CN115678853A - Urine circulating tumor cell collection method and system - Google Patents
Urine circulating tumor cell collection method and system Download PDFInfo
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- CN115678853A CN115678853A CN202211422906.3A CN202211422906A CN115678853A CN 115678853 A CN115678853 A CN 115678853A CN 202211422906 A CN202211422906 A CN 202211422906A CN 115678853 A CN115678853 A CN 115678853A
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Abstract
The invention provides a urine circulating tumor cell collecting and detecting method, which comprises the steps of obtaining a urine sample; carrying out cell capture on the urine sample to obtain undetermined circulating tumor cells; staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells; extracting the stained undetermined circulating tumor cells, and performing CCD scanning; and judging positive circulating tumor cells according to the scanning image and storing the scanning image. Can realize the storage and the continuous detection to large capacity urine sample through this application, and guaranteed the activity of cell in the large capacity urine sample, further can obtain more accurate testing result based on the continuous detection of large capacity sample, improve circulating tumor cell's positive rate, reduce false negative rate.
Description
Technical Field
The application relates to the field of medical equipment, in particular to a urine circulating tumor cell collection and detection method and system.
Background
With the continuous progress of society and the rapid development of economy, people no longer pay attention to whether the people are full of heat and solve the survival problem, but gather more eyes on health and safety.
In health and safety, the disease is the most easily overlooked, wherein the incidence of urinary tract tumors, such as bladder cancer, urothelial cancer, kidney cancer, etc., is increasing year by year, and the reasons for the disease are as follows: 1. for the work factor, the patient needs to be exposed to aromatic substances such as dyes, leather, rubber, paint, etc. for a long time; 2. in special habits, patients have habit or behavior of smoking for a long time, and smoking is one of the main pathogenic factors of urinary tract tumors; 3. in physical factors, the metabolism of the body of some patients is abnormal, and tryptophan substances cannot be metabolized normally, so that the synthesis of RNA and DNA of cells is influenced, and thus metabolites are excreted into the bladder under the action of the liver, and urinary tract tumors are easily induced; 4. for other reasons, similar to drug stimulation, it can induce the development of urinary tract tumors, because of certain conditions of the body, which require large doses of phenacetin drugs. The above factors are the main reasons for the annual increase of the incidence rate of the urinary tract tumor, so the early discovery and early treatment are the key to obtain a good prognosis of the tumor.
At present, the main targets for detecting Circulating Tumor Cells (CTCs) and circulating tumor DNA (ctDNA) are blood samples, but the blood samples need to be detected in professional medical institutions by using related medical technologies, and are monitored by follow-up visits at intervals of 3-6 months, while the tumor recurrence risk of devices at intervals of 3-6 months cannot be effectively monitored, so that monitoring samples and means capable of performing high-frequency and simple detection are needed. The urine detection can well replace blood sample detection due to the characteristics of no wound and easy sample acquisition, and becomes an important early warning index for early screening of the urinary tract tumor and a follow-up index after treatment. Furthermore, urine samples can be collected in large quantities, so that the urine samples become ideal samples for detecting CTCs, but at present, the most common urine detection is conventional urine detection, which is disposable urine small-volume detection, and the disposable urine detection with small volume cannot accurately obtain detection and monitoring results of tumors in urinary tracts.
Disclosure of Invention
In view of this, the present invention provides a method and a system for collecting urine circulating tumor cells, which aims to achieve the collection of large-volume samples, improve the accuracy of urinary tract tumor detection, and reduce the false negative rate.
In order to solve the above technical problems, the technical solution of the present invention is to provide a method and a system for collecting urine circulating tumor cells, comprising:
obtaining a urine sample;
carrying out cell capture on the urine sample to obtain undetermined circulating tumor cells;
staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells;
extracting the stained undetermined circulating tumor cells, and performing CCD scanning;
and judging positive circulating tumor cells according to the scanning image and storing the scanning image.
Optionally, the obtaining a urine sample comprises:
taking all urine collected by the urine collector within 24 hours as a urine sample;
and (4) carrying out cold storage and fresh keeping treatment on the urine sample collected within 24 hours by using a cold storage device.
Optionally, the performing cell capture on the urine sample includes:
and performing cell capture on the urine sample by using an enrichment device loaded with a capture ligand.
Optionally, the extracting the stained undetermined circulating tumor cell and performing CCD scanning include:
extracting the dyed tumor cells to be determined and circulating, and exciting color light of the dyed tumor cells to be determined and circulating;
and scanning the to-be-determined circulating tumor cells after the color light is excited by utilizing the frozen CCD scanner to obtain a to-be-determined circulating tumor cell scanning map.
Optionally, the determining positive circulating tumor cells from the scan and storing the scan comprises:
judging the cell population which presents the corresponding color light of the positive circulating tumor cells on the scanning image as the positive circulating tumor cells;
storing the scan in a database.
In addition, this application still provides a urine circulating tumor cell collecting and examining system, includes:
the sample collection module is used for obtaining a urine sample;
the cell enrichment module is used for capturing cells of the urine sample to obtain to-be-determined circulating tumor cells, and staining the to-be-determined circulating tumor cells to obtain stained to-be-determined circulating tumor cells;
the cell detection module is used for extracting the dyed undetermined circulating tumor cells and performing CCD scanning;
and the database module is used for judging positive circulating tumor cells according to the scanning image and storing the scanning image.
Optionally, the sample collection module comprises a urine storage unit, a refrigeration unit, a blending unit, a suction unit, a sterilization unit and an electromagnetic valve control unit,
wherein, urine storage unit is used for the storage of urine, cold-stored unit is used for guaranteeing the fresh degree of sample, the mixing unit is used for controlling the evenly distributed of sample, the suction unit is arranged in carrying the sample to the cell enrichment module, the disinfection and isolation unit is used for the washing of sample collection module, solenoid valve control unit is used for controlling the switch of solenoid valve.
Optionally, the cell enrichment module comprises a capture unit and a staining unit,
the capturing unit is used for capturing the tumor cells to be circulated, and the staining unit is used for staining the tumor cells to be circulated.
Optionally, the cell detection module comprises a fluorescence light-emitting unit, an automatic focusing unit, a freezing CCD scanning unit, and a data storage unit,
the fluorescence light-emitting unit is used for exciting color light of the stained tumor cells to be circulated, the automatic focusing unit is used for automatically focusing the freezing CCD scanning unit, the freezing CCD scanning unit is used for generating a scanned image of the tumor cells to be circulated, and the data storage unit is in communication connection with the database module and used for storing the scanned image of the tumor cells to be circulated.
The invention provides a urine circulating tumor cell collecting and detecting method, which comprises the steps of obtaining a urine sample; carrying out cell capture on the urine sample to obtain undetermined circulating tumor cells; staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells; extracting the stained undetermined circulating tumor cells, and performing CCD scanning; and judging positive circulating tumor cells according to the scanning image and storing the scanning image. Through the application, the storage and continuous detection of the large-capacity urine sample can be realized, the activity of cells in the large-capacity urine sample is ensured, a more accurate detection result can be obtained based on the continuous detection of the large-capacity sample, the positive rate of circulating tumor cells is improved, and the false negative rate is reduced.
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In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of steps of a urine circulating tumor cell collection method according to an embodiment of the present disclosure;
FIG. 2 is a schematic structural diagram of a urine circulating tumor cell collecting system according to an embodiment of the present disclosure;
FIG. 3 is a schematic structural diagram of a collection module in a urine circulating tumor cell collecting system according to an embodiment of the present disclosure;
fig. 4 is a schematic structural diagram of a cell enrichment module in a urine circulating tumor cell collecting system according to an embodiment of the present disclosure.
Detailed Description
In order to make those skilled in the art better understand the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The utility model discloses a domestic closestool rudiment based on, carry out the design of relevant system and method, make the closestool possess urine CTCs detection function, aim at can realize at home that high frequency, simple and convenient detection CTCs in the urine, ctDNA, be applied to the follow-up visit of urinary tract tumour, the problem phenomenon that the urine detection that meets in the prior art needs go to the hospital to detect convenient, not high frequency is got rid of, utilize more general closestool design to let urine CTCs detect, follow-up visit cycle shorten, effectively carry out prevention and management and control of urinary tract disease, the following will explain in detail a urine circulation tumour cell inspection method and system that realizes based on domestic closestool rudiment with the mode that the method combines with the system.
Referring to fig. 1, a schematic step diagram of a method for collecting urine circulating tumor cells according to an embodiment of the present application is shown.
S11, obtaining a urine sample.
The urine sample is extracted and is different from the small-volume sample collected by the existing disposable small-sample urine collector in the embodiment, and based on the collection module provided by the application, the embodiment can realize the collection of all urine samples within 24 hours and can ensure the freshness of the urine samples.
The collection module provided by the embodiment specifically comprises a storage unit, a refrigeration unit and a blending unit, wherein the storage unit is designed to be a storage container with a volume of 3000-5000ml, scientific data show that urine excreted by a normal human body or a urinary patient in one day is 300-4000ml, the storage unit can contain the whole amount of urine excreted by the human body in 24 hours in one day, the refrigeration unit is further used for controlling the space temperature of the storage unit, the space temperature of the storage unit is controlled to be 4-8 ℃, the urine in the storage unit is ensured to be in a refrigeration state at all times, the blending unit is used for enabling urine samples to be uniformly distributed, the refrigeration unit is matched to ensure the freshness of the urine samples, and the collection of large-capacity urine samples is realized.
In the practical operation process, the following detailed description is made with reference to fig. 3:
the utility model provides a collection module is except storage unit (1), cold-stored unit (2) and mixing unit (3), still include suction unit (4), disinfection and sterilization unit (5) and solenoid valve the control unit (9), collection module sets up in domestic closestool runner below, connect the runner mouth, set up solenoid valve (10) and solenoid valve (11) respectively, solenoid valve (10) set up in daily urine and excrement and urine discharge passage entrance, solenoid valve (11) set up in collection module entrance, when need not to collect the urine sample, only be used for daily domestic closestool normal use, close solenoid valve (11), open solenoid valve (10), excrement and urine and (or) urine are direct to be discharged through daily urine and excrement and urine discharge passage, when the urine sample is collected to needs, close solenoid valve (10), open solenoid valve (11), make the urine get into storage unit through collection module entrance and store in, domestic normal excretion and the mutual conflict of sample collection can be realized to such structural design, collection module's practicality and suitability have been improved.
Furthermore, the suction unit consists of a Z-shaped pipeline (7) and a suction pump (6), the Z-shaped pipeline is connected with the collection module and the cell enrichment module, an opening at one end of the Z-shaped pipeline connected with the collection module is arranged at the middle position of the storage unit, an electromagnetic valve (13) is arranged at the opening, a suction pump is arranged at one end of the Z-shaped pipeline connected with the cell enrichment module, when cell enrichment is carried out, the suction pump provides suction force to suck the urine sample in the storage unit into the cell enrichment module, when sample collection is finished and sample detection is needed, only the electromagnetic valve (13) is opened, all the other electromagnetic valves are closed, and the urine sample in the storage unit is sucked into the Z-shaped pipeline by the suction pump and then enters the cell enrichment module.
Further, after the urine sample is completely sucked, the storage unit needs to be cleaned, recycling is achieved, the electromagnetic valve (12) is arranged at an inlet of the cleaning pipeline and used for controlling the cleaning liquid and the sterile water for cleaning the pipeline to enter and discharging the waste liquid, when the storage unit needs to be cleaned, only the electromagnetic valve (12) is opened, the rest of the electromagnetic valves are closed, the cleaning liquid and the sterile water are injected from the cleaning pipeline, the storage unit after the sample is collected is sterilized by the aid of the sterilizing unit through ultrasonic cleaning (8) and ultraviolet sterilization, three-wheel cleaning is needed, the cleaning liquid is injected during the first round of cleaning, the sterile water is injected during the second round of cleaning and the third round of cleaning, the electromagnetic valve (12) is opened after each round of cleaning, and the waste liquid is discharged. The recycling of the collection module is realized through the design of the disinfection and sterilization unit, and the practicability of the collection module is enhanced.
S12, performing cell capture on the urine sample to obtain the tumor cells to be determined.
And S13, staining the circulating tumor cells to be determined to obtain the stained circulating tumor cells to be determined.
In this embodiment, when cell capture is performed on a urine sample, considering that the urine sample collected in this embodiment is a large-volume sample, which is different from a small-volume sample collected in a conventional disposable small sample container, for continuous detection of the large-volume sample, the cell enrichment module designed based on a microfluidic chip is proposed in this embodiment, and CTCs are enriched by using two physical and antibody capture modes, and epithelial cells in urine are restricted from passing through different size pores designed in the cell enrichment module, so as to further enhance the antibody capture efficiency, as shown in fig. 4, the cell enrichment module is a schematic structural diagram of the cell enrichment module proposed in this embodiment, and includes 6 sample flow channels, each of which includes an "inlet (111)", "outlet (112)", "shunt pipeline (113)", "fish scale" cell sieve (114) "," sample flow guide column (115) "," sample flow guide plate (116) "," large flow guide column (117) "," small flow column (118) "structure, so as to realize screening of the large-throughput sample, and the cell enrichment efficiency is reduced in the sample enrichment module.
The fish scale cell sieve array and the sample guide plate array are arranged in the sample flow channel (119) along the flow direction, the fish scale cell sieve is of a main structure for enriching CTCs, the sample guide plate has the functions of shunting and reducing the flow velocity in the flow channel, and all the sample flow channels are arranged in parallel and have the same size.
The cell enrichment module comprises a capturing unit and a staining unit, wherein the capturing unit is a capturing ligand which is loaded on the inner surface of the cell enrichment module and the surface of a fish scale cell sieve and is combined with a target component in a fluid, in the embodiment, the capturing ligand is a commercial streptavidin-biotin-EPCAM-GFP complex and is used for capturing CTCs (cytokine-induced killer) cells expressing EPCAM antigens in a sample as undetermined CTCs, and the staining unit is used for staining the undetermined CTCs captured in the capturing unit by adding CD45 and CK8/18 fluorescent antibodies.
The specific practical process is that, when enriching the sample, the suction pump provides power, simultaneously the suction pump can adjust the flow rate and pressure passing through the cell enrichment module, the urine sample enters the shunt pipeline through the inlet, then the sample flow rate is inhibited by the sample guide column to enter the sample flow channel, when passing through the scale cell sieve array, the CTCs cells in the urine sample can be captured by the prefabricated capture ligand loaded on the surface of the scale cell sieve, other liquids except the captured CTCs cells continue to enter the next layer of scale cell sieve through the sample guide plate until the sample flow channel flows out, at this time, the CTCs cells in the original urine sample are captured, the residual liquid flows to the cell enrichment module outlet after the flow rate is reduced by the flow column as waste liquid, the residual liquid flows out through the discharge channel, the waste liquid is discharged to daily urine and feces discharge channel, in the sample enrichment process, the solenoid valve (120) is in a whole-course open state, after the sample enrichment is finished, the waste liquid is completely discharged, all the solenoid valves are closed, the cell enrichment module is washed by using physiological saline, and CD45 and the CK8/18 fluorescent antibody is added through the inlet to dye the cells captured in the cell enrichment module after the CTCs for 30 minutes, and then the physiological saline is washed and the physiological saline for 3 times.
And S14, extracting the stained undetermined circulating tumor cells, and scanning by a CCD.
In this embodiment, the cell detection module performs fluorescence scanning on the tumor cells to be circulating captured by the capture unit in the cell enrichment module, so as to determine the tumor cells with positive circulation.
The cell detection module comprises a fluorescence light-emitting unit, an automatic focusing unit, a freezing CCD scanning unit and a data storage unit, wherein the fluorescence light-emitting unit is used for exciting color light of tumor cells to be circulated, the freezing CCD scanning unit is used for scanning the tumor cells to be circulated, the color light of the tumor cells to be circulated is excited, the automatic focusing unit is used for automatically focusing when the CCD scanner scans to increase imaging definition, and the data storage unit is in communication connection with the database module and used for storing a scanning image generated by the freezing CCD scanning unit.
The method comprises the specific operation steps that after sample cells are enriched, the cell enrichment module enriched with the cells is transmitted to a cell detection module, a fluorescence light-emitting unit is used for exciting color light of the tumor cells to be determined in circulation, imaging focusing before scanning is achieved through an automatic focusing unit, then a freezing CCD scanning unit is used for scanning the tumor cells to be determined in circulation, a generated scanning image is stored in a data storage unit, and finally the scanning image in the data storage unit is transmitted to a database module in communication connection with the data storage unit.
And S15, judging positive circulating tumor cells according to the scanning image and storing the scanning image.
In this embodiment, the database module is in communication connection with a data storage unit in the cell detection module and is used for data transmission, when the database module receives a scanogram of undetermined tumor cells transmitted by the data storage unit, the tumor cells undetermined in the scanogram are determined, and an EPCAM (+), CD45 (-), CK8/18 (+) cell group is determined as a positive CTCs cell, and after the positive circulating tumor cells are determined, the database module backups and stores the scanogram and analysis data, so that follow-up access and calling are facilitated.
In this embodiment, the solenoid valve control unit is configured to control the opening and closing of all the solenoid valves, including the solenoid valve (10), the solenoid valve (11), the solenoid valve (12), the solenoid valve (13), and the solenoid valve (120).
The application provides a urine circulating tumor cell collecting and detecting method, which comprises the steps of obtaining a urine sample; carrying out cell capture on the urine sample to obtain undetermined circulating tumor cells; staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells; extracting the stained undetermined circulating tumor cells, and performing CCD scanning; and judging positive circulating tumor cells according to the scanning image and storing the scanning image. The urine circulating tumor cell collecting system provided by the embodiment undoubtedly has the capabilities of collecting and storing samples with larger capacity, capturing and enriching CTCs with higher efficiency, and greatly reducing false negative results and improving the positive rate compared with the existing urine collecting device and urine detecting device.
It can be understood that, the logic of the present application is based on the fact that the existing sample detection such as urine sample and blood sample is sampling and detecting small sample amount during the collection and detection, which is easy to cause the detection error of small sample, especially for the detection of CTCs, the small sample cannot ensure the detection accuracy, therefore, it is necessary to provide a large-capacity sample collection module for collecting large-capacity sample, that is, the sample collection module provided in the present application, and after having the function of collecting large-capacity sample, the large-capacity sample can ensure the number of samples for continuous detection, which ensures the efficiency of sample detection, and it is necessary to consider that the activity of sample is ensured to support continuous detection, so that the large-capacity sample collection module can keep fresh continuously. The collection and inspection method and the system realize integrated treatment of CTCs detection through integrated collection, storage, fresh-keeping, enrichment and detection, and compared with the prior art, the collection of large-capacity samples is realized in collection; the preservation and the freshness preservation of large-capacity samples within 24 hours are realized on the aspect of storage and freshness preservation; the sample enrichment function of the large-flux sample continuous detection is realized in enrichment, the sample detection result can be more accurate based on the integration of the functions, and the misjudgment of the false negative rate sample is reduced. Meanwhile, based on the design concept of the household closestool prototype, the system is integrated in the household closestool, so that the CTCs can be detected more conveniently and rapidly, and the health management is further improved undoubtedly.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or system. Without further limitation, an element defined by the phrases "comprising a," "8230," "8230," or "comprising" does not exclude the presence of other like elements in a process, method, article, or system comprising the element.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and should be considered to be within the scope of the invention.
Claims (10)
1. A method for collecting urine circulating tumor cells, which is characterized by comprising the following steps:
obtaining a urine sample;
carrying out cell capture on the urine sample to obtain undetermined circulating tumor cells;
staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells;
extracting the stained undetermined circulating tumor cells, and performing CCD scanning;
and judging positive circulating tumor cells according to the scanning image and storing the scanning image.
2. The method for collecting urine circulating tumor cells according to claim 1, wherein the obtaining of the urine sample comprises:
taking all urine collected by the urine collector within 24 hours as a urine sample;
and (4) carrying out cold storage and fresh keeping treatment on the urine sample collected within 24 hours by using a cold storage device.
3. The urine circulating tumor cell collecting method as claimed in claim 1, wherein the cell capturing of the urine sample comprises:
and performing cell capture on the urine sample by using an enrichment device loaded with a capture ligand.
4. The method for collecting urine circulating tumor cells as claimed in claim 1, wherein the staining of the tumor cells to be circulating comprises:
staining the tumor cells to be circulated by using a fluorescent antibody;
and standing the stained undetermined circulating tumor cells for 30 minutes.
5. The method for collecting urine circulating tumor cells according to claim 1, wherein the step of extracting the stained undetermined circulating tumor cells and performing CCD scanning comprises:
extracting the dyed undetermined circulating tumor cells and exciting color light of the dyed undetermined circulating tumor cells;
and scanning the to-be-determined circulating tumor cells after the color light is excited by utilizing the frozen CCD scanner to obtain a to-be-determined circulating tumor cell scanning map.
6. The method for collecting urine circulating tumor cells according to claim 1, wherein the determining positive circulating tumor cells from the scan and storing the scan comprises:
judging the cell population which presents the corresponding color light of the positive circulating tumor cells on the scanning image as the positive circulating tumor cells;
storing the scan in a database.
7. A urine circulating tumor cell collecting and detecting system is characterized by comprising:
the sample collection module is used for obtaining a urine sample;
the cell enrichment module is used for capturing cells of the urine sample to obtain undetermined circulating tumor cells, and staining the undetermined circulating tumor cells to obtain stained undetermined circulating tumor cells;
the cell detection module is used for extracting the dyed undetermined circulating tumor cells and performing CCD scanning;
and the database module is used for judging positive circulating tumor cells according to the scanning image and storing the scanning image.
8. The urine circulating tumor cell collecting system of claim 7, wherein the sample collecting module comprises a urine storage unit, a refrigeration unit, a blending unit, a suction unit, a disinfection and sterilization unit and an electromagnetic valve control unit,
wherein, urine storage unit is used for the storage of urine, cold-stored unit is used for guaranteeing the fresh degree of sample, the mixing unit is used for controlling the evenly distributed of sample, the suction unit is arranged in carrying the sample to the cell enrichment module, the disinfection and isolation unit is used for the washing of sample collection module, solenoid valve control unit is used for controlling the switch of solenoid valve.
9. The urine circulating tumor cell collecting system of claim 7, wherein the cell enriching module comprises a capturing unit, a staining unit,
the capturing unit is used for capturing the tumor cells to be circulated, and the staining unit is used for staining the tumor cells to be circulated.
10. The urine circulating tumor cell collecting and inspecting system according to claim 7, wherein the cell detecting module comprises a fluorescence light emitting unit, an auto-focusing unit, a freezing CCD scanning unit, and a data storage unit,
the fluorescence light-emitting unit is used for exciting color light of the stained tumor cells to be circulated, the automatic focusing unit is used for automatic focusing of the freezing CCD scanning unit, the freezing CCD scanning unit is used for generating a scanning image of the tumor cells to be circulated, and the data storage unit is in communication connection with the database module and used for storing the scanning image of the tumor cells to be circulated.
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