparation method of the present invention is as follows:
Test kit one comprises following enrichment and differentiates two-part reagent.
Enriching section:
CS1 concentrates damping fluid (10 ×):
In every 1000mL water, containing 60gBSA, 5 bags PBS powder (2L/ bag), the EDTA of 100mL0.5M, 0.8mLProclin300.
CS2 concentrates storage liquid (10 ×):
In every 1000mL water, take 82.9gNH
4cl, 10gKHCO
3, 0.37gEDTA, water and 0.8mLProclin300, carry out sufficient stirring and dissolving, constant volume, is formulated as 10X concentrated solution.
CS3 separating medium:
Be the gradient centrifugation liquid dilution of 1.077 by density, test density in dilution, makes its density between 1.070-1.075.
Magnetic particle suspension:
CD45 antibody concentration is adjusted to 1mg/mL, carries out hatching 1h according to the ratio of 100uL:1mL with streptavidin immunomagnetic beads, be namely formulated as magnetic particle suspension.
Differentiate part:
CF1 stationary liquid:
Mixing PEG and dehydrated alcohol, make PEG final concentration be 1%, dehydrated alcohol final concentration is 50%.
10 × CF2 stationary liquid:
Take PBS as solvent, dissolve PFA powder, be formulated as the PFA concentrated solution of 5%.
Methane amide working fluid:
By the working fluid that the dilution of methane amide stoste is 50%.
20XSSC concentrates damping fluid:
In 1000mL water, add sodium-chlor 175.3g, ammonium citrate 88.2g.
EGFR/CEP7 probe:
Use methane amide, dextran sulfate sodium salt by EGFR and CEP7, be formulated as probe face liquid.
BSA powder:
Outsourcing packing.
CD45-AF594 fluorescence antibody:
CD45 antibody and AlexaFlour594 fluorescein lucifuge are hatched 1h, and fluorescein can be connected with CD45 antibody.
DAPI staining fluid:
Use anti-quencher Mountingmedium the DAPI stoste of 1mg/mL to be diluted according to 1:1000, be formulated as DAPI dyeing working fluid.
The using method of test kit one of the present invention, step is as follows:
(1) 0.1-5mL blood preparation is added in 50mL centrifuge tube, adds the CS1 working fluid that diluted to 45mL, centrifugal 5 minutes of 650 × g, inhale and abandon supernatant and remain about 12mL liquid;
(2) jog mixing, adds the CS2 cracking working fluid that dilute to 45mL, mixes centrifugal 5 minutes of 8-10 minute, 650 × g, and supernatant fluid is abandoned in suction;
(3) add the mixing of CS1 working fluid appropriate also jog, again add a certain amount of CS1 working fluid, add cleaned magnetic particle suspension 200uL, level shakes up (120rpm) 20 minutes;
(4) draw the rear liquid of all mixings, be superimposed on CS3 separating medium, centrifugal 5 minutes of 300 × g;
(5) the upper two-layer liquid of absorption except magnetic particle precipitation is in 15mL centrifuge tube, adds CS1 working fluid to 15mL, centrifugal 5 minutes of 950 × g, inhales and abandon supernatant;
(6) add 1mLCS1 working fluid, piping and druming mixes and is added in new 2mL centrifuge tube, magnetic separating 2 minutes.Transfer supernatant, in 1.5mL centrifuge tube, is placed on 15mL centrifuge tube, centrifugal 3 minutes of 3400rpm.
(7) supernatant is abandoned in suction, adds CF1 stationary liquid, and piping and druming mixing also smear, dries naturally;
(8) CF2 steady job liquid is added, timing 8-10 minute;
(9) inhale and abandon liquid, put into 2 × SSC dye vat 10 minutes of preheating;
(10) put into 75% successively, 85%, dehydrated alcohol 2-5 minute;
(11) add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C of 4-20 hour;
(12) take out sample, mounting material of tearing, puts into the methane amide working fluid of preheating, timing 15 minutes;
(13) 2 × SSC 5-10 minute is placed in;
(14) take out, add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reaction 1-5 hour;
(15) surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting.The present invention also provides another for the test kit two of the inventive method, comprises following composition:
Wherein, 10 ×, 20 × indicated concentration, namely this solution is the concentration of 10 times/20 times.
Test kit two comprises the reagent of following discriminating part.
Differentiate part:
CF1 stationary liquid:
Mixing PEG and dehydrated alcohol, make PEG final concentration be 1%, dehydrated alcohol final concentration is 50%.
10 × CF2 stationary liquid:
Take PBS as solvent, dissolve PFA powder, be formulated as the PFA concentrated solution of 5%.
Methane amide working fluid:
By the working fluid that the dilution of methane amide stoste is 50%.
20XSSC concentrates damping fluid:
In 1000mL water, add sodium-chlor 175.3g, ammonium citrate 88.2g.
EGFR/CEP7 probe:
Use methane amide, dextran sulfate sodium salt by EGFR and CEP7, be formulated as probe face liquid.
BSA powder:
Outsourcing packing.
CD45-AF594 fluorescence antibody:
CD45 antibody and AlexaFlour594 fluorescein lucifuge are hatched 1h, and fluorescein can be connected with CD45 antibody.
DAPI staining fluid:
Use anti-quencher Mountingmedium the DAPI stoste of 1mg/mL to be diluted according to 1:1000, be formulated as DAPI dyeing working fluid.
The using method of described test kit two, step is as follows:
(1) by the cell suspension that present method step (7) or additive method obtain, be applied on slide glass or diaphragm, and naturally dry.Add CF2 steady job liquid, timing 8-10 minute;
(2) inhale and abandon liquid, put into 2 × SSC dye vat 10 minutes of preheating;
(3) put into 75% successively, 85%, dehydrated alcohol 2-5 minute;
(4) add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C of 4-20 hour;
(5) take out sample, mounting material of tearing, puts into the methane amide working fluid of preheating, timing 15 minutes;
(6) 2 × SSC 5-10 minute is placed in;
(7) take out, add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reaction 1-5 hour;
(8) surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting.
Microscope can be utilized after mounting to carry out the differentiation of result, can differentiate CTC or other rare cells, obtain the number of EGFR amplification type CTC, the number of chromosome amplification type CTC, the overall number of EGFR/CEP7-CTC, EGFR gene amplification state simultaneously.
Note: in treating processes, visual practical situation, use the punching agent such as TritonX-100, Saponin (but being not limited to these two kinds punching agent) to carry out punching process, to reduce the resistance that probe enters cell to cell.
The present invention further provides the purposes of test kit of the present invention, namely at discriminating CTC simultaneously, this test kit can also measure EGFR/CEP7 result, for this reason, the invention reside in and provide test kit of the present invention at discriminating CTC simultaneously, EGFR/CEP7 result can also be measured to judge the application in the gene appearance that tumour patient is real-time.
Explanation of nouns:
Rare cell: the ratio of occupying in its all karyocytes in body fluid sample is less than 0.1%.Comprise CTC,
Circulating endothelial cells, fetal cell, tumor stem cell, stem cell, and some immunocyte etc.
Circulating tumor cell (CirculatingTumorCell, CTC): refer to the tumour cell entering into blood circulation.
Noun in form:
Embodiment
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1
Use the detection of test kit a pair blood sample
As shown in above-mentioned test kit using method 1-6 step, test 40 routine normal peoples, 40 routine lung benign diseases, 20 routine blood of patients with lung cancer samples, method, for be added in 50mL centrifuge tube by 3.2mL blood preparation, adds CS1 working fluid, centrifugal 5 minutes of 650 × g, inhales and abandons supernatant; Add CS2 working fluid cracking 8 minutes, centrifugal 5 minutes of 650 × g, inhale and abandon supernatant fluid; Again add a certain amount of CS1 working fluid, add magnetic particle suspension 200uL, shake up 20 minutes; Draw the rear liquid of all mixings, be superimposed on CS3 separating medium, centrifugal 5 minutes of 300 × g; The liquid of absorption except magnetic particle precipitation, in 15mL centrifuge tube, adds CS1 working fluid to 15mL, centrifugal 5 minutes of 950 × g, inhales and abandon supernatant; Add 1mLCS1 working fluid, piping and druming mixes and is added in new 2mL centrifuge tube, magnetic separating 2 minutes.Transfer supernatant, in 1.5mL centrifuge tube, is placed on 15mL centrifuge tube, centrifugal 3 minutes of 3400rpm.Supernatant is abandoned in suction, adds CF1 stationary liquid, and piping and druming mixing also smear, dries naturally; Add CF2 steady job liquid 8 minutes; Liquid is abandoned in suction, puts into 2 × SSC dye vat 10 minutes of preheating; Put into 75% successively, 85%, dehydrated alcohol dehydration 2 minutes; Add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C 4 hours; Take out sample, throw off cover glass, put into the methane amide working fluid 15 minutes of preheating; Be placed in 2 × SSC washing 5 minutes; Add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reacts 1 hour; Surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting.
Result shows, 7 routine EGFR amplification type CTC, median 4.5/3.2mL (scope 2-29) in pernicious patient, and all without detecting in normal people and optimum patient.
Embodiment 2
The preparation of test kit of the present invention
Test kit one:
CS1 concentrates damping fluid (10 ×):
In every 1000mL water, containing 60gBSA, 5 bags PBS powder (2L/ bag), the EDTA of 100mL0.5M, 0.8mLProclin300.
CS2 concentrates storage liquid (10 ×):
In every 1000mL water, take 82.9gNH
4cl, 10gKHCO
3, 0.37gEDTA, water and 0.8mLProclin300, carry out sufficient stirring and dissolving, constant volume, is formulated as 10X concentrated solution.
CS3 separating medium:
Be the gradient centrifugation liquid dilution of 1.077 by density, test density in dilution, makes its density between 1.070-1.075.
Magnetic particle suspension:
CD45 antibody concentration is adjusted to 1mg/mL, carries out hatching 1h according to the ratio of 100uL:1mL with streptavidin immunomagnetic beads, be namely formulated as magnetic particle suspension.
CF1 stationary liquid:
Mixing PEG and dehydrated alcohol, make PEG final concentration be 1%, dehydrated alcohol final concentration is 50%.
10 × CF2 stationary liquid:
Take PBS as solvent, dissolve PFA powder, be formulated as the PFA concentrated solution of 5%.
Methane amide working fluid:
By the working fluid that the dilution of methane amide stoste is 50%.
20XSSC concentrates damping fluid:
In 1000mL water, add sodium-chlor 175.3g, ammonium citrate 88.2g.
EGFR/CEP7 probe:
Use methane amide, dextran sulfate sodium salt by EGFR and CEP7, be formulated as probe face liquid.
BSA powder:
Outsourcing packing.
CD45-AF594 fluorescence antibody:
CD45 antibody and AlexaFlour594 fluorescein lucifuge are hatched 1h, and fluorescein can be connected with CD45 antibody.
DAPI staining fluid:
Use anti-quencher Mountingmedium the DAPI stoste of 1mg/mL to be diluted according to 1:1000, be formulated as DAPI dyeing working fluid.
Test kit two only comprises 5)-12) part, preparation method is identical.
Embodiment 3
Detect with test kit of the present invention a pair other body fluid sample
Illustrate: this test kit is not only applicable to blood, be also applicable to the detection of rare cell in other body fluid, such as hydrothorax, ascites, washing liquid, amniotic fluid etc., but be not limited to this several body fluid.
Use this test kit to detect 13 routine cells in pleural fluids from lung cancer cases, all find CEP7 amplification type positive cell, positive rate 100%.Wherein 4 examples find EGFR amplification type positive cell, and quantity is respectively: 15,27,29,57.
Embodiment 4
The CTC that test kit in this test kit two pairs of additive methods get detects
Illustrate: the component in test kit two, namely EGFR/CEP7 probe, CD45-AF594 fluorescence antibody, DAPI staining fluid etc., be also applicable to the CTC that additive method gets, such as filtration method, ChIP method etc., but be not limited to this two kinds of methods.
Using membrane filter method to process 7 routine peripheral blood of lung cancer patients samples, is the Ispore of 8um by diameter
tMmembraneFilters coordinate SWINNEX filter with the use of.Concrete grammar is, first 5mL blood preparation and 10mLPBS is mixed, is added in strainer, and by action of gravity natural subsidence, CTC is comparatively large because of volume, stays film surface, is transferred on standard glass slide by filter membrane.After drying, carry out dyeing process according to the working method of test kit two to CTC, method is as follows: by dried sample through CF2 steady job liquid 8 minutes; Liquid is abandoned in suction, puts into 2 × SSC dye vat 10 minutes of preheating; Put into 75% successively, 85%, dehydrated alcohol dehydration 2 minutes; Add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C 4 hours; Take out sample, throw off cover glass, put into the methane amide working fluid 15 minutes of preheating; Be placed in 2 × SSC washing 5 minutes; Add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reacts 1 hour; Surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting, and at fluorescence microscopy Microscopic observation.
Result is added up, and it is positive that 2 examples show CEP7 amplification type CTC, and 2 routine EGFR amplification types are positive.
Claims (10)
1. detect a method for EGFR/CEP7 gene appearance based on rare cell, it is characterized in that, said method comprising the steps of:
Step 1, obtains blood sample from patient or other contain the body fluid sample of rare cell, comprises CTC or other rare cells and leukocytic mixed cellularity group in sample;
Step 2, uses damping fluid diluted sample, centrifugal segregation blood plasma, and reclaims CTC or other rare cells;
Step 3, uses erythrocyte cracked liquid cracking, and centrifugal segregation red corpuscle also reclaims CTC or other rare cells;
Step 4, with the recovery cell blending incubation of magnetic particle with step 3 gained being coated with anti-human white corpuscle associated antigen, fully to combine with target white corpuscle to make magnetic particle and forms magnetic particle-white corpuscle mixture;
Step 5, centrifugal by magnetic particle-white corpuscle mixture and other cellular segregation, obtain containing CTC or other rare cells and a small amount of leukocytic mixed cellularity group;
Step 6, uses Immunofluorescence and the method combined of fluorescence in situ hybridization, measures the state of EGFR/CEP7, carry out the discriminating of CTC or other rare cells simultaneously.
2. method according to claim 1, is characterized in that, damping fluid described in step 2 is damping fluid that is close with density of blood, that comprise BSA and PBS, PH7-8, and wherein, erythrocyte cracked liquid described in step 3 is any one erythrocyte cracked liquid.
3. method according to claim 1, is characterized in that, the magnetic particle being coated with anti-human white corpuscle associated antigen described in step 4 be a kind of bag by the streptavidin magnesphere of anti-leukocyte common antigen (LCA) antibody, can securely in conjunction with white corpuscle.
4. method according to claim 1, is characterized in that, centrifugation medium described in step 5 is the density gradient centrifugation liquid of a kind of density between 1.070-1.080.
5. method according to claim 1, is characterized in that, uses the method for Immunofluorescence and fluorescence in situ hybridization described in step 6, and measure EGFR/CEP7, differentiate CTC or other rare cells, method is as follows:
(1) EGFR amplification type CTC: observe under sample being placed in fluorescent microscope respective channel, if find multiple EGFR signaling point distribution (>=3), and the signal that number is greater than CEP7 is counted, and without CD45 antigen presentation, then count a CTC positive cell, and be directly EGFR positive cell depending on this cell;
(2) chromosome amplification type CTC: count single karyocyte CEP7 signal number, if be more than or equal to 3 signaling points and without CD45 antigen presentation, then count a CTC positive cell;
(3) EGFR/CEP7-CTC overall number: record all EGFR amplification types and chromosome amplification type CTC number, summation is considered as EGFR/CEP7-CTC overall number;
(4) EGFR gene amplification state recording: if there is obvious and typical EGFR to increase in all specimen area, be then designated as EGFR amplification; If nothing, then by the result of EGFR and CEP7 number of signals ratio in CEP7 number of signals, average each nucleus in EGFR number of signals, average each nucleus in each nucleus average in all CTC cells.
6., for a test kit for claim 1 method, it is characterized in that comprising following composition:
CS1 concentrates damping fluid (10 ×)
CS2 concentrates storage liquid (10 ×)
CS3 separating medium
Magnetic particle suspension
CF1 stationary liquid
10 × CF2 stationary liquid
DAPI staining fluid
EGFR/CEP7 probe
CD45-AF594 fluorescence antibody
Methane amide working fluid
20 × SSC concentrates damping fluid
BSA。
7. the using method of test kit described in claim 6, is characterized in that, step is as follows:
(1) 0.1-5mL blood preparation is added in 50mL centrifuge tube, adds the CS1 working fluid that diluted to 45mL, centrifugal 5 minutes of 650 × g, inhale and abandon supernatant and remain about 12mL liquid;
(2) jog mixing, adds the CS2 cracking working fluid that dilute to 45mL, mixes centrifugal 5 minutes of 8-10 minute, 650 × g, and supernatant fluid is abandoned in suction;
(3) add the mixing of CS1 working fluid appropriate also jog, again add a certain amount of CS1 working fluid, add cleaned magnetic particle suspension 200uL, level shakes up (120rpm) 20 minutes;
(4) draw the rear liquid of all mixings, be superimposed on CS3 separating medium, centrifugal 5 minutes of 300 × g;
(5) the upper two-layer liquid of absorption except magnetic particle precipitation is in 15mL centrifuge tube, adds CS1 working fluid to 15mL, centrifugal 5 minutes of 950 × g, inhales and abandon supernatant;
(6) add 1mLCS1 working fluid, piping and druming mixes and is added in new 2mL centrifuge tube, magnetic separating 2 minutes.Transfer supernatant, in 1.5mL centrifuge tube, is placed on 15mL centrifuge tube, centrifugal 3 minutes of 3400rpm;
(7) supernatant is abandoned in suction, adds CF1 stationary liquid, and piping and druming mixing also smear, dries naturally;
(8) CF2 steady job liquid is added, timing 8-10 minute;
(9) inhale and abandon liquid, put into 2 × SSC dye vat 10 minutes of preheating;
(10) put into 75% successively, 85%, dehydrated alcohol 2-5 minute;
(11) add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C of 4-20 hour;
(12) take out sample, mounting material of tearing, puts into the methane amide working fluid of preheating, timing 15 minutes;
(13) 2 × SSC 5-10 minute is placed in;
(14) take out, add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reaction 1-5 hour;
(15) surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting.
8. for a test kit for claim 1 method, it is characterized in that, comprise following composition:
CF1 stationary liquid
10 × CF2 stationary liquid
DAPI staining fluid
EGFR/CEP7 probe
CD45-AF594 fluorescence antibody
Methane amide working fluid
20 × SSC concentrates damping fluid
BSA。
9. the using method of test kit described in claim 8, is characterized in that, step is as follows:
(1) by the cell suspension that present method step (7) or additive method obtain, be applied on slide glass or diaphragm, and naturally dry, add CF2 steady job liquid, timing 8-10 minute;
(2) inhale and abandon liquid, put into 2 × SSC dye vat 10 minutes of preheating;
(3) put into 75% successively, 85%, dehydrated alcohol 2-5 minute;
(4) add 10uLEGFR/CEP7 probe, covered, mounting also puts into hybridization instrument or other temperature regulating devices, 76 DEG C 5 minutes, 37 DEG C of 4-20 hour;
(5) take out sample, mounting material of tearing, puts into the methane amide working fluid of preheating, timing 15 minutes;
(6) 2 × SSC 5-10 minute is placed in;
(7) take out, add the CD45-AF594 fluorescence antibody (20uL antibody+180uL2%BSA) prepared, every slide 200uL, lucifuge reaction 1-5 hour;
(8) surplus liquid is abandoned in suction, and 0.2%BSA cleaning 1-3 time, adds DAPI counterstain 10uL, mounting.
10. claim 6 or 8 any one test kit is while mensuration rare cell, detects the application of EGFR/CEP7 gene appearance.
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| 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 |
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| CN102586189A (en) * | 2012-02-02 | 2012-07-18 | 厦门艾德生物医药科技有限公司 | Method for enriching specificity of circulating tumor 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 |
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