CN210846424U - Epithelial-mesenchymal hybrid circulating tumor cell detection kit - Google Patents

Abstract

The utility model discloses an epithelium interstitial mixed type circulating tumor cell detection kit, this kit, including kit box body and kit lid, kit box body inner wall is hugged closely and is had the heat preservation water barrier, kit box body in the bottom be equipped with cold box, be provided with end box on the cold box, be provided with a plurality of standing grooves and a plurality of hole of placing in the end box, be provided with spiral sample room and micro-fluidic chip in a plurality of standing grooves, a plurality of downthehole a plurality of reagent bottles that are provided with of placing, a plurality of reagent bottles form reagent bottle group. The kit is reasonable in design combination, economical in use and storage, convenient in transportation, and capable of being directly used for normal temperature environment for use, storage and transportation, overcomes the defects that the low-temperature preservation reagent is complex in low-temperature environment maintaining cold chain transportation, storage and use, saves energy consumption for storage, transportation and use, and ensures the effectiveness of reagent consumable material combination in the kit in the processes of enrichment, capture and detection of epithelial type, epithelial-mesenchymal mixed type and mesenchymal type CTCs.

Description

Epithelial-mesenchymal hybrid circulating tumor cell detection kit

Technical Field

The utility model relates to a kit and epithelium and stroma mixed type circulating tumor cell detection field, concretely relates to epithelium and stroma mixed type circulating tumor cell detection kit.

Background

Circulating Tumor Cells (CTCs) are Tumor Cells released into the peripheral blood circulation from solid tumors or metastases either spontaneously or as a result of diagnostic procedures, and are a generic term for various Tumor Cells present in the peripheral blood. CTCs are heterogeneous populations consisting primarily of epithelial CTCs, epithelial-mesenchymal hybrid CTCs, and mesenchymal CTCs subpopulations.

Metastatic spread of tumors is a major cause of cancer progression and associated death. CTCs play an important role in the process of tumor formation into distant metastases. If a malignant tumor patient has a large number of CTCs in the blood, this indicates that the prognosis is poor and the probability of tumor metastasis is increased. CTCs-induced tumor metastasis mainly involves two dynamic processes of Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Epithelial Transition (MET), wherein the EMT process involves tumor cells being shed from primary foci into blood to form Epithelial-Mesenchymal hybrid CTCs with metastatic invasiveness, and the MET process participates in the formation, stabilization and proliferation processes of distant metastases. The development of EMT and MET by the CTCs is accompanied by a transition between epithelial, mixed epithelial-mesenchymal and mesenchymal CTCs between different phenotypes, and the detection of CTCs in different subgroups in different stages of tumorigenesis (early stage, progressive stage and metastatic stage) has different clinical application values: the detection and analysis of the epithelial CTCs can be applied to the early auxiliary differential diagnosis, prognosis evaluation and other directions of tumors; the detection and analysis of the epithelial-mesenchymal mixed type and mesenchymal CTCs can guide the metastatic relapse monitoring, the curative effect monitoring and the like of tumor patients in the progressive stage and the metastatic stage. Therefore, the non-invasive acquisition and detection of different subsets of CTCs in peripheral blood of tumor patients have important clinical application values in clinical tumor early-stage auxiliary diagnosis, prognosis judgment, tumor treatment efficacy monitoring and evaluation, tumor metastasis and recurrence monitoring and early warning, individualized treatment guidance and improvement of survival states of tumor patients.

CTCs are present in very rare amounts in peripheral blood, accounting for 1/10 in peripheral blood leukocytes alone⁶～1/10⁷① mainly adopts the following 2 forms that the enrichment detection of CTCs relies on anti-EpCAM to enrich and capture CTCs, anti-cytokeratin (CK8, CK18, CK19 and the like) antibodies and CD45 to identify and detect CTCs (represented as CellSearch system), the method only aims at the enrichment and detection of epithelial CTCs and cannot detect mixed CTCs of mesenchymal phenotype and epithelial stroma, ② does not rely on capturing markers to enrich CTCs (membrane filtration, gradient centrifugation and the like), although the limitation of enrichment and capture markers is overcome, the enrichment specificity of the method is not high, CTCs with special biophysical characteristics can be missed and the CTCs with mixed epithelial stroma cannot be detected, therefore, the enrichment and detection of conventional CTCs are usually limited to the enrichment and detection of CTCs of single group and cannot detect mixed mesenchymal CTCs, and the clinical application of CTCs is limited to a great extent due to the influences of the characteristics, the heterogeneity and the like of epithelial and the clinical application of heterogeneous CTCs is limited.

For overcoming current CTCs enrichment detection technology defect limitation, the utility model designs a reasonable, use and deposit transportation and practice thrift convenient epithelium interstitial mixed type circulating tumor cell detect reagent box of design combination, ensures that epithelium interstitial mixed type, epithelium type and interstitial CTCs carry out the enrichment and catch and detect reagent combination performance to provide more reliable diagnosis, treatment and prognosis judgement evidence for the accurate treatment clinical application of the tumour based on CTCs and establish the basis.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide an epithelium interstitial mixed type circulating tumor cell detection kit, this kit design combination is reasonable, use deposit practice thrift, transport convenient can directly be used for normal atmospheric temperature environment to use the epithelium interstitial mixed type circulating tumor cell detection kit of preserving and transporting, has overcome the loaded down with trivial details low temperature environment of low-temperature preservation reagent and has maintained cold chain transportation and store and the use, practices thrift and stores the transportation and uses the energy consumption. The effectiveness of the reagent consumable combination in the epithelial-mesenchymal mixed circulating tumor cell detection kit in the enrichment capture and detection processes of the epithelial type, epithelial-mesenchymal mixed type and mesenchymal CTCs is ensured.

For making up the defect that the prior art can not effectively enrich and detect the mixed CTCs of epithelial and mesenchymal tissues, the utility model aims to further utilize the specific capture antibody combination of the mixed circulating tumor cells of epithelial and mesenchymal tissues and the reagent combination of the specific antibody to provide a simple and efficient detection mode of the mixed CTCs of epithelial and mesenchymal tissues.

The utility model provides a technical scheme that above-mentioned problem adopted is:

the epithelial-mesenchymal hybrid circulating tumor cell detection kit comprises a kit box body and a kit box cover matched with the kit box body, wherein a heat-insulating water-resisting layer is tightly attached to the inner wall of the kit box body, a cold box is arranged at the bottom in the kit box body, a bottom box is arranged on the cold box, a plurality of placing grooves and a plurality of placing holes are formed in the bottom box, spiral sample chambers and micro-fluidic chips are arranged in the plurality of placing grooves, a plurality of reagent bottles are arranged in the plurality of placing holes, and the plurality of reagent bottles form a reagent bottle group.

The utility model discloses in, the kit box body have the heat preservation water barrier, it is two-layer about divideing into by the base wall: the upper layer of the kit body is a reagent bottle comprising a base, a spiral sample chamber and a microfluidic chip placing layer, the lower layer is a cold box placing layer, the base is provided with a plurality of placing holes and placing grooves, the reagent bottles of the reagent bottle group are sequentially placed in the placing holes, and the spiral sample chamber and the microfluidic chip are placed in the placing grooves; the cold box placing layer is used for placing the cold box subjected to low-temperature freezing treatment; the reagent bottle group adopts an epithelial-mesenchymal mixed CTCs capturing detection reagent group which comprises an epithelial-mesenchymal mixed CTCs specific capturing antibody group contained in a reagent plate, an epithelial-mesenchymal mixed CTCs specific detecting antibody group with a fluorescent marker contained in the reagent bottle group and a reaction system auxiliary reagent. The cold box can maintain the whole low-temperature state of the kit in a normal-temperature environment to ensure the performance of the reagent, and the kit can be conveniently and quickly stored, transported and used at normal temperature. The sizes of the placing holes are matched with the sizes of the reagent bottles one by one, and the reagent bottles can be properly fixed; the placing groove is internally provided with a plurality of partitions, the size of each partition space corresponds to the external packing size of a single spiral sample chamber and a single micro-fluidic chip, the spiral sample chamber and the micro-fluidic chip are ensured to be properly clamped by the placing groove and cannot randomly move or fall out, and the spiral sample chamber and the micro-fluidic chip can be better protected and fixed.

The following is taken as the preferable technical proposal of the utility model:

the heat-insulating and water-resisting layer comprises pearl wool, an aluminum foil covered on the pearl wool and a PVC layer attached on the aluminum foil, namely the heat-insulating and water-resisting layer is a waterproof heat-insulating layer with two sides attached with PVC containing the pearl wool and the aluminum foil, and the PVC is Polyvinyl chloride (Polyvinyl chloride).

The heat preservation water barrier in the kit box body contains the cotton waterproof insulation layer who compounds the aluminium foil of pearl for two-sided laminating PVC, and the heat preservation ageing can reach more than 18 hours, can save the use environment temperature condition cooperation according to the transportation simultaneously and change cold box, and the longer time is maintained reagent group ensures the reagent performance with the low temperature state of consumptive material.

The base of the utility model is made of low-density foaming material. The low-density foaming material has the advantages of light weight, no pollution, convenient processing and molding, reusability, excellent shock resistance, impact resistance and moisture resistance, and can effectively fix and protect the reagent bottle group, the spiral sample chamber and the microfluidic chip. In addition, the foaming material has certain low-temperature heat preservation performance and can assist in maintaining the low-temperature state of the reagent.

Cold box for there is not ice, no long-term cold source of supplying of power repeatedly usable, can guarantee the cold source supply in kit use, storage and the transportation to continuously maintain low temperature environment.

The kit body is divided into an upper layer and a lower layer by the base partition, the upper layer is a reagent bottle group comprising the base, a spiral sample chamber and a microfluidic chip placing layer, and the lower layer is a cold box placing layer.

The hole of placing of base be the round hole with the end, the standing groove for leading to the groove. In practical application, the shape of the placing hole is designed according to the reagent bottle, the reagent bottle is vertically placed in the placing hole, and the round hole with the bottom is convenient to process and can better fit the size of the reagent bottle and protect the reagent bottle; the shape of the placing groove is designed according to the shapes of the spiral sample chamber and the external package of the microfluidic chip, the placing groove is a sheet-shaped or strip-shaped through groove, the production and the processing of the through groove surface are convenient on one hand, and on the other hand, the storage quantity of the spiral sample chamber and the microfluidic chip can be increased, and the space of the kit can be effectively utilized.

The reagent plate is a 96-hole plate containing epithelial-mesenchymal mixed CTCs specific capture antibody group freeze-dried powder, and a moisture-proof sealing aluminum film covers the 96-hole plate.

Reagent bottle group include cell separation liquid reagent bottle, washing buffer solution reagent bottle, antibody diluent reagent bottle, coloring agent A reagent bottle, coloring agent B reagent bottle, coloring agent C reagent bottle, coloring agent D reagent bottle, catch reinforcing agent reagent bottle, nucleic acid coloring agent reagent bottle, cell washing liquid reagent bottle, cell fixative reagent bottle, dyeing blocking agent A reagent bottle, dyeing blocking agent B reagent bottle and cell penetrating agent reagent bottle. The reagent bottle group is respectively filled with an epithelial-mesenchymal mixed CTCs specific detection antibody group with a fluorescent marker and a reaction system auxiliary reagent with corresponding names, and the reagent bottle group and the reaction system auxiliary reagent are sequentially arranged in the kit according to the sequence of experimental operation.

An enrichment detection method for epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cells adopts an epithelial-mesenchymal hybrid circulating tumor cell detection kit, wherein each reagent bottle in the reagent bottle group is respectively filled with an EpCAM (epithelial cell adhesion molecule) antibody marked by biotin, an anti-CSV (cell surface Vimentin) antibody marked by biotin, an anti-PanCK antibody marked by fluorescein, an anti-Vimentin antibody marked by fluorescein, an anti-CD 45 antibody marked by fluorescein, a PD-L1 primary antibody and a PD-L1 secondary antibody marked by fluorescein, and the method specifically comprises the following steps:

1) preparing a microfluidic immunocapture carrier of the biotin-labeled epithelial-mesenchymal mixed antibody;

the biotin-labeled epithelial-mesenchymal hybrid antibody adopts a biotin-labeled EpCAM (epithelial cell adhesion molecule) antibody and a biotin-labeled anti-CSV (cell surface vimentin) antibody;

2) separating and enriching epithelial-mesenchymal mixed CTCs;

3) adopting fluorescein labeled epithelial-mesenchymal mixed antibodies to carry out immunodetection on different subtypes of CTCs;

the fluorescein-labeled epithelial-mesenchymal hybrid antibody adopts a fluorescein-labeled anti-PanCK antibody, a fluorescein-labeled anti-Vimentin antibody and a fluorescein-labeled anti-CD 45 antibody;

4) performing PD-L1 phenotypic detection on the different subtype CTCs captured in the step 3) by using a PD-L1 primary antibody and a fluorescein labeled PD-L1 secondary antibody.

In the utility model, the combination of epithelial-mesenchymal specificity capturing and detecting antibody markers, specific immune check point antibody markers and capturing and dyeing enhancement liquid is combined with the micro-fluidic technology to enrich and detect the PD-L1 immunophenotypes of epithelial CTCs, mesenchymal CTCs and epithelial-mesenchymal mixed CTCs and different CTCs; wherein the epithelial-mesenchymal specific capture and detection antibody markers comprise: anti-EpCAM (epithelial cell adhesion molecule) antibody, anti-CSV (cell surface Vimentin) antibody, anti-PanCK antibody, anti-Vimentin (Vimentin) antibody, anti-CD 45 antibody; the specific immune checkpoint antibody markers include: anti-PD-L1 primary antibody and anti-PD-L1 secondary antibody; the method combines the epithelial-mesenchymal specificity capturing and detecting antibody marker, the specificity immune check point antibody marker, the capturing enhancement liquid and the dyeing enhancement liquid with the microfluidic technology, not only solves the problem of large blood volume of the existing CTCs detecting technology and improves the detecting sensitivity of the CTCs, but also simultaneously solves the technical defect that the existing CTCs detecting technology can not detect the epithelial-mesenchymal mixed CTCs which can not be enriched and detected, and not only can simply, efficiently and specifically enrich and detect the epithelial-mesenchymal mixed CTCs, but also can enrich and detect the epithelial and mesenchymal CTCs. The method is a method for enriching and detecting CTCs with epithelial type, interstitial type, epithelial-interstitial mixed CTCs and epithelial PD-L1 positive phenotype, epithelial-interstitial mixed PD-L1 positive phenotype and interstitial PD-L1 positive phenotype, which has small blood volume, is simple, efficient, sensitive and specific.

The following is taken as the preferable technical proposal of the utility model:

in the step 1), the mass percentage of the biotin-labeled EpCAM (epithelial cell adhesion molecule) antibody in the biotin-labeled epithelial-mesenchymal hybrid antibody is 35-60%; the mass percentage of the biotin-labeled anti-CSV (cell surface vimentin) antibody in the biotin-labeled epithelial-mesenchymal hybrid antibody is 25-50%.

Preferably, the microfluidic technology is microfluidic carrier chip technology; more preferably, the microfluidic device comprises an etched herringbone micro-vortex fluid channel top layer containing PDMS (polydimethylsiloxane) and a substrate containing a nano substrate coating structure.

The preparation of the microfluidic immunocapture carrier of the biotin-labeled epithelial-mesenchymal mixed antibody specifically comprises the following steps:

sucking a streptavidin solution by using a pipettor, adding a microfluidic carrier, incubating at 10-35 ℃ for 3-5 h, placing in a drying chamber with the environment humidity lower than 30% for drying for 20-28 h until no liquid remains in the microfluidic carrier, treating the microfluidic carrier by using ethanol, and immediately washing the microfluidic carrier by using a PBS buffer solution;

adding a mixed solution of an anti-EpCAM (epithelial cell adhesion molecule) antibody marked by biotin and an anti-CSV (cell surface vimentin) antibody marked by biotin into a microfluidic carrier, incubating for 25-35 min at 35-39 ℃, washing the microfluidic carrier by using a PBS (phosphate buffer solution), adding a capture enhancing solution into the microfluidic carrier, incubating for 0.5-1.5 h at 10-35 ℃, washing the microfluidic carrier by using the PBS buffer solution, completing the preparation of the microfluidic immunocapture carrier of the epithelial-mesenchymal mixed antibody marked by biotin, and placing the microfluidic immunocapture carrier into a wet box for later use.

The capture enhancing solution is a mixed solution containing 0.2-2% of surfactant, 0.5-10% of BSA (bovine serum albumin) and 88-99.3% of blocking serum;

in the step 2), the separation and enrichment of the epithelial-mesenchymal mixed CTCs specifically comprises the following steps:

diluting peripheral blood by using PBS (phosphate buffer solution), adding the peripheral blood above a porous barrier of a gradient centrifuge tube containing a monocyte separating medium, centrifuging, absorbing a PBMCs (mononuclear cells) layer, transferring the PBMCs (mononuclear cells) layer into a sterile centrifuge tube, washing the PBMCs (mononuclear cells) by using a cell cleaning solution, centrifuging, removing supernatant, adding a cell cleaning solution to gently resuspend the PBMCs (mononuclear cells), and finishing the preparation of a coarse separating medium of CTCs;

and (3) absorbing the crude separation liquid of the CTCs by using a micro-fluidic pump system, slowly injecting the crude separation liquid into the micro-fluidic immunocapture carrier of the biotin-labeled epithelial-mesenchymal mixed antibody prepared in the step 1) at an injection speed of 3-4 mL/h at a constant speed, absorbing PBS buffer solution to wash the micro-fluidic immunocapture carrier, completing the enrichment of the epithelial-mesenchymal mixed CTCs, and obtaining the micro-fluidic carrier enriched with the epithelial-mesenchymal mixed CTCs.

In the step 3), the immunodetection of the epithelial-mesenchymal mixed antibody marked by fluorescein on different subtypes of CTCs specifically comprises the following steps:

injecting a cell fixing solution into the microfluidic carrier enriched with the epithelial-mesenchymal mixed CTCs in the step 2), reacting for 15-25 min at 10-35 ℃, absorbing a PBS buffer solution to wash the microfluidic carrier, and completing cell fixation;

sucking the cell permeation liquid by a liquid transfer device, injecting the cell permeation liquid into the microfluidic carrier with the fixed cells, placing the microfluidic carrier in a light-proof wet box, acting for 8-12 min at 10-35 ℃, sucking PBS buffer solution to wash the microfluidic carrier, and completing cell permeation;

adding a mixed solution of a fluorescein-labeled anti-PanCK antibody, a fluorescein-labeled anti-Vimentin antibody, a fluorescein-labeled anti-CD 45 antibody, a staining blocking solution A, a staining blocking solution B and an antibody diluent into a cell-permeable microfluidic carrier, incubating at 10-35 ℃ in a dark place for 1.5-2.5 h, slowly microfluidizing the carrier by using a PBS (phosphate buffer solution), then absorbing a nucleic acid staining solution, injecting the nucleic acid staining solution into the microfluidic carrier, placing the microfluidic carrier in a dark wet box, incubating at 10-35 ℃ for 10-20 min, washing the microfluidic carrier by using the PBS buffer solution, completing the immunodetection of the epithelial CTCs, the epithelial-mesenchymal mixed CTCs and the mesenchymal CTCs, and observing the fluorescent color development condition by using an inverted fluorescence microscope.

Further preferably, the volume ratio of the fluorescein-labeled anti-PanCK antibody, the fluorescein-labeled anti-Vimentin antibody, the fluorescein-labeled anti-CD 45 antibody, the staining blocking solution a and the staining blocking solution B mixed is 1: 1: 1: 1: 1.

the staining blocking solution A is a solution containing an FC receptor blocking agent; the staining blocking solution B is a solution containing more than one (including one) of mouse serum, rabbit serum and sheep serum; the antibody diluent comprises a solution containing 2-10% of BSA (bovine serum albumin) and 0.3-5% of protein protection stabilizer by mass percentage.

Preferably, the protein protection stabilizer is a mixture of glycerol and a chemically inert high molecular polymer. More preferably, the chemically inert high molecular weight polymer is PEG.

In the step 4), the PD-L1 phenotype detection of the different subtype CTCs captured in the step 3) by adopting a PD-L1 primary antibody and a fluorescein labeled PD-L1 secondary antibody specifically comprises the following steps:

adding a mixed solution of the PD-L1 primary antibody, the staining blocking solution A, the staining blocking solution B and the antibody diluent into the microfluidic carrier which is reacted in the step 3), placing the microfluidic carrier in a wet box, incubating the microfluidic carrier for 1.5 to 2.5 hours at the temperature of 10 to 35 ℃ in a dark place, and washing the microfluidic carrier by using a PBS buffer solution;

slowly adding a fluorescein-labeled PD-L1 secondary antibody into the microfluidic carrier which has completed the PD-L1 primary antibody reaction by using an antibody diluent, placing the microfluidic carrier in a wet box, incubating for 1.5-2.5 h at 10-35 ℃ in a dark place, washing the microfluidic carrier by using a PBS buffer solution, completing the PD-L1 phenotype detection of different subtype CTCs, and observing the dyeing result by using an inverted fluorescence microscope.

The volume ratio of the PD-L1 primary antibody to the staining blocking solution A and the staining blocking solution B is 1: 2: 2; the volume percentage of the fluorescein-labeled PD-L1 secondary antibody to the fluorescein-labeled PD-L1 secondary antibody in the antibody diluent is 2-5%.

The anti-PanCK antibody, the anti-Vimentin antibody, the anti-CD 45 antibody, and the anti-PD-L1 secondary antibody are each labeled with fluorescein of different emission wavelengths that can be distinguished from each other. According to the known technical personnel in this field, in order to distinguish different specific detection antibodies, the different fluorescein labels adopted by the utility model can be completely distinguished under different filters by a fluorescence microscope. The labeled fluorescein can adopt any commonly used fluorescent dye in the field, more preferably, the labeled fluorescein comprises FITC (fluorescein isothiocyanate), PE (phycoerythrin), Alexa Fluor series molecules, PerCP (pleonanthrin-chlorophyll-protein complex), APC (allophycocyanin), TRITC (tetramethyl rhodamine isothiocyanate) and the like, and the fluorescent color development is different and can be distinguished from each other;

an enrichment detection method of epithelial-mesenchymal hybrid and PD-L1 positive circulating tumor cells comprises the following steps:

1) preparation of microfluidic immunocapture carrier adopting biotin-labeled epithelial-mesenchymal hybrid antibody

And (3) sucking 300 mu L of streptavidin solution by using a pipette, adding the microfluidic carrier, incubating for 4h at normal temperature, and placing in a drying chamber with the environmental humidity lower than 30% for drying for 24h until no liquid residue exists in the microfluidic carrier. The microfluidic carrier was treated 1 time with 300 μ L95% ethanol and immediately washed twice with PBS buffer.

Adding 200 mu L of mixed solution of a biotin-labeled anti-EpCAM (epithelial cell adhesion molecule) antibody and a biotin-labeled anti-CSV (cell surface vimentin) antibody into the microfluidic carrier, incubating for 30min at 37 ℃, slowly washing the microfluidic carrier twice by using 300 mu L of PBS buffer solution, adding 200 mu L of capture enhancing solution into the microfluidic carrier, incubating for 1h at room temperature, slowly washing the microfluidic carrier twice by using 300 mu L of PBS buffer solution, completing the preparation of the epithelial-mesenchymal hybrid capture microfluidic carrier, and placing the epithelial-mesenchymal hybrid capture microfluidic carrier into a wet box for later use.

2) Separation and enrichment of epithelial-mesenchymal mixed CTCs

Diluting 2mL of peripheral blood with 1: 1 PBS buffer, adding the peripheral blood to the upper part of a porous barrier of a gradient centrifuge tube containing 3mL of monocyte separating medium, centrifuging the peripheral blood for 10min at the room temperature of 1000 × g, carefully and slowly sucking a PBMCs (monocyte) layer, transferring the PBMCs into a sterile centrifuge tube, slowly washing the PBMCs by using 15mL of cell washing liquid, centrifuging the PBMCs for 10min at the room temperature of 300 × g, carefully removing supernatant, adding 1mL of cell washing liquid, and gently suspending the PBMCs to complete preparation of a coarse separating medium of CTCs.

And (3) sucking the 1mL of the coarse separation solution of the CTCs by using a micro-fluidic pump system, slowly injecting the coarse separation solution into the micro-fluidic carrier prepared in the step 1) at a constant speed at room temperature at an injection speed of 3.6mL/h, and then sucking 300 mu L of PBS buffer solution to slowly flush the micro-fluidic carrier twice to complete the enrichment of the epithelial-mesenchymal mixed CTCs.

3) Immunodetection of epithelial-mesenchymal hybrid CTCs

And (3) injecting 200 mu L of cell fixing solution into the microfluidic carrier enriched with the epithelial-mesenchymal mixed CTCs in the step 2), reacting at room temperature for 20min, and absorbing 300 mu L of PBS buffer solution to wash the microfluidic carrier twice to complete cell fixation.

And sucking 200 mu L of cell permeation solution by using a pipette, injecting the cell permeation solution into the microfluidic carrier with the fixed cells, placing the microfluidic carrier in a light-proof wet box for acting at room temperature for 10min, sucking 300 mu L of PBS buffer solution to wash the microfluidic carrier for three times, and completing cell permeation.

Adding 250 mu L of mixed solution of fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-CD 45 antibody, staining blocking liquid A, staining blocking liquid B and antibody diluent into the microfluidic carrier, incubating for 2h in a dark place at room temperature, slowly washing the microfluidic carrier twice with 300 mu L of PBS buffer solution, then sucking 150 mu L of nucleic acid staining solution, slowly injecting into the microfluidic carrier, incubating for 15min in a dark wet box at room temperature, slowly washing the microfluidic carrier twice with 300 mu L of PBS buffer solution, completing the immunodetection of the epithelial CTCs, the epithelial-mesenchymal mixed CTCs and the mesenchymal CTCs, and observing the fluorescent color development condition with an inverted fluorescence microscope.

4) PD-L1 phenotypic assay of different subtypes of CTCs

And (3) slowly adding 150 mu L of mixed solution of the PD-L1 primary antibody, the staining blocking solution A, the staining blocking solution B and the antibody diluent into the reacted microfluidic carrier in the step 3), placing the microfluidic carrier in a wet box, incubating the microfluidic carrier in the dark at room temperature for 2 hours, and slowly washing the microfluidic carrier three times by using 300 mu L of PBS buffer solution.

Diluting a fluorescein-labeled PD-L1 secondary antibody to 300 mu L by using an antibody diluent, slowly adding the diluted fluorescein-labeled PD-L1 secondary antibody into the microfluidic carrier which has completed the PD-L1 primary antibody reaction, placing the microfluidic carrier in a wet box, incubating for 2h in a dark room temperature, slowly washing the microfluidic carrier for three times by using 300 mu L PBS buffer solution, completing the PD-L1 phenotype detection of different subtype CTCs, and observing the dyeing result by using an inverted fluorescence microscope.

Preferably, the monocyte separation medium in the step 2) is a human lymphocyte separation medium containing Ficoll (Ficoll) and meglumine diatrizoate, and the density is 1.077 +/-0.001 g/mL; the gradient centrifugal tube is a centrifugal tube with a porous barrier partition plate inside, can assist in the separation of PBMCs, can effectively maintain the layered state of the PBMCs, and is convenient for the separation operation of CTCs; the cell washing solution is preferably a mixture containing a cell culture medium and 2% fetal bovine serum. The micro-fluid pump system is a micro-fluid pump which comprises a combination of a multi-way valve and a pipeline and can quantitatively control micro-fluid at a fixed speed through control software. More preferably, the microfluidic pump has a maximum volume of 500 microliters.

Preferably, the cell fixing solution in step 3) is an aqueous solution containing 2% PFA (paraformaldehyde); the cell permeation solution is preferably an aqueous solution containing 0.4% Triton X-100; the nucleic acid staining solution is a common cell nucleus staining solution, and is preferably one of Hoechst33342 or DAPI (4', 6-diamidino-2-phenylindole).

Compared with the prior art, the utility model has the advantages of as follows:

the utility model discloses in, kit structural design is reasonable, can directly maintain epithelial mesenchymal hybrid circulating tumor cell detection reagent, spiral sample room and micro-fluidic chip low temperature protection environment in the box in the reagent bottle group through built-in cold box, need not use and keep refrigerator, special transportation extranal packing material and transportation cold chain professional equipment and maintain low temperature environment, can directly implement the save and transport of reagent in normal atmospheric temperature environment, has practiced thrift transportation material and process energy consumption when stabilizing reagent performance, and the environmental protection is convenient, has reduced the cost of transportation; the kit has certain moisture resistance and impact resistance, good strength, difficult loss, recycling, environmental protection and saving. During detection, the kit can be used as a bracket for reagent bottle groups and consumables, and can directly provide a low-temperature protection environment without taking crushed ice to maintain the low-temperature environment of the reagent, thereby simplifying operation. The reagent bottle has reasonable combination layout and convenient taking, and is convenient for the enrichment capture and detection of epithelial type, epithelial-mesenchymal mixed type and interstitial CTCs.

The method combines the epithelial-mesenchymal specificity capturing and detecting antibody marker, the specificity immune check point antibody marker, the capturing enhancement liquid and the dyeing enhancement liquid with the microfluidic technology, not only solves the problem of large blood volume of the existing CTCs detecting technology and improves the detecting sensitivity of the CTCs, but also simultaneously solves the technical defect that the existing CTCs detecting technology can not detect the epithelial-mesenchymal mixed CTCs which can not be enriched and detected, and not only can simply, efficiently and specifically enrich and detect the epithelial-mesenchymal mixed CTCs, but also can enrich and detect the epithelial and mesenchymal CTCs. Simultaneously, the utility model discloses provide a PD-L1 immunophenotyping's of different grade type CTCs detection method with the mode that does not have the wound, more simple and direct economy has compensatied the defect that prior art detected PD-L1.

Furthermore, the utility model discloses a catch the combination of dyeing enhancement liquid, fully expose the antigenic determinant on CTCs cell membrane surface, increased the sensitivity and the specificity that CTCs caught and detected, exhibited CTCs fluorescence color development form better, do benefit to cell pathology form and phenotype analysis. The utility model provides a more reliable, economical and practical clinical application mode for noninvasive accurate diagnosis, treatment and prognosis judgment of tumors based on CTCs liquid biopsy.

Drawings

FIG. 1 is a schematic view of the kit assembly of the present invention;

FIG. 2 is a schematic diagram of the structure and the cold box of the kit body of the present invention;

FIG. 3 is a schematic view of the base and the consumable material arrangement and reagent plate of the reagent bottle of the present invention;

in FIGS. 1 to 3: 1. the kit comprises a kit body, 2, a kit box cover, 3, a heat-preservation water-resisting layer, 4, a base, 5, a reagent bottle group, 6, a spiral sample chamber, 7, a microfluidic chip, 8, a reagent plate, 9, a PVC layer, 10, an aluminum foil, 11, a pearl wool heat-preservation layer, 12, a cold box, 13, a washing buffer solution reagent bottle, 14, a cell separation solution reagent bottle, 15, a cell cleaning solution reagent bottle, 16, a capture reinforcing agent reagent bottle, 17, a cell fixing agent reagent bottle, 18, a cell penetrating agent reagent bottle, 19, an antibody diluent reagent bottle, 20, a staining blocking agent A reagent bottle, 21, a staining blocking agent B reagent bottle, 22, a staining agent A reagent bottle, 23, a staining agent B reagent bottle, 24, a staining agent C reagent bottle, 25, a staining agent D reagent bottle, 26, a nucleic acid staining agent reagent bottle, 27, a placing hole, 28 and a placing groove;

FIG. 4 is a schematic representation of the visualization of circulating tumor cells of the epithelial type;

FIG. 5 is a schematic of the visualization of stromal-type circulating tumor cells;

FIG. 6 is a schematic representation of the color development of epithelial-mesenchymal hybrid circulating tumor cells;

FIG. 7 is a color rendering schematic of positive expression of PD-L1 in circulating epithelial tumor cells;

FIG. 8 is a schematic color representation of positive expression of PD-L1 in circulating tumor cells of interstitial type;

FIG. 9 is a schematic diagram of the positive expression and color development of the epithelial-mesenchymal mixed circulating tumor cell PD-L1;

in FIGS. 4 to 9: the different groups of circulating tumor cells are all from clinical lung cancer samples, white arrows in the figure indicate different groups of CTCs, A is a merged image, B is a Hoechst33342 marker CTCs nuclear chromogenic image (actually blue), C is a CD45-PE chromogenic image (actually orange), D is a PanCk-PerCP chromogenic image (actually deep red), E is a Vimentin-FITC chromogenic image (actually green), and F is PD-L1-Alexa 647 (far infrared, and the microscopic scanning is endowed with yellow).

Detailed Description

The invention will be further explained by the embodiments with reference to the drawings.

Example 1 (epithelial-mesenchymal hybrid circulating tumor cell assay kit)

As shown in fig. 1-3, an epithelial-mesenchymal hybrid circulating tumor cell detection kit comprises a kit box body 1 and a kit box cover 2 matched with the kit box body 1, wherein a heat-insulating and water-proof layer 3 is tightly attached to the inner wall of the kit box body 1, a cold box 12 is arranged at the bottom in the kit box body 1, a bottom box 4 is arranged on the cold box 12, a plurality of placing grooves 28 and a plurality of placing holes 27 are arranged in the bottom box 4, a spiral sample chamber 6 and a microfluidic chip 7 are arranged in the plurality of placing grooves 28, a plurality of reagent bottles are arranged in the plurality of placing holes 27, and a plurality of reagent bottles form a reagent bottle group 25. The heat-insulating and water-resisting layer 3 comprises pearl wool 11, an aluminum foil 10 covered on the pearl wool 11 and a PVC layer 9 attached on the aluminum foil 10. The placement hole 27 of the base 4 is a bottomed circular hole, and the placement groove 28 is a through groove. A reagent plate 9 is placed on the base 4. Reagent plate 9 is a 96-well plate.

As shown in fig. 1 to 3, the present embodiment includes: a kit box body 1, a kit box cover 2 connected with one side edge of the kit box body 1, a heat-insulating water-resisting layer 3 consisting of a PVC layer 9, an aluminum foil 10 and a pearl wool heat-insulating layer 11 arranged on the inner layer of the kit box body 1, a partition base 4 arranged on the upper layer of the kit box body 1, a reagent bottle group 5 (comprising a washing buffer solution reagent bottle 13, a cell separation solution reagent bottle 14, a cell cleaning solution reagent bottle 15, a capture reinforcing agent reagent bottle 16, a cell fixing agent reagent bottle 17, a cell penetrating agent reagent bottle 18, an antibody diluent reagent bottle 19, a staining blocking agent A reagent bottle 20, a staining blocking agent B reagent bottle 21, a staining agent A reagent bottle 22, a staining agent B reagent bottle 23, a staining agent C reagent bottle 24, a staining agent D reagent bottle 25, a nucleic acid staining agent reagent bottle 26, a spiral staining agent 6 and a micro-fluidic staining agent chip 7 arranged in a placing groove 28 on the micro-fluidic base 4, reagent board 8, cold box 12 of placing in the lower floor of kit box body 1.

As shown in fig. 2 to 3, in the present embodiment, during actual storage and transportation, the cold box 12 subjected to low-temperature treatment is first placed at the bottom of the heat-insulating water-barrier layer 3 in the reagent box as a low-temperature source, the base 4 is placed above the cold box 12, the outer surface of the base 4 is attached to the heat-insulating water-barrier layer 3 and is appropriately clamped, then the washing buffer solution reagent bottle 13, the cell separation solution reagent bottle 14, the cell washing solution reagent bottle 15, the capture enhancer reagent bottle 16, the cell fixative reagent bottle 17, the cell permeation reagent bottle 18, the antibody diluent reagent bottle 19, the staining blocker a reagent bottle 20, the staining blocker B reagent bottle 21, the staining agent a reagent bottle 22, the staining agent B reagent bottle 23, the staining agent C reagent bottle 24, the staining agent D reagent bottle 25, and the nucleic acid staining agent reagent bottle 26 are sequentially placed in the placement holes 27 of the base from front to back, the spiral sample chamber 6 and the microfluidic chip 7 are sequentially placed in the placement groove 28, and finally, placing the reagent plate 8 on the uppermost layer of the heat-insulating and water-resisting layer 3 in the reagent box, covering the reagent box cover 2 to seal the opening of the reagent box body 1, and directly storing or transporting the reagent box by a conventional method.

As shown in fig. 1, in actual use, the reagent bottle group 5 can be used by directly opening the reagent bottle cap without taking out from the base 4, the base 2 is used as a reagent rack, and the cold box below the reagent rack is directly used as a low temperature source, so that the crushed ice does not need to be taken out to maintain the low temperature.

The above-mentioned detailed description of the technical solution and the beneficial effects of the present invention have been described in detail, it should be understood that the above is only the most preferred embodiment of the present invention, not used for limiting the present invention, any modification, supplement, equivalent replacement, etc. made within the principle scope of the present invention should be included within the protection scope of the present invention.

Example 2 (detection of circulating tumor cell typing of Lung cancer and detection of its expression of PD-L1)

An epithelial-mesenchymal mixed circulating tumor cell detection kit is adopted, and each reagent bottle in the reagent bottle group 5 is filled with a specific reagent required by detection;

materials: the micro-fluidic carrier adopts a micro-fluidic chip, and the micro-fluidic pump system adopts a circulating tumor cell analyzer (WY-C3000, produced by Hangzhou Huadelson biotechnology limited company)

1. Preparation of micro-fluidic immunocapture carrier of biotin-labeled epithelial-mesenchymal hybrid antibody

1.1. And (3) sucking 300 mu L of streptavidin solution with the concentration of 5 mu g/mL by using a pipette, adding the solution into the microfluidic chip, incubating at the normal temperature of 25 ℃ for 4h, and placing the chip in a drying chamber with the environmental humidity of lower than 30% for drying for 24h until no liquid residue exists in the microfluidic chip.

1.2. The microfluidic chip was filled with 300. mu.L of 95% ethanol by volume and immediately washed twice with 1 × PBS buffer.

1.3. 200 mu L of mixed solution of biotin-labeled anti-EpCAM (epithelial cell adhesion molecule) antibody and biotin-labeled anti-CSV (cell surface vimentin) antibody (the mass percentage of the mixed two antibodies is 50 percent of biotin-labeled anti-EpCAM antibody and 50 percent of biotin-labeled anti-CSV antibody), is added into a microfluidic carrier, incubated at 37 ℃ for 30min, and then the microfluidic chip is slowly washed twice by 300 mu L of PBS buffer solution.

1.4. Pipette 200. mu.L of capture enhancing solution into the microfluidic chip and incubate at 25 ℃ for 1 h. The capture enhancing solution is a mixed solution containing 0.4% by mass of Triton X-100, 3% by mass of BSA (bovine serum albumin) and 87% by mass of blocking serum, and the balance of water.

1.5. And (3) slowly washing the microfluidic carrier twice by using 300 mu L of PBS buffer solution to finish the preparation of the epithelial-mesenchymal mixed antibody capture microfluidic chip, and placing the microfluidic chip in a wet box for later use.

2. Separation and enrichment of epithelial-mesenchymal mixed CTCs

2.1 dilution of 2mL peripheral blood with 2mL PBS buffer, 4mL diluted blood samples were added to 3mL mononuclear cell-containing fractions

1077 of density gradient centrifugate

Centrifuging 1000 × g for 10min at room temperature of 25 ℃ above a porous barrier of a gradient centrifuge tube, wherein the monocyte separation liquid is a human lymphocyte separation liquid containing Ficoll (Ficoll) and meglumine diatrizoate, and the density is 1.077 +/-0.001 g/mL, and the gradient centrifuge tube is a centrifuge tube internally provided with a porous barrier partition plate, so that the separation of PBMCs can be assisted, the layered state of the PBMCs can be effectively maintained, and the separation operation of CTCs is facilitated;

2.2 the layer of PBMCs (monocytes) is gently aspirated slowly and transferred to a sterile centrifuge tube.

2.3 adding 15mL of cell cleaning solution into the centrifuge tube to slowly wash the PBMCs, centrifuging for 10min at the room temperature of 25 ℃ and 300 × g, wherein the cell cleaning solution is a mixed solution containing 98% of cell culture medium RPMI1640 mass percent and 2% of fetal bovine serum mass percent.

2.4 carefully remove the supernatant by pipette, add 1mL of cell wash and gently resuspend the PBMCs to complete the preparation of the crude isolate of CTCs.

2.5 using a micro-fluid pump system [ circulating tumor cell analyzer (WY-C3000) ] to absorb the 1mL of the crude separation liquid of the CTCs, injecting the crude separation liquid into the micro-fluid control chip prepared in the step 1 at room temperature of 25 ℃ at a constant speed of 3.6mL/h, then absorbing 300 muL of 1 × PBS buffer solution to slowly wash the micro-fluid control chip twice to complete the enrichment of the epithelial-mesenchymal mixed CTCs.

3. Immunodetection of epithelial-mesenchymal hybrid CTCs

3.1. A pipettor is used for injecting 200 mu L of cell fixing solution into the microfluidic chip enriched with the epithelial-mesenchymal mixed CTCs in the step 2, after the reaction is carried out for 20min at the room temperature of 25 ℃, 300 mu L of 1 × PBS buffer solution is absorbed to wash the microfluidic chip twice to complete the cell fixation, and the cell fixing solution adopts an aqueous solution containing 2% of PFA (paraformaldehyde) in percentage by mass;

3.2. sucking 200 mu L of cell permeation liquid by a liquid transfer machine, injecting the cell permeation liquid into the microfluidic chip which is fixed by the cells, placing the microfluidic chip in a light-proof wet box for acting at room temperature for 10min, sucking 300 mu L of 1 × PBS buffer solution to wash the microfluidic chip for three times, and completing cell permeation, wherein the cell permeation liquid adopts aqueous solution containing 0.4 mass percent of Triton X-100;

3.3. 10 mu L of fluorescein PerCP-labeled anti-PanCK antibody, 10 mu L of fluorescein FITC-labeled anti-Vimentin antibody, 10 mu L of fluorescein PE-labeled anti-CD 45 antibody, 10 mu L of staining blocking solution A, 10 mu L of staining blocking solution B and 200 mu L of antibody diluent are mixed uniformly and then added into the microfluidic chip, and the microfluidic chip is incubated for 2 hours at room temperature and 25 ℃ in the dark. The dye blocking solution A is a solution containing an FC receptor blocking agent; the staining blocking solution B is a solution containing mouse serum and rabbit serum; the antibody diluent is a solution containing 2 mass percent BSA and 0.5 mass percent protein protection stabilizer. The protein protection stabilizer is glycerol and PEG.

3.4. The microfluidic chip was slowly rinsed twice with 300 μ L of 1 × PBS buffer by pipetting.

3.5. And then, 150 mu L of nucleic acid staining solution Hoechst33342 is absorbed and slowly injected into the microfluidic chip, the microfluidic chip is placed in a light-proof wet box and incubated for 15min at the room temperature of 25 ℃, 300 mu L of PBS buffer solution is used for slowly washing the microfluidic carrier twice, the immunodetection of the epithelial CTCs, the epithelial-mesenchymal mixed CTCs and the mesenchymal CTCs is completed, and the fluorescence color development condition is observed by using an inverted fluorescence microscope.

Interpretation of the observations by inverted fluorescence microscopy of CTCs typing

4.1. Phenotype of epithelial CTCs, fig. 4: hoechst33342+, PanCK +, Vimentin-, CD 45-;

4.2. phenotype of mesenchymal CTCs, fig. 5: hoechst33342+, PanCK-, Vimentin +, CD 45-;

4.3. epithelial-mesenchymal mixed CTCs phenotype, fig. 6: hoechst33342+, PanCK +, Vimentin +, CD45-

5. PD-L1 phenotypic assay of different subtypes of CTCs

5.1. Respectively sucking 6 mu L of PD-L1 primary antibody, 12 mu L of staining blocking solution A, 12 mu L of staining blocking solution B and 120 mu L of antibody diluent by a pipette, uniformly mixing, slowly adding into the reacted microfluidic chip in the step 3, and incubating for 2h in a humid box at room temperature in the dark

5.2. The microfluidic chip was slowly rinsed three times with 300 μ L of 1 × PBS buffer.

5.3. Diluting 6 mu L of the PD-L1 secondary antibody marked by the fluorescein Alexa 647 to 300 mu L by 294 mu L of antibody diluent, slowly adding the diluted solution into a 5.2 microfluidic chip which completes the PD-L1 primary antibody reaction, and placing the microfluidic chip in a humid box to incubate for 2 hours at the room temperature of 25 ℃ in the dark;

5.4. and (3) slowly washing the microfluidic carrier three times by using 300 mu L of PBS buffer solution to finish the PD-L1 phenotype detection of different subtype CTCs, and observing the dyeing result of PD-L1 by using an inverted fluorescence microscope.

Interpretation of the results of inverted fluorescence microscope observations of CTCs typing

Epithelial CTCs of PD-L1 positive phenotype, fig. 7: hoechst33342+, PanCK +, Vimentin-, CD45-, PD-L1 +;

interstitial CTCs of PD-L1 positive phenotype, fig. 8: hoechst33342+, PanCK-, Vimentin +, CD45-, PD-L1 +;

epithelial-mesenchymal mixed CTCs of PD-L1 positive phenotype, fig. 9: hoechst33342+, PanCK +, Vimentin +, CD45-, PD-L1 +;

example 3CTCs enrichment assay potency comparison experiment

Comparative example: the difference from example 2 is that in this example, a group a (capture antibody only using anti-EpCAM antibody capture detection only using anti-PanCK antibody and anti-CD 45 antibody) and a group b (capture antibody only using anti-CSV antibody detection antibody only using anti-CSV antibody and anti-CD 45 antibody) were set according to the conventional technical method to perform comparative experiments with the present technical scheme.

Materials: 1) breast cancer cell lines BT474, MCF7, SKBR3 and MDAMB231 at different stages; 2)6 portions of 2ml healthy human peripheral blood.

Cell suspensions of BT 474150, MCF 7150, SKBR 3150 and MDAMB231150 were mixed to 6 parts of a suspension containing about 600 cells, and each of the 6 parts was added to 2ml of peripheral blood of a healthy person to prepare a mock blood sample.

Following the procedure in example 2: control group a prepared microfluidic immunocapture carriers with 200 μ L of biotin-labeled anti-EpCAM (epithelial cell adhesion molecule) antibody, and detection antibodies were anti-PanCK and anti-CD 45 antibody 1: 1 mass percent; and (3) preparing a microfluidic immunocapture carrier by using 200 mu L of biotin labeled anti-CSV (cell surface vimentin) antibody in the group b of the comparison group, wherein the detection antibody adopts anti-CSV and anti-CD 45 antibody 1: 1 mass percent; experimental group c microfluidic immunocapture vectors of epithelial-mesenchymal hybrid antibodies were prepared using the method of example 2; the separation, enrichment, detection and interpretation of the observation results of the 3 groups of CTCs are performed by the method of example 2, each experiment is repeated 1 time, and the results include the capture efficiency of the epithelial CTCs, the capture efficiency of the mesenchymal CTCs and the capture efficiency of the epithelial-mesenchymal hybrid CTCs,% of the capture efficiency is the number of cells on the chip/the number of cells put into the chip.

Specific results are shown in table 1, and the results show that: the capture efficiency of the group c on different types of CTCs completely by the method of example 2 is higher than that of the group a and the group b respectively by the traditional single enrichment and antibody detection method.

TABLE 1

Note: total capture rate of CTCs: -average capture rate of epithelial CTCs + average capture rate of interstitial CTCs + average capture rate of epithelial-interstitial mixed CTCs

Parts not described in detail in this specification are prior art using structures and principles known to those skilled in the art.

The above description is within the reach of a person skilled in the art.

Furthermore, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and those skilled in the art should understand that the technical solutions and the inventive concepts of the present invention should be equally replaced or changed within the scope of the appended claims.

Claims (6)

1. The epithelial-mesenchymal hybrid circulating tumor cell detection kit is characterized in that a heat-insulating water-resisting layer is tightly attached to the inner wall of the kit body, a cold box is arranged at the bottom in the kit body, a bottom box is arranged on the cold box, a plurality of placing grooves and a plurality of placing holes are formed in the bottom box, spiral sample chambers and microfluidic chips are arranged in the placing grooves, a plurality of reagent bottles are arranged in the placing holes, and the reagent bottles form a reagent bottle group.

2. The epithelia-mesenchyme mixed type circulating tumor cell detection kit of claim 1, wherein the heat preservation and water-proof layer comprises pearl wool, an aluminum foil covered on the pearl wool and a PVC layer attached on the aluminum foil.

3. The epithelial-mesenchymal hybrid circulating tumor cell detection kit according to claim 1, wherein the kit body is divided into an upper layer and a lower layer by a base partition, the upper layer is a reagent bottle group comprising the base, a spiral sample chamber and a microfluidic chip placement layer, and the lower layer is a cold box placement layer.

4. The epithelia-mesenchyme mixed type circulating tumor cell detection kit as claimed in claim 3, wherein the placement hole of the base is a bottomed round hole, and the placement groove is a through groove.

5. The epithelial-mesenchymal hybrid circulating tumor cell detection kit according to claim 3, wherein a reagent plate is placed on the base.

6. The kit for detecting epithelial-mesenchymal hybrid circulating tumor cells according to claim 5, wherein the reagent plate is a 96-well plate.

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