CN109781975B - Reagent and method for enriching circulating rare cells - Google Patents
Reagent and method for enriching circulating rare cells Download PDFInfo
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- CN109781975B CN109781975B CN201711121110.3A CN201711121110A CN109781975B CN 109781975 B CN109781975 B CN 109781975B CN 201711121110 A CN201711121110 A CN 201711121110A CN 109781975 B CN109781975 B CN 109781975B
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Abstract
The present invention relates to reagents and methods for enriching Circulating Rare Cells (CRC). The invention discloses a method for separating leukocytes in blood by using a preferred CD marker combination so as to enrich CRC, and the method is a negative sorting method. The method can greatly improve the identification accuracy of the cells, realize the breakthrough of high-purity CRC sorting, and the obtained sample is more favorable for being directly applied to subsequent genome analysis. Meanwhile, the invention also provides a microfluid detection device applied to the method.
Description
Technical Field
The invention belongs to the field of oncology, and particularly relates to a reagent for enriching circulating rare cells, application thereof and a method for enriching circulating rare cells.
Background
The current individualized treatment scheme for cancer depends on tumor information in tissue specimens, but tissue specimens are difficult to obtain in clinical practice, especially in the treatment process and in the early stage of cancer metastasis. Therefore, finding an alternative to tissue specimens is currently the most urgent requirement for cancer therapy monitoring and drug resistance mechanism research.
Circulating Tumor Cells (CTCs) are cells released from a primary tumor that carry biological information unique to the tumor. CTC as an easily-obtained tumor information carrier can provide a sample for dynamically monitoring the change of biological characteristics of the tumor due to the convenience and timeliness of peripheral blood detection; scientific research also shows that CTC is an independent tumor prognosis and prediction factor, and dynamic monitoring of the CTC number of a patient can timely understand the disease progression of the patient and the therapeutic intervention effect. Therefore, the circulating tumor cell detection technology is developed and perfected, so that the circulating tumor cell detection technology can detect various types of tumors more sensitively and monitor the disease conditions of patients in early and middle stages, a large amount of treatment cost can be saved, the survival quality of the patients is improved, and the circulating tumor cell detection technology has great social significance.
Circulating tumor cells are contained in Circulating Rare Cells (CRC), and the technical nature of CRC detection and isolation is how CRC can be identified from a raw blood sample and then isolated from other blood cells. Current CRC separation techniques fall into two main categories.
The first category of methods distinguishes and separates CRC and other cells based on the physical characteristics of CTCs, such as size, deformability, and electrical impedance. However, at the cellular morphological level, CRC morphologies vary greatly in size from patient to patient for different types of cancer, from patient to patient for the same cancer, and even from patient to patient in the same patient. In addition, although some CRC sizes are significantly larger than white blood cells, there are still many CRC sizes and morphologies that differ little from normal white blood cells. Thus, other than cell size-based separation has been successfully used for some known large-size CRCs, particularly CRC-forming cell microblocks, the remaining separation techniques have not been fully case-validated. In particular, the physical mechanism is not clear, and has not been widely verified and accepted.
The second category of methods is based on protein markers on the surface of CRC, positive enrichment of CRC using magnetic beads or certain microfluidic structures for certain known cell markers (e.g. using EpCAM), or negative depletion of leukocytes (e.g. using CD 45). However, the disadvantages of these two types of CRC separation methods are also very significant. Forward enrichment based on specific markers although higher CRC purity (> 1%) is easily obtained, its extraction rate strongly depends on the expression level of certain CRC specific markers and is not conducive to CRC that does not express or under express the protein. CRC with very aggressive capacity undergoes transformation from epithelial cells to mesenchymal cells (EMT). The phenomenon is the disappearance of epidermal cell features (e.g., EpCAM) on the cell, while mesenchymal marker proteins are expressed. Thus, EpCAM-based CRC isolation techniques cannot enrich this class of critically important cells. For example, the currently only commercial CellSearch system can only effectively detect late-stage cancer patients with several cancers (such as breast cancer, colon cancer and prostate cancer), and the technology is difficult to effectively detect some tumors with high morbidity and mortality (such as lung cancer, liver cancer and stomach cancer). In addition, CellSearch has a detection rate of less than 10% for early and mid-stage patients with cancer. In addition, marker-based forward isolation techniques often require immobilization, perforation and cytoplasmic keratin labeling of the cells. Cells have lost activity after this process and are not available for many subsequent assays such as cell culture and drug action.
Compared with positive separation, the negative separation technology adopts a leukocyte characteristic marker to strive to remove leukocytes to the maximum extent. The whole process is independent of tumor cell markers, and various types of CRC can be retained to the maximum extent. However, the existing single leukocyte markers cannot completely mark and remove leukocytes, so that although the extraction rate of the remaining CRC is high, the purity is not ideal, usually below 0.1%, and the direct genotype analysis of a sample cannot be carried out.
Therefore, the current detection technology of CRC still has the defects of low detection sensitivity, less tumor type adaptation, complex device operation and high detection cost.
Disclosure of Invention
The invention aims to provide a reagent and a method for enriching Circulating Rare Cells (CRC).
In a first aspect of the invention, there is provided a method for enriching Circulating Rare Cells (CRC) from a blood sample (ex vivo sample), characterized in that said method comprises:
separating and removing red blood cells from the blood sample;
labeling leukocytes in a blood sample by using an antibody carrying a recognizable signal, and labeling nucleated cells by using a fluorescent dye;
cells are detected by a device capable of identifying identifiable signals and fluorescent dyes, nucleated cells without leucocyte markers are separated, and finally enriched circulating rare cells are obtained.
In a preferred embodiment, the antibody comprises: anti-CD 45 antibody.
In another preferred embodiment, the antibody is an antibody combination comprising: anti-CD 45 antibodies, CD14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies, and anti-CD 36 antibodies.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In another preferred embodiment, the device for identifying the identifiable signal and the fluorescent dye is a microfluidic detection device comprising:
a microfluidic structure (7) including a single-cell channel (11) allowing a single cell to pass at a stable flow rate, and a plurality of single-cell channels being present to allow shunting of the single cell;
the fluorescence detection component (5) is used for screening each cell according to different fluorescence signals, rapidly confirming the identity of each cell and judging whether the cell is sorted in the next step or not;
and the microjet generator (6) comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component (5) and initiates a microthermal bubble.
In another preferred embodiment, the identifiable signals include (but are not limited to): FITC, PE, APC, Rhodamine B, Alexa 555; preferably FITC.
In another preferred embodiment, the fluorescent dyes include (but are not limited to): DAPI or hoechst; preferably DAPI.
In another preferred example, the method for separating and removing the red blood cells is lysis of lysate or density gradient centrifugation; or
Separating and removing red blood cells from a blood sample by treating the blood sample with an antibody carrying a recognizable signal; preferably, the identifiable signal applied to the isolated depleted red blood cells is different from the identifiable signal applied to the depleted white blood cells.
In another aspect of the invention, there is provided the use of an antibody for isolating leukocytes from a blood sample when the sample is enriched for circulating rare cells; the antibodies include anti-CD 45 antibodies; preferably, the antibody is an antibody combination comprising: anti-CD 14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies, and anti-CD 36 antibodies.
In a preferred embodiment, the use is a non-diagnostic and a non-therapeutic use.
In another aspect of the present invention, there is provided a microfluidic detection device comprising:
a microfluidic structure (7) comprising a single-cell channel (11) allowing passage of a single cell at a steady flow rate, and a plurality of single-cell channels being present to allow shunting of the single cell;
the fluorescence detection component (5) is used for screening each cell according to different fluorescence signals, rapidly confirming the identity of each cell and judging whether the cell is sorted in the next step or not;
and the microjet generator (6) comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component (5) and initiates a microthermal bubble.
In a preferred embodiment, the microfluidic detection device includes:
a substrate (8);
a microjet generator (6) located on the substrate;
a microfluidic structure (7) located on the microjet generator (6);
a cover plate (10) on the microfluidic structure (7);
preferably, the microjet generator (6) is present in a protective layer (9) between the substrate (8) and the microfluidic structure (7).
In another preferred embodiment, the protective layer (9) is a silicon dioxide protective layer.
In another aspect of the present invention, there is provided a kit for enriching circulating rare cells from a blood sample, comprising said microfluidic detection device;
an antibody carrying a recognisable signal; the antibodies include anti-CD 45 antibodies; preferably, the antibody is an antibody combination comprising: anti-CD 14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies, and anti-CD 36 antibodies; and
a fluorescent dye that labels nucleated cells.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
Fig. 1, CRC microfluidic detection system architecture based on negative sorting.
Wherein 1 is a sample inlet;
2 is a residual cell collecting tube;
3 is a CRC collecting pipe;
4 cells (red DAPI-FITC +/-cells, blue DAPI + FITC + cells, orange DAPI + FITC-cells);
5 is CRC fluorescence detection component;
6 is a micro-jet generator;
7 is a microfluidic structure.
FIG. 2 shows the processing steps of the micro-thermal bubble jet chip, and the whole steps are sequentially subjected to steps a), b), c) and d).
Wherein 6 is a micro-jet generator;
7 is a microfluidic structure;
8 is a substrate;
9 is a protective layer;
10 is a cover plate;
11 is a single cell channel;
wherein the protective layer is a silicon dioxide protective layer.
FIG. 3, flow assay results for unstained control samples.
FIG. 4, flow assay results for CD45-FITC and DAPI stained specimens.
Figure 5, flow assay results of samples stained with multiple CD fluorescent antibodies and DAPI.
In FIGS. 3 to 5, FSC indicates the relative size of cells, and larger values of FSC indicate larger cells; SSC indicates the complexity of the interior of the cell, and a larger value of SSC indicates more particles in the interior of the cell. The region R1 represents lymphocytes, the region R2 represents monocytes and granulocytes, and the region R3 represents undiluted red blood cells, platelets and cell debris. FL1 represents the DAPI detection channel and FL2 represents the FITC detection channel. Total events represent the Total number of sample cells in the sample during flow detection, and Gated events represent the corresponding number of cells in the R1, R2 and R3 regions, respectively.
FIG. 6 shows the results of immunofluorescence staining assay of KB cell-spiked blood samples sorted by the detection system;
(A) incorporation of 20 KB cells;
(B) incorporation into 50 KB cells;
after blood samples spiked with KB cells were sorted by the test system, the cells enriched in positive collection tubes included: DAPI+Represents all cells sorted into positive collection tubes; FITC represents leukocytes that were misclassified into positive collection tubes; DAPI+FITC-Pan-Keratin-Indicating leukocytes which are not labeled with fluorescent antibodies of the CD series; Pan-Keratin+Represents KB cell; DAPI+FITC-Pan-Keratin+I.e., CRC cells, and KB cells are cancer cells herein, incorporated into blood samples to mimic CRC cells.
Detailed Description
In view of the problems and difficulties of the existing products and technologies, the present inventors have conducted extensive studies and have revealed a method for enriching Circulating Rare Cells (CRC) by separating leukocytes from blood using a preferred combination of CD markers, which is a negative sorting method. The method can greatly improve the identification accuracy of the cells, realize the breakthrough of high-purity CRC sorting, and the obtained sample is more favorable for being directly applied to subsequent genome analysis. Meanwhile, the invention also provides a microfluid detection device applied to the method.
As used herein, the Circulating Rare Cells (CRC) include Circulating Tumor Cells (CTCs).
Method for enriching circulating rare cells
The inventor of the present invention has made an effort to research the separation of CRC from blood, and found in the early stage of research that if a forward separation method is used to directly separate CRC from blood, the separation efficiency is not high, and more CRC still exists in blood, which can only be roughly characterized, but cannot be accurately characterized and quantified. If a negative separation method is adopted, the efficiency of removing white blood cells after red blood cells are separated from a sample is not ideal, and a final product often has more white blood cells.
The present inventors have conducted extensive studies with respect to the problem that leukocytes are difficult to be efficiently eliminated, and the number of human leukocyte surface antigens (CD) is considerable, and is more than 100. In the past, people generally used a single CD to sort leukocytes, namely CD 45. The present inventors have found that when an anti-CD 45 antibody is used to bind and isolate leukocytes, the isolated product still contains more leukocytes. Therefore, the present inventors have analyzed and screened against a large number of CDs, from which CD45, CD14, CD55, CD66b, CD90, CD33, and CD36 were selected as combined targets. Thus, in the most preferred method of the invention, anti-CD 45 antibodies, anti-CD 14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies and anti-CD 36 antibodies are used in combination to isolate leukocytes from a blood sample to obtain an enriched circulating rare cell product. Sorting with the combined antibody can reduce the proportion of unlabeled leukocytes very significantly.
Accordingly, the present invention discloses a method for enriching circulating rare cells from a blood sample, said method comprising: separating and removing red blood cells from the blood sample; labeling leukocytes in a blood sample by using an antibody carrying a recognizable signal, and labeling nucleated cells by using a fluorescent dye; cells are detected by a device capable of identifying identifiable signals and fluorescent dyes, nucleated cells without leucocyte markers are separated, and finally enriched circulating rare cells are obtained. In a preferred embodiment of the invention, the antibody is a combination of antibodies, including an anti-CD 45 antibody, an anti-CD 14 antibody, an anti-CD 55 antibody, an anti-CD 66b antibody, an anti-CD 90 antibody, an anti-CD 33 antibody and an anti-CD 36 antibody.
Methods for separating and removing red blood cells from a blood sample can employ separation methods known in the art. In a preferred embodiment of the present invention, erythrocytes are removed by centrifugation, which is differential centrifugation. As another preferred mode of the present invention, red blood cells are isolated and removed from a blood sample by treating the blood sample with an antibody carrying a recognizable signal; preferably, the identifiable signal applied to the isolated depleted red blood cells is different from the identifiable signal applied to the depleted white blood cells. For example, the identifiable signal applied to the isolation of the removed red blood cells is signal 1, and the identifiable signal applied to the removal of white blood cells is signal 2.
In the present invention, the term "identifiable signal" refers to a marker or label that can generate different signals under the irradiation of a light source to distinguish cells. In a preferred embodiment of the present invention, the identifiable signal is a fluorescent label. More preferably, the identifiable signal is selected from (but not limited to): FITC, DAPI, PE, APC, Rhodamine B, Alexa 555. In a preferred embodiment of the present invention, the identifiable signal is FITC.
The purpose of the cell marker portion is to distinguish blood cells from CRC as accurately as possible by fluorescent staining. Before the on-machine detection of the blood sample, all cells in the sample are stained with multiple markers, or a whole blood sample is processed to remove most of red blood cells (such as density gradient centrifugation or red blood cell lysis), and then white blood cells and CRC are stained. For example, in example 2, leukocytes and CRC are nuclear stained with nuclear dyes (e.g., DAPI) to differentiate (exclude) small residual red blood cells, platelets, and cell membrane debris. On the other hand, staining was performed with a corresponding fluorescent antibody (e.g., CD-FITC) against a variety of specific leukocyte surface antigens to maximally label all leukocytes. Cells with DAPI + FITC will be interpreted as CRC and cells with DAPI + FITC + will be interpreted as leukocytes.
In a preferred embodiment of the present invention, when circulating rare cells are enriched from a blood sample, after leukocytes are separated and removed, the enriched circulating rare cells are obtained by further sorting using the microfluidic detection device of the present invention.
The labeled cells are input through the inlet of the microfluidic detection device, and serial cell flow of single cells is continuously and stably generated by adopting the microfluidic technology. The cells in the cell stream may pass individually through an optical detection window and a subsequent cell sorting unit.
Compared with other CRC sorting technologies, the method based on negative sorting and the microfluid detection device have the following main advantages:
(1) in the cell marking part, after the multiple CD markers are adopted to mark the leucocytes, the leucocytes are negatively removed, on one hand, the left CRC of all types can be sorted without omission, the detection sensitivity and the sorting efficiency of the CRC can be greatly improved, and the problem of false negative possibly caused by the fact that a certain specific CRC marker is relied on to identify the CRC is avoided. On the other hand, the optimal multiple leukocyte marker of the invention overcomes the problem of excessive residual leukocytes caused by using a single marker (usually CD45), is favorable for greatly improving the identification accuracy of cells, realizes the breakthrough of high-purity CRC sorting, and enables a sample to be used for subsequent genome analysis.
(2) The detection and sorting of the cells are operated on single cells, and the sorting accuracy can be greatly improved. The whole process does not need cell immobilization, permeabilization or keratin dyeing, has small mechanical damage effect on cells, the sorted cells still keep cell activity, can be directly cultured in vitro, and is used for researching the generation of a tumor drug resistance mechanism and guiding the individualized treatment of tumors.
(3) The detection method has enough elasticity, the parameter combination can be adjusted by a user according to specific conditions, and different detection parameters are set aiming at different types of cancers, so that the detection system has wider application range and can be suitable for the detection of more types of cancers or different subtypes of cancers. And the separation (positive or negative) can be adjusted according to the CRC type or user requirements.
(4) The detection and the sorting are integrated in a totally closed system, and the sample pollution can be avoided. The preparation process is simple, complex chemical modification on the internal structure is not needed, no microcolumn or hole and other complex structures are needed, and the cell is not easily blocked in the sorting process.
(5) High miniaturization, integration and automation, low cost, simple and convenient operation and strong adaptability to clinical samples.
The application direction of the detection system has two aspects: in the aspect of scientific research, high-purity active CRC cells can be sorted, and the most direct research sample is provided for the drug resistance mechanism and the metastasis mechanism of tumors. In clinical application, an automatic CRC detection platform suitable for most cancer types is provided for the current extremely urgent cancer detection requirement; provides a diagnosis basis for clinic, dynamically monitors the change of the tumor in treatment, and provides prognosis evaluation for patients.
Microfluidic detection device
The inventor finds that when the conventional FACS instrument is used for sorting the cells, the cells are seriously damaged, so that the qualitative and quantitative detection effects are not ideal. Thus, the present inventors have also optimized the apparatus for performing cell sorting.
The invention relates to a micro-fluid detection system applied to CRC detection based on negative sorting, the general structure of which is shown in FIG. 1, and the system comprises: a microfluidic structure 7 including a single cell channel allowing a single cell to pass at a stable flow rate, and a plurality of single cell channels to allow shunting of the single cell; the fluorescence detection component 5 is used for screening each cell according to different fluorescence signals, rapidly confirming the identity of each cell and judging whether the cells are sorted in the next step or not; and the microjet generator 6 comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component 5 and initiates a microthermal bubble.
Cells of different identities are shunted to different collection vessels via different single cell channels.
Reagent kit
Based on the new findings of the present inventors, the present invention also provides a kit for enriching circulating rare cells from a blood sample, comprising the microfluidic detection device of the present invention; an antibody carrying a recognisable signal; the antibody is a combination of an anti-CD 45 antibody, an anti-CD 14 antibody, an anti-CD 55 antibody, an anti-CD 66b antibody, an anti-CD 90 antibody, an anti-CD 33 antibody, and an anti-CD 36 antibody; and, a fluorescent dye that labels nucleated cells.
For the convenience of the skilled person, the kit of the invention further comprises instructions for use of the method for enriching a blood sample for circulating rare cells. For example, the aforementioned method of the present invention can be described in the specification.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBrook et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Biological material and apparatus
Erythrocyte lysates (R1010) were purchased from beijing solibao technologies ltd.
The KB cell line was purchased from the shanghai cell bank of the chinese academy.
Fluorescent antibodies CD45-FITC (REF MHCD4501, LOT 1669391A), CD14-FITC (REF MHCD1401, LOT 767825I), CD90-FITC (REF A15761, LOT 521928), CD33-FITC (REF A15763, LOT 521680), CD36-FITC (REF MA1-19771, LOT QH2076195) and the nuclear dye DAPI (REF D1306, LOT 876632) are all available from ThermoFisher corporation.
CD55-FITC (REF MA1-19573, LOT QK2121353) was purchased from Pierce.
CD66b-FITC (REF555724, LOT12597) was purchased from BD corporation.
Pan-Keratin (C11) -Alexa555 (#3478) was purchased from Cell signalling.
The remaining reagents were purchased from Sigma Aldrich.
The hardware components associated with the CRC microfluidic detection system were purchased from belgian microelectronics research center (IMEC).
Example 1 preparation of CRC microfluidic detection System
The core of the hardware part of the CRC microfluid detection system is a miniature metal conductor, and the sorting signal of the CRC fluorescence detection component 5 is received to trigger a micro-thermal bubble emitted from a micro-jet generator 6, so that fluid in a micro-thermal bubble jet unit cavity is driven to be ejected out of the cavity, and CRC right passing through a sorting node is pushed to enter a positive channel.
The fabrication process is shown in fig. 2, and is carried out by first providing a substrate 8, processing a metal conductor (e.g. aluminum or copper) for generating micro-thermal bubbles on a silicon or glass substrate using photolithography techniques, and depositing a protective layer (e.g. silicon dioxide) thereon (fig. 2, steps a), b)). At the same time, the microfluidic structure is fabricated on another cover glass 9 and bonded to the previously fabricated metal conductor device (fig. 2, steps c), d)). After the body portion is completed, it is enclosed in a cartridge containing the input and output portions.
The device has simple principle and easy processing, can be applied to fluid sorting of any cell, and adopts the micro-processing technology to process so as to ensure the precision, the production scale and the cost of the device.
Example 2 screening of fluorescent-labeled antibody against leukocytes
Aiming at the mixed sample of CRC and leucocyte after pretreatment, the key is how to effectively remove leucocyte to the maximum extent by adopting multiple markers, and the recognition rate and specificity of CRC are improved. The present inventors examined the labeling efficiency of multiple CD fluorescent antibodies on leukocytes using a flow cytometer.
Two 3mL samples of healthy human were taken, the experimental procedure was as follows:
(1) removing red blood cells: diluting whole blood with PBS (phosphate buffer solution) of the same volume, slowly adding the diluted whole blood into a centrifuge tube filled with histopaque of the same volume, and centrifuging the whole blood at 2000rpm for 30 min; after blood centrifugation, obtaining a cell layer between plasma and a red blood cell layer;
(2) washing: gently taking out the cells of the cell layer in the step (1), adding an equal amount of PBS for washing, and centrifuging for 15min at 600 g; 500 μ L PBS resuspended cell pellet;
(3) and (3) fluorescent antibody staining: one sample is marked with leucocytes by selecting the following combination of antibodies: CD45-FITC, CD14-FITC, CD55-FITC, CD66b-FITC, CD90-FITC, CD33-FITC, CD36-FITC, each 10. mu.L; another sample, CD45-FITC alone, 10. mu.L; incubating at room temperature in dark for 40 min;
(4) nuclear staining marks nucleated cells: DAPI, 5 μ L, incubating at room temperature in dark for 2 min; all nucleated cells, including leukocytes and CRC cells, were stained;
(5) washing: washing with 1mL PBS for 3 times, and centrifuging at 600g for 10 min;
(6) and (3) detection: resuspending the cell pellet with 500. mu.L PBS, and detecting with flow cytometer (BD LSR II);
(7) meanwhile, a sample which is not subjected to cell staining treatment is used as a negative control, and the flow detection voltage and the threshold line are adjusted to make 99% of the cell fluorescence signal value negative.
The results are shown in FIGS. 3 to 5.
The above results show that the ratio of the number of unlabeled leukocytes (sum of UL regions of R1, R2, and R3) to the total number of sample cells was 0.04% when only CD45-FITC was used for staining, and that the ratio of the number of unlabeled leukocytes to the total number of sample cells was 0.01% when multiple CD fluorescent antibodies were used for staining.
The above results indicate that the majority of leukocytes can be distinguished from CRC by labeling multiple CD fluorescent antibodies, which helps to obtain high purity CRC.
Further experiments showed that the unlabelled fraction of the CD45-FITC + CD90-FITC combination was 0.04%, the unlabelled fraction of the CD45-FITC + CD34-FITC combination was 0.03%, and the unlabelled fraction of the CD45+ CD50 was 0.03%, all the unlabelled fractions being higher than the combinations described for CD45-FITC, CD14-FITC, CD55-FITC, CD66b-FITC, CD90-FITC, CD33-FITC, and CD 36-FITC.
Example 3 establishment of CRC microfluidic detection System of the invention
The microfluidic detection device of the present invention was set up as in fig. 1. The microfluidic detection device comprises a microfluidic structure 7, which comprises a single cell channel, allowing single cells to pass through at a stable flow rate, wherein the single cell channel starts from a sample inlet 1, a plurality of single cell channels exist at the downstream of the sample inlet to allow the diversion of the single cells, one channel is communicated with a CRC collection tube 3, and the other channel or channels are communicated with a residual cell collection tube 2; the fluorescence detection component 5 can discriminate each cell according to different fluorescence signals, quickly confirm the identity of each cell and judge whether to sort each cell in the next step; and the microjet generator 6 comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component 5, triggers a microthermal bubble and promotes shunting of cells.
In sorting, a flow of stained living cells 4 enters the single cell channel from the sample inlet 1 of the microfluidic device of fig. 1, passes through the fluorescence detection assembly 5, the fluorescence detection assembly 5 can identify the difference of the staining marks carried by the cells, and transmits the identified information to the microjet generator 6, if the cells are not CRC, the cells are not sorted, and the cells are directly collected to the remaining cell collection tube 2 in the middle (or above). If the cell is judged to be CRC, sorting is started, the flow direction of the cell is changed, the cell enters a positive channel and is collected by a CRC collecting pipe 3, and therefore enrichment of CRC is achieved.
Example 4 Performance testing of negative sorting-based CRC microfluidic detection System
A3 mL healthy human blood sample is taken and respectively doped with 20 KB cells and 50 KB cells (human oral epidermoid carcinoma cells), a tumor patient blood sample containing CRC is simulated, the sorting performance of the detection system of the invention on CRC is examined, and the experimental steps are as follows:
(1) removing red blood cells: diluting whole blood with PBS (phosphate buffer solution) of the same volume, slowly adding the diluted whole blood into a centrifuge tube filled with histopaque of the same volume, and centrifuging the whole blood at 2000rpm for 30 min; after blood centrifugation, obtaining a cell layer between plasma and a red blood cell layer;
(2) washing: gently taking out the cells in the cell layer, adding an equal amount of PBS for washing, and centrifuging at 600g for 15 min; PBS resuspended cell pellet.
(3) And (3) fluorescent antibody staining: the following combinations of antibodies were selected to label leukocytes: CD45-FITC, CD14-FITC, CD55-FITC, CD66b-FITC, CD90-FITC, CD33-FITC, CD36-FITC, 10. mu.L each, were incubated at room temperature for 40min in the absence of light.
(4) Nuclear staining marks nucleated cells: DAPI, 5. mu.L, incubated 2min at room temperature in the dark. All nucleated cells, including leukocytes and CRC cells, were stained.
(5) Cell sorting: the cell suspension was passed through the CRC microfluidic detection system of the present invention at a flow rate of 1,000 cells/sec, and the cells sorted to the positive channel were collected into a collection tube.
(6) Cell identification: the enriched cells in the positive collection tube are subjected to staining identification according to the following steps, and the sorting recovery rate and purity of the detection system are calculated.
And (4) dripping: adding 100 mu L PBS into the collection tube, resuspending the cell precipitate, completely dripping the cell suspension into the labeled area on the glass slide, and drying in an oven at 37 ℃; placing in a staining jar filled with PBS for hydration for 2 min;
membrane penetration: taking out the sample from the staining jar, adding 100.0 μ L of 0.1% Triton X-100, and performing membrane penetration at room temperature for 10 min;
washing: the sample was washed 2 times in a staining jar with PBS;
and (3) sealing: taking out the sample from the staining jar, adding 100.0 μ L of 1% BSA, and blocking at room temperature for 30 min;
antibody incubation: blocking solution was removed, and the KB cell specific Pan-Keratin (C11) -Alexa555 antibody, 0.05 ml/plate (1/25), was added and incubated at room temperature in the absence of light for 60 min.
Washing: the sample was washed 2 times in a staining jar with PBS;
sealing: taking out the sample from the staining jar, adding an anti-fluorescence quenching mounting solution, and mounting the sample by using a cover glass;
and (3) detection: the fluorescent signal was observed under a fluorescent microscope (OLYMPUS).
The results are shown in FIG. 6 and Table 1.
TABLE 1
The results show that the sorting recovery rate and the purity of the positive cells (CRC) by adopting the CRC microfluid detection system based on negative screening are not lower than 80% and not lower than 10%.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (12)
1. A method for enriching circulating rare cells from a blood sample, the method comprising:
separating and removing red blood cells from the blood sample;
labeling leukocytes in a blood sample by using an antibody carrying a recognizable signal, and labeling nucleated cells by using a fluorescent dye;
detecting cells by a device capable of identifying identifiable signals and fluorescent dyes, separating out nucleated cells without leucocyte markers, and finally obtaining enriched circulating rare cells; the device capable of identifying the identifiable signal and the fluorescent dye is a microfluidic detection device;
the antibody is an antibody combination which is: anti-CD 45 antibodies, anti-CD 14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies, and anti-CD 36 antibodies.
2. The method of claim 1, wherein the microfluidic detection device comprises:
a microfluidic structure (7) including a single-cell channel (11) allowing a single cell to pass at a stable flow rate, and a plurality of single-cell channels being present to allow shunting of the single cell;
the fluorescence detection component (5) is used for screening each cell according to different fluorescence signals, rapidly confirming the identity of each cell and judging whether the cell is sorted in the next step or not;
and the microjet generator (6) comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component (5) and initiates a microthermal bubble.
3. The method of claim 1, wherein said identifiable signal comprises: FITC, PE, APC, Rhodamine B or Alexa 555.
4. The method of claim 3, wherein the identifiable signal is FITC.
5. The method of claim 1, wherein the fluorescent dye comprises: DAPI or hoechst.
6. The method of claim 1, wherein the fluorescent dye is DAPI.
7. The method of claim 1, wherein the separation to remove red blood cells is lysate lysis or density gradient centrifugation; or
Red blood cells are isolated and removed from a blood sample by treating the blood sample with an antibody that carries a recognizable signal.
8. The method of claim 1, wherein the identifiable signal applied to the isolated depleted red blood cells is different from the identifiable signal applied to the depleted white blood cells.
9. Use of an antibody for isolating leukocytes from a blood sample when enriching circulating rare cells in the sample; the antibody is an antibody combination comprising: anti-CD 45 antibodies, anti-CD 14 antibodies, anti-CD 55 antibodies, anti-CD 66b antibodies, anti-CD 90 antibodies, anti-CD 33 antibodies, and anti-CD 36 antibodies.
10. A kit for enriching circulating rare cells from a blood sample, comprising a microfluidic detection device comprising: a microfluidic structure (7) including a single-cell channel (11) allowing a single cell to pass at a stable flow rate, and a plurality of single-cell channels being present to allow shunting of the single cell; the fluorescence detection component (5) is used for screening each cell according to different fluorescence signals, rapidly confirming the identity of each cell and judging whether the cell is sorted in the next step or not; the microjet generator (6) comprises a miniature metal conductor, receives the sorting signal of the fluorescence detection component (5) and initiates microheat bubbles;
an antibody carrying a recognisable signal; the antibody is an antibody combination comprising: anti-CD 45, anti-CD 14, anti-CD 55, anti-CD 66b, anti-CD 90, anti-CD 33, and anti-CD 36 antibodies; and
a fluorescent dye that labels nucleated cells.
11. The kit of claim 10, wherein the microfluidic detection device comprises:
a substrate (8);
a microjet generator (6) located on the substrate;
a microfluidic structure (7) located on the microjet generator (6);
a cover sheet (10) positioned on the microfluidic structure (7).
12. A kit according to claim 11, characterized in that the microjet generator (6) is present in a protective layer (9) between the substrate (8) and the microfluidic structure (7).
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