CN212066756U - Device for in-vivo capturing fetal eukaryotic red blood cells from maternal body - Google Patents
Device for in-vivo capturing fetal eukaryotic red blood cells from maternal body Download PDFInfo
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- CN212066756U CN212066756U CN201922100583.6U CN201922100583U CN212066756U CN 212066756 U CN212066756 U CN 212066756U CN 201922100583 U CN201922100583 U CN 201922100583U CN 212066756 U CN212066756 U CN 212066756U
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Abstract
The utility model discloses a device of catching foetus eukaryotic red blood cell from parent in-situ, the device includes push-pull device, empty section of thick bamboo container, puncture head and polymer seal wire, empty section of thick bamboo container both ends opening, puncture head is connected to one end, and push-pull device is placed to the other end, push-pull device and the inner wall sealing contact of empty section of thick bamboo container to can slide along the inner wall of empty section of thick bamboo container, the polymer seal wire passes inside the puncture head to connect push-pull device's bottom. Adopt the utility model discloses the device can realize making the contact of short time with maternal peripheral blood, just can catch the rare foetal nucleated erythrocyte of quantity to can catch foetal nucleated erythrocyte in vivo, normal position and catch, avoided a large amount of extractions maternal peripheral blood, and use low efficiency methods such as centrifugation to enrich, separate foetal nucleated erythrocyte because of consuming time long, spend high scheduling problem, the method is with low costs, weak point consuming time, catches efficiently.
Description
Technical Field
The utility model relates to a device for capturing fetal eukaryotic red blood cells from a mother body in situ, which belongs to the technical field of medical instruments.
Background
Cells with the quantity lower than 100 per milliliter of blood are called rare cells, and a plurality of types of rare cells have important research values, wherein Circulating Tumor Cells (CTCs) and fetal Nucleated Red Blood Cells (NRBCs) have wide prospects in clinical diagnosis and treatment and are widely researched. The fetal nucleated red blood cells contain complete genetic information of the fetus, are convenient for genetic and molecular biological analysis, and are richer in maternal peripheral blood in the early pregnancy than other fetal cells. Therefore, NRBCs have important research significance in the field of prenatal diagnosis of fetuses, and whether the fetuses have congenital malformation or genetic diseases, such as Down syndrome, gene copy number variation and the like, can be diagnosed by detecting NRBCs genes.
Currently, in order to separate and enrich NRBCs, a widely used method is to separate blood from a human body, and to separate NRBCs based on a physical method or an immunoaffinity method by utilizing the difference in size, density, deformability, and cell surface expression of specific antigens, etc., between the NRBCs and normal blood cells. Such as Nagy et al (naggy G R, BAN Z, SIPOS F, et al, isolation of epsilon-halo-chain positive magnetic cells with micro-manipulation for preattal di agnosis [ J ]. preattal di agnosis,2005,25(5): 398-. The patent document with the Chinese patent application publication number of CN108795685A discloses a method for capturing and releasing fetal nucleated red blood cells by utilizing a microfluidic chip, the patent utilizes the microfluidic chip containing a micro-channel and a specific target substance to realize the specific capture of the fetal nucleated red blood cells, and further utilizes optical cutting to cut off a connecting arm connected with the target cells to realize the release of the target cells; the patent document with the publication number of CN108535228A of Chinese invention patent application utilizes the difference of fetal nucleated erythrocyte volume and surface antigen, combines the micro-fluidic technology and the immunoaffinity effect through a microarray structure chip with specific geometric arrangement, and realizes the capture and enrichment of fetal cells with different sizes; the patent document with the Chinese patent application publication number of CN105063181A discloses a method for enriching NRBCs by a density gradient centrifugation method, adding the NRBCs on a substrate chip modified with antibodies for standing and capturing, and then identifying a fetus Y chromosome by a FISH probe to separate fetus nucleated red blood cells from the peripheral circulating blood of a pregnant woman for noninvasive prenatal diagnosis; the patent document with the Chinese patent application publication number of CN105420086A discloses a single cell positioning microporous membrane, application and a single cell automatic acquisition device, wherein the device disperses single cells in a positioning microporous filter membrane, and opens the corresponding filter membrane after automatic identification by a fluorescence microscope so that the cells fall into a collection hole to achieve the purpose of separation; the Chinese patent application publication No. CN101310025A discloses a system and method for enriching analytes, which increases the concentration of target cells by 10000 times by forming a microarray enrichment analysis system instrument; chinese patent application publication nos. CN102884170A and CN107604039A disclose a micro-fluidic sorter for detecting and separating cells, which realizes the separation of NRBCs from other cells by the difference in cell deformability in a linear or spiral narrow flow channel in the micro-fluidic sorter; the patent document with the Chinese patent application publication number of CN108546676A discloses a method for separating and enriching rare cells in peripheral blood by a tunnel magnetic field technology, wherein magnetic beads and target cell cells are connected in cell incubation through antibody-antigen interaction, and then the enrichment and separation are realized by flowing through a tunnel magnetic field; the patent document with the Chinese patent application publication number of CN106119189A discloses a separation and purification method of fetal nucleated red blood cells and a kit for separating and enriching NRBCs by centrifugation and magnetic beads marked by antibodies; the Chinese patent application publication No. CN103797368A discloses a method for detecting fetal nucleated red blood cells, which comprises forming a compound by a CD147 antibody and a blood sample containing NRBCs, and screening and detecting under a microscope after density gradient centrifugation and enrichment; the patent document with Chinese patent application publication number CN103103161A discloses a separation and purification method of fetal nucleated red blood cells and a Down syndrome screening kit, wherein magnetic beads marked by CD54 antibodies are combined with NRBCs and then separated by a magnetic separator; the patent document with the Chinese patent application publication number of CN107312748A discloses a method for sorting fetal nucleated red blood cells, wherein nano-microspheres are connected to NRBCs through antibody-antigen action so as to enlarge the density difference with other cells, and then are enriched by centrifugal separation; the patent document with Chinese patent application publication No. CN105043831A discloses a nano material for capturing nucleated red blood cells, wherein CD147 antibody is connected to a base film formed by spreading nanoparticles on a glass substrate through streptavidin, and the base film is contacted with NRBCs enriched by a gradient centrifugation method and identified by an immunofluorescence staining method; the patent document with the Chinese patent application publication number of CN104977284A discloses a method for capturing and identifying fetal nucleated red blood cells, which comprises the steps of firstly preparing a nano basement membrane for modifying an antibody and connecting biotin, enriching NRBCs by a gradient centrifugation method and then contacting the basement membrane, and identifying by a double-positive immunofluorescence staining method; chinese patent application publication No. CN108220453A discloses a noninvasive prenatal paternity test method and a kit thereof, wherein optimized technologies such as density gradient centrifugation, immunomagnetic bead negative selection, immunofluorescence, laser microdissection, single cell amplification and the like are combined together to form a system identification method, fetal nucleated red blood cells in the peripheral blood of a pregnant woman are successfully separated, and the fetal nucleated red blood cells are successfully applied to paternity test; the Chinese patent application publication No. CN107312706A discloses a biological reaction carrier for biological sample extraction, wherein carboxyl/amino is modified on the inner wall of a reaction cavity (sphere, hemisphere, groove, micro-channel, capillary), and then an antibody is connected to contact with a cell suspension to realize the extraction of NRBCs; the patent document with the Chinese patent application publication number of CN106488981A discloses a preparation method of fetal Nucleated Red Blood Cells (NRBCS) for diagnostic test, which comprises the steps of firstly obtaining cell fractions containing NRBCs by density gradient centrifugation and enrichment, further carrying out flow cytometry sorting or sorting by micromanipulation after carrying out fluorescence labeling after magnetic activated cell sorting; the patent document with the Chinese patent application publication number of CN103602632A discloses a technical scheme for separating fetal nucleated red blood cells from peripheral blood of a pregnant woman by a centrifugal separation method; the patent document with the Chinese invention patent application publication number of CN104662424A discloses a method for selectively capturing and releasing circulating cells through stimulation on a nano-structure device, wherein a nanowire array is constructed on a substrate, one end of a nanowire is fixed, the other end of the nanowire is connected with an antibody, a temperature-sensitive response material layer is arranged between the antibody and the nanowire, the temperature-sensitive response material layer has a compact structure at a first temperature to capture cells, and the temperature-sensitive response material layer is unfolded to release the captured cells at a second temperature; the patent document with the publication number of CN108753573A in the Chinese invention patent application discloses a method for capturing and identifying fetal nucleated red blood cells in a microfluidic chip, which enables nanoparticles to flow through the microfluidic chip so as to form a coating on the inner wall, modify streptavidin, and graft a biologically modified antibody for NRBCs sorting.
At present, most methods for separating and enriching NRBCs are in vitro capture, more peripheral blood needs to be extracted, in-situ capture in vivo is difficult to realize, the sample volume is small, and the blood volume effectively contacting the periphery of a parent body is small; furthermore, because NRBCs are present in less amounts in maternal peripheral blood (less than 100 per ml), the in vitro capture uncertainty is higher than that of in vivo in situ capture; furthermore, in vitro capture needs to be performed in a specific experimental site, and the captured cells are difficult to store or transport without damage, which is not favorable for downstream gene detection and analysis.
SUMMERY OF THE UTILITY MODEL
Utility model purpose: the main object of the utility model is to provide a device of catching foetal eukaryotic red blood cell from the maternal in situ, the device can realize the in situ of human rare cell and catch, obtains foetal nucleated red blood cell through subsequent elution step.
The technical scheme is as follows: in order to achieve the above purpose, the utility model adopts the following technical scheme:
the device comprises a push-pull device, an empty cylinder container, a puncture head and a polymer guide wire, wherein the two ends of the empty cylinder container are open, one end of the empty cylinder container is connected with the puncture head, the push-pull device is placed at the other end of the empty cylinder container, the push-pull device is in sealing contact with the inner wall of the empty cylinder container and can slide along the inner wall of the empty cylinder container, and the polymer guide wire penetrates through the interior of the puncture head and is connected with the bottom of the push-pull device.
Preferably, the push-pull device comprises a push-pull rod and a piston which are connected together, and the piston is in sealing contact with the inner wall of the empty cylinder container. The puncture head can be detached.
The push-pull device can also be designed in an integrated way, the hollow cylinder container can be in a cylindrical shape or a square cylinder shape, and the opening sizes of the two ends of the hollow cylinder container can be the same or different. The puncture head can be detached or can be integrally designed with the empty cartridge container.
Preferably, the polymer guide wire is made of a high molecular material, and a carboxyl group is connected to the surface of the polymer guide wire.
The high polymer material is selected from natural high polymer materials or artificially synthesized high polymer materials, and the natural high polymer materials are selected from cellulose or protein, preferably cellulose; the synthetic polymer material is selected from polyamide, polyester, polyurethane, polyacrylate or polyether amide, and is further selected from polyamide 6, polyamide 66, polyamide 610, polyamide 1010, polyamide 1212, polyethylene glycol terephthalate, polybutylene terephthalate, polylactic acid, lactic acid-glycolic acid copolyester, polycaprolactone, polyether polyurethane, polyester polyurethane, polyacrylate fibers obtained by copolymerizing methyl methacrylate and other acrylate monomers, or various polyether-polyamide copolymers, preferably polyamide 6, polyamide 66, polyamide 610, polyamide 1010 or polyethylene glycol terephthalate.
The polymer guide wire surface is connected with carboxyl groups by adopting a chemical grafting method, a surface coating method and the like, and preferably adopting a chemical grafting method. The carboxyl group is a carboxyl group with zwitterionic property, namely, the carboxyl group is connected to form an anionic group, and simultaneously, a quaternary ammonium group with cationic property is also connected to the same chemical structure, and the monomer for grafting also has an acrylate or methacrylate structure. A further preferred chemical structure is a carboxybetaine (meth) acrylate structure having the general formula wherein the preferred structure is R ═ CH3Carboxybetaine methacrylate (CBMA) of 1, n, and 1.
R＝H,CH3,n＝1,2,3,...,m＝1,2,3,...
Namely, the polymer guide wire is a high molecular material guide wire with the surface connected with Carboxyl Betaine Methacrylate (CBMA) oligomer.
Preferably, the push-pull device has a movable distance of 1-20cm, the empty cartridge container has a capacity of not more than 2mL, the puncture tip inner diameter is not less than 0.3mm, and the polymer guide wire has a diameter of 0.1-0.3mm and a length of 1-20 cm.
The method for capturing the fetal nucleated red blood cells in situ by using the device comprises the following steps:
1) sucking the solution containing the fetal nucleated red blood cell antibody into an empty cylinder container through a push-pull device, connecting the antibody to a polymer guide wire, and then discharging the solution;
2) the normal saline is sucked into the empty cylinder container through the push-pull device, so that the polymer guide wire is soaked in the normal saline, then the puncture head is inserted into a subcutaneous blood vessel, the normal saline is discharged through the push-pull device, so that the polymer guide wire is exposed in the blood, and the device is pulled out after the polymer guide wire is fully contacted with the blood;
3) and (4) taking down the polymer guide wire which is fully contacted with blood, and eluting to obtain the polymer guide wire.
The fetal nucleated red blood cell antibody includes but is not limited to CD34, CD71 or Hoechst33258, and the preferred antibody is CD 71. The antibody is linked before capturing the cells, without prior linking, thereby avoiding antibody failure during storage.
Preferably, the method of attaching the antibody to the polymeric guidewire (4) uses a carbodiimide/N-hydroxysuccinimide coupling reaction (EDC/NHS) attachment.
Preferably, the blood is contacted sufficiently for a period of time of 1 to 60 minutes, preferably 10 to 30 minutes.
The principle of the method available for elution in step 3) is a well-known method in cell biology, and the fetal nucleated red blood cells obtained by elution can be further used for genetic analysis and detection, so that all genetic information of a fetus can be obtained.
The principle of capturing fetal nucleated red blood cells using the device is as follows:
the polymer guide wire penetrates through the puncture head and is tightly connected with the push-pull device, and the guide wire can move back and forth through the movement of the push-pull device. When the push-pull device is arranged at the end closest to the puncture head, a part of the guide wire is exposed outside the device, and when the push-pull device is arranged at the end farthest from the puncture head, the guide wire is completely retracted in the device. When the guide wire is connected with the device, a complete device for capturing the fetal nucleated red blood cells from the mother in situ is obtained.
The utility model discloses the device, design benefit, wherein polymer seal wire nature is soft, can drift along with the blood flow in internal blood vessel, can not adhere to the vascular wall on to with more blood contact, realize and the specificity of rare nucleated erythrocyte catch. Furthermore, the utility model discloses the device can make the connection of antibody simple and easy, can let the antibody connect just realize through the alternate suction of several kinds of solutions before the puncture, lets the connection of apparatus and antibody (medicine) realize through simple operation on-the-spot. Therefore, the antibody which can be connected with the instrument and can be kept active only under special conditions (low temperature and short time) can be avoided during the manufacture of the instrument, and the inactivation of the antibody during later storage and transportation and use can be avoided.
The technical effects are as follows: compared with the prior art, adopt the utility model discloses the device can realize making the contact of short time with maternal peripheral blood, just can catch the rare foetus nucleated red blood cell of quantity for further genetic analysis detects etc. can learn foetal genetic information, thereby provides the basis for the screening of early genetic disease. Furthermore, utilize the utility model provides a device can catch foetal nucleated erythrocyte in physically, normal position and catch, has avoided a large amount of extractions maternal peripheral blood to use inefficiency methods such as centrifugation to enrich, separate foetal nucleated erythrocyte and therefore long, spend high grade problem, the method is with low costs, weak point consuming time, catches efficiently.
Drawings
FIG. 1: the utility model discloses the overall structure sketch map of foetus nucleated red blood cell device is caught to normal position.
FIG. 2: the embodiment 2 of the utility model provides an overall structure schematic diagram of fetus nucleated red blood cell device is caught to normal position, wherein: a is the state that the functionalized guide wire is protected in the device, B is the intermediate state of a part of the guide wire pushing device, and C is the state that the guide wire pushing device is exposed to the external environment.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A device for capturing fetal nucleated red blood cells in situ is shown in figure 1 and comprises a push-pull device 1, an empty cylinder container 2, a puncture head 3 and a polymer guide wire 4, wherein two ends of the empty cylinder container 2 are open, one end of the empty cylinder container is connected with the puncture head 3, the other end of the empty cylinder container is provided with the push-pull device 1, the push-pull device 1 is in sealing contact with the inner wall of the empty cylinder container 2 and can slide along the inner wall of the empty cylinder container 2, and the polymer guide wire 4 penetrates through the interior of the puncture head 3 and is connected with the bottom of the push-pull device 1.
The push-pull device 1 comprises a push-pull rod 1-1 and a piston 1-2 which are connected together, and the piston 1-2 is in sealing contact with the inner wall of the hollow cylinder container 2. The puncture head 3 is detachable.
The empty container 2 may be cylindrical or square-cylindrical, and the opening sizes at both ends may be the same or different.
The polymer guide wire 4 is made of high molecular material, and the surface of the polymer guide wire is connected with carboxyl groups.
The movable distance of the push-pull device 1 is 1-20cm, the empty cylinder container 2 has the capacity of not more than 2mL, the inner diameter of the puncture head 3 is not less than 0.3mm, and the polymer guide wire 4 has the diameter of 0.1-0.3mm and the length of 1-20 cm.
Example 2
As a specific embodiment of the present invention, in this embodiment, a device for capturing fetal nucleated red blood cells in situ made using a syringe or the like, as shown in fig. 2, comprises a push rod 1-1, a piston 1-2, an empty cylinder container 2, a needle 3 and a polymer guide wire 4 (corresponding to the push rod 1-1, the piston 1-2, the empty cylinder container 2, the puncture head 3 and the polymer guide wire 4 in the embodiment 1 respectively), the two ends of the hollow cylinder container 2 are open, one end is connected with a detachable needle 3, the other end is provided with a push rod 1-1 and a piston 1-2 which are connected together, the piston 1-2 is in sealing contact with the inner wall of the hollow cylinder container 2, and can slide along the inner wall of the hollow cylinder container 2 under the push and pull of the push rod 1-1, and the polymer guide wire 4 passes through the needle 3 and is connected with the bottom of the piston 1-2.
The back-and-forth movement distance of the push rod 1-1 is 1-20cm, the empty cylinder container 2 has a capacity of not more than 2mL, the inner diameter of the needle 3 is not less than 0.3mm, the polymer guide wire 4 has a diameter of 0.1-0.3mm and a length of 1-20cm, and the piston 1-2 is a soft piston.
The polymer guide wire 4 is connected with the CBMA oligomer on the surface thereof through chemical bonds, so that the surface of the guide wire has carboxyl.
The assembly process of the device of the utility model needs to be realized in an aseptic environment, and the combined device is finally used for the human body after the sterilization of the known sterilization method in the medical appliance industry.
Example 3
A method for capturing fetal nucleated red blood cells in situ by using a polymer guide wire comprises the following specific steps:
taking a nylon 6 guide wire with the diameter of 0.25mm and the length of 15cm, washing the nylon 6 guide wire, placing the nylon 6 guide wire into formic acid for soaking for 1 hour to activate the surface, initiating a grafting reaction by using a combined initiator of ammonium ceric nitrate and concentrated sulfuric acid, reacting the grafting reaction with a CBMA solution, and connecting a CBMA oligomer on the surface of the nylon guide wire to obtain the carboxylated nylon guide wire 5.
A1 mL syringe with a standard specification is provided with an injection needle 4 (inner diameter: 0.41mm) with an inner diameter of 22G, and the state shown in FIG. 2 is that the push rod 1-1 is pulled to draw the piston 1-2 out of the empty cylinder container 2 and separate from the push rod 1-1. A previously prepared carboxylated nylon guidewire 5 is taken through the needle 3 and the empty cartridge container 2. A small hole is pricked on the piston 1-2 by a needle, and the small hole passes through the guide wire 4 and is fixed by knotting. And (3) remounting the piston 1-2 on the push rod 1-1, loading the piston into the empty cylinder container 2, pushing the push rod 1-1 to be in a state C in the drawing, observing the state of the guide wire 4 to be normal, cutting off redundant guide wires according to the requirement, pulling the push rod 1-1 to be in a state A in the drawing, and finishing the assembly of the device. The length of the guidewire 4 in the final device is 12.5 cm.
Preparing a mixed solution of 1, 4-dioxane and sterile pure water, and fully dissolving EDC/NHS in the mixed solution to obtain EDC/NHS activated solution. 1mL of the prepared EDC/NHS activation solution was extracted with an assembled syringe, gently shaken at room temperature for 1 hour, and then the activation solution was pushed out and washed 3 times with physiological saline. Using the activated syringe to extract 1mL of CD71 antibody solution with specific concentration, and standing for reaction at 4 ℃ for 12 hours; after the injector connected with the antibody is washed by the physiological saline for 3 times, 1mL of physiological saline is extracted to store the nylon guide wire containing the antibody; after air in the injector is exhausted, the injector is gently inserted into the subcutaneous blood vessel along the telecentric direction by the needle head, the injector push rod is slowly pushed out to ensure that the nylon guide wire is exposed in the blood and fully contacts the peripheral blood along the blood flow direction, the push rod is pulled back to withdraw the guide wire into the device after the indwelling for 20min, and the guide wire sample capturing the fetal nucleated red blood cells is obtained by pulling out the injector.
The guide wire sample is washed, trypsinized and fixed on a glass slide, the fetal nucleated red blood cells are confirmed to be obtained by capture through fluorescence labeled in situ hybridization (FISH) detection, and the green fluorescence signal of the X chromosome and the red fluorescence signal of the Y chromosome can be seen in cells, so that the cells are proved to be from the baby boy, namely the captured cells are cells of the fetus but not cells of the mother body.
Claims (7)
1. The device for in-vivo capturing of fetal eukaryotic blood cells from a mother body is characterized by comprising a push-pull device (1), an empty tube container (2), a puncture head (3) and a polymer guide wire (4), wherein the empty tube container (2) is open at two ends, one end of the empty tube container is connected with the puncture head (3), the other end of the empty tube container is provided with the push-pull device (1), the push-pull device (1) is in sealing contact with the inner wall of the empty tube container (2) and can slide along the inner wall of the empty tube container (2), and the polymer guide wire (4) penetrates through the interior of the puncture head (3) and is connected with the bottom of the push-pull device (1).
2. The device for in vivo capture of fetal eukaryotic blood cells from a mother according to claim 1, wherein said push-pull device (1) comprises a push-pull rod (1-1) and a piston (1-2) connected together, the piston (1-2) being in sealing contact with the inner wall of the empty cartridge container (2).
3. The device for in situ capture of foetal eukaryotic red blood cells from a maternal body according to claim 1, characterised in that said empty cartridge container (2) has a cylindrical or square cylindrical shape with the same or different size of the openings at the two ends.
4. The device for in vivo capture of fetal eukaryotic blood cells from a mother according to claim 1, wherein the piercing head (3) is detachable or integrated with the empty cartridge container (2).
5. The device for in situ capture of fetal eukaryotic blood cells from a maternal host as claimed in claim 1 wherein said polymeric guidewire (4) is made of a polymer material and has a surface attached with a carboxyl group.
6. The device for in situ capture of fetal eukaryotic blood cells from a mother according to claim 1, wherein the polymer guide wire (4) is a high molecular material guide wire with surface attached Carboxyl Betaine Methacrylate (CBMA) oligomer.
7. The device for in vivo capture of fetal eukaryotic blood cells from a mother according to claim 1 wherein the push-pull device (1) has a movable distance of 1-20cm, the empty cartridge container (2) has a capacity of no more than 2mL, the puncture head (3) has an inner diameter of no less than 0.3mm, the polymeric guidewire (4) has a diameter of 0.1-0.3mm and a length of 1-20 cm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201922100583.6U CN212066756U (en) | 2019-11-29 | 2019-11-29 | Device for in-vivo capturing fetal eukaryotic red blood cells from maternal body |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201922100583.6U CN212066756U (en) | 2019-11-29 | 2019-11-29 | Device for in-vivo capturing fetal eukaryotic red blood cells from maternal body |
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