阅读说明：本技术 体外环境中细胞捕获及检测方法 (Method for capturing and detecting cells in-vitro environment ) 是由 许宝芝 王卿卿 于 2019-10-09 设计创作，主要内容包括：本发明提供了一种体外环境中细胞的捕获及检测方法,其中的细胞捕获方法包括对血液进行处理后细胞采集,还包括对血液外的其它体液或混合有体液的混合液体多次离心,留下最终获得的沉淀物,向最终获得的沉淀物中加入完全培养液,混合均匀得到沉淀悬浮液,再将采样器的采集区浸泡于悬浮液中捕获细胞。本发明可通过采样器的细胞采集区在体外环境中对细胞进行捕获。(The invention provides a method for capturing and detecting cells in an in vitro environment, wherein the method for capturing the cells comprises the steps of collecting the cells after processing blood, and also comprises the steps of centrifuging other body fluids except the blood or mixed liquid mixed with the body fluids for a plurality of times to leave precipitates finally obtained, adding complete culture solution into the precipitates finally obtained, uniformly mixing to obtain precipitate suspension, and soaking a collecting area of a sampler in the suspension to capture the cells. The invention can capture cells in an in vitro environment through the cell collection region of the sampler.)

1. A method of capturing cells in an in vitro environment, comprising: the cell capturing method is used for capturing cells in a body fluid or a mixed fluid mixed with the body fluid in an in vitro environment, and comprises the following steps:

s1. if the body fluid is blood, the cell capturing method comprises anticoagulating blood, immersing the collecting region of the sampler with the cell collecting region in blood for cell capturing, or lysing red blood cells to remove red blood cells, removing white blood cells, and immersing the collecting region of the sampler with the cell collecting region in blood for cell capturing;

s2, if the body fluid is not blood or the cell capturing is performed with a mixed fluid mixed with the body fluid, the cell capturing method includes:

step s21. centrifuging the body fluid and obtaining a precipitate;

step s22, adding the complete culture solution into the precipitate finally obtained in the step s21, and uniformly mixing to obtain a precipitate suspension;

and step s23, soaking the collection area of the sampler with the cell collection area in the suspension obtained in the step s22, and putting the whole sampler on a shaker to adsorb in a constant temperature environment so as to collect the cells from the collection area.

2. The method of claim 1, wherein the method comprises: step s21 includes:

step s211, taking a part of body fluid or mixed liquid, and centrifuging at room temperature;

step s212, removing the supernatant after centrifugation, and leaving a precipitate;

step s213, mixing a part of body fluid with the precipitate in the step s212, and centrifuging at room temperature;

step s214. repeating step s212 and step s213 until all body fluids are centrifuged;

step s215. after the last centrifugation in step s214, the supernatant is removed, leaving the precipitate finally obtained.

3. The method of claim 2, wherein the method comprises: in s2, each centrifugation step and adsorption step were performed in low adsorption centrifuge tubes.

4. The method of claim 2, wherein the method comprises: before the step s23, the method further comprises the step of performing blow-mixing and homogenizing on the suspension obtained in the step s22.

5. The method of claim 2, wherein the method comprises: in steps s211 and s213, a horizontal centrifuge is used for centrifugation, and in step s23, the whole is placed on a three-dimensional shaking table for adsorption.

6. The method of claim 5, wherein the method comprises: in step s23, the sampler is inverted once every time the threshold time is set for adsorption.

7. The method for capturing cells in an in vitro environment according to any one of claims 1 to 6, wherein: s2 further includes:

and step s24, after adsorption is finished, placing the collection area of the sampler in a pre-cooled fixing solution for cell fixing.

8. A method of detecting a cell, comprising: the detection method is used for detecting the cells captured by the cell capturing method in the in vitro environment according to any one of claims 1 to 7, and the detection method at least comprises the steps of detecting cell proteins by performing immunofluorescence staining on the captured cells, extracting DNA of the captured cells and performing whole genome amplification to perform gene detection including but not limited to digital PCR, fluorescence quantitative PCR and gene second generation sequencing.

Technical Field

The invention relates to the technical field of cell capture and detection, in particular to a cell capture method in an in vitro environment, and also relates to a detection method of captured cells.

Background

In pathological or molecular biological diagnosis, human cells are often collected for subsequent detection and treatment due to the need of etiological judgment or pathological culture. At present, there are two types of collection of human cells, in vivo collection and in vitro collection, and most of the in vivo collection is, for example, collection of cells in human circulating blood by capturing. However, the in vivo collection method is a method of directly performing relevant operations on a human body, and there are still many inconveniences in implementation, so that trying to capture and collect cells in an in vitro environment by using body fluid discharged from the human body becomes a better alternative, but the existing method of capturing cells in an in vitro environment has many steps, needs to perform cell treatment for many times, has long treatment time, and is easy to damage cells.

Disclosure of Invention

In view of the above, the present invention aims to provide a simple and short-time cell capture method in an in vitro environment, so as to capture and collect cells in the in vitro environment while maintaining the integrity of the cells.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a cell capturing method in an in vitro environment for capturing cells in a body fluid or a mixed fluid mixed with a body fluid in an in vitro environment, comprising:

s1. if the body fluid is blood, the cell capturing method comprises anticoagulating blood, immersing the collecting region of the sampler with the cell collecting region in blood for cell capturing, or lysing red blood cells to remove red blood cells, removing white blood cells, and immersing the collecting region of the sampler with the cell collecting region in blood for cell capturing;

s2, if the body fluid is not blood or the cell capturing is performed with a mixed fluid mixed with the body fluid, the cell capturing method includes:

step s21. centrifuging the body fluid and obtaining a precipitate;

step s22, adding the complete culture solution into the precipitate finally obtained in the step s21, and uniformly mixing to obtain a precipitate suspension;

and step s23, soaking the collection area of the sampler with the cell collection area in the suspension obtained in the step s22, and putting the whole sampler on a shaker to adsorb in a constant temperature environment so as to collect the cells from the collection area.

Further, step s21 includes:

step s211, taking a part of body fluid or mixed liquid, and centrifuging at room temperature;

step s212, removing the supernatant after centrifugation, and leaving a precipitate;

step s213, mixing a part of body fluid with the precipitate in the step s212, and centrifuging at room temperature;

step s214. repeating step s212 and step s213 until all body fluids are centrifuged;

step s215. after the last centrifugation in step s214, the supernatant is removed, leaving the precipitate finally obtained.

Further, in s2, each of the centrifugation step and the adsorption step is performed in a low adsorption centrifuge tube.

Further, before the step s23, the method further comprises a step of subjecting the suspension obtained in the step s22 to blow-mixing and homogenizing.

Further, in steps s211 and s213, a horizontal centrifuge is used for centrifugation, and in step s23, the whole is placed on a three-dimensional shaking table for adsorption.

Further, the sampler is inverted every time the threshold time is set for adsorption in step s23.

Further, s2 includes:

and step s24, after adsorption is finished, placing the collection area of the sampler in a pre-cooled fixing solution for cell fixing.

Compared with the prior art, the invention has the following advantages:

the body fluid cell capturing method of the present invention can collect cells by using the sampler after processing blood, and can collect cells to reduce cell loss by separating supernatant from other body fluids other than blood or mixed fluids mixed with body fluids. Meanwhile, the activity of the cells can be guaranteed by the added complete culture medium, so that the cells can be captured by the cell collecting area of the sampler on the basis, and the cells in the body fluid can be collected in an in-vitro environment.

In addition, the invention can ensure the effective collection of the cells to be trapped by realizing the complete separation of the body fluid once part at a time and for a plurality of times through the design.

Another object of the present invention is to propose a cell detection method for detecting cells captured by the cell capture method in an in vitro environment as described above, and the detection method at least comprises detection of cellular proteins by immunofluorescent staining of the captured cells, and extraction of DNA of the captured cells and whole genome amplification for gene detection including but not limited to digital PCR, fluorescent quantitative PCR and gene second generation sequencing.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

Example one

This example relates to a method of capturing cells in an in vitro environment.

In the cell capturing method of the present embodiment, cells in a body fluid obtained from a subject or a mixed fluid mixed with the body fluid are captured and collected in an in vitro environment, so that the collected cells can be subjected to operations such as corresponding subsequent culture and detection.

In terms of overall design, the method for capturing cells in the extracorporeal environment of the present embodiment is based on the difference of cell-carrying media, and includes two parts s1 and s2, where part s1 is directed to directly capture a large class of body fluid, i.e., cells in blood, and part s2 is directed to capture other classes of body fluid than blood, and cells in a mixed fluid mixed with body fluid. It should be noted that, in general, other body fluids than blood may include urine, ascites and pleural effusion, and the mixed fluid mixed with body fluids may be surgical irrigating fluid obtained during surgery of a subject, such as peritoneal irrigating fluid or cystoscopic lavage fluid.

Based on this, the cell capturing method of the present embodiment mainly includes:

for s1, where the body fluid is blood, the cell capture method includes anticoagulating the blood, and immersing the collection region of the sampler with the cell collection region in the blood for cell capture, or lysing the red blood cells to remove the red blood cells, removing the white blood cells, and immersing the collection region of the sampler with the cell collection region in the blood for cell capture.

For s2, i.e., the body fluid is not blood, or the cell trapping is performed with a mixed fluid mixed with the body fluid, the cell trapping method includes the following steps:

step s21. centrifuging the body fluid and obtaining a precipitate;

step s22, adding the complete culture solution into the precipitate finally obtained in the step s21, and uniformly mixing to obtain a precipitate suspension;

and step s23, soaking the collection area of the sampler with the cell collection area in the suspension obtained in the step s22, and putting the whole sampler on a shaker to adsorb in a constant temperature environment so as to collect the cells from the collection area.

The step s21 may further include the following steps:

step s211, taking a part of body fluid or mixed liquid, and centrifuging at room temperature;

step s212, removing the supernatant after centrifugation, and leaving a precipitate;

step s213, mixing a part of body fluid with the precipitate in the step s212, and centrifuging at room temperature;

step s214. repeating step s212 and step s213 until all body fluids are centrifuged;

step s215. after the last centrifugation in step s214, the supernatant is removed, leaving the precipitate finally obtained.

It should be noted that, as a broader description of the meaning of the body fluid according to the present invention, the body fluid described in the cell capture method of the present embodiment generally refers to a fluid sample obtained from a subject suffering from a certain symptom (including a subject not treated or already treated), or a subject suspected to suffer from a certain symptom, and particularly, the symptom or suspected symptom of the subject is preferably determined by analyzing cells in the body fluid to obtain the required judgment information. The subject is typically a mammal, which includes, for example, a dog, cat, cow, cat, rodent, such as a mouse, or primate, such as a human.

As a more general implementation, the subject is a human being in the present embodiment, and it should be noted that the body fluid obtained from the subject generally includes blood, lymph, saliva, mucus, sputum, pus, urine, feces, gastrointestinal secretions, as well as cochlear fluid, synovial fluid, cerebrospinal fluid, tears, vitreous humor, semen, vaginal secretions, mammary secretion, etc. In the present embodiment, the capturing method will be specifically described below by taking the cell capture from human urine as an example.

In this embodiment, as a preferred embodiment, in the section s2, each of the centrifugation step and the adsorption step can be performed in a low adsorption centrifuge tube. It is also generally preferred that the suspension of step s22 is further subjected to a blow-and-mix homogenization process prior to step s23 to ensure the collection of cells by the collection region on the sampler.

In addition, as a preferred embodiment, it should be noted that the centrifugation in the above steps s211 and s213 can be performed by a horizontal centrifuge, and the adsorption in the step s22 can be performed on a three-dimensional shaking table, which can ensure that the adsorption solution is sufficiently contacted with the collection area, and can avoid the condition of low adsorption amount compared with the case of using a horizontal shaking table. In addition, in step s23, the sampler can be optionally turned over once every time the threshold time is set for adsorption, so that the whole peripheral wall surface of the collection area can be in good contact with the suspension, thereby being beneficial to improving the success rate of cell capture. The threshold time may be selected according to actual application, and the capture time is about 30min in the general capture method based on the embodiment.

After the cells are captured by the sampler, the cells captured on the sampling area should be immobilized to ensure that the cells are stably adsorbed on the surface of the collection area. For this purpose, in this embodiment, after the adsorption is completed, the collection area of the sampler is placed in a pre-cooled fixing solution for cell fixation. The fixing solution may be a conventional low-temperature solution capable of fixing cells, and is selected by the operator according to the detection conditions.

The cell capture and collection process of the present embodiment will be described in detail below, taking urine obtained from a human body as a body fluid to capture bladder epithelial cells as an example.

At this time, the human body from which the urine is obtained, that is, the subject, is a tumor patient. And when the capture is carried out, the low adsorption tube adopts two types of 2.0ml low adsorption EP tube and 50ml low adsorption EP tube, and the specific information of the two types of low adsorption EP tubes is respectively as follows: protein Lobind Tube 2.0ml, Eppendorf Tubes, Order No.: 0030108132, respectively; protein Lobind Tube 50ml, Eppendorf Tubes, Order No.: 0030122240.

in addition, the horizontal centrifuge can be a centrifuge commonly used in biological detection and experiments, the three-dimensional shaking table can be a shaking table with model number of QR-24 of Shanghai Waren instruments and meters, the constant temperature environment during constant temperature adsorption can be obtained through a constant temperature incubator, and the constant temperature incubator can be an incubator with model number of BPH-9162 of Shanghai Hengscience instruments and meters.

In this particular collection example, the sampler for cell capture may be a sampling needle product of the present applicant and be equipped with a silicone plug for collection along with the sampling needle. Besides the above devices, a liquid-transferring gun and a matched gun head, an indwelling needle and the like are used in the specific collection process, and the liquid-transferring gun and the matched gun head, the indwelling needle and the like are all prepared by adopting corresponding conventional devices. In addition, for the capture of cells from urine, the complete medium used was specifically L-15 complete medium containing the components L-15 cell culture medium + 10% FBS fetal bovine serum.

In the specific capture, it should be noted that the obtained urine sample should be centrifuged and cell-adsorbed within 2 hours. The specific process is as follows:

first, preparation of adsorption solution

Firstly, pouring a part of the obtained urine into 1 50ml low-adsorption centrifuge tube, centrifuging at room temperature for 5min under the centrifugal force of 500 g;

step two, pouring off the supernatant, and leaving about 1-2ml without touching the sediment carefully;

thirdly, adding the residual urine sample into the centrifugal tube in the second step, and centrifuging at room temperature for 5min at a centrifugal force of 500 g;

and fourthly, repeating the second step and the third step until all urine samples are centrifuged, so that cells in all urine are collected in the same centrifugal tube, and the loss of the cells is minimized.

Fifth, after the final centrifugation, the supernatant was decanted and care was taken not to touch the pellet.

Sixthly, adding 1.0ml of prepared L-15 complete culture solution along the tube wall, slightly blowing and uniformly mixing, and then transferring the precipitate suspension into a prepared 2.0ml EP tube;

and seventhly, sucking 0.5ml of L-15 complete culture solution, adding the L-15 complete culture solution into the 50ml centrifugal tube in the fifth step, washing the centrifugal tube, transferring the centrifugal tube into the 2.0ml EP tube in the sixth step, appropriately supplementing the L-15 complete culture medium to enable the final volume to be 2.0ml, and then blowing, beating and uniformly mixing.

Secondly, constant temperature adsorption

Firstly, blowing and uniformly mixing the prepared adsorption solution again;

and secondly, the sampling needle passes through the silica gel plug through the retention needle, and the silica gel plug is sleeved on the low adsorption tube filled with the adsorption liquid, so that the collection area on the sampling needle is soaked in the adsorption liquid. And then the whole body is placed on a 3D shaking table for adsorption, the adsorption is carried out for 30min at the temperature of 37 ℃, and the sampling needle is turned over every 5min in the adsorption process, so that the adsorption of the bladder epithelial cells is completed.

After adsorption, the part of the sampling region of the sampling needle where the cells are collected is put into pre-cooled methanol at 4 ℃ for fixation for 5 min. The fixed sampling needle can be put into the preservation solution of the pretreatment kit for pathological analysis for transportation or preservation according to requirements.

Example two

This example relates to the detection of bladder epithelial cells captured in example one.

The detection of the captured bladder epithelial cells of the present example is specifically carried out by immunofluorescent staining of the captured cells for detection of cellular proteins. More specifically, the method for detecting cellular proteins by immunofluorescence staining specifically comprises the following steps:

a. putting the trapping area of the sampler with the captured cells into sealing liquid for sealing;

b. after the sealing is finished, putting the collection area of the sampler into the cleaning solution for soaking;

c. placing the cleaned collection area of the sampler into an anti-antibody staining solution for staining, and cleaning the collection area of the sampler after staining;

d. placing the cleaned collection area of the sampler into a secondary antibody staining solution for staining, and cleaning the collection area of the sampler after staining;

e. putting the cleaned collection area of the sampler into a nuclear staining solution for incubation staining, and cleaning the collection area of the sampler after staining;

f. fixing the cleaned sampler on a clamping device, performing photographing microscopic examination on the acquisition area of the sampler by using a fluorescence microscope with a camera, and judging the acquisition result by using the bladder epithelial cell identification criterion.

The method takes the staining and interpretation of the captured EpCAM-expressing bladder epithelial cells or the bladder cancer marker CK20 as an example, wherein reagents and related instruments needed to be prepared during detection are as follows:

reagent:

quality control product:

three-color quality control products: two kinds of cells with blue-green signals and blue-red signals after dyeing;

CK20/P53 quality control product: cells with blue-green-violet signals after staining.

Equipment:

tweezers, a timer, a pipette tip (200 mu L), and a centrifuge tube rack.

Microscopic examination equipment:

an upright fluorescence microscope with a black and white camera (e.g., Axio Imager, Carl Zeiss AG);

fluorescence microscope filter setting: e.g. a zeiss filter 49 (blue); 52 (green); 64 (purple); 50 (red);

a sampler clamping device (the rotary detection table in the patent CN205133584U granted by the applicant can be adopted).

In combination with the above steps a to f, the specific detection process is as follows:

putting one end of the sampling area of the sampler into the sealing liquid, starting to seal, timing for 20-120min, such as 30min, and recording the sealing start-stop time. Opening the cleaning solution before the closing time is over, reversing and mixing uniformly, placing the sampling area of the sampler into the soaking tank for 1-15min, for example 1min, and taking out the sampler and twisting the sampler with fingers for 3-5 times.

Under conditions strictly avoiding sunlight and light, the following dyeing process is carried out:

1. taking out an anti-antibody staining solution within the closed time of the sampling area of the sampler, and gently blowing and uniformly mixing the anti-antibody staining solution by using a 200 mu L liquid transfer gun;

2. placing the sampling area of the sampler into an anti-antibody staining solution, timing for 20min-24h, such as 60min, and recording the staining start-stop time;

3. opening 3 tubes of new cleaning solution before the dyeing of the primary antibody is finished, reversing and uniformly mixing, and sequentially putting the sampling areas of the sampler into the cleaning solution after the dyeing of the primary antibody is finished;

4. before the dyeing time of the primary antibody dyeing solution is finished, opening the secondary antibody dyeing solution, and gently blowing and uniformly mixing the secondary antibody dyeing solution by using a 200 mu L pipette gun;

5. placing the sampling area of the sampler into a secondary antibody staining solution, timing for 20-120min, such as 60min, and recording the staining start-stop time;

6. opening 3 tubes of new cleaning solution before the secondary antibody dyeing is finished, reversing and uniformly mixing, and sequentially putting the sampling areas of the sampler into the cleaning solution after the secondary antibody dyeing is finished;

7. taking out the nuclear staining solution, gently blowing and uniformly mixing by using a 200 mu L liquid transferring gun, putting the sampling area of the sampler into the nuclear staining solution, incubating for 1-15min (for example, 5 min) at a fixed time, and recording the starting time and the stopping time;

8. opening a new tube of cleaning solution before the nuclear staining is finished, and soaking the sampling area of the sampler in the cleaning solution after the nuclear staining is finished.

The method comprises the steps of judging results after dyeing, wherein the judging process specifically comprises the steps of opening a microscope program, calibrating or skipping calibration, fixing a dyed sampler on a clamping device, adjusting by a microscope coarse focusing screw to enable a sampling area of the sampler to move to a position about 5mm away from an objective lens, opening fluorescence of the microscope, cutting to a green channel, moving a microscope handle to enable the green fluorescence to be irradiated to a central area of the sampling area of the sampler to generate intense reflection, putting down a baffle plate to protect eyes, switching to a blue fluorescence channel to find the outline of the sampling area of the sampler under the microscope through an eyepiece, moving to the center of a visual field, switching to the green channel, and when a target of suspected cell morphology is found, switching to the channel to observe the superposition condition of green and blue signals. If the pull rod is pulled out to click for previewing, the exposure time is adjusted (see interpretation standard), and the picture is ensured to be shot under the state of not overexposure.

Observing the red channel of the photograph, the finding is a CTC if the red signal is absent, or is weak and uniformly colored without distinct boundaries, and a blood cell-related target if the red signal is present and has cellular morphology. The sampler is moved slowly to check each field of view (only the cells in the center are read) avoiding repeated counting until microscopic examination is complete, at which point the sampling area of the sampler can be returned to the storage tube for storage at-20 ℃.

Wherein (1) one example of an interpretation criterion is as follows:

recommended exposure time at shooting (III light source):

and (3) dyeing results:

(2) the criteria for identifying epithelial cells may be, for example:

should have the morphology of the cell;

cells may be polymorphic (large cells, irregular cell shape, aggregation or overlapping of several cells, etc.);

the result of nuclear staining is positive;

CD45 was negative;

CK and/or EpCAM is positive;

the cell diameter is more than or equal to 4 mu m;

the coloration of the core can be distinguished significantly from the coloration of CK/EpCAM.

Criteria for identifying CK20 positive epithelial cells: while meeting the criteria for epithelial cells, it should also meet the positive criteria for P53 and/or CK 20.

(3) The case of no interpretation is:

there is a significant cell morphology, with very large nuclei (CTCs are usually large in size), however,

CK/EpCAM, CD45 has no signal at all, or,

all positive (CK/EpCAM and CD45 positive);

the target size is like a cell, located on a blood clot or on a large dye clot.

The cell detection method of the present embodiment, in combination with the in vitro cell collection of the first embodiment, can perform the required correlation detection on the collected cells, and can provide a more effective cell diagnosis approach.

EXAMPLE III

This embodiment relates to the capture and detection of fetal cells in the peripheral blood of pregnant women.

The cell capturing method of the embodiment is based on the s1 part in the first embodiment, and specifically, a certain amount of pregnant woman peripheral blood is captured and collected, and in the capturing process, the collected peripheral blood is subjected to anticoagulation treatment, and then the collecting area of the sampler with the cell collecting area is soaked in the blood for cell capturing. Alternatively, the blood may be subjected to red blood cell lysis to remove red blood cells, then to leukocyte removal, and then to cell trapping by immersing the collection area of the sampler having the cell collection area in the blood. The trapping time is 10-60 min.

After the trapping is finished, the cells captured on the collecting area are fixed, namely the collecting area is placed in a precooled fixing liquid for cell fixing. Cells fixed on the collection area can be subjected to gene detection including but not limited to digital PCR, fluorescent quantitative PCR and second-generation sequencing by immunofluorescent staining for detection of protein levels and extraction of DNA of captured cells and whole genome amplification.

Specifically, the above method for detecting cellular proteins by immunofluorescence staining specifically comprises the following steps:

a. putting the trapping area of the sampler with the captured cells into sealing liquid for sealing;

b. after the sealing is finished, putting the collection area of the sampler into the cleaning solution for soaking;

c. placing the cleaned collection area of the sampler into an anti-antibody staining solution for staining, and cleaning the collection area of the sampler after staining;

d. placing the cleaned collection area of the sampler into a secondary antibody staining solution for staining, and cleaning the collection area of the sampler after staining;

e. putting the cleaned collection area of the sampler into a nuclear staining solution for incubation staining, and cleaning the collection area of the sampler after staining;

f. fixing the cleaned sampler on a clamping device, performing photographing microscopic examination on an acquisition area of the sampler by using a fluorescence microscope with a camera, and judging a capture result by using a fetal cell identification criterion.

Taking the example of staining and interpreting the captured fetal cells (fetal nucleated red blood cells and trophoblast cells) expressing proteins such as CD71, GPA, CD36, HbF, TER119, HLA-G, CD14, etc., the reagents and related instruments needed to be prepared for detection are as follows:

reagent:

quality control product:

three-color quality control products: two kinds of cells with blue-green signals and blue-red signals after dyeing;

CK20/P53 quality control product: cells with blue-green-violet signals after staining.

Equipment:

tweezers, a timer, a pipette tip (200 mu L), and a centrifuge tube rack.

Microscopic examination equipment:

an upright fluorescence microscope with a black and white camera (e.g., Axio Imager, Carl Zeiss AG);

fluorescence microscope filter setting: e.g. a zeiss filter 49 (blue); 52 (green); 64 (purple); 50 (red);

a sampler clamping device (the rotary detection table in the patent CN205133584U granted by the applicant can be adopted).

In combination with the above steps a to f, the specific detection process is as follows:

putting one end of the sampling area of the sampler into the sealing liquid, starting to seal, timing for 20-120min, such as 30min, and recording the sealing start-stop time. Opening the cleaning solution before the closing time is over, reversing and mixing uniformly, placing the sampling area of the sampler into the soaking tank for 1-15min, for example 1min, and taking out the sampler and twisting the sampler with fingers for 3-5 times.

Under conditions strictly avoiding sunlight and light, the following dyeing process is carried out:

1. taking out an anti-antibody staining solution within the closed time of the sampling area of the sampler, and gently blowing and uniformly mixing the anti-antibody staining solution by using a 200 mu L liquid transfer gun;

2. placing the sampling area of the sampler into an anti-antibody staining solution, timing for 20min-24h, such as 60min, and recording the staining start-stop time;

3. opening 3 tubes of new cleaning solution before the dyeing of the primary antibody is finished, reversing and uniformly mixing, and sequentially putting the sampling areas of the sampler into the cleaning solution after the dyeing of the primary antibody is finished;

4. before the dyeing time of the primary antibody dyeing solution is finished, opening the secondary antibody dyeing solution, and gently blowing and uniformly mixing the secondary antibody dyeing solution by using a 200 mu L pipette gun;

5. placing the sampling area of the sampler into a secondary antibody staining solution, timing for 20-120min, such as 60min, and recording the staining start-stop time;

6. opening 3 tubes of new cleaning solution before the secondary antibody dyeing is finished, reversing and uniformly mixing, and sequentially putting the sampling areas of the sampler into the cleaning solution after the secondary antibody dyeing is finished;

7. taking out the nuclear staining solution, gently blowing and uniformly mixing by using a 200 mu L liquid transferring gun, putting the sampling area of the sampler into the nuclear staining solution, incubating for 1-15min (for example, 5 min) at a fixed time, and recording the starting time and the stopping time;

8. opening a new tube of cleaning solution before the nuclear staining is finished, and soaking the sampling area of the sampler in the cleaning solution after the nuclear staining is finished.

After dyeing, the results were interpreted, and observed and photographed using a fluorescence microscope. And observing a red channel of the shot picture, and if the red signal does not exist or the red signal is weaker and is uniformly colored without an obvious boundary, observing a green channel and a purple channel, and judging whether the target cell is the target cell. If a red signal is present, it is excluded as a target cell. The sampler is moved slowly and each field is examined (only the cells in the center are read) avoiding repeated counts until microscopic examination is complete. At this point, the sampling area of the sampler may be placed back in the storage tube and stored at-20 ℃.

Wherein (1) one example of an interpretation criterion is as follows:

recommended exposure time at shooting (III light source):

and (3) dyeing results:

(2) the criteria for identifying fetal cells may be, for example:

should have the morphology of the cell;

cells may be polymorphic (large cells, irregular cell shape, aggregation or overlapping of several cells, etc.);

the result of nuclear staining is positive;

CD45 was negative;

CD14/CD71/CD36 are positive;

GPA positive (for nucleated red blood cells) or HLA-G positive (for trophoblast cells)

The cell diameter is more than or equal to 4 mu m;

the coloration of the nucleus can be distinguished significantly from the coloration of CD14/CD71/CD 36.

As with the second embodiment, the cell detection method of the present embodiment can perform the required correlation detection on the collected cells when combined with the in vitro cell collection of the first embodiment, and thus can provide a more effective cell diagnosis approach.

Example four

The present example relates to the detection of captured cells, such as the aforementioned captured bladder epithelial cells or fetal cells, and specifically the detection of the present example is the extraction of DNA from the captured cells and whole genome amplification, but also gene detection including, but not limited to, digital PCR, fluorescent quantitative PCR, and gene-sequencing by second generation.

The gene assay for the cell assay of this example generally comprises the following steps:

a, cutting a collection area with cells captured by a collector into a plurality of sections and placing the sections into a PCR tube;

b, adding cell lysate into the PCR tube for cell lysis, and performing enzyme inactivation after cell lysis;

c, adding a pre-amplification reaction mixed solution for gene amplification;

d, purifying and recovering the gene product amplified by the PCR;

and e, carrying out digital PCR, fluorescent quantitative PCR or gene second-generation sequencing detection on the recovered gene product.

Specifically, for the cell gene detection of this embodiment, taking the collected circulating epithelial cells as an example, the PCR amplification may be, for example, a single-cell whole genome amplification kit of jiangsu yikang gene technology ltd, and the recovery of the amplification product may be a PCR product recovery kit of beijing tiangen biochemical technology ltd, and for other methods such as cell lysis, digital PCR, fluorescent quantitative PCR, or second-generation sequencing of genes, reference may be made to related technical means applied in the prior art, which is not described in detail in this embodiment.

Similarly, the cell detection method of the present embodiment can perform the required relevant detection on the collected cells when combined with the in vitro cell collection of the first embodiment, thereby providing a more effective cell diagnosis approach.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.