CN111235104A - Method and separator for separating and purifying nucleated red blood cells of pregnant woman peripheral blood fetus - Google Patents
Method and separator for separating and purifying nucleated red blood cells of pregnant woman peripheral blood fetus Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to the field of cell biology detection, and particularly relates to a separation and purification method and a separation instrument for fetal nucleated red blood cells. The method comprises the following steps: (1) culturing a CD71 positive cell line (K562) and detecting the binding force and specificity of an antibody; (2) establishing a capture system by using the CTCBIOPSY detection system and K562 cells and optimizing capture conditions by using umbilical cord blood; (3) separating and collecting fetal nucleated red blood cells: the microsphere coupled with the CD71 polyclonal antibody is used for expanding the diameter of target cells, and nucleated red blood cells in the peripheral blood of the pregnant women are separated by means of the CTCBIOPSY detection system and the filtering principle. The method has the advantages of high capture efficiency, low cost, simple operation steps, short time consumption and high sensitivity.
Description
Technical Field
The invention belongs to the field of cell biology detection, and particularly relates to a separation and purification method and a separation instrument for fetal nucleated red blood cells.
Background
Birth defects refers to a general term for structural, functional or metabolic abnormalities of the body of an infant before birth, and generally includes congenital structural deformity, chromosome abnormality, inherited metabolic disease, and functional abnormalities such as blindness, deafness and intellectual disability. In 9 months 2012, the "chinese birth defect prevention and treatment report" issued by the ministry of health of the former country indicates that the incidence of the chinese birth defects is about 5.6%, about 90 ten thousand birth defects are newly added each year, and about 25 ten thousand birth defects are clinically and obviously seen at birth. Birth defects are the leading causes of early abortion, premature birth, dead fetus, perinatal death, and infant death, and can lead to long-term illness, shortened life span, and even lifelong disability in children. Birth defects not only seriously jeopardize the survival and the life quality of children, but also bring great economic burden to families and society, and become public health problems influencing the population quality and the population health level. Therefore, prenatal screening and prenatal diagnosis are important for preventing birth defects.
Prenatal diagnosis refers to the diagnosis of some genetic diseases or congenital malformations of the fetus before birth by combining genetic detection and image examination results, and prenatal diagnosis technology can be used for detecting birth defects such as chromosome number abnormality diseases, gene copy number variation diseases, monogenic diseases and the like. Currently, prenatal diagnosis is classified into invasive prenatal diagnosis and noninvasive prenatal diagnosis according to the difference of sampling methods. Among them, invasive prenatal diagnosis methods mainly include amniocentesis, chorionic puncture, and cord blood sampling, and although the result accuracy is high, these invasive means have certain risks to both the mother and the fetus, which may cause vaginal bleeding, amniotic fluid leakage, fetal membrane rupture, infection, or fetal abortion and other complications.
Fetal free DNA, RNA, and fetal cells have been found in maternal peripheral blood to provide the possibility for noninvasive prenatal diagnosis. Since the discovery of free DNA fragments derived from fetuses in maternal plasma in 1997, free fetal DNA in maternal peripheral blood has been the key material for non-invasive prenatal diagnosis, and in 2008, the use of free fetal DNA for making a diagnosis of 21-trisomy syndrome was achieved using a new generation of high throughput sequencing technology. Thereafter, the noninvasive prenatal detection technology based on the DNA of the free fetus in the peripheral blood of the pregnant woman is subjected to large-scale scientific research and clinical verification, and the technology is rapidly developed. However, the use of fetal-free DNA has its own drawbacks, as shown in: (1) in addition to free DNA of fetal origin, maternal DNA is present in large amounts in maternal blood, and DNA derived from fetuses accounts for about 3-20% of the total amount, and the individual differences are large; (2) half of the fetal genome is of maternal origin, so the source of DNA is currently relatively difficult to distinguish; (3) fetal free DNA in maternal blood is fragmented and does not cover the complete genome of the fetus, so it is currently mainly aimed at the detection of chromosomal abnormalities. Therefore, the free DNA in the peripheral blood of the pregnant woman can not detect the requirements and contents of chromosome microdeletion, micro-duplication, monogenic disease detection and the like, and the items are more elaborate.
For the above reasons, researchers have shifted the focus of research to maternal peripheral blood to look for another genetic component derived from the fetus, fetal cells. Unlike free DNA in peripheral blood, fetal cells contain intact nuclei, carrying the entire fetal genomic information. The fetal cells separated from the peripheral blood of the pregnant woman are used for whole genome sequencing, not only can detect the aneuploidy of fetal chromosomes and the deletion and insertion of chromosomes, but also can effectively detect monogenic diseases and the like caused by SNP of a single locus, so the fetal cells in the peripheral blood of the pregnant woman are the best materials for carrying out noninvasive prenatal diagnosis. It has now been found that foetal cells which can pass from the placenta into the peripheral blood of the mother are: trophoblast cells, lymphocytes, fetal nucleated red blood cells.
The fetal nucleated red blood cells have complete genetic information, are rich in the peripheral blood of the pregnant woman in the early pregnancy period, have short cell survival period and basically disappear after the delivery of the infant for several days; has obvious morphological characteristics, has some cell surface markers (such as CD71, GPA, CD 147) and some intracellular markers such as embryonic or fetal (gamma-) hemoglobin, and can be enriched and identified by some positive antibodies such as CD71, GPA, fetal hemoglobin and the like. Therefore, nucleated red blood cells have been considered as the first target cells for prenatal diagnosis compared to other fetal cells circulating in maternal blood, but the number of nucleated red blood cells in maternal blood circulation is very small, which affects the application of the nucleated red blood cells in prenatal diagnosis.
At present, methods for separating and extracting fetal nucleated red blood cells from peripheral blood of pregnant women include a microfluidic technology, density gradient centrifugation, fluorescence activated cell sorting, magnetic activated cell sorting, a nanotechnology, an avidin separation method and the like. The prior art system has the problems of low capture efficiency, high cost, complex operation steps, long time consumption, low sensitivity, poor specificity and the like, and limits the application of the system in clinic.
The invention content is as follows:
the invention provides a method and a separator for separating nucleated red blood cells of a pregnant woman peripheral blood fetus, the method adopts a microsphere method coupled with a CD71 polyclonal antibody, separates the nucleated red blood cells in the peripheral blood by using a CTCBIOPSY detection system according to the size of cells and a filtering principle, and the method has the advantages of high capture efficiency, low cost, simple operation steps, short time consumption and high sensitivity.
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) culturing CD71 positive cell line K562 cells and detecting antibodies;
(2) establishing a capture system: preparing microspheres and coupling antibodies; obtaining an optimal capture condition by using K562 cells; optimizing the optimal capture conditions by using the cord blood;
(3) separating and collecting fetal nucleated red blood cells.
The step (1) comprises the culture of a CD71 positive cell line K562 cell and the detection of an antibody. The method specifically comprises the following steps: culturing a CD71 positive cell line K562; and selecting a CD71 polyclonal antibody and a monoclonal antibody to perform an immunofluorescence experiment, and verifying the binding force of the antibodies.
The step (2) of establishing a capture system comprises the following steps:
respectively coupling S1 microspheres with the polyclonal antibody obtained in the step (1);
s2 cell capture: adding microspheres coupled with polyclonal antibodies in S1 into peripheral blood of the women who are not pregnant with the cells in the step (1), incubating, separating by using a CTCBIOPSY detection system, and observing the capture condition of the cells;
s3 clinical collection of umbilical cord blood, adding microspheres with the optimal S2 capture conditions into the umbilical cord blood, incubating, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
s4 determining fetal origin of the captured cells: the captured cells were subjected to immunofluorescence staining with CD147, CD45, DAPI to determine the fetal origin of the cells.
The microspheres in the step S1 are: one of ferroferric oxide microspheres or PS microspheres; the concentration of CD71 antibody in the microspheres was: 15-20. mu.g/ml.
In step S2, the incubation conditions are as follows: the suspension instrument was rotated at room temperature for 20r/min and incubated for 30 min.
The step (3) of separating and collecting the fetal nucleated red blood cells comprises the following steps:
j1 peripheral blood collection: collecting peripheral blood of pregnant woman in 11-28 weeks, and adding erythrocyte lysate.
Centrifuging and collecting cell sediment;
j2 is added into the cell precipitate resuspension according to the capture system in the step (2), and then the cells are incubated, separated by using the CTCBIOPSY detection system, and collected and sorted the fetal nucleated red blood cells.
The step (3) further comprises: the identification of the fetal origin of the cells comprises the following specific steps: and identifying by using immunofluorescence and Y chromosome specific sequences.
The separator prepared by the method is adopted.
The CTCBIOPSY detection system and the corresponding filter separate nucleated red blood cells in peripheral blood according to the size of cells by using a filtering principle, the filter used by the system has the aperture of 8 mu m, and the diameter of cells is increased by combining microspheres with the cells by using specific antibodies, and the filtration and the separation are carried out.
The invention has the advantages of
The operation steps are simple, the time consumption is short, and the sensitivity is high; mature red blood cells can be effectively decomposed by using the red blood cell lysate, hemolysis is reduced, and the efficiency of separating nucleated red blood cells is improved.
The specific antibody is selected so that the microspheres bind to the cells increasing the diameter of the cells and the desired cells are isolated by simple filtration.
Detailed Description
CTCBIOPSY detection system and corresponding diagnostic reagent: on the basis of the ISET (isolation by size of epithelial cells) method, the technical system for detecting CTCs (platelet-rich plasma cells) by the subject group of Shandong province's drug research institute is improved in 2010, a separation technical system for circulating epithelial cells in peripheral blood is established, and corresponding detection equipment (CTCBIOPSY detection system and corresponding diagnostic reagents) is developed. The separation of nucleated red blood cells of a pregnant woman in peripheral blood and a fetus is a sub-topic of the project of important research and development in Shandong province, namely establishment and application of a peripheral blood circulation epithelial cell capture and target gene expression/single cell sequencing technology system.
The erythrocyte lysate used in the examples is commercially available Solarbio R1010.
Example 1
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) culturing a CD71 positive cell line (K562) and verifying the binding force of the antibody;
① culturing the CD71 positive cell line (K562);
② immunofluorescence technique for verifying the binding capacity of the CD71 antibody, namely selecting CD71 polyclonal antibody (ab8403), CD71 monoclonal antibody (ab214039), CD45 mouse monoclonal antibody (ab 8216), CD147 polyclonal antibody (ab 64616), goat polyclonal antibody secondary antibody to mouse (ab 150116) and goat polyclonal antibody secondary antibody to rabbit (ab 150077) to perform immunofluorescence experiment to verify the binding capacity of the antibodies;
(2) establishment of Capture System
③ the type and diameter of the microsphere are 1 μm ferroferric oxide microsphere;
④ obtaining the best capture condition by using K562 cells, coupling 1 μm ferroferric oxide microspheres with CD71 polyclonal antibody, adding the coupled microspheres into the peripheral blood of a woman who does not have the cell pregnancy in the step (1) (the concentration of the CD71 antibody in the microspheres is 15 μ g/ml), rotating a suspension instrument at room temperature for 20r/min to incubate for 30min, observing the microspheres under a microscope, and observing the binding condition of the antibody and the cells;
⑤ verification of the best capture condition by using umbilical cord blood, namely adding microspheres (20 mu l of 1 mu m ferroferric oxide microspheres coupled with CD71 polyclonal antibody and incubated for 30min at room temperature by a rotary suspension apparatus at 20 r/min) into 5ml of umbilical cord blood, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
⑥ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) separating and collecting fetal nucleated red blood cells
⑦ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑧ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑨ adding microspheres with the best capture condition (20 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at room temperature by rotating a suspension apparatus at 20 r/min) after the cell precipitates are resuspended, then separating by using a CTCBIOPSY detection system, collecting and sorting fetal nucleated red blood cells;
⑩ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Example 2
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) culturing a CD71 positive cell line (K562) and verifying the binding force of the antibody;
① culturing the CD71 positive cell line (K562);
② immunofluorescence technique for verifying the binding capacity of the CD71 antibody, namely selecting CD71 polyclonal antibody (ab8403), CD71 monoclonal antibody (ab214039), CD45 mouse monoclonal antibody (ab 8216), CD147 polyclonal antibody (ab 64616), goat polyclonal antibody secondary antibody to mouse (ab 150116) and goat polyclonal antibody secondary antibody to rabbit (ab 150077) to perform immunofluorescence experiment to verify the binding capacity of the antibodies;
(2) establishment of Capture System
③ the type and diameter of the microsphere are 1 μm ferroferric oxide microsphere;
④, obtaining the optimal capture condition by using K562 cells, namely coupling 1 mu m ferroferric oxide microspheres with CD71 polyclonal antibodies, then combining the cells in the step (1) (the concentration of the CD71 antibodies in the microspheres is 20 mu g/ml) and incubating the cells for 30min at room temperature by a rotary suspension instrument at 20r/min, observing the combination condition of the microspheres and the antibodies with the cells under a microscope, separating the cells by using a CTCBIOPSY (chemical mechanical control of molecular dynamics) detection system, and observing the capture condition of the cells;
⑤ verification of the best capture condition by using umbilical cord blood, namely adding microspheres (20 mu l of 1 mu m ferroferric oxide microspheres coupled with CD71 polyclonal antibody and incubated for 30min at room temperature by a rotary suspension apparatus at 20 r/min) into 5ml of umbilical cord blood, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
⑥ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) separating and collecting fetal nucleated red blood cells
⑦ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑧ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑨ adding microspheres with the best capture condition (20 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at room temperature by rotating a suspension apparatus at 20 r/min) after the cell precipitates are resuspended, then separating by using a CTCBIOPSY detection system, collecting and sorting fetal nucleated red blood cells;
⑩ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Example 3
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) culturing a CD71 positive cell line (K562) and verifying the binding force of the antibody;
① culturing the CD71 positive cell line (K562);
② immunofluorescence technique for verifying the binding capacity of the CD71 antibody, namely selecting CD71 polyclonal antibody (ab8403), CD71 monoclonal antibody (ab214039), CD45 mouse monoclonal antibody (ab 8216), CD147 polyclonal antibody (ab 64616), goat polyclonal antibody secondary antibody to mouse (ab 150116) and goat polyclonal antibody secondary antibody to rabbit (ab 150077) to perform immunofluorescence experiment to verify the binding capacity of the antibodies;
(2) establishment of Capture System
③ the type and diameter of the microsphere are 1 μm ferroferric oxide microsphere;
④ obtaining the best capture condition by using K562 cells, coupling 1 μm ferroferric oxide microspheres with CD71 polyclonal antibody, adding the coupled microspheres into the peripheral blood of a woman who does not have the cell pregnancy in the step (1) (the concentration of the CD71 antibody in the microspheres is 20 μ g/ml), rotating a suspension instrument at room temperature for 20r/min to incubate for 30min, observing the microspheres under a microscope, and observing the binding condition of the antibody and the cells;
⑤ verifying the optimal capture condition by using umbilical cord blood, adding microspheres (20 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at 37 ℃) into the umbilical cord blood, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
⑥ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) clinical testing
⑦ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑧ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑨ adding the microspheres (20 mu l of 1 mu m ferroferric oxide microspheres coupled with CD71 polyclonal antibody and incubated for 30min at 37 ℃) under the conditions after the cell sediment is resuspended, then separating by utilizing a CTCBIOPSY test system, and collecting and sorting fetal nucleated red blood cells;
⑩ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Example 4
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) culturing a CD71 positive cell line (K562) and verifying the binding force of the antibody;
① culturing the CD71 positive cell line (K562);
② immunofluorescence technique for verifying the binding capacity of the CD71 antibody, namely selecting CD71 polyclonal antibody (ab8403), CD71 monoclonal antibody (ab214039), CD45 mouse monoclonal antibody (ab 8216), CD147 polyclonal antibody (ab 64616), goat polyclonal antibody secondary antibody to mouse (ab 150116) and goat polyclonal antibody secondary antibody to rabbit (ab 150077) to perform immunofluorescence experiment to verify the binding capacity of the antibodies;
(2) establishment of Capture System
③ the type and diameter of the microsphere are 300nm PS microsphere;
④ obtaining the best capture condition by using K562 cells, coupling 300nm PS microspheres with CD71 polyclonal antibody, adding the coupled PS microspheres into the peripheral blood of a woman who does not have the cell pregnancy in the step (1) (the concentration of the CD71 antibody in the microspheres is 15 mu g/ml), rotating a suspension instrument at room temperature for 20r/min to incubate for 30min, observing the binding condition of the microspheres and the antibody and the cells under a microscope, separating by using a CTCBIOPSY detection system, and observing the capture condition of the cells;
⑤ verifying the optimal capture condition by using umbilical cord blood, namely adding microspheres (20 mu l of 300nmPS microspheres coupled with CD71 polyclonal antibody and incubating for 30min at room temperature by rotating a suspension apparatus at 20 r/min) under the condition into the umbilical cord blood, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
⑥ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) clinical testing
⑦ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑧ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑨ resuspending the cell precipitate, adding microspheres (20 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at 37 ℃) under the above conditions, separating by using a CTCBIOPSY detection system, and collecting and sorting fetal nucleated red blood cells;
⑩ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Example 5
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) CD71 antibody cell culture and detection
① culturing the CD71 positive cell line (K562);
② immunofluorescence technique for verifying the binding capacity of the CD71 antibody, namely selecting CD71 polyclonal antibody (ab8403), CD71 monoclonal antibody (ab214039), CD45 mouse monoclonal antibody (ab 8216), CD147 polyclonal antibody (ab 64616), goat polyclonal antibody secondary antibody to mouse (ab 150116) and goat polyclonal antibody secondary antibody to rabbit (ab 150077) to perform immunofluorescence experiment to verify the binding capacity of the antibodies;
(2) establishment of Capture System
③ the type and diameter of the microsphere are 1 μm ferroferric oxide microsphere;
④ obtaining the best capture condition by using K562 cells, namely adding 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody into the peripheral blood of a woman who is not pregnant with the cells in the step (1) (the concentration of CD71 antibody in the microspheres is 20 μ g/ml), rotating a suspension instrument at room temperature for 20r/min to incubate for 30min, observing the microspheres under a microscope, and the binding condition of the antibody and the cells;
⑤ verification of the best capture condition by using umbilical cord blood, adding microspheres (200 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at room temperature with a rotary suspension apparatus at 20 r/min) into umbilical cord blood under the above condition, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
⑥ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) separating and collecting fetal nucleated red blood cells
⑦ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑧ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑨ resuspending the cell precipitate, adding microspheres (200 μ l of 1 μm ferroferric oxide microspheres coupled with CD71 polyclonal antibody, incubating for 30min at 37 ℃) under the above conditions, separating by using a CTCBIOPSY detection system, and collecting and sorting fetal nucleated red blood cells;
⑩ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Comparative example 1
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) CD71 antibody cell culture and detection
① culturing the CD71 positive cell line (K562);
(2) establishment of Capture System
② the type and diameter of the microsphere are 1 μm ferroferric oxide microsphere;
③ binding the microspheres with the cells, namely binding the microspheres with the cells in the step (1), and observing the binding condition of the cells under a microscope;
④ obtaining the best capture condition by using umbilical cord blood, namely adding 20 mul of antibody-free 1 mu m ferroferric oxide microspheres into the umbilical cord blood, incubating for 30min at room temperature by rotating a suspension instrument for 20r/min, separating by using a CTCBIOPSY test system, and observing the capture condition of cells;
⑤ determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(3) clinical testing
⑥ collecting peripheral blood of pregnant woman in 11-28 weeks;
⑦ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑧ resuspending the cell precipitate, adding microspheres (20 μ l of 1 μm ferroferric oxide microspheres, incubated for 30min at 37 ℃) under the above conditions, separating by using a CTCBIOPSY detection system, and collecting and sorting fetal nucleated red blood cells;
⑨ identifying the fetal origin of the target cells by immunofluorescence and Y chromosome specific sequence identification.
Comparative example 2
A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus comprises the following specific operation steps:
(1) establishment of Capture System
① observing the capture condition by using the umbilical cord blood, namely collecting the umbilical cord blood, separating by using the CTCBIOPSY detection system, and observing the capture condition of the cells;
② determining the fetal origin of the captured cells, wherein the captured cells are subjected to CD147, CD45 and DAPI immunofluorescent staining to determine the fetal origin of the cells;
(2) clinical testing
③ collecting peripheral blood of pregnant woman in 11-28 weeks;
④ adding 3 times of erythrocyte lysate into peripheral blood, centrifuging, and collecting cell precipitate;
⑤, separating by using a CTCBIOPSY detection system, and collecting and sorting the fetal nucleated red blood cells;
as a result:
| item | Retention Rate of nucleated Red blood cells |
| Example 1 | More than half of the total |
| Example 2 | Half of the total |
| Example 3 | Pipe for blocking |
| Example 4 | Half of the total |
| Example 5 | Half of the total |
| Comparative example 1 | Less than half of the total |
| Comparative example 2 | ＜5% |
Claims (8)
1. A method for separating nucleated red blood cells of a pregnant woman peripheral blood fetus is characterized by comprising the following steps: the specific operation steps are as follows
(1) Culturing CD71 positive cell line K562 cells and detecting antibodies;
(2) establishing a capture system: preparing microspheres and coupling antibodies; obtaining an optimal capture condition by using K562 cells; optimizing the optimal capture conditions by using the cord blood;
(3) separating and collecting fetal nucleated red blood cells.
2. The separation method according to claim 1, characterized in that: culturing CD71 positive cell line K562 cells and detecting antibodies in the step (1); the method specifically comprises the following steps: culturing a CD71 positive cell line K562; and selecting a CD71 polyclonal antibody and a monoclonal antibody to perform an immunofluorescence experiment, and verifying the binding force of the antibodies.
3. The separation method according to claim 1, characterized in that: the step (2) of establishing a capture system comprises the following steps:
s1 microspheres are respectively coupled with the polyclonal antibody in the step (1);
s2 obtaining optimal capture conditions: adding microspheres coupled with polyclonal antibodies in S1 into peripheral blood of the non-pregnant women containing the cells in the step (1), incubating, separating by using a CTCBIOPSY detection system, and observing the capture condition of the cells;
s3 clinical collection of umbilical cord blood, adding microspheres with the optimal S2 capture conditions into the umbilical cord blood, incubating, separating by using a CTCBIOPSY detection system, and observing the capture condition of cells;
s4 determining fetal origin of the captured cells: the captured cells were subjected to immunofluorescence staining with CD147, CD45, DAPI to determine the fetal origin of the cells.
4. The separation method according to claim 3, wherein the microspheres of step S1 are: one of ferroferric oxide microspheres with the particle size of 1 mu m or PS microspheres with the particle size of 300 nm; the concentration of CD71 antibody in the microspheres was: 15-20. mu.g/ml.
5. The separation method according to claim 3, wherein in step S2, the incubation conditions are as follows: the suspension instrument was rotated at room temperature for 20r/min and incubated for 30 min.
6. The separation method according to claim 1, characterized in that: the step (3) of separating and collecting the fetal nucleated red blood cells comprises the following steps:
j1 peripheral blood collection: collecting peripheral blood of pregnant women after 11-28 weeks of pregnancy, adding erythrocyte lysate, centrifuging, and collecting cell precipitate;
adding the J2 cell precipitate into the capture system obtained in the step (2) after resuspension, separating by using the CTCBIOPSY detection system, and collecting and sorting the fetal nucleated red blood cells.
7. The separation method according to claim 1, wherein the step (3) further comprises: the identification of the fetal origin of the cells comprises the following specific steps: and identifying by using immunofluorescence and Y chromosome specific sequences.
8. A separator prepared by the method of claim 1.
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