CN206396221U - The equipment that a kind of high flux fast Acquisition purifies circulating tumor cell - Google Patents
The equipment that a kind of high flux fast Acquisition purifies circulating tumor cell Download PDFInfo
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- CN206396221U CN206396221U CN201621375772.4U CN201621375772U CN206396221U CN 206396221 U CN206396221 U CN 206396221U CN 201621375772 U CN201621375772 U CN 201621375772U CN 206396221 U CN206396221 U CN 206396221U
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Abstract
The utility model provides the equipment that a kind of high flux fast Acquisition purifies circulating tumor cell, belongs to biological technical field.The high flux fast Acquisition purifies the device of circulating tumor cell, including：Filtering module；The filtering module includes filter, is arranged on filter interior and filter membrane, negative pressure device with filter inner wall sealing；The negative pressure device is connected to the exit end of the filter, for making the inside of the filter keep negative pressure；The filter membrane is used for when the negative pressure device makes the inside of the filter keep negative pressure, circulating tumor cell is retained, and the reagent being automatically added to via the reagent sample introduction module is dyed and cleaned, sample liquid is input to filter and negative pressure device and can automatically controlled by the device, and then cause the process of membrane retention and dye cycle tumour cell to have automaticity high, high flux (increase number of filters is that can be achieved) and other effects.
Description
Technical field
The utility model is related to biological technical field, swollen in particular to a kind of purification circulation of high flux fast Acquisition
The device and method of oncocyte.
Background technology
Circulating tumor cell (CTC, Circulating Tumor Cell) is that all kinds of tumours being present in peripheral blood are thin
The general designation of born of the same parents.In the evolution of tumour, because spontaneous or operation of diagnosis and treatment is de- from entity tumor focus (primary tumor, transfer stove)
Fall, most of CTC occurs apoptosis or swallowed after peripheral blood is entered, and minority, which can escape and anchor, develops into transfer stove,
Increase malignant tumor patient mortality risk, played an important role in the invasion and attack transfer of tumour.
Circulating tumor cell is shedded into the circulatory system, and many results of study have confirmed its diagnosis in metastases
There is potential value with terms of prognosis, medicament research and development, individualized treatment and its exploration mechanism of tumor metastasis.Therefore, special,
Sensitively detection circulating tumor cell is extremely important, can not only more accurately assess the prognosis of tumor patient, also help
Formulate personalized therapy program.
According to the approach of Nasopharyngeal neoplasms, the tumour cell circulated in blood or lymphatic vessel is defined as circulating tumor
Cell (circulating tumor cell, CTC), and if the cell mass of wherein stem cell recruitment formation is referred to as circulation and swollen
The micro- bolt of knurl (circulating tumor mocroemboli, CTM).CTM is tumour cell " collective migration " behavior, because of it
Apoptosis can be resisted, ability of cell proliferation is kept and there is higher metastatic potential.However, circulating tumor is thin in peripheral blood
The number of born of the same parents is less (1 CTC/106~107 monocyte), is precisely separated highly difficult.
CellSearch systems, which are that current FDA is the only approved, is used for the technical equipment of clinical enrichment and detection and analysis, is one
Semi-automated techniques are planted, have gathered the separation detection technique of immunological magnetic bead sorting technology and immunocytochemical method.It is marked with anti-
The magnetic bead of EpCAM antibody is combined with target cell, is retained under additional magnetic fields, then using fluorescence labeling (CK8/
18/19, DAPI, CD45) distinguish CTC and haemocyte, then using semi-automatic fluorescence microscope detection and analysis cell size and
Form, finally determines CTC.
But, the method complex operation, cost is very high, and flux is low, limits its answering when clinical Large-scale Screening is detected
With.Further, since there is a number of Non-cancerous epithelial cell (circulation epithelial cell) in peripheral blood, most of evils are added
Property circulating tumor cell due to epithelial-mesenchymal change, so as to lost epithelium antigen EpCAM, thus can not be detected.Therefore
When CTC is counted for assessing tumour to the reaction for the treatment of, tumor recurrence risk and tumor screening, these problems just seem special
It is unimportant.Further, since multistep cell marking and processing can make cell mass discrete, so that immunomagnetic isolation method can not
For detecting the higher CTM that shifts risk.
Another CTC enrichment method uses density gradient separation principle, and the density of the various cells in blood is not
Together, centrifuge, make certain close using a kind of specific density and after being bordering on isotonic solution (layering liquid) and the same test tube of sample addition
The cell of degree is distributed by corresponding density gradient, so that various cells in blood be separated.Cell dye can be carried out after separation
Color, immune labeled etc. are further analyzed.The OncoQuick methods (German Greiner companies) set up on this basis,
With a kind of special 50mL test tube, built-in porous barrier, it is density gradient separation liquid that it is lower, and sample is placed in into barrier when using
On so as to avoiding mix with separating liquid before centrifugation, compared with preceding (CellSearch systems), be more easy to divide from granulocyte
Tumour cell is separated out, so as to be more beneficial for further analysis.
The method is limited in that because Partial tumors cell can adjourn to plasma layer or stay in red blood cell or neutrophil leucocyte
In, the loss of the tumour cell in separation process is not only easily caused, and if blood anticoagulant is incomplete, micro clots then may be used
Tumour cell can be made to drop down onto the bottom of Graded Density liquid.Therefore the method sensitiveness it is relatively low and dependent on tumour cell characteristic, from
The many factors such as time, the temperature of the heart.
Vona in 2000 etc. reports a kind of according to cell size, passes through the side of filtering directly enrichment epithelial tumour cell
Method (isolationby size of epithelial tumor cells, ISET).The method can make less lymphocyte and
Neutrophil leucocyte passes through, and larger tumour cell is then arrested on film.This method is not only simple to operate, and relatively quicker
Sense, can avoid loaded down with trivial details multi-step from separating destruction and the loss of the rare cells caused, and the CTM with high metastatic potential also can
It is enriched with and is counted.The cell being enriched to can carry out cytology dyeing, or pass through immune labeled, FISH
The method such as (fluorescence in situ hybridization, FISH), TUNEL analyzes its antigen, aneuploidy and thin
Born of the same parents' apoptosis etc..But, the current fado is operated using laboratory hand, and automaticity is low, and flux is low, and error is big, and easily
Cause cross pollution, be especially susceptible to interfere in follow-up molecular Biological Detection.In addition, long-term manually operated to laboratory technician
Health also result in the threat of potentiality.
In summary, the method for purification circulating tumor cell common in presently relevant technology there is complex operation, it is automatic
Change degree is low, flux is low and sensitiveness is relatively low (many factors such as characteristic, time, the temperature of centrifugation dependent on tumour cell)
Etc. technical problem.
In view of this, it is special to propose the utility model.
Utility model content
First purpose of the present utility model is to provide the device that a kind of high flux fast Acquisition purifies circulating tumor cell,
The device is used for fast Acquisition and purifies circulating tumor cell, with automaticity height, high flux, is difficult to cause cross pollution etc.
Effect.
In order to realize above-mentioned purpose of the present utility model, spy uses following technical scheme：
The utility model provides the device that a kind of high flux fast Acquisition purifies circulating tumor cell：The high flux is fast
The device of speed capture purification circulating tumor cell includes：Filtering module；
The filtering module includes filter, is arranged on filter interior and the filter membrane with filter inner wall sealing, negative pressure
Device；The negative pressure device is connected to the exit end of the filter, for making the inside of the filter keep negative pressure；Institute
Stating filter membrane is used to, when the negative pressure device makes the inside of the filter keep negative pressure, circulating tumor cell be retained.
This high flux fast Acquisition that the utility model is provided purifies the device of circulating tumor cell, and that includes filtering
Module, and in the filtering module, include filter, be arranged on filter interior and the filter membrane with filter inner wall sealing and
It is connected to the exit end negative pressure device of the filter；During capture purification circulating tumor cell, in advance by sample
Liquid is input to filter interior, and negative pressure device can cause filter interior formation negative pressure, and cause sample liquid to pass through filter membrane,
Discharged from the port of export of filter.During sample liquid passes through filter membrane, the circulating tumor cell contained by sample liquid is due to it
Volume is big compared with other blood cells, and then can be by membrane retention, it is achieved thereby that high flux fast Acquisition purification circulating tumor is thin
The effect of born of the same parents；It can be automatically controlled due to sample liquid is input into filter and negative pressure device, therefore membrane retention is circulated
The process of tumour cell has automaticity high, high flux (increase number of filters is that can be achieved) and other effects.And at the same time
When operating multiple filters, the pipeline that each filter and filtered solution are flowed through is independent pipeline, and no cross contamination, filtered solution can
It is collected separately for other analyses or is directly discharged into the waste collecting device.
Optionally, the device of the high flux fast Acquisition purification circulating tumor cell also includes：Reagent sample introduction module and
First negative pressure pump；
The reagent sample introduction module is connected with the filter, and first negative pressure pump is arranged on the reagent and enters original mold
On the connecting pipeline of block and the filter.
After the sample liquid filtering in filter, typically the circulating tumor cell being trapped on filter membrane can all be carried out
The operations such as cleaning, dyeing, to meet the requirement of subsequent experimental analysis.Therefore, in order to further improve cleaning and dyeing
Automaticity, has been preferably provided with reagent sample introduction module and the first negative pressure pump.In use, in the presence of the first negative pressure pump, examination
Reagent in agent sample introduction module smoothly can be imported into filter, so as to be highly convenient for realizing the cleaning of sample liquid, dyeing
Operation.
Optionally, the filter is multiple, and on the connecting pipeline of all filters and first negative pressure pump
It is provided with a reagent distributor.
In order to realize the effect of batch processing sample liquid, filter preferably to be multiple, and all filters with
A reagent distributor is provided with the connecting pipeline of first negative pressure pump；Original mold can will be entered from reagent by reagent distributor
The reagent of block input is assigned in multiple filters.It is more highly preferred to, multiple filters (generally 8-10) can pass through one
Individual fixed support is realized and is wholely set, and forms the filter of one group of fixation.When treating capacity is larger, multigroup mistake can be set parallel
Filter.
For the structure of filter, it is preferred that filter includes body, sealing ring, can place the support of filter membrane, and filter membrane is set
Put on filter holder, sealing ring causes filter membrane and filter holder, the inner wall sealing of body.
Optionally, the reagent sample introduction module includes cleaning fluid sample storage bottle, dyeing liquor sample storage bottle and preserves liquid sample storage bottle；
The cleaning fluid sample storage bottle, the dyeing liquor sample storage bottle and the preservation liquid sample storage bottle are connected by a multichannel
Head is connected with first negative pressure pump.
Generally, for the circulating tumor cell being trapped after filtering, it can be cleaned, be dyed and Seal and preservation more
If the operation of (not using immediately), therefore, for reagent sample introduction module, it preferably includes cleaning fluid sample storage bottle, dyeing liquor storage
Sample bottle and preservation liquid sample storage bottle；And cleaning fluid sample storage bottle, dyeing liquor sample storage bottle and preservation liquid sample storage bottle are connected by a multichannel
Joint (being easy to multiple sample storage bottles being connected with reagent distributor) is connected with the first negative pressure pump.
Optionally, the cleaning fluid sample storage bottle, the dyeing liquor sample storage bottle and the preservation liquid sample storage bottle lead to more with described
Channel valve is respectively arranged with the connecting pipeline of road connector.
The flow direction of multiple sample storage bottle interior reagents can be easily controlled by multiple channel valves, be further increasing
Automaticity.
Optionally, in addition to waste collecting device and the second negative pressure pump；The outlet of the filter and the waste collection
Device is connected；And overfall is additionally provided with the tube wall of the filter, the overfall passes through with the waste collecting device
Overflow pipe is connected；Second negative pressure pump is arranged on the overflow pipe.
Because the outlet of filter is connected with the waste collecting device, therefore sample liquid after filtering and from sample storage
Bottle outflow reagent can be collected using after finishing by waste collecting device.It is additionally, since overflow pipe and the second negative pressure pump is (more excellent to be
Peristaltic pump, vavuum pump) setting, operate start when, start the second negative pressure pump, can actively prevent due to filter membrane block caused by
Sample liquid and reagent solution overflow, so that pollution when avoiding high flux operation to greatest extent between sample, while having avoided
Harmful effect of the malicious harmful substance to instrument and operating personnel.
Optionally, in addition to programmable logic controller；
The programmable logic controller and all channel valves, the negative pressure device, first negative pressure
Pump, second negative pressure pump, the multichannel connector and the reagent distributor are connected, and for controlling the channel valve
Door, the negative pressure device, first negative pressure pump, second negative pressure pump, the multichannel connector and the reagent point
The open/close state of orchestration.
The channel valve, the negative pressure device, first negative pressure pump, institute are controlled by programmable logic controller
The open/close state of the second negative pressure pump, the multichannel connector and the reagent distributor is stated, be further increasing whole
Automaticity in circulating tumor cell acquisition procedure, reduces the manually operated error that may result in and reagent can be right
Human body causes the defect of potential hazard.
Optionally, the filter membrane is high molecular polymerization microporous barrier, and its aperture is 5-10 μm.
The port of export of negative pressure device is connected with for filter, the filtration head of taper is preferably provided with, each filter
Filtration head is all connected by independent filtered solution delivery pipe filtered solution collecting pipe or with waste collecting device, if filtered solution is collected
In filtered solution collecting pipe, due between each sample and per being independent operation between batch sample, same batch is avoided to greatest extent
Filtered solution can still be used in cross pollution between sample between different batches sample, therefore filtered solution collecting pipe carries out other realities
Test analysis.In addition, be preferably transparent or semitransparent high molecular polymerization microporous barrier for filter membrane, or black non-fluorescent background
High molecular polymerization microporous barrier, the average pore size of microporous barrier is between 5-10 μm, the flowing of sample liquid or reagent in filter,
Negative pressure is formed by negative pressure device (more excellent is peristaltic pump, vavuum pump, plunger pump, syringe pump) to realize, and 5-10 μm of high score
Sub- polymeric micro-porous film can realize good circulating tumor cell retention.
Optionally, the dyeing liquor sample storage bottle on the connecting pipeline of the reagent distributor with being provided with particulate filter；
The particulate filter is between the channel valve on the dyeing liquor sample storage bottle and the connecting pipeline.
Optionally, the negative pressure device is any one in peristaltic pump, vavuum pump, plunger pump and syringe pump.
The method for purifying circulating tumor cell using the device of described high flux fast Acquisition purification circulating tumor cell,
Comprise the following steps：
Blood sample is delivered in filter, starting negative pressure device makes the filter formation negative pressure, and makes blood sample
During this passes through filter membrane, contained tumour cell is trapped on filter membrane.
It is preferred that, this method specifically includes following steps：
1) after, sample liquid is fixed, add into filter, start negative pressure device so that sample liquid is through filter membrane, and institute
The circulating tumor cell contained is trapped；
2), the circulating tumor cell being trapped within filter membrane is cleaned using phosphate buffer successively, dyeing liquor is utilized
Dyeing, using phosphate buffer clean and using preserve liquid preserve, produce, wherein, dyeing liquor be DAPI, AO, PI,
Any one in hoechst, syb green, fluorescent labeled antibody, Yihong methylene blue and Rui Shi-Ji's nurse Sa.
This method realizes the retention and dyeing of circulating tumor cell by way of negative pressure leaching, and whole operation is simply easy
OK, controllability is strong.
Compared with prior art, the beneficial effects of the utility model are：
(1), the device overall structure link up it is compact, be fast and effeciently capture purification circulating tumor cell provide protect
Circulating tumor cell height automation capture can be achieved in barrier, the structural advantage based on the device.
(2) separation, cleaning and the dyeing of circulating tumor cell, can be achieved by automatically controlling whole device and preserve
Deng operation, overcoming whole acquisition procedure in the prior art needs a large amount of manual hand manipulations and that may be present to experimenter
Potential hazard and the low defect of capture rate.
(3), due to the particular design of filter, pipeline and negative pressure device so that between each sample and per between batch sample
It is independent operation, the cross pollution between different batches sample between same batch sample, therefore filtered solution is avoided to greatest extent
Again after collecting, can still be used for carries out other experimental analyses, has greatly widened the application of equipment so that 1 pipe sample enters
The multinomial detection and analysis of row is possibly realized.
(4), the combination of the apparatus and method, overcomes the behaviour of manual capture circulating tumor cell common in the art
The had flux of work is low, error is big, and easily causes cross pollution, is particularly easy to make in follow-up molecular Biological Detection
Into the defect of interference.
Brief description of the drawings
, below will be to embodiment in order to illustrate more clearly of the utility model embodiment or technical scheme of the prior art
Or the accompanying drawing used required in description of the prior art is briefly described.
The structural representation of the device for the high flux fast Acquisition purification circulating tumor cell that Fig. 1 provides for the utility model
Reference：
0- reagent sample storage bottles, 1- reagent sample introduction modules, 2- filtering modules, the anti-spilled module of 3- actives, 4- automatically controls mould
Block, 5- waste collecting devices；01 is cleaning fluid sample storage bottle, and 02 is dyeing liquor sample storage bottle, and 03 is preserves liquid sample storage bottle, and 0X is to expand
Reagent bottle is filled, 111-11X is reagent delivery pipe, and 121-12X is channel valve, and 13 be multichannel connector, and 14 be the first negative pressure
Pump, 15 reagent distributors；21 be filter fixed support, and 22 be filter, and 23 be filter membrane, 24 filtered solution delivery pipes, and 25 be negative
Pressure device, 26 be filtered solution delivery pipe clips, and 27 be sealing-plug, and 221 be body, and 223 be sealing ring, and 224 be that can place filter
The support of film, 225 be filtration head, and 222 be overfall, and 31 be overflow pipe, and 32 be the second negative pressure pump, and 41 be programmable logic control
Device processed, 42 be control panel；
Fig. 2 is the result figure for being obtained and being purified circulating tumor cell using equipment of the present utility model.
Embodiment
Embodiment of the present utility model is described in detail below in conjunction with embodiment, but those skilled in the art
It will be understood that, the following example is merely to illustrate the utility model, and is not construed as limiting scope of the present utility model.Embodiment
In unreceipted actual conditions person, the condition advised according to normal condition or manufacturer carries out.Agents useful for same or the unreceipted system of instrument
Standby manufacturer person, is the conventional products that can be obtained by commercially available purchase.
Fig. 1 is refer to, this high flux fast Acquisition that the utility model is provided purifies the device of circulating tumor cell, bag
Include filtering module 2；Filtering module 2 include filter 22, be arranged on the inside of filter 22 and with the filter of the inner wall sealing of filter 22
Film 23, negative pressure device 25；Negative pressure device 25 is connected to the exit end of filter 22, and the inside for making filter 22 keeps negative
Pressure；Filter membrane 23 is used to, when negative pressure device 25 makes the inside of filter 22 keep negative pressure, circulating tumor cell be retained.
In more preferred scheme, the device of high flux fast Acquisition purification circulating tumor cell also includes：Reagent
The negative pressure pump 14 of sample introduction module 1 and first；Reagent sample introduction module 1 is connected with filter 22, and the first negative pressure pump 14 is arranged on reagent
On the connecting pipeline of sample introduction module 1 and filter 22.Filter 22 is multiple, and the negative pressure pump 14 of all filters 22 and first
A reagent distributor 15 is provided with connecting pipeline.
In order to further improve the automaticity of cleaning and dyeing after filtering to the circulating cells of retention, preferably
There is provided the negative pressure pump 14 of reagent sample introduction module 1 and first.In use, in the presence of the first negative pressure pump 14, reagent sample introduction module 1
In reagent can smoothly be imported into filter 22, so as to be highly convenient for realizing cleaning, the dying operation of sample liquid.In order to
Realize the effect of batch processing sample liquid, filter 22 is preferably to be multiple, and the negative pressure pump 14 of all filters 22 and first
Connecting pipeline on be provided with a reagent distributor 15；It will can be inputted by reagent distributor 15 from reagent sample introduction module 1
Reagent is assigned in multiple filters 22.
Above-mentioned more preferred scheme basis on, it is preferred that reagent sample introduction module 1 include cleaning fluid sample storage bottle 01,
Dyeing liquor sample storage bottle 02 and preservation liquid sample storage bottle 03；
Cleaning fluid sample storage bottle 01, dyeing liquor sample storage bottle 02 and preserve liquid sample storage bottle 03 by a multichannel connector 13 with
First negative pressure pump 14 is connected.Cleaning fluid sample storage bottle 01, dyeing liquor sample storage bottle 02 and preservation liquid sample storage bottle 03 and reagent distributor 15
Connecting pipeline on be respectively arranged with channel valve.It can easily control to try inside multiple sample storage bottles by multiple channel valves
The flow direction of agent, further increasing the automaticity of device.
On the basis of the above method, it is more highly preferred to, the high flux fast Acquisition purifies the dress of circulating tumor cell
Put and further comprise the negative pressure pump 32 of waste collecting device 5 and second；The outlet of filter 22 is connected with waste collecting device 5；And
Overfall 222 is additionally provided with the tube wall of filter 22, overfall 222 is connected with waste collecting device 5 by overflow pipe 31；The
Two negative pressure pumps 32 are arranged on overflow pipe 31.
So, after filtering sample liquid and from sample storage bottle outflow reagent using can be by waste collecting device after finishing
5 collect or are collected into again in filtered solution collecting pipe.It is additionally, since the negative pressure pump 32 of overflow pipe 31 and second (more excellent to be compacted
Dynamic pump, vavuum pump) setting, operate start when, start the second negative pressure pump 32, can actively prevent to cause because filter membrane 23 is blocked
Sample liquid and reagent solution overflow so that avoid to greatest extent high flux operation when sample between pollution, avoid simultaneously
Venomous injurant confrontation instrument and the harmful effect of operating personnel.
In addition, in order to realize further Automated condtrol, on the basis of above-mentioned highly preferred scheme, preferably
, also set up programmable logic controller 41；Programmable logic controller and all channel valves, negative pressure device 25,
First negative pressure pump 14, the second negative pressure pump 32, multichannel connector 13 and reagent distributor 15 are connected, and for control passage valve
Door, negative pressure device 25, the first negative pressure pump 14, the second negative pressure pump 32, the ON/OFF of multichannel connector 13 and reagent distributor 15
State.Being preferably provided with based on more than, further increasing the automaticity in whole circulating tumor cell acquisition procedure,
The defect of potential hazard can be caused to experimenter by reducing the manually operated error that may result in and reagent.
In addition, in above-mentioned all schemes, in order to realize a large amount of retentions to circulating weight cell, it is preferred that filter membrane
For high molecular polymerization microporous barrier, (more specifically, it is polycarbonate membrane or polyester film；More excellent aperture production model is etching method),
And its aperture is 5-10 μm.
Fig. 1 is refer to, in another technical scheme of the present utility model, high flux fast Acquisition purification circulating tumor is thin
Born of the same parents' equipment includes：Reagent sample storage bottle 0, reagent sample introduction module 1, filtering module 2, actively anti-spilled module 3, automatic control module 4,
Waste collecting device 5；
Reagent sample storage bottle 0 is made up of multiple reagent bottles, respectively to stored sample processing, dyeing, cleaning, fixed, preservation
When the reagent that needs；(but being not limited only to this) shown in accompanying drawing 1, wherein, 01 is cleaning fluid sample storage bottle, and 02 is dyeing liquor sample storage
Bottle, 03 is preserves liquid sample storage bottle, and 0X is extendible reagent bottle (is, for example, fixer sample storage bottle, preserves liquid sample storage bottle etc.), can root
Need to increase some related reagent bottles (at most can expand to 10 sample storage bottles) according to dyeing is extracted.
Reagent sample introduction module 1 includes：Reagent delivery pipe the 111-11X, -12X of channel valve 121, multichannel connector 13,
One negative pressure pump 14, reagent distributor 15.Reagent delivery pipe 111-11X has a plurality of, to connect reagent sample storage bottle 0 and filter
- the 12X of channel valve 121 is respectively arranged with 22, and every reagent delivery pipe, can be square by multiple -12X of channel valve 121
Just the flow direction of multiple reagent sample storage bottle (01-0X) interior reagents is controlled, automaticity is further increasing.
In use, in the presence of the first negative pressure pump 14, the reagent in reagent sample storage bottle (01-0X) can be smooth in order
Be directed respectively into single or multiple filters 22, so as to automatically realize the cleaning of sample liquid, dying operation.Due to from
Sample liquid is filtered to retain circulating tumor cell, to the circulating tumor cell that various reagents are inputted to multiple 22 pairs of retentions of filter
All steps for being cleaned, fixed, being dyed can be automatically controlled, therefore the full mistake of membrane retention dye cycle tumour cell
Journey has automaticity high, high flux (increase number of filters is that can be achieved) and other effects.
Reagent sample introduction module 1 is connected with reagent sample storage bottle (01-0X), and every kind of reagent is from separate agent delivery pipe (111-
11X), reagent distributor 1.5 is entered by multichannel connector 13；It is (more excellent for wriggling that reagent flows through the first negative pressure pump 14
Pump, plunger pump, vavuum pump, syringe pump) form negative pressure promotion reagent liquid flowing；On the separate agent transfer pipeline of every kind of reagent
Be equipped with channel valve (121-12X, more excellent be magnetic valve), to control by the reagent of the passage it is open and close when
Between；Reagent distributor 15 has positioning and distribution function, the reagent entered in distributor can be evenly distributed into filtering module 2
In multiple filters 22；
Particulate filter 1121 is provided with dyeing liquor passage 112, to filter formed in dyeing liquor due to crystallization
Grain thing, reduces the interference to stained cells to greatest extent；All channel valves, negative pressure pump, multichannel connector 13 and examination
Agent distributor 15 is connected with programmable logic controller 41, and by program control.
Filtering module 2 includes：Filter fixed support 21, filter 22, filter membrane 23, filtered solution delivery pipe 24, negative pressure dress
Put 25；8-10 filter 22 can be fixed on one set filter fixed support 21 simultaneously, many set filter fixed supports 21 pass through
Combining form is convenient to be increased；Filter 22 by body 221, sealing ring 223, can place filter membrane support 224, filter it is first 225 groups
Into；The body of filter 22 is provided with overfall 222, and the overfall 222 is connected with the anti-overflow flow module 3 of active, to prevent by
Sample caused by being blocked in filter membrane 23 and reagent solution overflow.
Body 221 and filter membrane 23, the support 224 that filter membrane can be placed, the first 225 assembling combination formation filter 22 of filtration；Sealing
Circle 223 is used to the space between sealed tube body 221 and filter membrane 23, the support 224 that can place filter membrane, is easy to follow-up negative-pressure operation；
The filtration first 225 of each filter 22 is connected with independent filtered solution delivery pipe 24, and same batch sample is avoided to greatest extent
Between cross pollution between different batches sample, filtered solution delivery pipe 24 is finally connected with waste collecting device 5, or with other filters
Liquid collecting pipe is crossed to be connected；Filter membrane 23 is transparent or semitransparent high molecular polymerization microporous barrier, or black non-fluorescent background
High molecular polymerization microporous barrier, the average pore size of microporous barrier is between 5-10 μm；Sample reagent is filtered and filtered solution flowing, by negative
Pressure device 25 (more excellent is peristaltic pump, vavuum pump) forms negative pressure and promotes reagent liquid flowing；Negative pressure device 25 is patrolled with programmable
Controller 41 is collected to be connected, and by program control.
Actively anti-spilled module 3 includes overflow pipe 31, the second negative pressure pump 32.The body 221 of overflow pipe 31 and filter 22
On overfall 222 be connected, be finally connected with waste collecting device 5；The second negative pressure pump 32 is connected on overflow passage (more excellent to be compacted
Dynamic pump, vavuum pump), start negative-pressure operation when sample operations start, actively prevent because filter membrane 23 blocks caused sample
Overflowed with reagent solution, so that pollution when avoiding high flux operation to greatest extent between sample, while avoiding poisonous and harmful
Harmful effect of the material to instrument and operating personnel；Second negative pressure pump 32 is connected with programmable logic controller 41, and is compiled
Journey programme-control.
Waste collecting device 5 and filtered solution delivery pipe 24, overflow pipe 31 are connected, built-in pathogenic microorganism inactivation reagent (compared with
Excellent is sodium hypochlorite, alcoholic solution etc.) to inactivate into the micro- life of cause of disease in the filtered solution or overflowing liquid of waste collecting device
Thing, so as to effectively be protected to operating environment and personnel.
Automatic control module 4 is to control reagent sample introduction module 1, filtering module 2, actively anti-spilled module 3 is programmable
Formula logic controller 41, programmable logic controller 4.1 can be connected with computer or control panel 42, to be pressed according to program
Order and each controller of time-controllable, or change operation sequence and time by specific requirement.
Next, with reference to the content of the above, purifying circulating tumor to utilization high flux fast Acquisition of the present utility model thin
The method of born of the same parents' equipment fast Acquisition circulating tumor cell enumerates specific examples below：
The method for this fast Acquisition circulating tumor cell that the utility model is provided specifically includes the steps：
1st, the filtering of blood sample (sample liquid)：
A) paraformaldehyde solutions of 3ml 4% are added in 2ml peripheral blood samples, are handled 10 minutes, in human peripheral blood
Cell is fixed, while being inactivated to possible pathogenic microorganism.
B) disposable or be added portionwise in the filter 22 assembled, the liquid level added every time in after-filter 2.2 should be low
In the bottom of the overfall 222 of filter 22；
C) start negative pressure device 25, and continuous service 10-15 minutes, blood sample is completely extended across filter under suction function
Film 23, enables circulating tumor cell to be trapped in film surface by microporous barrier.
In this step, because the diameter of circulating tumor cell is more than red blood cell and most blood karyocytes, because
This, circulating tumor cell is able to be trapped in the surface of filter membrane 23 by microporous barrier, and red blood cell and blood karyocyte then pass through micropore
Film eventually enters into waste collecting device 5 by independent filtered solution delivery pipe 24, and inactivates reagent by filtration by pathogenic microorganism
Pathogenic microorganism inactivation that may be present in liquid, or filtered solution is collected into other filtered solution collecting pipes, for other detections point
Analysis.Fixer can be any reagent for having fixed cell sample to act on, more excellent for aldehydes fixers such as paraformaldehyde, formaldehyde；Or
Person, it is more excellent for methanol, ethanol and without aldehydes fixer.
2nd, on microporous barrier circulating tumor cell cleaning：
A) after the completion of blood sample filtration step, the second negative pressure pump 32 (being always on thereafter) is started immediately, while the
One negative pressure pump 14；
B) cleaning fluid channel valve 121 is opened, cleaning fluid phosphate buffer (pH7.4) is acted in the first negative pressure pump 14
Under, enter cleaning fluid conveying pipeline road 111 from cleaning fluid sample storage bottle 01, cleaning fluid is again by multichannel connector 13 and reagent distribution
Device 15 is evenly distributed in multiple filters 22；
C) after the first negative pressure pump 14 is opened 1-2 minutes, negative pressure device 25 is again turned on, makes the cleaning fluid in filter 22
Through filter membrane, the cleaning action to filter membrane 23 and the cell retained thereon is played.
Wherein, in above-mentioned step, a length of 5 minutes during the unlatching of the negative pressure pump 14 of cleaning fluid channel valve 121 and first,
Subsequent cleaning fluid channel valve 121 is closed, and the first negative pressure pump 14 stops；And negative pressure device 25 must be opened, when a length of 10-15 point
Clock, until all cleaning fluids emptying in filter 22.
In addition, cleaning fluid is in addition to selection phosphate buffer (pH7.4), can also be physiological saline, 5% glucose solution
Deng.
3rd, on filter membrane circulating tumor cell dyeing：
A) after the completion of cleaning step, suspend negative pressure device 25, be again turned on the first negative pressure pump 14, while opening dyeing liquor
Channel valve 122；
B) dyeing liquor is Rui Shi-Ji's nurse Sa dyeing liquor (dyeing liquor classification is selected according to actual conditions) in the first negative pressure
Under pump 14 is acted on, enter dyeing liquor conveyance conduit 112 from dyeing liquor sample storage bottle 02, particulate filter 1121 is then passed through, through many
Passage connector 13 and reagent distributor 15 are evenly distributed in multiple filters 22；
C) after negative pressure device 25 suspends 15-20 minutes, negative pressure device 25 is again turned on, makes the dyeing liquor in filter 22
Through filter membrane 23, play a part of to the cell dyeing on filter membrane 23, until all dyeing liquors emptying in filter 22.
In above-mentioned steps, a length of 5 minutes during the unlatching of the negative pressure pump 14 of dyeing liquor channel valve 122 and first, with poststaining
Liquid channel valve 122 is closed, and the first negative pressure pump 14 stops；And negative pressure device 25 continues to stop after the stopping of the first negative pressure pump 14
15-20 minutes, subsequent negative pressure device 25 was opened, when it is a length of 1-2 minutes, until filter 22 in all dyeing liquors emptying.
It is noted that except Rui Shi-Ji's nurse Sa dyeing liquor, the dyeing liquor in the step can also be nucleus and/or
Cytoplasm fluorescent staining liquid, more excellent is the dyeing liquors such as DAPI, AO, PI, hoechst, syb green, can also be various eucaryons
Cell dyeing liquid, e.g., Yihong methylene blue liquid.
4th, circulating tumor cell fluorescent labeled antibody dyeing (optional) on filter membrane
To further discriminate between leucocyte and circulating tumor cell on filter membrane, the CD45 antibody of fluorescence labeling is can select to filter membrane
On circulating tumor cell carry out negative choosing dyeing.
A) before step 3 or after the completion of, suspend negative pressure device 25, be again turned on the first negative pressure pump 14, at the same open it is glimmering
Signal CD45 antibody staining liquid channel valves 12X；
B) fluorescence labeling CD45 antibody stainings liquid is under the effect of the first negative pressure pump 14, from fluorescence labeling CD45 antibody staining liquid
Sample storage bottle 0X enters dyeing liquor conveyance conduit 11X, and multiple mistakes are evenly distributed to through multichannel connector 13 and reagent distributor 15
In filter 22；
C) after negative pressure device 25 suspends 60-90 minutes, negative pressure device 25 is again turned on, makes the fluorescence mark in filter 22
Remember that CD45 antibody stainings liquid passes through filter membrane 23, play a part of marking dyeing to the cell antibody on filter membrane 23, until filter
All dyeing liquors emptying in 22.
In above-mentioned steps, a length of 1 minute during the unlatching of the negative pressure pump 14 of dyeing liquor channel valve 122 and first, with poststaining
Liquid channel valve 12X is closed, and the first negative pressure pump 14 stops；Negative pressure device 25 continues to stop 60- after the stopping of the first negative pressure pump 14
It is 90 minutes, later on, when it is a length of 1-2 minutes, until filter 22 in all dyeing liquors emptying.
It is noted that except fluorescence labeling CD45 antibody staining liquid, the fluorescent labeled antibody dyeing liquor of the step may be used also
To be selected according to tissue characteristics to be analyzed or cancer cell-types.
5th, cleaning step after dyeing：
The concrete operations of the step are consistent with step 2, and wash number is twice, therefore not to repeat here.
6th, cell preserves step (optional) on film：
If a) stained cells are not immediately used to subsequent analysis, to ensure eucaryotic cell structure, coloration result and intracellular nucleic
The stabilization of acid, can use this step；
B) after dyeing after the completion of cleaning step, suspend negative pressure device 25, be again turned on the first negative pressure pump 14, open simultaneously
Preserve liquid channel valve (marked as 123 in figure)；
C) liquid is preserved under the effect of the first negative pressure pump 14, is entered preservation liquid conveyance conduit 113 from liquid sample storage bottle 03 is preserved, is protected
Liquid storage is evenly distributed in multiple filters 22 by multichannel connector 13 and reagent distributor 15；
D) after preservation liquid enters filter 22 and can cover filter membrane 23, all negative pressure pumps and negative pressure device are closed, is used
Filtered solution delivery pipe clips 26 and overflow pipe clips 33 are closed, after closing, and whole filter 22 is removed, in filtering
Filled at the top of device after sealing-plug 27, filter 22 is placed in 4-25 DEG C of preservation；
E) in above-mentioned steps, it can be any water-soluble or ethanol mountant to preserve liquid, more excellent for anti-fluorescent quenching envelope
Solution, polyvinyl alcohol, polyvinyl alcohol and water that piece liquid, glycerine, glycerine are mixed with water or other buffer solutions by different proportion or its
Solution that his buffer solution is mixed by different proportion etc.；
F) cell after being preserved using this step, before subsequent counter and purification analysis is carried out, preferably again using clear
Wash step 1 time, cleaning fluid used can be permeabilization buffer or the ethanol such as the neutrality given in cleaning step, and liquid is preserved to rear to reduce
It is continuous to analyze the possibility interfered.
7th, circulating tumor cell is counted and purification step on film：
A) in order to carry out analysis of accounts to circulating tumor cell, the filter membrane 23 after dyeing is taken out from filter 22, put down
It is layered on clean slide or lining form, is then placed in digital pathology scanning device, filter membrane is swept from suitable wavelength
Retouch, due to existing on the karyomorphism of nucleated blood cell in the circulating tumor cell and blood after dyeing and surface fluorescence mark
Very big difference, can conveniently be distinguished, therefore can carry out analysis of accounts to the result after scanning；
B) filter membrane is placed on lining form after the end of scan, then circulating tumor cell is used under laser microprobe dating instrument and swashed
Light is cut down, and is collected respectively in different centrifuge tubes, for follow-up molecular biological analysis；
C) for the suspicious nucleated blood cell around circulating tumor cell, to prevent it to the circulating tumor cell after cutting
Molecular biological analysis interfere (nucleated blood cell be very easy to circulating tumor cell molecular biological analysis is caused significantly
Interference), innovative punctures technology, destruction suspicious cells and nucleic acid molecules using single beam high power laser before cutting, maximum
Limit prevents non-specific amplification and interference.
Test example
Multiple sample liquid are handled simultaneously to the method that circulating tumor cell is purified with above-mentioned high flux fast Acquisition, determined
The rejection and collimation of circulating tumor cell, concrete operation method are as follows：
S1：1000 HeLa Cells of scheduled volume are added in 2ml phosphate buffers, then by this practicality
Machine is filtered in novel operating method, while determining 10 identical samples；
S2：Take pictures counting, cell retention are carried out to cell on film with digital pathology system and analysis of accounts software after filtering
Rate=(count results/1000 on film) × 100%.
As a result (referring to table 1) is shown, multiple samples can be handled using the utility model apparatus and method, cell
It is good that rejection is above collimation between 75%, and sample.
Table 1：The rejection and collimation of tumour cell when handling multiple samples simultaneously
| Filter number | 1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# |
| Cell retention rate | 78.2% | 75.9% | 79.3% | 80.6% | 76.7% | 76.1% | 78.9% | 79.1% | 81.3% | 75.1% |
In addition, in fig. 2 it is shown that the result of circulating tumor cell is obtained and purified using equipment of the present utility model：
The part irised out by black curve is the micro- bolt of the circulating tumor being trapped on filter membrane；The small figure in right side is big using single beam before cutting
Power laser punctures technology and destroyed after the suspicious nucleated blood cell in periphery, the micro- bolt of circulating tumor purified with microdissection technology；
As seen in Figure 2, the device for this purification circulating tumor cell that the utility model is provided is realized to circulating tumor cell
Preferable rejection effect.
To sum up, the device that the utility model is provided, during sample liquid passes through filter membrane, the circulation contained by sample liquid
Tumour cell is big compared with other blood cells due to its volume, and then can realize the purification of high flux fast Acquisition by membrane retention
The effect of circulating tumor cell；It can be automatically controlled due to sample liquid is input into filter and negative pressure device, therefore filter
The process of film retention circulating tumor cell has automaticity high, high flux (can be achieved by increasing number of filters) etc.
Effect.
Finally it should be noted that：Various embodiments above is only limited to illustrate the technical solution of the utility model, rather than to it
System；Although the utility model is described in detail with reference to foregoing embodiments, one of ordinary skill in the art should
Work as understanding：It can still modify to the technical scheme described in foregoing embodiments, or to which part or complete
Portion's technical characteristic carries out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from this practicality
The scope of new each embodiment technical scheme.
Claims (10)
1. a kind of high flux fast Acquisition purifies the device of circulating tumor cell, it is characterised in that the high flux fast Acquisition
The device of purification circulating tumor cell includes：Filtering module；
The filtering module includes filter, is arranged on filter interior and filter membrane, negative pressure device with filter inner wall sealing；
The negative pressure device is connected to the exit end of the filter, for making the inside of the filter keep negative pressure；The filter
Film is used to, when the negative pressure device makes the inside of the filter keep negative pressure, circulating tumor cell be retained.
2. high flux fast Acquisition according to claim 1 purifies the device of circulating tumor cell, it is characterised in that described
The device of high flux fast Acquisition purification circulating tumor cell also includes：Reagent sample introduction module and the first negative pressure pump；
The reagent sample introduction module is connected with the filter, and first negative pressure pump be arranged on the reagent sample introduction module with
On the connecting pipeline of the filter.
3. high flux fast Acquisition according to claim 2 purifies the device of circulating tumor cell, it is characterised in that described
Filter is multiple, and all filters on the connecting pipeline of first negative pressure pump with being provided with a reagent distributor.
4. high flux fast Acquisition according to claim 3 purifies the device of circulating tumor cell, it is characterised in that described
Reagent sample introduction module includes cleaning fluid sample storage bottle, dyeing liquor sample storage bottle and preserves liquid sample storage bottle；
The cleaning fluid sample storage bottle, the dyeing liquor sample storage bottle and the preservation liquid sample storage bottle by a multichannel connector with
The first negative pressure pump connection.
5. high flux fast Acquisition according to claim 4 purifies the device of circulating tumor cell, it is characterised in that described
On the connecting pipeline of cleaning fluid sample storage bottle, the dyeing liquor sample storage bottle and the preservation liquid sample storage bottle and the multichannel connector
It is respectively arranged with channel valve.
6. high flux fast Acquisition according to claim 5 purifies the device of circulating tumor cell, it is characterised in that also wrap
Include waste collecting device and the second negative pressure pump；
The outlet of the filter is connected with the waste collecting device；And it is additionally provided with overflow on the tube wall of the filter
Mouthful, the overfall is connected with the waste collecting device by overflow pipe；Second negative pressure pump is arranged on the overflow pipe
On.
7. high flux fast Acquisition according to claim 6 purifies the device of circulating tumor cell, it is characterised in that also wrap
Include programmable logic controller；
The programmable logic controller and all channel valves, the negative pressure device, first negative pressure pump, institute
The second negative pressure pump, the multichannel connector and the reagent distributor is stated to be connected, and for controlling the channel valve, institute
State negative pressure device, first negative pressure pump, second negative pressure pump, the multichannel connector and the reagent distributor
Open/close state.
8. the high flux fast Acquisition according to claim any one of 1-6 purifies the device of circulating tumor cell, its feature
It is, the filter membrane is high molecular polymerization microporous barrier, and its aperture is 5-10 μm.
9. high flux fast Acquisition according to claim 5 purifies the device of circulating tumor cell, it is characterised in that described
Dyeing liquor sample storage bottle on the connecting pipeline of the reagent distributor with being provided with particulate filter；
The particulate filter is between the channel valve on the dyeing liquor sample storage bottle and the connecting pipeline.
10. high flux fast Acquisition according to claim 5 purifies the device of circulating tumor cell, it is characterised in that institute
It is any one in peristaltic pump, vavuum pump, plunger pump and syringe pump to state negative pressure device.
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| CN201621107073 | 2016-10-09 | ||
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018064933A1 (en) * | 2016-10-09 | 2018-04-12 | 陈静 | Apparatus for high-throughput rapid trapping of circulating tumor cells and method for purifying circulating tumor cells |
| CN108126522A (en) * | 2017-12-21 | 2018-06-08 | 深圳汇芯生物医疗科技有限公司 | The method of target particles in separating chips, separator and separation liquid sample |
| CN112608820A (en) * | 2020-12-15 | 2021-04-06 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
-
2016
- 2016-12-15 CN CN201621375772.4U patent/CN206396221U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018064933A1 (en) * | 2016-10-09 | 2018-04-12 | 陈静 | Apparatus for high-throughput rapid trapping of circulating tumor cells and method for purifying circulating tumor cells |
| CN108126522A (en) * | 2017-12-21 | 2018-06-08 | 深圳汇芯生物医疗科技有限公司 | The method of target particles in separating chips, separator and separation liquid sample |
| CN112608820A (en) * | 2020-12-15 | 2021-04-06 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
| CN112608820B (en) * | 2020-12-15 | 2022-04-15 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
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