CN114457139A

CN114457139A - Cell detection method and kit

Info

Publication number: CN114457139A
Application number: CN202111602579.5A
Authority: CN
Inventors: 施奇惠; 吴旭波; 张子缘
Original assignee: Central Hospital Of Minhang District Shanghai
Current assignee: Central Hospital Of Minhang District Shanghai
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-05-10

Abstract

The invention provides a cell detection method and a kit, wherein the cell detection method comprises the following steps: obtaining cells to be detected; carrying out fluorescent staining on the cells to be detected; carrying out fluorescence detection on the cells to be detected after the fluorescence staining; and judging whether the cell to be detected is the target cell or not based on the fluorescence signal. The cell detection method of the invention firstly carries out fluorescence staining on the cells to be detected and then carries out fluorescence detection, and has the advantages of high detection sensitivity, high accuracy and the like.

Description

Cell detection method and kit

Technical Field

The invention relates to the technical field of cell detection, in particular to a cell detection method and a cell detection kit.

Background

For exfoliated cells (such as bile duct exfoliated cells) needing to be detected, the existing detection technology generally has the problems of poor sensitivity, low accuracy and the like.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a cell detection method, which is used for solving the problems in the prior art.

The invention provides a cell detection method, which comprises the following steps:

obtaining cells to be detected;

carrying out fluorescent staining on the cells to be detected;

carrying out fluorescence detection on the cells to be detected after the fluorescence staining;

and judging whether the cell to be detected is the target cell or not based on the fluorescence signal.

Optionally, before the fluorescence staining of the cells to be detected, the method further comprises: and enriching the cells to be detected.

Optionally, after the enriching of the cells to be detected and before the performing of the fluorescent staining of the cells to be detected, the method further comprises:

providing an addressable microwell array chip or glass sheet;

dispersing the enriched cells to be detected on the addressable micropore array chip or the glass sheet and fixing.

Optionally, the performing fluorescent staining on the cells to be detected comprises: and carrying out fluorescent staining on the cells to be detected by using a fluorescence-labeled hexokinase 2 antibody and a fluorescence-labeled cytokeratin antibody.

Optionally, the performing fluorescent staining on the cells to be detected further comprises: and carrying out fluorescent staining on the cells to be detected by using a fluorescent-labeled leukocyte marker antibody and a cell nucleus stain.

Optionally, after determining whether the cell to be detected is a target cell based on the fluorescence signal, if the target cell is detected, the method further includes:

obtaining the target cell;

performing whole genome amplification on the target cells;

and performing gene copy number variation detection, whole exome sequencing or gene Panel detection on the target cells after the whole genome is amplified.

The invention also provides a kit, which comprises at least one of a fluorescence-labeled hexokinase 2 antibody, a fluorescence-labeled cytokeratin antibody, a fluorescence-labeled leukocyte marker antibody and a cell nucleus stain, and is used for carrying out fluorescence staining on the cells to be detected in the cell detection method in any scheme.

As described above, the cell detection method of the present invention has the following advantageous effects: the cell detection method of the invention firstly carries out fluorescence staining on the cells to be detected and then carries out fluorescence detection, and has the advantages of high detection sensitivity, high accuracy and the like.

Drawings

FIG. 1 is a flowchart of the cell detection method of the present invention.

FIG. 2 is a graph showing the staining of HK2 fluorescent antibody on cells to be detected in the cell detection method of the present invention.

FIG. 3 is a CK fluorescent antibody staining pattern of cells to be detected in the cell detection method of the present invention.

FIG. 4 is a DAPI (nuclear dye) staining pattern of cells to be detected in the cell detection method of the present invention.

Fig. 5 is a bright field image of the corresponding target cell of fig. 2, 3, 4.

FIG. 6 is a fluorescence channel distribution diagram of target cells in the cell detection method of the present invention, and cells HK2 positive/CK positive/CD 45 negative/DAPI positive are exfoliated tumor cells, wherein the HK2 and CK positive thresholds are the mean of the fluorescence values of leukocytes plus five-fold standard deviation.

FIG. 7 shows the single-cell copy number variation detection of exfoliated tumor cells detected by the cell detection method of the present invention, which confirms that the rare tumor cells identified based on the HK2/CK marker are tumor cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

Referring to fig. 1, the present invention provides a cell detection method, including:

s10: obtaining cells to be detected;

s20: carrying out fluorescent staining on the cells to be detected;

s30: carrying out fluorescence detection on the cells to be detected after the fluorescence staining;

s40: and judging whether the cell to be detected is the target cell or not based on the fluorescence signal.

The cell detection method of the invention has the following beneficial effects: the cell detection method of the invention firstly carries out fluorescence staining on the cells to be detected and then carries out fluorescence detection, and has the advantages of high detection sensitivity, high accuracy and the like.

As an example, before the fluorescence staining of the cells to be detected, the method further comprises: and enriching the cells to be detected.

As an example, after the enriching of the cells to be detected and before the fluorescence staining of the cells to be detected, the method further includes:

providing an addressable microwell array chip or glass sheet;

Specifically, the cells to be detected after enrichment treatment are prepared into cell suspension, and the cell suspension is spread on the addressable micropore array chip or the glass sheet in a single cell form; where the cells to be detected comprise cells in a bile sample, the enrichment treatment comprises reducing the number of red blood cells (if any), white blood cells (if any) and other impurities in the bile sample.

As an example, the performing fluorescent staining on the cells to be detected includes: the cells to be detected were fluorescently stained using a fluorescently labeled hexokinase 2(HK2) antibody and a fluorescently labeled Cytokeratin (CK) antibody.

Specifically, HK2 is a tumor cell metabolism marker, and CK is an epithelial cell marker.

More specifically, the fluorescently labeled hexokinase 2(HK2) antibody may also be a combination comprising an HK2 antibody and a fluorescently labeled HK2 antibody.

As an example, the cells to be detected may be cells in a cultured bile sample or a harvested bile sample. The positive judgment of the fluorescence detection result is determined according to a threshold value, the fluorescence detection result is positive above the threshold value and negative below the threshold value, and the threshold value can be confirmed by adopting a method and a technical means which are commonly used in the field, for example, the average value of HK2/CK fluorescence signals of all white blood cells in fluorescence detection plus five standard deviations is used as a threshold value for judging the HK2 high expression/CK positive.

Further, the fluorescence staining of the cells to be detected further comprises: the cells to be detected are fluorescently stained using a fluorescently labeled leukocyte marker (CD45) antibody and a nuclear stain (DAPI). Cells that met high expression of HK2/CK positive/white blood cell marker negative/nuclear staining were exfoliated tumor cells.

Specifically, the leukocyte marker antibody is an antibody aiming at a cell membrane surface protein CD45, and the cell nucleus stain is a cell nucleus fluorescent dye 4', 6-diamidino-2-phenylindole (DAPI). The average value of the HK2/CK fluorescence values of CD45 positive cells in the fluorescence detection plus five-fold standard deviation is used as a threshold value for judging the HK2 high expression/CK positivity.

The kit adopts HK2/CK as a marker combination, and identifies the exfoliated tumor cells through the marker combination of HK2/CK/CD45 (leukocyte marker)/DAPI (cell nucleus stain), thereby improving the detection sensitivity and specificity. We also further verified the reliability of suspected tumor cells detected by this marker combination by single cell sequencing.

In one example, after determining whether the cell to be detected is a target cell based on the fluorescence signal, if the target cell is detected, the method further includes:

obtaining the target cell;

performing whole genome amplification on the target cells;

and (3) carrying out gene Copy Number Variation (CNV) detection, Whole Exome Sequencing (WES) or gene Panel detection on the target cells after the whole genome is amplified.

Specifically, when the cell detection method of the present invention is used for detecting exfoliated cells in a bile sample, the cell detection method may include the following steps:

(a) pretreating and enriching a human bile sample;

(b) dispersing all cells in the enriched sample on an addressable micropore array chip or a glass sheet;

(c) fixing all cells on the micropore array chip or the glass sheet;

(d) performing marker-based fluorescent staining and imaging on all cells after fixation;

(e) and identifying suspected exfoliated tumor cells according to the threshold value of the fluorescence signal or by adopting an artificial intelligence mode, and counting after manual examination.

(f) Separating and recovering the identified tumor cells by using a micromanipulation technology;

(g) after the single tumor cell or a plurality of combined tumor cells are subjected to genome-wide amplification aiming at separation and recovery, gene Copy Number Variation (CNV) detection, Whole Exome Sequencing (WES) or gene Panel detection is carried out.

More specifically, the cell detection method of the present invention may comprise the steps of:

(a) taking a bile sample of a bile duct cancer patient: collecting bile samples of patients with cholangiocarcinoma according to the conventional steps specified by the manual on the premise of obtaining informed consent of the patients;

(b) pretreatment and enrichment: if the collected bile has bleeding, the sample contains more red blood cells, and simple cell enrichment, namely lysis and red blood cell removal, is required, and in addition, other enrichment steps are not generally required;

(c) cell plating: spreading the cells on an addressable microwell array chip or glass slide;

(d) cell fixation, membrane penetration and staining: fixing and penetrating a membrane of the cells, and then performing a staining treatment to identify exfoliated tumor cells in human bile, wherein the staining reagent is a certain concentration of fluorescently-labeled HK2 antibody substances, CK antibody substances, CD45 antibody substances and cell nucleus stain DAPI;

(e) imaging and image analysis: performing fluorescence analysis to preliminarily judge the cells with high expression of HK 2/positive CK/negative CD 45/positive DAPI as the exfoliated tumor cells, wherein the HK2 and CK positive threshold values are the average value of the fluorescence value of the white blood cells plus five times of standard deviation;

(f) removal of the cells of interest (target): obtaining target cells through various devices such as a micromanipulator;

(g) gene amplification and sequencing: single cell sequencing or other detection means known in the art to determine rare exfoliated tumor cells is performed to determine whether or not they are exfoliated tumor cells.

Wherein the fluorescent-labeled HK2/CK/CD45 antibody substance is a combination of HK2/CD45/CK antibody directly labeled by fluorescein or other fluorescent substances (such as quantum dots) or primary antibody of non-labeled HK2/CD45/CK and secondary antibody labeled by fluorescein or other fluorescent substances.

Further, in order to reduce or eliminate the interference of some non-cellular impurities (such as cell debris, bubbles, non-cellular particles, etc.) having an adsorbing effect on the fluorescent-labeled antibody substance of HK2/CK, which are introduced in the test sample or during the treatment process, on the fluorescence analysis, embodiments of the method of the present invention further include staining the cells of the sample with a nuclear Dye (DAPI) simultaneously, before or after the HK2/CK staining. In this case, HK2 high-expression/CK positive cells, which were also nuclear staining positive, were initially judged as exfoliated tumor cells.

Although the present invention is established based on a bile sample, it is contemplated by those skilled in the art upon reading this specification that the methods and kits of the present invention may also be suitable for detecting Circulating Tumor Cells (CTCs) from a peripheral blood sample of a patient with biliary duct cancer. In the case of peripheral blood, in which the number of blood cells is high and the number of CTCs is minimal, it is known in the art that subsequent detection steps such as staining, fluorescence imaging, etc., such as removal of erythrocytes by erythrocyte lysis, removal of leukocytes by density gradient centrifugation, or enrichment procedures based on CD45 antibody-coated magnetic spheres or partial removal of leukocytes, can be suitably performed after pretreatment, enrichment procedures.

The following examples illustrate the overall process and analysis results for specific human bile exfoliated tumor cell identification using the above detection methods to demonstrate the effectiveness and advantages of the present invention.

In this embodiment, the method includes the steps of:

(a) mixing the bile sample with Hank's Balanced Salt Solution (HBSS) in a volume ratio of 1:1, centrifuging (4 ℃, 300g, 10min, centrifuge brake: acceleration 9, deceleration 9), discarding the supernatant, adding 10mLHBSS to resuspend cells, centrifuging (4 ℃, 300g, 10min, centrifuge brake: acceleration 9, deceleration 9), discarding the supernatant, adding 1mLHBSS to resuspend cells;

(b) adding 1mL of cell suspension into a funnel of a slide preparation bin, putting the slide into a flail machine, centrifuging (4 ℃, 200g, 3min), discarding liquid in the slide preparation bin, and adding a proper amount of HBSS into the slide preparation bin to clean the slide;

(c) carefully rotating the slide making bin, taking out the slide glass, keeping the cells in a liquid environment, immediately fixing the cell plating range by using immunohistochemical strokes, absorbing the liquid in the defined range by using a pipette gun, adding 200 mu L of 2% Paraformaldehyde (PFA) for fixing the cells at room temperature for 10min, and adding 300 mu L of Phosphate Buffer Solution (PBS) to clean the slide glass for 3-5 times after absorbing the fixing liquid;

(d) adding 200 μ L of 0.5% Triton X-100 membrane for treating for 15min to increase permeability of cell membrane to antibody, washing with PBS for 3-5 times, adding 100 μ L of mixed solution of 3% BSA and 10% goat serum, sealing at room temperature for 1 hr, and washing with PBS for 2 times;

(e) add 200. mu.L of antibody mix: the antibody contains 1% PE-labeled CK antibody (murine, ThermoFisher), 1% APC-labeled CD45 antibody (murine, ThermoFisher) and 1% HK2 primary antibody (rabbit, Abcam), and is incubated at 4 ℃ overnight;

(f) after absorbing the antibody mixed solution, washing the mixture for 5 times by PBS, adding 100 mu L of a mixed solution of 3% BSA and 10% goat serum, sealing the mixture for 1h at room temperature, washing the mixture for 2 times by PBS, adding 200 mu L of a 0.25% FITC-labeled goat anti-rabbit secondary antibody (ThermoFisher company), incubating the mixture for 1h at room temperature, washing the mixture for 5 times by PBS, adding 100 mu L of LDAPI stock solution, incubating the mixture for 5-10min at room temperature, and washing the mixture for 5 times by PBS after the incubation is finished;

(g) the high-speed fluorescence imaging device imaged (results are shown in fig. 2, fig. 3, fig. 4, fig. 5 and fig. 6), and analyzed the scanning results, wherein the HK2/CK positive threshold is the mean of the fluorescence values of the white blood cells on the slide plus five standard deviations. The judgment standard of CD45 positive and DAPI positive is that the color development is positive; FIG. 6 includes a HK2 fluorescence subchannel map of 6 target cells, a CK fluorescence subchannel map of 6 target cells, a CD45 fluorescence subchannel map of 6 target cells, and a DAPI fluorescence subchannel map of 6 target cells;

(h) target tumor single cells (HK2 high expression/CK positive/CD 45 negative/DAPI positive) are accurately separated and recovered by a micromanipulation platform, and single cell whole genome amplification is completed by using a commercial kit MALBAC (Yikang gene, China). The amplified product of the whole genome of the tumor single cell is directly used for constructing a whole genome sequencing library. Tumor single cell whole genome amplification products were recovered by purification using AMPure XP beads (Beckman Coulter, usa), whole genome sequencing library construction was done using the ultratmdanalibrary Prep Kit (New England Biolabs, uk), sequencing library concentration and quality were evaluated using qubit 3.0(ThermoFisherScientific, usa) and Agilent2100Bioanalyzer (Agilent, usa), respectively, and whole genome sequencing was performed using HiseqXten (Illumina, usa) sequencing platform, using PE150 sequencing strategy.

FIG. 7 shows the single cell Copy Number Variation (CNV) detection of exfoliated tumor cells in bile of patients with cholangiocarcinoma, which confirms that the rare tumor cells positively identified based on HK2/CK marker are tumor cells.

In bile sample experiments of cholangiocarcinoma patients, all samples detected rare tumor cells, target single cells were accurately recovered by means of a micromanipulation platform and subjected to single cell amplification and sequencing studies, and the results are shown in fig. 7, and HK2 high-expression/CK positive/CD 45 negative/DAPI positive cells were confirmed to be tumor cells.

It should be noted that, in FIG. 7, the numbers 1 to 22 at the top indicate 22 autosomes of 1 to 22, X indicates the X chromosome, and each staining pattern gives a fluorescent staining pattern of each channel of 6 positive cells; the left ordinate in FIG. 7 is the chromosome copy number, and the right ordinate is the number of positive cells; in FIG. 7, a chromosome copy number of more than 2 indicates that amplification of the chromosome copy number, a chromosome copy number of 2 or less indicates that the chromosome copy number is normal, and a chromosome copy number of less than 2 indicates that deletion of the chromosome copy number.

In conclusion, the results suggest that the method has higher sensitivity and reliability and can be used for detecting the exfoliated tumor cells in the bile sample of the cholangiocarcinoma patient.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes 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 specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.

Claims

1. A method of detecting a cell, comprising:

obtaining cells to be detected;

carrying out fluorescent staining on the cells to be detected;

2. The cell detection method according to claim 1, characterized in that: the method also comprises the following steps before the fluorescence staining of the cells to be detected: and enriching the cells to be detected.

3. The cell detection method according to claim 2, characterized in that: after the enriching of the cells to be detected and before the fluorescence staining of the cells to be detected, the method further comprises the following steps:

providing an addressable microwell array chip or glass sheet;

4. The cell detection method according to claim 1, characterized in that: the fluorescence staining of the cells to be detected comprises: and carrying out fluorescent staining on the cells to be detected by using a fluorescence-labeled hexokinase 2 antibody and a fluorescence-labeled cytokeratin antibody.

5. The cell detection method according to claim 4, characterized in that: the fluorescence staining of the cells to be detected further comprises: and carrying out fluorescent staining on the cells to be detected by using a fluorescent-labeled leukocyte marker antibody and a cell nucleus stain.

6. The cell detection method according to any one of claims 1 to 5, characterized in that: after judging whether the cell to be detected is the target cell based on the fluorescence signal, if the target cell is detected, the method further comprises the following steps:

obtaining the target cell;

performing whole genome amplification on the target cells;

7. A kit, comprising: at least one of a fluorescence-labeled hexokinase 2 antibody, a fluorescence-labeled cytokeratin antibody, a fluorescence-labeled leukocyte marker antibody, and a cell nucleus stain for use in the cell detection method according to any one of claims 1 to 6 for fluorescence staining the cells to be detected.