阅读说明：本技术 循环肿瘤细胞阴性富集检测方法 (Negative enrichment detection method for circulating tumor cells ) 是由 温冬 金炜翔 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种循环肿瘤细胞阴性富集检测方法,所述循环肿瘤细胞阴性富集检测方法包括：向含有全血样本的第一离心管中加入红细胞特异性抗体和白细胞特异性抗体的组合,以使全血样本中红细胞和白细胞耦联在一起；利用密度梯度离心法去除所述全血样本中耦联在一起的红细胞和白细胞,以实现循环肿瘤细胞阴性富集。基于本发明的循环肿瘤细胞阴性富集检测方法可以有效提高单核细胞层中CTCs的纯度,从而降低后续鉴定工作的复杂程度,提高鉴定的效率和准确率。(The invention provides a circulating tumor cell negative enrichment detection method, which comprises the following steps: adding a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to a first centrifuge tube containing a whole blood sample to couple red blood cells and white blood cells in the whole blood sample together; and removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method so as to realize negative enrichment of the circulating tumor cells. The circulating tumor cell negative enrichment detection method based on the invention can effectively improve the purity of CTCs in the monocyte layer, thereby reducing the complexity of subsequent identification work and improving the efficiency and accuracy of identification.)

1. A negative enrichment detection method for circulating tumor cells is characterized by comprising the following steps:

s1: adding a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to a first centrifuge tube containing a whole blood sample to couple red blood cells and white blood cells in the whole blood sample together;

s2: and removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method so as to realize negative enrichment of the circulating tumor cells.

2. The method according to claim 1, wherein the step of performing S2 specifically comprises the steps of:

placing the first centrifuge tube on a shaking table for incubation;

adding density gradient centrifugate into a second centrifuge tube with an insert, and transferring the incubated whole blood sample in the first centrifuge tube into the second centrifuge tube;

placing the second centrifuge tube in a centrifuge for centrifugation, wherein a mononuclear cell layer in the centrifuged whole blood sample is positioned above the plug-in unit, and red blood cells and white blood cells which are coupled together are positioned below the plug-in unit;

and pouring the sample positioned above the plug in the second centrifuge tube after centrifugation into a third centrifuge tube.

3. The method for detecting negative enrichment of circulating tumor cells according to claim 2, further comprising the following steps after performing S2:

s3: the CTCs are marked by using cell fluorescent antibody suspension staining, and are identified by cell staining and morphology based on fluorescence signals acquired by scanning.

4. The method of claim 3, wherein in S3, the cellular fluorescent antibody comprises: anti-human EpCAM cell surface epithelial cell adhesion molecule antibodies, anti-human CK cytokeratin antibodies, nuclear dye DAPI, and anti-human CD45 leukocyte specific antibodies.

5. The method for detecting negative enrichment of circulating tumor cells according to claim 4, wherein the collection of fluorescence signals is performed by a fluorescence scanner and the CTCs are identified based on cell staining and morphology in S3.

6. The method of claim 5, wherein the CTCs are defined as cells that meet DAPI + and CK/EpCAM + and are characterized by CD 45-staining and cytomorphological features.

7. The method according to claim 3, wherein the step of performing S3 specifically comprises the steps of:

adding cell diluent into the third centrifuge tube, mixing uniformly in sequence, and centrifuging;

removing supernatant from a sample in a third centrifugal tube after centrifugation, adding a cell fixing solution for fixing and permeabilizing, then adding a cell washing solution, and centrifuging;

discarding the supernatant, adding a cell nucleus dye, cytokeratin, EpCAM and CD45 into the third centrifuge tube, and incubating and dyeing in dark;

replenishing cell washing liquor into the third centrifuge tube again, and centrifuging;

discard the supernatant, transfer the sample from the third centrifuge tube to a 96-well reading plate, scan and image analyze.

8. The method of claim 7, wherein the nuclear dye is DAPI.

Technical Field

The invention relates to the technical field of biology, in particular to a circulating tumor cell negative enrichment detection method.

Background

The Circulating Tumor Cells (CTCs) negative enrichment detection method means that the purpose of enriching the CTCs in blood is achieved by removing the blood Cells except the CTCs in the blood, the removal method mainly adopts a density gradient centrifugation mode, namely, after the blood is mixed with a medium (density gradient centrifugate), the Cells in the blood are separated and layered according to the density of the blood by centrifugal force, and a blood sample subjected to gradient density centrifugation is divided into 4 layers which are sequentially from top to bottom: plasma, monocytes, centrates (density gradient centrates) and red and white blood cells; the CTCs as the monocytes can be positioned in the monocyte layer, so that the aim of enriching the CTCs in the blood can be achieved only by extracting the monocyte layer.

However, the existing CTCs negative enrichment detection method has the following defects:

1) in the existing method, the negative enrichment of CTCs can completely extract a sample of a monocyte layer, and the layer contains a large amount of white blood cells, so that the enrichment purity of CTCs is not high enough, the complexity of subsequent identification work is increased, and the identification efficiency and accuracy are influenced;

2) the negative enrichment and stratification effects of CTCs are related to blood samples, particularly erythrocytes, after density gradient centrifugation, some blood samples can generate erythrocyte upgoing and cannot be strictly distinguished from a monocyte layer, so that the final enrichment purity of CTCs is reduced, and subsequent identification work is seriously interfered;

3) when red blood cells are shifted upwards in a blood sample, mixed red blood cells in the mononuclear cell layer are usually removed in a way of red blood cell splitting, so that the operation has side effect on the integrity of other mononuclear cells, and a cleaning step is added due to red blood splitting, so that the recovery rate of final cells is influenced;

4) because the CTCs negative enrichment detection method comprises the steps of enriching CTCs from blood and the subsequent dyeing identification process, the process is long, and multiple links of cleaning and tube replacement are required, in the complete process, the loss of cells can be caused, and the final detection sensitivity is influenced.

Aiming at the defects of the CTCs negative enrichment detection method in the prior art, the technical personnel in the field are always searching for a solution.

Disclosure of Invention

The invention aims to provide a circulating tumor cell negative enrichment detection method to solve the defects of the CTCs negative enrichment detection method in the prior art.

In order to solve the technical problems, the invention provides a circulating tumor cell negative enrichment detection method, which comprises the following steps:

s1: adding a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to a first centrifuge tube containing a whole blood sample to couple red blood cells and white blood cells in the whole blood sample together;

s2: and removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method so as to realize negative enrichment of the circulating tumor cells.

Optionally, in the method for detecting negative enrichment of circulating tumor cells, the step of executing S2 specifically includes the following steps:

placing the first centrifuge tube on a shaking table for incubation;

adding density gradient centrifugate into a second centrifuge tube with an insert, and transferring the incubated whole blood sample in the first centrifuge tube into the second centrifuge tube;

placing the second centrifuge tube in a centrifuge for centrifugation, wherein a mononuclear cell layer in the centrifuged whole blood sample is positioned above the plug-in unit, and red blood cells and white blood cells which are coupled together are positioned below the plug-in unit;

and pouring the sample positioned above the plug in the second centrifuge tube after centrifugation into a third centrifuge tube.

Optionally, in the method for detecting negative enrichment of circulating tumor cells, after performing S2, the method further includes the following steps:

s3: the CTCs are marked by using cell fluorescent antibody suspension staining, and are identified by cell staining and morphology based on fluorescence signals acquired by scanning.

Optionally, in the method for detecting negative enrichment of circulating tumor cells, in S3, the cytofluorescent antibody comprises: anti-human EpCAM cell surface epithelial cell adhesion molecule antibodies, anti-human CK cytokeratin antibodies, nuclear dye DAPI, and anti-human CD45 leukocyte specific antibodies.

Optionally, in the method for detecting the negative enrichment of the circulating tumor cells, in S3, the fluorescence signal is collected by a fluorescence scanner, and CTCs are identified according to cell staining and morphology.

Alternatively, in the method for detecting the negative enrichment of circulating tumor cells, the cells which meet DAPI + and CK/EpCAM + and have CD 45-staining characteristics and cell morphology characteristics are defined as CTCs.

Optionally, in the method for detecting negative enrichment of circulating tumor cells, the step of executing S3 specifically includes the following steps:

adding cell diluent into the third centrifuge tube, mixing uniformly in sequence, and centrifuging;

removing supernatant from a sample in a third centrifugal tube after centrifugation, adding a cell fixing solution for fixing and permeabilizing, then adding a cell washing solution, and centrifuging;

discarding the supernatant, adding a cell nucleus dye, cytokeratin, EpCAM and CD45 into the third centrifuge tube, and incubating and dyeing in dark;

replenishing cell washing liquor into the third centrifuge tube again, and centrifuging;

discard the supernatant, transfer the sample from the third centrifuge tube to a 96-well reading plate, scan and image analyze.

Optionally, in the negative enrichment detection method for circulating tumor cells, the nuclear dye is DAPI.

In the method for detecting the negative enrichment of the circulating tumor cells provided by the invention, the method for detecting the negative enrichment of the circulating tumor cells comprises the following steps: adding a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to a first centrifuge tube containing a whole blood sample to couple red blood cells and white blood cells in the whole blood sample together; and removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method so as to realize negative enrichment of the circulating tumor cells. The circulating tumor cell negative enrichment detection method based on the invention can effectively improve the purity of CTCs in the monocyte layer, thereby reducing the complexity of subsequent identification work and improving the efficiency and accuracy of identification.

Drawings

FIG. 1 is a flow chart of a method for detecting negative enrichment of circulating tumor cells according to an embodiment of the present invention;

FIG. 2 is a graph showing the results of a linear regression analysis of all sample results in accordance with one embodiment of the present invention;

FIG. 3 is a graph of a lower limit test fit curve detected in an embodiment of the present invention;

FIG. 4 is a scatter plot of the recovery rates of spiked cells for high and low concentration sample assays in accordance with an embodiment of the present invention.

Detailed Description

The method for detecting the negative enrichment of circulating tumor cells provided by the invention is further described in detail by combining the figures and specific examples. Advantages and features of the present invention will become apparent from the following description and from the claims. It is to be noted that the drawings are in a very simplified form and are not to precise scale, which is merely for the purpose of facilitating and distinctly claiming the embodiments of the present invention.

The present invention will be described in more detail with reference to the accompanying drawings, in order to make the objects and features of the present invention more comprehensible, embodiments thereof will be described in detail below, but the present invention may be implemented in various forms and should not be construed as being limited to the embodiments described.

Please refer to fig. 1, which is a schematic structural diagram of the negative enrichment detection method for circulating tumor cells according to the present invention. As shown in fig. 1, the negative enrichment detection method for circulating tumor cells comprises:

first, step S1 is performed to add a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to the first centrifuge tube containing the whole blood sample to couple red blood cells and white blood cells in the whole blood sample together;

next, step S2 is executed, in which red blood cells and white blood cells coupled together in the whole blood sample are removed by using a density gradient centrifugation method to achieve negative enrichment of circulating tumor cells;

wherein, executing S2 specifically includes the following steps:

placing the first centrifuge tube on a shaking table for incubation;

adding density gradient centrifugate into a second centrifuge tube with an insert, and transferring the incubated whole blood sample in the first centrifuge tube into the second centrifuge tube;

placing the second centrifuge tube in a centrifuge for centrifugation, wherein a mononuclear cell layer in the centrifuged whole blood sample is positioned above the plug-in unit, and red blood cells and white blood cells which are coupled together are positioned below the plug-in unit;

and pouring the sample positioned above the plug-in piece in the second centrifugal tube after centrifugation into a third centrifugal tube, namely pouring the mononuclear cell layer positioned above the plug-in piece into the third centrifugal tube.

The insert is a transparent plastic baffle with a micropore in the middle, red blood cells are arranged below the insert after density gradient centrifugation, a mononuclear cell layer is arranged above the insert, the density gradient centrifuge tube can be directly inverted, liquid above the insert is completely poured out, and a red blood cell layer sample below the insert is blocked; compare in and remove to absorb monocyte layer sample with the rifle head, take the centrifuging tube of plug-in components like this promptly convenience of customers and draw monocyte sample, can maximize the collection efficiency who guarantees the monocyte again.

Has the advantages that: after red blood cells and white blood cells in a whole blood sample are coupled together based on S1, most white blood cells can be isolated to the bottommost layer below the middle insert plate of the second centrifugal tube along with the cells by adopting a density gradient centrifugation method, so that the problem that some blood samples can have red blood cells flee up to cause the red blood cells to be mixed in a mononuclear cell layer and cannot be strictly distinguished in the existing detection method after the density gradient centrifugation is avoided, and the purity of the sample of the mononuclear cell layer is effectively improved.

Next, step S3 is performed to label CTCs with cytofluorescent antibody suspension staining and identify CTCs by cell staining and morphology based on the fluorescence signal acquired by scanning. Preferably, the cytofluorescent antibody comprises: anti-human EpCAM cell surface epithelial cell adhesion molecule antibodies, anti-human CK cytokeratin antibodies, nuclear dye DAPI, and anti-human CD45 leukocyte specific antibodies.

The explanation for the terms involved in S3 is as follows:

1) CTCs: circulating Tumor Cells, refers to Tumor Cells that have been shed from a solid Tumor into the peripheral circulation system.

2) CD 45: cluster of differentiation 45, a common antigen of leukocytes, all of which are expressed, is a transmembrane protein with a large molecular weight.

3) EpCAM: epitopic cell addition molecules, Epithelial cell adhesion molecules, Epithelial-derived cancer cell surface markers.

4) CK: cytokeratin, Cytokeratin and CK family members are numerous and widely distributed, and are common markers of tumor cells of epithelial origin.

5) DAPI: 4',6-diamidino-2-phenylindole, fluorescent dye capable of strongly combining with DNA and commonly used for fluorescence microscope observation.

Specifically, in S3, the fluorescence signal is collected by a fluorescence scanner, and CTCs are identified based on cell staining and morphology. In this example, the criteria for identifying CTCs are: cells that met DAPI + and CK/EpCAM + and CD 45-staining and cell morphology were defined as CTCs. Here the "+" symbol represents a fluorescent signal, i.e. CK/EpCAM + means CK or EpCAM has a fluorescent signal; the "-" symbol represents no fluorescence signal.

Further, executing S3 specifically includes the following steps:

adding cell diluent into the third centrifuge tube, mixing uniformly in sequence, and centrifuging;

removing supernatant from a sample in a third centrifugal tube after centrifugation, adding a cell fixing solution for fixing and permeabilizing, then adding a cell washing solution, and centrifuging;

discarding the supernatant, adding a cell nucleus dye, cytokeratin, EpCAM and CD45 into the third centrifuge tube, and incubating and dyeing in dark;

replenishing cell washing liquor into the third centrifuge tube again, and centrifuging;

discard the supernatant, transfer the sample from the third centrifuge tube to a 96-well reading plate, scan and image analyze.

In conclusion, the circulating tumor cell negative enrichment detection method provided by the invention adopts the steps of adding the combination of the erythrocyte-specific antibody and the leukocyte-specific antibody into the whole blood sample to adsorb erythrocytes and leukocytes, and removing most of erythrocytes and leukocytes in the blood sample by combining the density gradient centrifugation method, so that the problems of the labels 1), 2) and 3) in the background art can be effectively solved.

On the other hand, in order to solve the problem of reference numeral 4) mentioned in the background art, the solution adopted by the present invention is: the cell adsorption prevention treatment is carried out before all consumables (test tubes, gun heads and the like) contacting the whole blood sample are used, the loss rate of cells in the operation process is effectively reduced, and the sensitivity of the CTCs detection method based on negative enrichment is further improved.

In addition, for better understanding of the method for detecting negative enrichment of circulating tumor cells of the present invention, the following examples are listed, and the details of the examples are as follows:

1.1 healthy human blood collected in EDTA-2K anticoagulation tubes using EpCAM/CK expressing tumor cells, such as human breast cancer SKBR3 cells, were used as samples for analytical performance evaluation. The number of incorporated SKBR3 cells can be accurately counted by microscopy.

1.2 clinical Whole blood samples suitable for Collection Using EDTA-2K anticoagulation tubes

1.3 accuracy verification and determination of measurement Range

The accuracy of the detection method was evaluated using a recovery experiment, i.e., a known number of SKBR3 cells were added to the negative matrix blood, and the ability of the detection method to accurately determine the number of added cells was evaluated, with the results expressed as recovery.

The recovery rates were calculated separately by detecting different numbers of incorporated cells (0-250) and the optimal measurement range for the method was determined by linear regression analysis. The recovery rate was x 100% as the number of positive cells/number of incorporated cells recovered.

1.3.1 verification protocol

Approximately 250, 100, 50, 10 and 0 SKBR3 cells were added to 5 healthy donor blood samples each day, repeated for 3 consecutive days. Recovery was obtained by dividing the number of observed tumor cells by the number of added tumor cells. The average yield was retrieved as the average recovery. And obtaining the measurement range of the CTCs negative enrichment detection method through linear regression analysis.

1.3.2 determination of results

And if the average recovery rate is more than 60%, the requirement of the verification scheme is met. The range of linearity that can achieve the desired recovery is the measurement range of the method.

1.3.3 test results

See table 1 for the mean recovery data for all samples in each spiked cell group; a graph of the results of the linear regression analysis of all sample results data is shown in fig. 2.

TABLE 1 mean recovery data for all samples of each spiked cell group

Group of	Mean	CV	N
					250	71.1％	5.5％	3
100	72.9％	8.5％	3
				50	82.3％	11.4％	3
10	83.8％	14.4％	3
				0	0％	0％	3

The test results are shown in table 1, the lowest sample recovery rate in the test results is 66%, and the average recovery rate of all samples is more than 70%.

All sample results data were subjected to linear analysis (results of linear analysis are shown in fig. 2), regression equation: y ═ 0.706X + 2.0662; r²0.99. In the range of 0-250 cells, the recovery data of all samples exceed 60%, and the linear relationship is good, so 0-250 cells are the measurement range of the method.

1.4 minimum detection Limit verification

Adding different amounts of SKBR3 cells into a negative matrix blood sample for detection, calculating the detection rate of each cell level, and taking the lowest cell level with the detection rate of 100% positive (the number of detected CTCs is more than or equal to 1) as the lowest detection limit.

1.4.1 verification scheme

Approximately 3, 2, and 1 SKBR3 cells were added to the stromal blood sample, respectively, and repeated 10 times. The lowest cell level with a positive detection rate of 100% was taken as the lowest detection limit.

1.4.2 determination of results

The detection rate of each incorporated cell level was calculated separately, and the lowest cell level at which the positive detection rate reached 100% was determined as the lowest detection limit.

1.4.3 test results

The lower limit test was performed in 8 time periods, 3, 2, 1 SKBR3 cells were incorporated each time, and 3 tests were performed in 10 replicates in total, with the test results shown in fig. 3. The detection rate for all samples was 100% when the spiked cells were 1.

1.5 specificity verification

The specificity of the analysis is expressed as "negative detection rate", i.e.the number of negative (detection of no CTC) results in the total number of tests, which are carried out by multiple tests of blood samples from healthy donors without a history of tumors.

1.5.1 verification protocol

About 10 healthy human whole blood samples were tested daily for a total of 20 samples in 2 trials. After enriching CTCs, the method carries out specific antibody combination staining, CK and EpCAM are used for identifying CTCs, DAPI is used for determining cell staining, and CD45 is used for distinguishing white blood cells. Non-specific staining was therefore excluded by a comprehensive judgment of various antibody combinations and nuclear staining.

1.5.2 determination of results

The negative detection rate of 10 times of determination results is not lower than 90%.

1.5.3 test results

According to the actual sample condition, the specificity detection is completed for 2 times of tests totally, and 20 samples are counted, and the detection results are all negative, namely the specificity of the samples is 100%.

1.6 repeatability verification

And evaluating the repeatability of the detection method by adopting a recovery experiment, namely adding a known number of SKBR3 cells into negative matrix blood, carrying out repeated detection, detecting the recovery rate of the cells, and evaluating the repeatability of the detection method by comparing CV of the recovery rate under the repeated detection.

1.6.1 verification protocol

About 50 SKBR3 cells were spiked into 10 healthy donor blood samples and about 100 SKBR3 cells were spiked into another 10 healthy donor blood samples, followed by CTCs detection. The average value of 10 detection results of high concentration (doped with 100 SKBR3) and low concentration (doped with 50 SKBR3) is calculated respectivelyAnd variance SD according to the formula:

and calculating the coefficient of variation CV of the high and low concentration detection results.

1.6.2 determination of results

The high-concentration sample and the low-concentration sample are respectively and repeatedly detected for 10 times, and the coefficient of variation CV of the detection result is required to be less than or equal to 20 percent.

1.6.3 test results

A total of 20 validation tests were completed and the results are shown in figure 4. The average recovery of all samples was greater than 60%. The low-concentration sample is repeatedly detected for 10 times, the average recovery rate is 70%, the coefficient of variation CV is 13%, and the repeatability requirement is met; the high-concentration sample is repeatedly detected for 10 times, the average recovery rate is 71%, the coefficient of variation CV is 15%, and the repeatability requirement is met.

In summary, in the method for detecting the negative enrichment of circulating tumor cells provided by the present invention, the method for detecting the negative enrichment of circulating tumor cells comprises: adding a combination of red blood cell-specific antibodies and white blood cell-specific antibodies to a first centrifuge tube containing a whole blood sample to couple red blood cells and white blood cells in the whole blood sample together; and removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method so as to realize negative enrichment of the circulating tumor cells. From the above, the circulating tumor cell negative enrichment detection method based on the invention can effectively improve the purity of CTCs in the monocyte layer, thereby reducing the complexity of subsequent identification work and improving the efficiency and accuracy of identification.

The above description is only for the purpose of describing the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention, and any variations and modifications made by those skilled in the art based on the above disclosure are within the scope of the appended claims.