CN113777089A - Antibody-independent circulating tumor cell detection method - Google Patents
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Abstract
The invention discloses an antibody-independent circulating tumor cell detection method, which comprises the following steps: step 1, taking a proper amount of peripheral blood solution of a detected person, removing red blood cells and white blood cells, and removing impurities for removing the red blood cells and the white blood cells through a cell filter to obtain a circulating tumor cell enriched extracting solution; the invention has the following beneficial effects that the fluorescent electron microscope is adopted to be matched with a pathological image analysis system, then the antibody solution adapted to the circulating tumor cells is mixed with the marked circulating tumor cells, and the combined quantity is recorded again, so that the determination precision is improved, and the multi-class interference is prevented; the cell filter is adopted to remove the residual impurities of the red blood cells and the white blood cells, so that the subsequent treatment is convenient and the definition of the circulating tumor cells in the peripheral blood solution is improved; the agglutinin is added to lead the circulating tumor cells to be gathered, improve the concentration ratio, facilitate the observation and reduce the possibility of missed detection.
Description
Technical Field
The invention belongs to the technical field of cell detection, and particularly relates to an antibody-independent circulating tumor cell detection method.
Background
Antibodies that recognize a unique feature of a particular foreign object, referred to as an antigen;
clinically, it is always a difficult question to answer clearly to judge whether a patient has a tumor, which may cause misdiagnosis for many patients; say, pancreatic cancer often finds that it is advanced after diagnosis? This is because at an early stage, the diagnosis of pancreatic cancer is very difficult; in the blood of many patients difficult to be diagnosed, if circulating tumor cells can be detected, the method has positive significance for matching with the conclusion of tumor diagnosis of doctors;
for routine detection of tumors, e.g., imaging; in the case of a tumor of less than one centimeter, the physician does not consider it abnormal; it can be seen through domestic and foreign studies that, not to say, a centimeter, many tumors have tumor cells entering blood circulation under the condition of 2-4 mm, and from this point of view, it has the meaning that cannot be underestimated for early diagnosis;
numerous experiments have demonstrated that the appearance of CTCs is closely linked to the prognosis of patients with advanced cancer and can generally only be predicted by determining the number and general appearance of circulating tumor cells when performing prospective tests, and for this reason, it is imperative to propose methods for the detection of circulating tumor cells based on a single combined independent (repetitive) property to improve the accuracy of the defined type.
Disclosure of Invention
Technical problem to be solved
In order to overcome the defects of the prior art, an antibody-independent circulating tumor cell detection method is provided to solve the problems of the background art, and the beneficial effect of accurately determining the number of circulating tumor cells by combining specificity is achieved.
(II) technical scheme
The invention is realized by the following technical scheme: the invention provides an antibody-independent circulating tumor cell detection method, which comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, removing red blood cells and white blood cells, and removing impurities for removing the red blood cells and the white blood cells through a cell filter to obtain a circulating tumor cell enriched extracting solution;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope, adding agglutinin, and standing for a period of time to enable the circulating tumor cells to gather;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
and 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the patient, and providing assistance for determining the sex of the cancer.
Further, the specific operation flow in step 7 is as follows: when the circulating tumor cell types with a large number are one, directly mixing a proper amount of treatment solvent adapted to the circulating tumor cells with the marked circulating tumor cells;
when the circulating tumor cells with a large number of types are various, firstly taking one of the treating solvents with a proper amount of adaptation circulating tumor cells to be mixed with the marked circulating tumor cells, observing in the step 8 in advance, then adding another proper amount of treating solvent with adaptation circulating tumor cells, and observing in the step 8 again.
Further, the step 1 of removing the red blood cells adopts a non-hypotonic lysis method.
Further, the specific operation of removing leukocytes in step 1 is as follows: the method comprises the steps of firstly removing leukocytes by an immunomagnetic bead method, and then further killing the leukocytes in the enriched extracting solution by immunotoxin.
Further, the step 2 of separating the circulating tumor cells specifically comprises the following steps: and (2) directly marking all cells in the enriched extracting solution in the step (1) by using a DNA probe in a solution fluorescence in-situ hybridization mode, marking and standing for a period of time, and separating the marked enriched extracting solution into circulating tumor cells by using a flow cytometer.
Further, the DNA aptamers capable of specifically recognizing the tumor cells are provided with a plurality of groups.
Further, the standing time for mixing the antibody solution adapted to the circulating tumor cells and the labeled circulating tumor cells in the step 7 is more than 30 seconds.
(III) advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
(1, through adopting the fluorescence electron microscope to cooperate with the pathological image analysis system, obtain the circulating tumor cell type and accurate cell number of preliminary observation, mix by the antibody solution of adaptation circulating tumor cell with the circulating tumor cell of mark, the DNA aptamer through specificity discernment tumor cell selects tumor cell, the magnetic responsiveness of magnetic particle is enlargied through the polymerization of DNA simultaneously, improve the display, carry out the combination or the destruction condition of observing processing solvent and marked circulating tumor cell by the fluorescence electron microscope cooperation pathological image analysis system, record the quantity of combination or destruction again, improve and confirm the precision, reached through adopting the combination, destroy the beneficial effect of characteristic come the accurate determination circulating tumor cell number, prevent multiclass interference.
(2) removing the residual impurities of the red blood cells and the white blood cells from the peripheral blood solution from which the red blood cells and the white blood cells are removed by using a cell filter, so that the subsequent separation treatment of the circulating tumor cells is facilitated, and the definition of the circulating tumor cells in the peripheral blood solution is improved.
(3, after the circulating tumor cells are placed at the observation position of a fluorescence electron microscope, the agglutinin is added, so that the circulating tumor cells are gathered, the concentration ratio is improved, the observation is convenient, and the possibility of missed detection is reduced.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a flow chart of the detection method of the present invention;
FIG. 2 is a schematic diagram of antibody assembly of an antibody solution of the present invention;
FIG. 3 is a schematic representation of the binding of an antibody solution of the present invention to circulating tumor cells.
Detailed Description
Example 1:
as shown in FIGS. 1 to 3, the present invention provides an antibody-independent method for detecting circulating tumor cells, comprising: the method comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, and removing red blood cells and white blood cells to obtain a circulating tumor cell enrichment extract;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
step 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the detected person, and providing assistance for determining the sex of the cancer;
the method comprises the steps of obtaining the type of circulating tumor cells and the accurate cell number of primary observation by matching a fluorescence electron microscope with a pathological image analysis system, mixing an antibody solution adapted to the circulating tumor cells with the marked circulating tumor cells, selecting the tumor cells by a DNA (deoxyribonucleic acid) adapter for specifically recognizing the tumor cells, amplifying the magnetic responsiveness of magnetic particles through the polymerization of DNA, improving the display performance, observing the combination condition of a treatment solvent and the marked circulating tumor cells by matching the fluorescence electron microscope with the pathological image analysis system, recording the combined number again, improving the determination precision, and achieving the beneficial effect of accurately determining the number of the circulating tumor cells by adopting combination and destroying specificity.
Example 2:
as shown in FIGS. 1 to 3, the present invention provides an antibody-independent method for detecting circulating tumor cells, comprising: the method comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, removing red blood cells and white blood cells, and removing impurities for removing the red blood cells and the white blood cells through a cell filter to obtain a circulating tumor cell enriched extracting solution;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
step 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the detected person, and providing assistance for determining the sex of the cancer;
example 2 is superior to example 1 in that the peripheral blood solution from which the red blood cells and the white blood cells are removed is subjected to removal of residual impurities from the red blood cells and the white blood cells by using a cell filter, thereby facilitating subsequent separation treatment of the circulating tumor cells and improving the definition of the circulating tumor cells in the peripheral blood solution.
Example 3:
as shown in FIGS. 1 to 3, the present invention provides an antibody-independent method for detecting circulating tumor cells, comprising: the method comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, and removing red blood cells and white blood cells to obtain a circulating tumor cell enrichment extract;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope, adding agglutinin, and standing for a period of time to enable the circulating tumor cells to gather;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
step 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the detected person, and providing assistance for determining the sex of the cancer;
example 3 is superior to example 1 in that after the circulating tumor cells are placed at the observation site of the fluorescence electron microscope, lectin is added to aggregate the circulating tumor cells, improve the concentration ratio, facilitate observation, and reduce the possibility of missed detection.
Example 4:
as shown in FIGS. 1 to 3, the present invention provides an antibody-independent method for detecting circulating tumor cells, comprising: the method comprises the following steps:
the invention provides an antibody-independent circulating tumor cell detection method, which comprises the following steps: the method comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, removing red blood cells and white blood cells, and removing impurities for removing the red blood cells and the white blood cells through a cell filter to obtain a circulating tumor cell enriched extracting solution;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope, adding agglutinin, and standing for a period of time to enable the circulating tumor cells to gather;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
and 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the patient, and providing assistance for determining the sex of the cancer.
Wherein, the specific operation flow in the step 7 is as follows: when the circulating tumor cell types with a large number are one, directly mixing a proper amount of treatment solvent adapted to the circulating tumor cells with the marked circulating tumor cells;
when the circulating tumor cells with a large number of types are various, firstly taking one of the treating solvents with a proper amount of adaptation circulating tumor cells to be mixed with the marked circulating tumor cells, observing in the step 8 in advance, then adding another proper amount of treating solvent with adaptation circulating tumor cells, and observing in the step 8 again.
Wherein, the step 1 of removing the red blood cells adopts a non-hypotonic lysis method.
Wherein, the specific operation of removing the white blood cells in the step 1 is as follows: the method comprises the steps of firstly removing leukocytes by an immunomagnetic bead method, and then further killing the leukocytes in the enriched extracting solution by immunotoxin.
Wherein, the operation of separating the circulating tumor cells in the step 2 is as follows: and (2) directly marking all cells in the enriched extracting solution in the step (1) by using a DNA probe in a solution fluorescence in-situ hybridization mode, marking and standing for a period of time, and separating the marked enriched extracting solution into circulating tumor cells by using a flow cytometer.
Wherein, the DNA aptamers capable of specifically recognizing the tumor cells are provided with a plurality of groups.
Wherein the standing time for mixing the antibody solution adapted to the circulating tumor cells and the labeled circulating tumor cells in the step 7 is 30 seconds or more.
The cell filter (cell filtering screen) is generally made by combining a polypropylene frame and a special nylon screen, the holes of the cell filter are punched by a special processing cell filter laser punching machine, the working principle of the cell filter laser punching is that laser beams passing through a laser are highly concentrated in space and time, the diameter of a light spot can be reduced to a micron order, so that high power density is obtained, and almost any material can be subjected to laser punching;
a flow cytometer is a device for automatically analyzing and sorting cells;
fluorescence in situ hybridization, which is a new technology combining non-radioactive molecular biology and cytogenetics developed on the basis of radioactive in situ hybridization technology in the end of the 20 th century and the 80 th era, and is a new in situ hybridization method formed by replacing isotope labeling with fluorescent labeling;
the pathological image analysis system is also called as a microscopic medical image workstation; microscopic image software; a pathological graphic analysis system; a pathology graphic reporting system; a pathological medical imaging workstation; a pathology image-text workstation; a pathology workstation; a pathology image workstation; pathology graphics workstation software, etc.; the system adopts advanced image processing technology and high-precision hardware configuration, realizes colorization, automation and intellectualization from acquisition, measurement and processing of system signals to printing output, and has the advantages of simple and convenient operation, strong image processing function, intelligent image analysis, high image definition, fast printing of image-text reports, strong database management function and the like; provides an epoch-making advanced tool for clinical pathology, pharmacological pathology and all technologists using microscopes.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. An antibody-independent method for detecting circulating tumor cells, comprising: the method comprises the following steps:
step 1, taking a proper amount of peripheral blood solution of a detected person, removing red blood cells and white blood cells, and removing impurities for removing the red blood cells and the white blood cells through a cell filter to obtain a circulating tumor cell enriched extracting solution;
step 2, separating the enriched extracting solution processed in the step 1 from circulating tumor cells;
step 3, placing the circulating tumor cells separated in the step 2 at the observation position of a fluorescence electron microscope, adding agglutinin, and standing for a period of time to enable the circulating tumor cells to gather;
step 4, acquiring a distribution image of circulating tumor cells at the observation position by using a fluorescence electron microscope;
step 5, classifying cell types in the pathological images by using a fluorescent electron microscope in cooperation with a pathological image analysis system through artificial intelligence, and counting the cell amount;
step 6, if the cell count obtained in the step 5 is large, calculating one or more circulating tumor cells with the same type and large number to determine the approximate type of the circulating tumor cells;
and 7, mixing an appropriate amount of antibody solution matched with one or more kinds of circulating tumor cells in the step 6 with the marked circulating tumor cells in the observation position of the fluorescence electron microscope in the step 3, and standing for a period of time, wherein the antibody of the antibody solution: the magnetic nano-particles with the super-paramagnetic property and the DNA aptamer capable of specifically recognizing the tumor cells are assembled by taking DNA molecules as templates;
step 8, observing the combination condition of the antibody solution and the marked circulating tumor cells by the cooperation of a fluorescence electron microscope and a pathological image analysis system, recording the number of combination reactions, and improving the determination precision;
and 9, periodically repeating the steps 1 to 8 to test the change of the number of the circulating tumor cells in the peripheral blood solution of the patient, and providing assistance for determining the sex of the cancer.
2. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: the specific operation flow in the step 7 is as follows: when the circulating tumor cell types with a large number are one, directly mixing a proper amount of treatment solvent adapted to the circulating tumor cells with the marked circulating tumor cells;
when the circulating tumor cells with a large number of types are various, firstly taking one of the treating solvents with a proper amount of adaptation circulating tumor cells to be mixed with the marked circulating tumor cells, observing in the step 8 in advance, then adding another proper amount of treating solvent with adaptation circulating tumor cells, and observing in the step 8 again.
3. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: and (3) removing the red blood cells in the step 1 by adopting a non-hypotonic lysis method.
4. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: the specific operation of removing the white blood cells in the step 1 is as follows: the method comprises the steps of firstly removing leukocytes by an immunomagnetic bead method, and then further killing the leukocytes in the enriched extracting solution by immunotoxin.
5. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: the specific operation of the step 2 for separating the circulating tumor cells is as follows: and (2) directly marking all cells in the enriched extracting solution in the step (1) by using a DNA probe in a solution fluorescence in-situ hybridization mode, marking and standing for a period of time, and separating the marked enriched extracting solution into circulating tumor cells by using a flow cytometer.
6. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: the DNA aptamers capable of specifically recognizing the tumor cells are provided with a plurality of groups.
7. The method of claim 1, wherein the antibody-independent assay for circulating tumor cells comprises: the standing time for mixing the antibody solution adapted to the circulating tumor cells and the labeled circulating tumor cells in the step 7 is more than 30 seconds.
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| CN115094121A (en) * | 2022-07-04 | 2022-09-23 | 厦门骁科码生物科技有限公司 | Liquid phase cell in-situ hybridization method |
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| CN1454313A (en) * | 2000-07-14 | 2003-11-05 | 免疫公司 | Increased separation efficiency via controlled aggregation of magnetic nanoparticles |
| CN103154740A (en) * | 2010-10-14 | 2013-06-12 | 维里德克斯有限责任公司 | Methods and kits for the detection of circulating tumor cells in pancreatic patients using polyspecific capture and cocktail detection reagents |
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