CN211026389U - A centrifuging tube for circulating tumor cell enrichment - Google Patents
A centrifuging tube for circulating tumor cell enrichment Download PDFInfo
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- CN211026389U CN211026389U CN201921695573.5U CN201921695573U CN211026389U CN 211026389 U CN211026389 U CN 211026389U CN 201921695573 U CN201921695573 U CN 201921695573U CN 211026389 U CN211026389 U CN 211026389U
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Abstract
the utility model discloses a centrifuging tube for enrichment of circulating tumor cell, including one go up body, a filter core connecting seat, a filter membrane, a sealing washer and body once, utilize the utility model discloses only need through the centrifugation, can follow effectual enrichment to circulating tumor cell in the blood, do not need special instrument, the operation is quick, simple and convenient, according to adaptation centrifuge's configuration, 8-16 samples can be handled to a centrifugation, and the processing flux is high, and every sample can handle the liquid that reaches 10m L capacity as much.
Description
Technical Field
The utility model particularly relates to a centrifuging tube for circulating tumor cell enrichment.
Background
Cancer metastasis is the leading cause of death in most cancer patients, and an important way for cancer metastasis is through Circulating Tumor Cells (CTCs) in the blood. Circulating tumor cells are a general term for various tumor cells existing in peripheral blood, mainly fall off from solid tumor foci (primary foci and metastatic foci) into blood due to spontaneous or diagnosis and treatment operations, are closely related to distant metastasis and recurrence of tumors and disease progression, and are first discovered by Thomas Ashworth in 1869. Unlike tissue blood detection, blood detection has the advantages of easy acquisition, small wound, repeated performance and easy acceptance by patients, and currently, blood circulation tumor cell detection plays an important role in early tumor discovery, prognosis judgment, recurrence monitoring, drug resistance monitoring, curative effect evaluation, personalized medication guidance and the like.
However, because the number of CTCs in peripheral blood is very small, patients with advanced, metastatic tumors also have only 1-10 CTCs per ml of peripheral blood, about 400-1000 million leukocytes, and about 50 hundred million erythrocytes, it is critical to enrich them for detection of CTCs. The current CTC enrichment methods are mainly divided into two main categories, physical methods and biochemical methods. The first type is an affinity-based biochemical method, which generally relies on the combination of an antigen on the surface of a cell membrane and an antibody coupled on a separation medium to achieve the purpose of separating and capturing CTC, the purity of the CTC enriched by the method is high, but the capture sensitivity is low due to the heterogeneity of the CTC, insufficient antibody affinity and the like, and in addition, the combination of the antigen and the antibody can trigger the change of a signal path network inside a cell, so that the reliability of downstream analysis is seriously challenged, and the application of the enrichment method is limited. The second type is a physical method, which separates CTCs from blood cells by density gradient centrifugation, filtration, microfluidics, electrophoresis and other means based on differences in physical properties, such as size, deformability, density, dielectricity and the like, between tumor cells and normal blood cells.
The existing CTC enrichment method based on filtration needs special instruments, is expensive, or needs complex pretreatment on body fluid, and is complex to operate, or only can process one or two samples each time, the flux is not high, or each sample can only process a small amount of liquid, and the capacity is not high.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide a centrifuging tube for circulating tumor cell enrichment.
The utility model discloses a concrete technical scheme as follows:
A centrifuge tube for the enrichment of circulating tumor cells, comprising:
An upper tube body having an upper opening and a lower opening, the upper opening having an upper cover;
The filter element connecting seat is provided with a central through hole, the size of the central through hole is matched with the lower opening of the upper tube body, and the edge of the central through hole extends upwards to form an annular support;
A filter membrane;
A seal ring;
And a lower tube body having an upper opening;
The lower extreme adaptation of going up the body connects the upper end of filter core connecting seat, and the under shed of going up the body passes through the sealed intercommunication of sealing washer and above-mentioned central through-hole, and the filter membrane clamp is located between the under shed and the cyclic annular support of body, and the lower extreme of filter core connecting seat is connected to the upper shed adaptation of body down.
In a preferred embodiment of the present invention, the lower opening of the upper pipe body is smaller than the upper opening of the upper pipe body.
In a preferred embodiment of the invention, the pore size of the filter membrane is 5-10 μm.
in a preferred embodiment of the present invention, the upper pipe body has a volume of 1 to 20m L.
in a preferred embodiment of the present invention, the lower pipe body has a volume of 1 to 20m L.
The utility model discloses an in a preferred embodiment, the lower extreme of upper pipe body links to each other through threaded connection structure, buckle connection structure or cup joint structure with the upper end of filter core connecting seat.
In a preferred embodiment of the present invention, the upper opening of the lower tube body is connected to the lower end of the filter element connecting seat through a screw connection structure, a buckle connection structure or a sleeve connection structure.
The utility model has the advantages that:
1. utilize the utility model discloses only need through the centrifugation, can follow effectual enrichment to circulating tumor cell in the blood, do not need special instrument, the operation is quick, simple and convenient, according to the configuration of adaptation centrifuge, 8-16 samples can be handled to centrifugation, and the throughput is high, and every sample can handle the liquid up to 10m L capacity.
2. The utility model discloses after centrifugal filtration, its filter membrane can be dismantled, and the cell that the enrichment was arrived on the filter membrane can carry out various low reaches experiments, like nucleic acid extraction, fluorescence PCR, second generation sequencing, fluorescence in situ hybridization, immunohistochemistry, immunofluorescence etc. extensive applicability.
Drawings
Fig. 1 is an exploded perspective view of embodiment 1 of the present invention.
Fig. 2 is a perspective sectional view of embodiment 1 of the present invention.
Fig. 3 is a perspective sectional view of the filter element connecting seat according to embodiment 1 of the present invention.
Fig. 4 is an exploded perspective view of embodiment 2 of the present invention.
Fig. 5 is a perspective sectional view of embodiment 2 of the present invention.
Fig. 6 is a perspective sectional view of the filter element connecting seat according to embodiment 2 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1
As shown in fig. 1 to 3, a centrifuge tube 1 for enriching circulating tumor cells includes an upper tube 11, a filter element connecting seat 12, a filter membrane 13, a sealing ring 14 and a lower tube 15.
an upper tube body 11 having a diameter of 28mm and a volume of not more than 20m L, and having an upper opening and a lower opening, the upper opening having an upper cap 110, the lower opening of the upper tube body 11 being smaller than the upper opening of the upper tube body 11;
The filter element connecting seat 12 is provided with a central through hole 120, the size of the central through hole 120 is matched with the lower opening of the upper tube body 11, and the edge of the central through hole 120 extends upwards to form an annular support 121;
The filter membrane 13 is made of polycarbonate, the aperture is 8 mu m, and the diameter is 13 mm;
a lower tube body 15, 28mm in diameter, having an upper opening, with a volume not exceeding 20m L;
The lower end of the upper pipe body 11 is adapted to be connected with the upper end of the filter element connecting seat 12, the lower opening of the upper pipe body 11 is hermetically communicated with the central through hole 120 through a sealing ring 14, the filter membrane 13 is clamped between the lower opening of the upper pipe body 11 and the annular support 121, and the upper opening of the lower pipe body 15 is adapted to be connected with the lower end of the filter element connecting seat 12. Specifically, the lower end of the upper pipe body 11 is connected with the upper end of the filter element connecting seat 12 through a threaded connection structure. The upper opening of the lower pipe body 15 is connected with the lower end of the filter element connecting seat 12 through a threaded connection structure.
The utility model discloses a use method does: open upper cover 110, add the peripheral blood sample to body 11 of making progress, cover upper cover 110 again and return to the upper shed of body 11, will the utility model discloses put into horizontal centrifuge and centrifugation, peripheral blood leucocyte, erythrocyte, liquid pass through the centrifugation, flow into down the body 15 through lower open-end, filter membrane 13 and central through-hole 120 of body 11 in proper order, and circulation tumor cell is held back on filter membrane 13, realizes the enrichment of circulation tumor cell.
The utility model discloses an evaluation method of efficiency of enrichment effect as follows:
(1) human lung cancer cell lines H3122-GFP (carrying the A L K gene fusion mutation) and HCC78-GFP (carrying the ROS1 gene fusion mutation) cultured in vitro, both of which carry a green fluorescent protein GFP tag, were harvested and observed under the FITC channel of a fluorescence microscope, whereas peripheral blood leukocytes and erythrocytes were not observed under this channel.
(2) H3122 cells and HCC78 cells were each diluted to the appropriate cell density with serum-free medium.
(3) peripheral blood was collected from 3 healthy adult volunteers using EDTA anticoagulant blood tubes, 20m L were collected from each volunteer and divided into 4 groups of 5m L.
(4) Each group was spiked with-5H 3122 cells, -15H 3122 cells, -5 HCC78 and-15 HCC78 cells, respectively, and carefully mixed.
(5) Adding equal volume of 4% paraformaldehyde solution into each group, mixing, and fixing at room temperature for 20 min.
(6) Adding the mixed solution of the blood and the paraformaldehyde solution into an upper tube body 11 of the circulating tumor cell enrichment centrifugal tube 1, and covering a tube cover of the centrifugal tube 1;
(7) Placing the centrifuge tube 1 into a horizontal centrifuge, and centrifuging for 10min at 800 g;
(8) the centrifuge tube 1 was removed, disassembled, the filter 13 was carefully removed with forceps, placed face up on a slide, and the enriched H3122 cells and HCC78 cells were observed and counted under the FITC channel of the fluorescence microscope to calculate the enrichment efficiency (number of enriched tumor cells/number of incorporated cells × 100%), and the results are shown in table 1.
(9) the filter 13 on the slide was transferred to a 1.5-m L EP tube, and RNA was extracted using an RNA extraction kit (cat # 8.0224101X036G) from Xiamen Alder biomedical science and technology Co.
(10) RT-PCR detection is carried out on each group of samples by using a human EGFR/A L K/ROS1 gene mutation joint detection kit (cargo number: 8.01.25501W008A) of Xiamen Ed biological medicine science and technology Co., Ltd, and the detection results are shown in the following table 2.
TABLE 1 enrichment efficiency test results of circulating tumor cell enrichment centrifuge tube 1
TABLE 2 circulating tumor cell enrichment centrifuge tube 1RT-qPCR detection results
As can be seen from Table 1, the utility model discloses an overall enrichment efficiency is 84.5%, and enrichment efficiency between the different cell lines does not have the significance difference (82.8% VS 86.1%, P value is 0.343), and the enrichment efficiency of doping 5 tumor cells and doping between 15 tumor cells does not have the significance difference (84.4% VS 84.5%, P value is 0.989), shows the utility model discloses enrichment efficiency is higher to enrichment efficiency is not influenced by cell type and the cell quantity of doping, and the enrichment effect is more stable.
As can be seen from Table 2, the downstream nucleic acid extraction and RT-PCR detection are carried out after the enriched cells of the utility model are used, 5 tumor cells and 15 tumor cells can be detected when being mixed into 5m L blood, which indicates that the detection capability of the detection method of the utility model can reach 1 CTC/m L blood depending on the classification.
Example 2
As shown in fig. 4 to 6, a centrifuge tube 1 for enriching circulating tumor cells includes an upper tube 11, a filter element connecting seat 12, a filter membrane 13, a sealing ring 14 and a lower tube 15.
an upper tube body 11 having a diameter of 16mm and a volume of not more than 6m L, and having an upper opening and a lower opening, the upper opening having an upper cap 110, the lower opening of the upper tube body 11 being smaller than the upper opening of the upper tube body 11;
The filter element connecting seat 12 is provided with a central through hole 120, the size of the central through hole 120 is matched with the lower opening of the upper tube body 11, and the edge of the central through hole 120 extends upwards to form an annular support 121;
The filter membrane 13 is made of polycarbonate, the aperture is 6.5 mu m, and the diameter is 13 mm;
a lower tube body 15, having a diameter of 16mm and an upper opening, having a volume not exceeding 6m L;
The lower end of the upper pipe body 11 is adapted to be connected with the upper end of the filter element connecting seat 12, the lower opening of the upper pipe body 11 is hermetically communicated with the central through hole 120 through a sealing ring 14, the filter membrane 13 is clamped between the lower opening of the upper pipe body 11 and the annular support 121, and the upper opening of the lower pipe body 15 is adapted to be connected with the lower end of the filter element connecting seat 12. Specifically, the lower end of the upper pipe body 11 is connected with the upper end of the filter element connecting seat 12 through a threaded connection structure. The upper opening of the lower pipe body 15 is connected with the lower end of the filter element connecting seat 12 through an interference sleeving structure.
The utility model discloses a use method does: open upper cover 110, add the peripheral blood sample to body 11 of making progress, cover upper cover 110 again and return to the upper shed of body 11, will the utility model discloses put into horizontal centrifuge and centrifugation, peripheral blood leucocyte, erythrocyte, liquid pass through the centrifugation, flow into down the body 15 through lower open-end, filter membrane 13 and central through-hole 120 of body 11 in proper order, and circulation tumor cell is held back on filter membrane 13, realizes the enrichment of circulation tumor cell.
The utility model discloses an evaluation method of efficiency of enrichment effect as follows:
Evaluation of enrichment efficiency, only 15 tumor cells were tested for incorporation, the procedure was as in example 3, and the results are shown in Table 3.
TABLE 3 enrichment efficiency test results of the circulating tumor cell enrichment centrifuge tube 1
As can be seen from Table 3, the total enrichment efficiency of the utility model is 86.1%, which shows that the enrichment efficiency of the utility model is higher.
The FISH detection capability evaluation method comprises the following specific operation steps:
(1) human lung cancer cell line H3122 (which carries the a L K gene fusion mutation) and HCC78 (which carries the ROS1 gene fusion mutation) were harvested in vitro cultures.
(2) H3122 cells and HCC78 cells were each diluted to the appropriate cell density with serum-free medium.
(3) peripheral blood 6m L was collected from 1 healthy adult volunteer using EDTA anticoagulant blood collection tubes and divided into 2 groups of 3m L each.
(4) Separately incorporate 5H 3122 cells and 5 HCC78 cells, carefully mix.
(5) Adding equal volume of 4% paraformaldehyde solution, mixing, and fixing at room temperature for 10 min.
(6) Adding the mixed solution of blood and paraformaldehyde solution into the upper tube 11, and covering the upper cover 110;
(7) Placing the centrifuge tube 1 into a horizontal centrifuge, and centrifuging for 10min at 800 g;
(8) The centrifuge tube 1 is taken out, disassembled, the filter membrane 13 is carefully taken out by a pair of tweezers, the filter membrane is reversely buckled on a polylysine positive electricity loading glass sheet from the front side, and the glass sheet is placed at 37 ℃ for 30min, so that the cells on the filter membrane 13 are adsorbed and transferred onto the glass sheet.
(9) Fixing with 10% neutral formalin solution for 20min, and washing with water.
(10) Treating with 0.1% Triton X-100 for 5min, and washing with water.
(11) digesting with 0.05mg/m L pepsin for 2min, and washing with water.
(12) gradient ethanol dehydration, adding A L K fragmentation probe and ROS1 fragmentation probe respectively, denaturation at 85 deg.C for 5min, and hybridization at 37 deg.C overnight.
(13) Wash with 0.1% NP-40 wash and water.
(14) Dehydrating with gradient ethanol, and adding DAPI counterstain solution.
(15) cells positive for a L K and ROS1 were observed and counted under a fluorescent microscope.
the test result shows under the mirror, incorporates 10m L peripheral blood of 5H 3122 cells, through the utility model discloses carry out the FISH after the enrichment and detect, can observe 3A L K FISH positive cells (being H3122 cells), incorporate 10m L peripheral blood of 5 HCC78 cells, can observe 4 ROS1 FISH positive cells (being HCC78 cells), demonstrate to use the utility model discloses the CTC of enrichment can be used to low reaches FISH and detect.
the utility model is used for enriching CTC and detecting fluorescence RT-PCR of 10m L peripheral blood of 70 patients with advanced NSC L C, and the experimental steps are as follows:
(1) centrifuging 10m L peripheral blood at 2000g for 10min, transferring upper layer plasma for ctDNA detection, and supplementing PBS buffer solution to lower layer blood cells to 10m L;
(2) an equal volume (10m L) of 4% paraformaldehyde solution was added, carefully mixed, and fixed at room temperature for 20 min.
(3) Adding the mixed solution of blood and paraformaldehyde solution into the upper tube body 11, covering the upper cover 110, putting the centrifugal tube 1 into a horizontal centrifuge, and centrifuging for 10min at 800 g;
(4) taking out the centrifuge tube 1, dismantling, carefully taking out the filter membrane 13 by using forceps, and putting the filter membrane into the centrifuge tube 1 of 1.5m L;
(5) CTC RNA was extracted using an RNA extraction kit (cat # 8.0224101X036G) from Xiamen Alder biomedical science and technology, Inc.
(6) RT-PCR detection is carried out by using a human EGFR/A L K/ROS1 gene mutation joint detection kit (cargo number: 8.01.25501W008A) of Xiamen Ed biomedical science and technology Co., Ltd, and the fusion state of the A L K and the ROS1 genes is detected.
the detection results show that 3 cases of A L K fusion positive and 1 case of ROS1 fusion positive are detected in CTC enriched by peripheral blood samples of 70 cases of advanced NSC L C patients, the positive detection ratios are respectively 4.3% and 1.4%, and the positive detection ratios are consistent with the reported A L K positive rate of 4-8% and ROS1 positive rate of 1-2% of NSC L C patients, and the circulating tumor cell enrichment centrifugal tube 1 disclosed by the invention also has good detection capability on clinical samples.
The above description is only a preferred embodiment of the present invention, and therefore the scope of the present invention should not be limited by this description, and all equivalent changes and modifications made within the scope and the specification of the present invention should be covered by the present invention.
Claims (7)
1. A centrifuging tube for circulating tumour cell enrichment which characterized in that: the method comprises the following steps:
An upper tube body having an upper opening and a lower opening, the upper opening having an upper cover;
The filter element connecting seat is provided with a central through hole, the size of the central through hole is matched with the lower opening of the upper tube body, and the edge of the central through hole extends upwards to form an annular support;
A filter membrane;
A seal ring;
And a lower tube body having an upper opening;
The lower extreme adaptation of going up the body connects the upper end of filter core connecting seat, and the under shed of going up the body passes through the sealed intercommunication of sealing washer and above-mentioned central through-hole, and the filter membrane clamp is located between the under shed and the cyclic annular support of body, and the lower extreme of filter core connecting seat is connected to the upper shed adaptation of body down.
2. The centrifuge tube of claim 1, wherein: the lower opening of the upper pipe body is smaller than the upper opening of the upper pipe body.
3. The centrifuge tube of claim 1, wherein: the aperture of the filter membrane is 5-10 μm.
4. the centrifuge tube of claim 1, wherein the upper tube body has a volume of 1-20m L.
5. the centrifuge tube of claim 1, wherein the lower tube body has a volume of 1-20m L.
6. The centrifuge tube of any one of claims 1 to 5, wherein: the lower end of the upper pipe body is connected with the upper end of the filter element connecting seat through a threaded connection structure, a buckle connection structure or a sleeving connection structure.
7. The centrifuge tube of any one of claims 1 to 5, wherein: the upper opening of the lower pipe body is connected with the lower end of the filter element connecting seat through a threaded connection structure, a buckle connection structure or a sleeving connection structure.
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| CN201921695573.5U CN211026389U (en) | 2019-10-11 | 2019-10-11 | A centrifuging tube for circulating tumor cell enrichment |
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| CN201921695573.5U CN211026389U (en) | 2019-10-11 | 2019-10-11 | A centrifuging tube for circulating tumor cell enrichment |
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