CN106635995B - Negative enrichment method for circulating tumor cells - Google Patents
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Abstract
The invention relates to the field of molecular biology, in particular to a negative enrichment method of circulating tumor cells. The density gradient centrifugation based on the lymphocyte separation tube is adopted to carry out pretreatment on the blood sample, so that the operation steps of separating leucocytes are simplified, the enrichment time of circulating tumor cells is shortened, and the whole operation can be finished within 2.5 hours; the number of the left white blood cells is 30-50% of the original number of the white blood cells of the blood sample, the left white blood cells are marked with self-made negative enrichment immunomagnetic beads CD45 and are removed, the using amount of the immunomagnetic beads is reduced, and the cost of negative enrichment of circulating tumor cells is greatly saved; the invention adopts the lymphocyte separation tube to pretreat the blood sample, improves the negative enrichment method of the circulating tumor cells, effectively improves the stability of enrichment operation, and ensures that the recovery rate of the circulating tumor cells reaches more than 85 percent; the invention also has the advantage of high sensitivity, and can stably detect the circulating tumor cells in a sample with extremely small quantity.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a negative enrichment method of circulating tumor cells.
Background
Circulating Tumor Cells (CTCs) are tumor cells that are released into the peripheral blood circulation from solid tumors or metastases, either spontaneously or as a result of medical procedures, and are the primary cause of distant metastasis in malignant patients. Metastasis is statistically a cause of death in more than 90% of tumor patients. In recent years, with the continuous improvement of related technologies of some basic disciplines, CTC detection is used as a novel noninvasive diagnosis tool, and compared with traditional tissue biopsy, the CTC detection has the advantages of being noninvasive, capable of being obtained for multiple times, capable of being monitored in real time and the like, so that the detection of CTC is vividly called as 'liquid biopsy', has important clinical research and application values, and has become a hot spot of clinical research. A number of clinical studies have demonstrated that: by detecting the circulating tumor cells, the early diagnosis, prognosis judgment, postoperative recurrence monitoring, individualized treatment, rapid evaluation of chemotherapeutic drugs, drug resistance monitoring and the like of various tumors can be effectively carried out.
A large number of clinical studies in recent 10 years show that the detection of the number of CTCs contributes to the prognostic evaluation of patients with colon cancer, liver cancer, breast cancer and prostate cancer; when the CTC has epithelial-mesenchymal transition (EMT) and over-expresses epithelial cell adhesion molecule (EpCAM), the prognosis of tumor patients is often poor,
clinical studies have shown that 90% of patients with early-stage cancer can be cured. However, due to the limitation of the conventional detection technology, the population for detecting cancer at the early stage only accounts for a few, the number of cancer patients at the early stage (stage I) in China accounts for about 5-10% of the total number of patients, 70% of the patients are detected at the late stage, and the 5-year survival rate of the patients is only 10-30%. The world health organization pointed out three principles of cancer prevention as early as 2006: early detection, early diagnosis and early treatment, and thus, the early detection is the key for preventing and treating the cancer. However, in the conventional detection means of tumors, such as CT or nuclear magnetic resonance examination, the diagnosis of tumors smaller than 5 mm is difficult, and pancreatic cancer and lung cancer are often found to be in middle and advanced stages after diagnosis. It has now been found that many tumors already can be examined in the blood for Circulating Tumor Cells (CTCs) at 1 mm. Therefore, the early detection of micrometastases not only has important significance for judging the recurrence and prognosis of the tumor, but also has great value for guiding clinical treatment. However, the clinical tumor discovery and diagnosis at present highly depends on the imaging and the examination of serum tumor markers, so that the metastasis or recurrence of the tumor is difficult to discover at an early stage, and the curative effect is difficult to reflect in time. Therefore, the conventional screening and diagnosis method for tumors cannot meet the clinical requirements, and a novel method for screening and diagnosing cancers, which is safe, high in accuracy and convenient to operate, is urgently needed in clinic.
Circulating tumor cells in the blood are present in lower amounts in the blood and generally need to be enriched beforeCan be effectively detected and analyzed. Since the cells in blood are of various types, it is not easy to identify tumor cells in blood with absolute accuracy. CTC is extremely rare in peripheral blood of tumor patients, about 1-10/mL, and the concentration of all blood cells in peripheral blood of normal adults is 109Per mL, the concentration of leukocytes is 4 to 10 × 106and/mL. The scarcity of cell numbers well below that of other types in blood makes CTCs extremely challenging in blood detection.
CTC detection typically involves two parts, CTC enrichment and CTC identification, usually by an enrichment step to remove the majority of normal cells, and then re-identifying the resulting cells as indeed CTCs. The enrichment method of CTC is mainly divided into two main categories of physical method and biochemical method. CTCs are identified primarily by cellular morphology or analysis at the nucleic acid molecular level.
The enrichment method of CTC is mainly divided into two main categories of physical method and biochemical method. The physical method is based on the difference of the physical properties of the tumor cells and normal cells, such as size, deformability, density, dielectricity and the like, and separation and capture are carried out by the action of external force fields, such as a magnetic field, a fluid field, an electric field and the like. Biochemical methods generally rely on the binding of an antigen on the surface of a cell membrane to an antibody coupled to a separation medium for separation and capture.
The enrichment technology applied to CTC at the present stage mainly comprises: centrifugation based on cell density, filtration membrane filtration based on cell volume (ISET), immunomagnetic bead (MACS), CTC-Chip microfluidic Chip technology, etc.
The existing density gradient centrifugation method has low specificity, low sensitivity, low recovery rate and low stability, is easy to cause the loss of tumor cells, depends on various factors such as the characteristics of the tumor cells, the centrifugation time, the temperature and the like, and has very limited future development potential as a single CTC separation means. Although the ISET method is simple and convenient to operate, low in price, high in flux, capable of simultaneously processing samples in large batches and short in time consumption, the possibility that specificity is lacked and tumor cells are missed (less than or equal to 8um) exists, and the ISET method cannot become a future development trend and trend.
The most extensive applications of CTC in the current circulating tumor cell enrichment are positive enrichment methods, including immunomagnetic bead positive enrichment and microfluidic chip positive enrichment.
The defects of applying the micro-fluidic chip to rare cell sorting mainly show that the micro-fluidic chip is long in time consumption, poor in effect and damaged in cell activity. In addition, flux and purity are often two mutually restricted indexes in most chips, and an increase in flux means a decrease in purity, and vice versa, and how to combine the two is another challenge in the development of microfluidic sorting technology. Finally, considering that the captured cells may contain different subtypes or different growth stages of the same type of cells, the subsequent phenotypic analysis has important significance for clinical diagnosis, which puts high demands on the activity of the captured cells. However, extrusion of microstructures in the micro-channels, a high-shear flow field, an external electric field, an external sound field and the like can cause certain damage to the activity of cells, and troubles are caused to subsequent analysis.
Chinese patent application No. 201110161224.7 discloses a reagent and a kit for quantitative analysis of circulating tumor cells, which can be used in conjunction with flow cytometry to obtain the measurement result of circulating tumor cells by designing a probe suitable for detecting circulating tumor cells.
The immunomagnetic bead method is the most widely applied CTC separation method at present, mainly refers to a positive enrichment method, has the advantages of simplicity, convenience, easy operation and high separation purity, but has the defects of low recovery rate and missed detection. Currently, a number of immunomagnetic bead CTC studies have been isolated on tumor epithelial antigens and EpCAM is considered to be the currently most optimal tumor biomarker, but there are studies that suggest that CTCs of most malignancies lose EpCAM antigen. This means that there is a possibility that this method will miss detection of invasive CTCs. In fact, it was found that only 70% of the 134 tumor cells with different tissue types expressed EpCAM, but there was a difference in the expression intensity. At present, there is no tumor biomarker with 100% specificity.
However, the circulating tumor cell positive enrichment method has the defects of lack of CTC specific markers, difficult monoclonal antibody screening, low recovery rate, omission (tumor cells which cannot be identified by the antibody are ignored) and the like, and the detection result is influenced. Because CTC lacks a specific marker, EpCAM (epithelial cell adhesion molecule) magnetic beads are generally adopted to mark and separate CTC at present, and instruments such as CellSearch and the like developed based on the principle are already put into the market to operate. However, this method failed to detect CTCs that express low and no EpCAM. It has been shown that isolation of CTCs based on this principle would underestimate their numbers and lose a subset of CTCs with high metastatic and proliferative potential. A typical example is that CTCs that undergo epithelial-to-mesenchymal transition (EMT) express little or no EpCAM subpopulations because of their high migratory capacity, which is considered to be one of the most prominent causes of metastasis.
Compared with the positive enrichment method of the immunomagnetic beads, the negative enrichment method of the CTC immunomagnetic beads has more advantages. The technology utilizes immunomagnetic beads combined with CD45 monoclonal antibodies to specifically combine with CD45 antigen (leukocyte common antigen) on the surface of leukocytes, carries out magnetic labeling on the leukocytes, removes the leukocytes through an external magnetic field, and enriches all subgroups and types of CTC independent of CTC surface markers. The CTC immunomagnetic bead negative enrichment technology has technical advantages in isolating all types of CTCs, while being expected to be useful for CTC enrichment of all types of solid tumors since it is independent of CTC surface markers.
Therefore, the development of a separation process that does not rely on CTC surface markers is a promising technology. On the other hand, the immunomagnetic bead method of the negative enrichment strategy indirectly enriches rare epithelial cells by effectively removing leukocytes of CD45+ or other cells of peripheral blood such as macrophages and platelets of CD61+, and overcomes the defect of the EpCAM positive enrichment strategy caused by EMT to some extent. In addition, the circulating tumor cells obtained by the negative enrichment strategy also have the advantages of no marker, high cell activity and the like.
As described above, although each of the various CTC isolation methods reported has unique detection advantages, problems such as low recovery rate of circulating tumor cells (70% -80%), no availability of active CTCs, difficulty in biomarker selection, complicated isolation techniques, etc. are still revealed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a negative enrichment method of circulating tumor cells, and solves the problems of low recovery rate of the circulating tumor cells, incapability of obtaining active circulating tumor cells, difficult selection of biomarkers and complex separation technology in the existing circulating tumor cell separation method.
In order to solve the technical problems, the invention adopts the technical scheme that: a method of negative enrichment of circulating tumor cells comprising the steps of:
s1: mixing density gradient separation liquid (Ficoll bag plus, GE Health) and blood sample of tumor patient in lymphocyte separation tube, adding washing buffer solution, and centrifuging for the first time;
s2: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube after the first centrifugation to a first centrifuge tube, performing second centrifugation, sucking the supernatant, adding erythrocyte lysate, and standing;
s3: centrifuging the first centrifuge tube after standing for the third time, sucking out the supernatant, adding a magnetic bead incubation buffer solution, adding negative enrichment immunomagnetic beads, uniformly mixing, and incubating for 20-40min, wherein the negative enrichment immunomagnetic beads are combined with a target leukocyte surface broad-spectrum marker CD 45;
s4: placing the incubated first centrifuge tube on a magnetic frame for standing, transferring all supernatant into a second centrifuge tube, and washing the second centrifuge tube with washing buffer solution;
s5: and (3) centrifuging the second centrifuge tube for the fourth time, sucking the supernatant of the second centrifuge tube, adding a cell fixing solution into the second centrifuge tube, resuspending, coating the cell fixing solution on a glass slide, and drying overnight to obtain the dry circulating tumor cells.
The invention has the beneficial effects that: the negative enrichment method of the circulating tumor cells disclosed by the invention adopts density gradient centrifugation on the basis of the lymphocyte separation tube to carry out pretreatment on a blood sample, simplifies the operation steps of separating white blood cells, shortens the enrichment time of the circulating tumor cells, and can finish the whole operation within 2.5 h; the number of the left white blood cells after treatment is 30-50% of the original number of the white blood cells of the blood sample, the left white blood cells are marked with the self-made negative enrichment immunomagnetic beads CD45 and are removed, the using amount of the immunomagnetic beads is reduced, and the cost of negative enrichment of circulating tumor cells is greatly saved; the invention adopts the lymphocyte separation tube to pretreat the blood sample, improves the negative enrichment method of the circulating tumor cells, effectively improves the stability of enrichment operation, and ensures that the recovery rate of the circulating tumor cells reaches more than 85 percent; the invention also has the advantage of high sensitivity, and can stably detect the circulating tumor cells in a sample with extremely small quantity.
Drawings
FIG. 1 is a histogram of the enrichment and recovery of circulating tumor cells by a negative enrichment method of circulating tumor cells and a conventional density gradient centrifugation method according to an embodiment of the present invention;
FIG. 2 is a bar graph of the enrichment and recovery rate of different types of tumor cells by a negative enrichment method for circulating tumor cells according to an embodiment of the present invention;
FIG. 3 is a diagram showing the results of immunofluorescence assay after enrichment of SKOV3 cells by a negative enrichment method for circulating tumor cells according to an embodiment of the present invention;
FIG. 4 is a bar graph of the mean recovery of various numbers of NCI-H226 circulating tumor cells by a method of negative enrichment of circulating tumor cells, in accordance with an embodiment of the present invention;
FIG. 5 is a linear regression plot of the recovery rates of various numbers of NCI-H226 circulating tumor cells by a method of negative enrichment of circulating tumor cells in accordance with an embodiment of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
Abbreviations and key term definitions to which the present invention relates:
CTC: circulating tumor cells;
CD 45: leukocyte surface common antigen;
CK-FITC: fluorescein isothiocyanate coupled cytokeratin antibodies;
CD 45-PE: phycoerythrin protein conjugated CD45 antibody;
DAPI: 4', 6-diamidino-2-phenylindole.
The most key concept of the invention is as follows: the density gradient centrifugation on the basis of the lymphocyte separation tube is adopted to carry out pretreatment on the blood sample, so that the leukocyte separation operation steps are simplified, the enrichment time of the circulating tumor cells is shortened, the left leukocytes are marked with leukocytes and removed by self-made negative enrichment immunomagnetic beads CD45, the dosage of the immunomagnetic beads is reduced, and the cost of the negative enrichment of the circulating tumor cells is greatly saved.
A method of negative enrichment of circulating tumor cells comprising the steps of:
s1: mixing density gradient separation liquid (Ficoll bag plus, GE Health) and blood sample of tumor patient in lymphocyte separation tube, adding washing buffer solution, and centrifuging for the first time;
s2: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube after the first centrifugation to a first centrifuge tube, performing second centrifugation, sucking the supernatant, adding erythrocyte lysate, and standing;
s3: centrifuging the first centrifuge tube after standing for the third time, sucking out the supernatant, adding a magnetic bead incubation buffer solution, adding negative enrichment immunomagnetic beads, uniformly mixing, and incubating for 20-40min, wherein the negative enrichment immunomagnetic beads are combined with a target leukocyte surface broad-spectrum marker CD 45;
s4: placing the incubated first centrifuge tube on a magnetic frame for standing, transferring all supernatant into a second centrifuge tube, and washing the second centrifuge tube with washing buffer solution;
s5: and (3) centrifuging the second centrifuge tube for the fourth time, sucking the supernatant of the second centrifuge tube, adding a cell fixing solution into the second centrifuge tube, resuspending, coating the cell fixing solution on a glass slide, and drying overnight to obtain the dry circulating tumor cells.
The negative enrichment principle of the circulating tumor cells of the invention is as follows: the invention discloses a detection method of circulating tumor cells, which mainly comprises the steps of removing red blood cells and a large number of neutrophils by using density gradient centrifugation, further removing residual red blood cells by red blood cell lysis, incubating obtained white blood cells with self-made negative enrichment immunomagnetic beads (targeted white blood cell surface broad spectrum marker CD45), removing white blood cells by magnetic field adsorption, collecting supernatant parts which are not combined with the magnetic beads, smearing after centrifugation, and air drying and flaking to obtain dry circulating tumor cells.
According to the negative enrichment method of the circulating tumor cells, the blood sample is pretreated by adopting density gradient centrifugation on the basis of the lymphocyte separation tube, so that the operation steps of separating white blood cells are simplified, the enrichment time of the circulating tumor cells is shortened, and the whole operation can be completed within 2.5 hours; the number of the left white blood cells is 30-50% of the original number of the white blood cells of the blood sample, the left white blood cells are marked with self-made negative enrichment immunomagnetic beads CD45 and are removed, the using amount of the immunomagnetic beads is reduced, and the cost of negative enrichment of circulating tumor cells is greatly saved; the invention adopts the lymphocyte separation tube to pretreat the blood sample, improves the negative enrichment method of the circulating tumor cells, effectively improves the stability of enrichment operation, and ensures that the recovery rate of the circulating tumor cells reaches more than 85 percent; the invention also has the advantage of high sensitivity, and can stably detect the circulating tumor cells in a sample with extremely small quantity.
Further, the S1 specifically includes: adding a density gradient separation solution into the lymphocyte separation tube, and performing instantaneous centrifugation to enable the density gradient separation solution to be completely centrifuged to the bottom of the tube; 3mL of tumor patient blood sample is added into a lymphocyte separation tube filled with density gradient separation liquid, more than 3mL of washing buffer solution is added, and the first centrifugation is carried out, wherein the first centrifugation condition is 25 ℃ and 800g/15 min.
Further, the S2 specifically includes: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube after the primary centrifugation into a first centrifuge tube, carrying out secondary centrifugation under the conditions of 25 ℃ and 300g/10min, sucking the supernatant, adding 5mL of erythrocyte lysate, and standing for 5 min.
Further, the S3 specifically includes: and (3) centrifuging the first centrifuge tube after standing for the third time, wherein the third centrifugation condition is 25 ℃ and 300g/10min, absorbing the supernatant, adding 5mL of magnetic bead incubation buffer solution, adding 200uL of negative enrichment immunomagnetic beads, blowing, uniformly mixing, vertically and uniformly mixing at a low speed of 4 ℃, incubating for 30min, and combining the negative enrichment immunomagnetic beads with a targeted leukocyte surface broad-spectrum marker CD 45.
Further, the S4 specifically includes: and (3) placing the incubated first centrifuge tube on a magnetic frame, standing for 5min, completely transferring the supernatant into a second centrifuge tube, and washing the second centrifuge tube for 2 times by using a washing buffer solution.
Further, the S5 specifically includes: and (3) centrifuging the second centrifuge tube for the fourth time, sucking the supernatant until the residual volume is 100uL, adding 100uL of cell fixing solution into the second centrifuge tube, resuspending, coating the cell fixing solution on a glass slide, and drying overnight at 25 ℃ to obtain dry circulating tumor cells, wherein the centrifugation for the fourth time is performed at 25 ℃ and 600g/5 min.
Further, the negative enrichment method of the circulating tumor cells further comprises the following steps:
s6: dried circulating tumor cells were identified and fluorescently counted using multicolor immunofluorescence techniques.
Further, the dye adopted by the multicolor immunofluorescence technology is CK-FITC, CD45-PE or DAPI.
Comparative example 1
A certain amount of SiHa cells are mixed in peripheral blood of a healthy person to simulate a blood sample containing circulating tumor cells of a cervical cancer patient, and the blood sample is diluted in proportion to obtain 2 x 104The cell suspension is accurately counted under a fluorescence microscope and adjusted to 100/5 uL, the counted cell suspension is mixed into 3mL of peripheral blood to respectively prepare peripheral blood samples containing 100 SiHa cells/3 mL, the blood samples are taken as the blood samples of tumor patients, and after the blood samples are respectively treated according to the following three blood pretreatment schemes (A, red blood cell lysis; B, density gradient centrifugation; C, red blood cell lysis + density gradient centrifugation), the steps of the embodiment 1 are respectively carried out to carry out negative enrichment on circulating tumor cells; the erythrocyte removal efficiency and the leukocyte removal efficiency were evaluated.
The erythrocyte removal efficiency and the leukocyte removal efficiency of the different blood pretreatment protocols are shown in table 1.
TABLE 1 erythrocyte and leukocyte depletion efficiencies of different blood pretreatment protocols
| Blood pretreatment protocol | Efficiency of erythrocyte removal | Efficiency of leukocyte depletion |
| Erythrocyte lysis | ～98～99.5％ | >99.5％ |
| Density gradient centrifugation | >99.5％ | ～99.7-99.9％ |
| Erythrocyte lysis + density gradient centrifugation | >99.99％ | ～99.7-99.9％ |
As shown in Table 1, the erythrocyte removal efficiency and the leukocyte removal efficiency are highest by adopting a pretreatment mode of erythrocyte lysis and density gradient centrifugation.
Example 1
A method of negative enrichment of circulating tumor cells comprising the steps of:
s1: adding a density gradient separation solution into the lymphocyte separation tube, and performing instantaneous centrifugation to enable the density gradient separation solution to be completely centrifuged to the bottom of the tube; adding 3mL of tumor patient blood sample into a lymphocyte separation tube filled with density gradient separation liquid, adding more than 3mL of washing buffer solution, and carrying out primary centrifugation under the conditions of 25 ℃ and 800g/15 min;
s2: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube subjected to the first centrifugation to a first centrifuge tube, carrying out second centrifugation under the conditions of 25 ℃ and 300g/10min, sucking the supernatant, adding 5mL of erythrocyte lysate, and standing for 5 min;
s3: centrifuging the first centrifuge tube after standing for the third time, wherein the centrifugation condition for the third time is 25 ℃ and 300g/10min, absorbing the supernatant, adding 5mL of magnetic bead incubation buffer solution, adding 200uL of negative enrichment immunomagnetic beads, blowing, uniformly mixing, vertically and uniformly mixing at a low speed of 4 ℃, and incubating for 30min, wherein the negative enrichment immunomagnetic beads are combined with a targeted leukocyte surface broad-spectrum marker CD 45;
s4: placing the incubated first centrifuge tube on a magnetic frame, standing for 5min, transferring all the supernatant into a second centrifuge tube, and washing the second centrifuge tube for 2 times by using a washing buffer solution;
s5: centrifuging the second centrifuge tube for the fourth time, sucking out the supernatant until the residual volume is 100uL, adding 100uL of cell fixing solution into the second centrifuge tube, re-suspending, coating the cell fixing solution on a glass slide, and drying at 25 ℃ overnight to obtain dry circulating tumor cells, wherein the centrifugation for the fourth time is at 25 ℃ and 600g/5 min;
s6: the dried circulating tumor cells were identified and fluorescently counted using multicolor immunofluorescence using CK-FITC, CD45-PE or DAPI.
Example 2
Referring to fig. 1, a certain amount of SiHa cells are doped into peripheral blood of a healthy person to simulate a blood sample containing circulating tumor cells of a cervical cancer patient, and the enrichment efficiency of the established circulating tumor cell negative enrichment method on the circulating tumor cells is evaluated, and the specific monitoring steps are as follows:
1. cultured and digested SiHa cells were pre-labeled with Green fluorescence using Celltracker Green as describedProportional dilution to obtain 2X 104The cell suspension is accurately counted under a fluorescence microscope and adjusted to 100/5 uL;
2. the counted cell suspension is mixed into 3mL of peripheral blood to respectively prepare peripheral blood samples containing 100 SiHa cells/3 mL;
3. performing negative enrichment of circulating tumor cells on a peripheral blood sample containing 100 SiHa cells/3 mL respectively according to the following two schemes;
A. traditional density gradient centrifugation; B. lymphocyte separation tube density gradient centrifugation (modified scheme)
4. Peripheral blood samples were enriched and tested using the circulating tumor negative enrichment method of example 1, and the enrichment efficiency of the circulating tumor cell negative enrichment method was evaluated.
As shown in figure 1, the recovery rate of SiHa cells obtained by adopting a lymphocyte separation tube density gradient centrifugation combined with a circulating tumor negative enrichment method reaches 89%, and is remarkably higher than that of SiHa cells obtained by a traditional density gradient centrifugation method by 76%.
Example 3
Referring to fig. 2 and fig. 3, a certain amount of ovarian cancer SKOV-3, cervical cancer SiHa cells and breast cancer SKBR3 cells are doped into peripheral blood of a healthy person, and whether the circulating tumor cell negative enrichment method according to the scheme is suitable for circulating tumor cell detection of different tumor types is evaluated, and the specific detection steps are as follows:
1. in this example, 100 ovarian cancer SKOV-3, cervical cancer SiHa cells, breast cancer SKBR3 cells were incorporated into peripheral blood of healthy people to simulate blood samples containing circulating tumor cells from different tumor patients, cells of different tumor types obtained by culturing and digesting were pre-labeled with Green fluorescence using cellmarker Green, and cell suspensions of different concentrations were obtained by dilution in proportion, and then accurately counted under a fluorescence microscope.
2. The counted cell suspension was spiked into 3mL of peripheral blood to prepare 100 different types of tumor cells per 3mL of peripheral blood sample.
3. Peripheral blood samples were enriched and tested using the circulating tumor negative enrichment method of example 1.
As shown in FIG. 2, the recovery rates of different types of tumor cells are all over 85% by adopting the negative enrichment method of circulating tumor.
Example 4
Referring to FIG. 4, the method for enriching circulating tumor cells of example 1 was used to evaluate the enriching efficiency of circulating tumor cells by using the blood sample obtained by doping different numbers of NCI-H226 cells into peripheral blood of healthy people, and the specific detection steps are as follows:
1. in this example, non-small cell lung cancer NCI-H226 cells were used to simulate a blood sample containing circulating tumor cells from a patient with lung cancer. The NCI-H226 cells obtained by culture and digestion are pre-labeled by green fluorescence with CelltrackerGreen, and are diluted in proportion to obtain cell suspensions with different concentrations, and the cell suspensions are accurately counted under a fluorescence microscope.
2. The counted cell suspensions were spiked into 3ml of peripheral blood to prepare peripheral blood samples containing 6, 9, 15, 30, 90, 300 NCI-H226 cells/3 ml, respectively.
3. By adopting the negative enrichment kit for the circulating tumor cells disclosed by the invention, the circulating tumor cells in the peripheral blood sample are enriched by using the method, after air drying and flaking, the green fluorescent cells of CellTracker + are counted, and the enrichment efficiency and sensitivity of the negative enrichment method for the circulating tumor cells are evaluated.
From fig. 4, the recovery rates of circulating tumor cell enrichment of peripheral blood samples without using number of NCI-H226 cells by the circulating tumor cell negative enrichment method of the present invention reach higher levels, wherein the average recovery rates of peripheral blood samples containing 9, 15, and 30 NCI-H226 cells/3 ml are all above 85%, and from fig. 5, the overall recovery rate of the overall enrichment of circulating tumor cells with different numbers is 77.66%, which indicates that the circulating tumor cell enrichment method of the present invention has higher sensitivity.
In conclusion, the method for negatively enriching the circulating tumor cells, provided by the invention, has the advantages that the blood sample is pretreated by adopting density gradient centrifugation on the basis of the lymphocyte separation tube, the operation steps of separating the white blood cells are simplified, the enriching time of the circulating tumor cells is shortened, and the whole operation can be completed within 2.5 hours; the number of the left white blood cells is 30-50% of the original number of the white blood cells of the blood sample, the left white blood cells are marked with self-made negative enrichment immunomagnetic beads CD45 and are removed, the using amount of the immunomagnetic beads is reduced, and the cost of negative enrichment of circulating tumor cells is greatly saved; the invention adopts the lymphocyte separation tube to pretreat the blood sample, improves the negative enrichment method of the circulating tumor cells, effectively improves the stability of enrichment operation, and ensures that the recovery rate of the circulating tumor cells reaches more than 85 percent; the invention also has the advantage of high sensitivity, and can stably detect the circulating tumor cells in a sample with extremely small quantity.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (9)
1. A method for negative enrichment of circulating tumor cells, comprising the steps of:
s1: mixing the density gradient separation liquid and a blood sample of a tumor patient in a lymphocyte separation tube, adding a washing buffer solution, and performing primary centrifugation;
s2: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube after the first centrifugation to a first centrifuge tube, performing second centrifugation, sucking the supernatant, adding erythrocyte lysate, and standing;
s3: centrifuging the first centrifuge tube after standing for the third time, sucking out the supernatant, adding a magnetic bead incubation buffer solution, adding negative enrichment immunomagnetic beads, uniformly mixing, and incubating for 20-40min, wherein the negative enrichment immunomagnetic beads are combined with a target leukocyte surface broad-spectrum marker CD 45;
s4: placing the incubated first centrifuge tube on a magnetic frame for standing, transferring all supernatant into a second centrifuge tube, and washing the second centrifuge tube with washing buffer solution;
s5: and (3) centrifuging the second centrifuge tube for the fourth time, sucking the supernatant of the second centrifuge tube, adding a cell fixing solution into the second centrifuge tube, resuspending, coating the cell fixing solution on a glass slide, and drying overnight to obtain the dry circulating tumor cells.
2. The method of claim 1, further comprising the steps of:
s6: dried circulating tumor cells were identified and fluorescently counted using multicolor immunofluorescence techniques.
3. The method for negative enrichment of circulating tumor cells according to claim 1, wherein S1 is specifically: adding a density gradient separation solution into the lymphocyte separation tube, and performing instantaneous centrifugation to enable the density gradient separation solution to be completely centrifuged to the bottom of the tube; 3mL of tumor patient blood sample is added into a lymphocyte separation tube filled with density gradient separation liquid, more than 3mL of washing buffer solution is added, and the first centrifugation is carried out, wherein the first centrifugation condition is 25 ℃ and 800g/15 min.
4. The method for negative enrichment of circulating tumor cells according to claim 1, wherein S2 is specifically: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube after the primary centrifugation into a first centrifuge tube, carrying out secondary centrifugation under the conditions of 25 ℃ and 300g/10min, sucking the supernatant, adding 5mL of erythrocyte lysate, and standing for 5 min.
5. The method for negative enrichment of circulating tumor cells according to claim 1, wherein S3 is specifically: and (3) centrifuging the first centrifuge tube after standing for the third time, wherein the third centrifugation condition is 25 ℃ and 300g/10min, absorbing the supernatant, adding 5mL of magnetic bead incubation buffer solution, adding 200uL of negative enrichment immunomagnetic beads, blowing, uniformly mixing, vertically and uniformly mixing at a low speed of 4 ℃, incubating for 30min, and combining the negative enrichment immunomagnetic beads with a targeted leukocyte surface broad-spectrum marker CD 45.
6. The method for negative enrichment of circulating tumor cells according to claim 1, wherein S4 is specifically: and (3) placing the incubated first centrifuge tube on a magnetic frame, standing for 5min, completely transferring the supernatant into a second centrifuge tube, and washing the second centrifuge tube for 2 times by using a washing buffer solution.
7. The method for negative enrichment of circulating tumor cells according to claim 1, wherein S5 is specifically: and (3) centrifuging the second centrifuge tube for the fourth time, sucking the supernatant until the residual volume is 100uL, adding 100uL of cell fixing solution into the second centrifuge tube, resuspending, coating the cell fixing solution on a glass slide, and drying overnight at 25 ℃ to obtain dry circulating tumor cells, wherein the centrifugation for the fourth time is performed at 25 ℃ and 600g/5 min.
8. The method of claim 2, wherein the multicolor immunofluorescence technique employs a dye that is CK-FITC, CD45-PE, or DAPI.
9. The method of claim 1, comprising the steps of:
s1: adding a density gradient separation solution into the lymphocyte separation tube, and performing instantaneous centrifugation to enable the density gradient separation solution to be completely centrifuged to the bottom of the tube; adding 3mL of tumor patient blood sample into a lymphocyte separation tube filled with density gradient separation liquid, adding more than 3mL of washing buffer solution, and carrying out primary centrifugation under the conditions of 25 ℃ and 800g/15 min;
s2: transferring the upper layer solution of the baffle plate in the lymphocyte separation tube subjected to the first centrifugation to a first centrifuge tube, carrying out second centrifugation under the conditions of 25 ℃ and 300g/10min, sucking the supernatant, adding 5mL of erythrocyte lysate, and standing for 5 min;
s3: centrifuging the first centrifuge tube after standing for the third time, wherein the centrifugation condition for the third time is 25 ℃ and 300g/10min, absorbing the supernatant, adding 5mL of magnetic bead incubation buffer solution, adding 200uL of negative enrichment immunomagnetic beads, blowing, uniformly mixing, vertically and uniformly mixing at a low speed of 4 ℃, and incubating for 30min, wherein the negative enrichment immunomagnetic beads are combined with a targeted leukocyte surface broad-spectrum marker CD 45;
s4: placing the incubated first centrifuge tube on a magnetic frame, standing for 5min, transferring all the supernatant into a second centrifuge tube, and washing the second centrifuge tube for 2 times by using a washing buffer solution;
s5: centrifuging the second centrifuge tube for the fourth time, sucking out the supernatant until the residual volume is 100uL, adding 100uL of cell fixing solution into the second centrifuge tube, re-suspending, coating the cell fixing solution on a glass slide, and drying at 25 ℃ overnight to obtain dry circulating tumor cells, wherein the centrifugation for the fourth time is at 25 ℃ and 600g/5 min;
s6: the dried circulating tumor cells were identified and fluorescently counted using multicolor immunofluorescence using CK-FITC, CD45-PE or DAPI.
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| CN107326045A (en) * | 2017-07-20 | 2017-11-07 | 杭州京北生物科技有限公司 | A kind of plasmid and the detection screening technique using the plasmid for detecting screening circulating tumor cell |
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| CN107656044A (en) * | 2017-09-25 | 2018-02-02 | 亚能生物技术（深圳）有限公司 | The detection kit and detection method of a kind of circulating tumor cell |
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| CN110456077A (en) * | 2019-09-12 | 2019-11-15 | 太仓极光克赛生物技术有限公司 | A method of the immunomagnetic beads enrichment detection CTCs of coupling anti-EpCAM antibody |
| CN112881647A (en) * | 2021-01-12 | 2021-06-01 | 何惠端 | Preparation method of quality control sample by combining CTC negative enrichment method with imFISH detection technology |
| CN114964996A (en) * | 2022-06-10 | 2022-08-30 | 江苏格诺生物科技有限公司 | Method for negatively enriching circulating tumor cells by using immunomagnetic beads |
| CN115125212A (en) * | 2022-06-17 | 2022-09-30 | 成都诺医德医学检验实验室有限公司 | Peripheral blood circulation tumor cell organoid culture method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087775A (en) * | 2015-07-24 | 2015-11-25 | 北京莱尔生物医药科技有限公司 | Method and related kit for detecting c-MET/CEP7 gene status based on rare cells |
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
-
2017
- 2017-03-10 CN CN201710140338.0A patent/CN106635995B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
| CN105087775A (en) * | 2015-07-24 | 2015-11-25 | 北京莱尔生物医药科技有限公司 | Method and related kit for detecting c-MET/CEP7 gene status based on rare cells |
Non-Patent Citations (2)
| Title |
|---|
| 利用微流控免疫磁珠分选仪高效富集外周血循环肿瘤细胞;邱林等;《第三军医大学学报》;20160330;第38卷(第6期);第634-637页 * |
| 肝细胞癌循环肿瘤细胞与肿瘤转移和预后关系的研究;王真等;《药学实践杂志》;20150325;第33卷(第2期);第159-162页 * |
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