CN108646034B - Method for determining rare cells in cell population - Google Patents
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Abstract
The invention relates to the field of biological statistical analysis, in particular to a rare cell interpretation method in a cell population, which calculates a cell ratio R (R is a positive signal value F of a cell/a negative signal value F' of the cell) by detecting different expression levels of a certain target point of a cell line,and analyzing the positive cell group ratio and the negative cell group ratio by using an ROC curve to obtain a ratio threshold value Rt. Simultaneous calculation of fluorescence threshold
(the average value of F of the cell group) + ns (s is the standard deviation of F of the cell group), obtaining the fluorescence threshold Ft, and judging whether the cells are negative or positive according to the relation between the ratio R and Rt and the relation between F and Ft. Compared with the prior art, the method is simple, fast, high in accuracy and good in consistency.
Description
Technical Field
The invention relates to the field of biological statistical analysis, in particular to a rare cell interpretation method in a cell population.
Background
Rare cell interpretation in cell populations is a common goal in the biological or medical fields; common applications are, for example, the search of Circulating Tumor Cells (CTCs) from blood cells.
The circulating tumor cells are tumor cells which are dropped from the tissue part of the primary tumor focus and enter the blood circulation, and a large number of experiments prove that the detection of CTC is helpful for monitoring the recurrence and metastasis of the tumor, judging the prognosis of patients and guiding the postoperative treatment of the patients[1,2]. The detection and analysis methods of CTCs are many, and the common techniques comprise flow cytometry analysis, gene chip, reverse transcription-polymerase chain reaction, immunofluorescence technique and the like[3]. At present, products on the market for CTCs immunofluorescence detection mainly comprise a Cellsearch detection system of Qiangsheng company, a CTC-Biopsy detection system of Wuhan Yongzhiyou medical science and technology, Inc., and the like.
The criteria for determining tumor cells by the Cellsearch detection system are generally: 1) having a typical cell morphology, 2) having a nuclear morphology, 3) the cell morphology is distinguished from a leukocyte morphology, 4) CK staining is positive, 5) CD45 staining is negative, 6) the proportion of morphology is greater than 4 x 4 μm, 7) CK staining is at least 3 times greater than background color, 8) the cell membrane CK staining area is at least 50% greater, 9) cell membrane CK staining is more uniform than spot cell membrane CK staining areaDyeing of figures[4]. Although the analysis system introduces a computer analysis method, the tumor cells can be rapidly judged by the aid of a computer, the result still needs to be rechecked by an analyst, the given judgment index is not quantified by numbers, the subjective impression of the analyst is still greatly depended on, and the result misjudgment is easily caused by information loss based on simple computer assistance. There is also a study of the interpretation of HER2 positive cells based on the cellsearch detection system using the relative ratio (cell HER2 staining fluorescence/cell area)/(background fluorescence intensity/cell area)[5]. This method, although defined directly for HER2 positive cells with relatively simple values, is susceptible to background staining and results are prone to false positives.
The CTC-Biopsy detection system is used for judging CTC based on pathological states of cells, collects and summarizes CTC cell morphological standards proposed by a large number of current cytopathology experts, and simultaneously carries out a large number of immunohistochemical verification works together with the cytopathology experts in a plurality of hospitals nationwide, but the system has strict requirements on an analyst, requires the analyst to be strictly trained and have certain level of pathological knowledge to judge results, and in actual operation, at least two analyst strict analysis results are often required to be referred to for the same result, which is a challenge for clinical examination and is not small[6]. Just like the current clinical immunohistochemistry as gold standard, the interpretation method is also established based on a large amount of cytopathology knowledge, but in clinical practice, the requirements on the interpretation readers are extremely strict, and different analysts have certain differences in interpretation of the same result.
Therefore, it is very important to establish a simple, fast and accurate method for judging immunofluorescence positive cells.
Reference to the literature
[1]E Crowley,NF Di,F Loupakis,A Bardelli.(2013)Liquid biopsy:monitoring cancer-genetics in the blood.Nature Reviews Clinical Oncology 10(8):472.
[2]G Brock,E Castellanos-Rizaldos,L Hu,et al.(2015)Liquid biopsy forcancer screening,patient stratification and monitoring.Translational CancerResearch 4(3).
[3]Zhang Z,Ramnath N and Nagrath S.(2015)Current status of CTCs asliquid biopsy in lung cancer and future directions.Front Oncol.5:209.
[4]KC Andree,G Van Dalum,LWMM Terstappen.(2016)Challenges incirculating tumor cells detection by the cellsearch system.Molecular Oncology10(3):395-407.
[5]Ignatiadis M,Rothe′F,Chaboteaux C,Durbecq V,Rouas G,et al.(2011)HER2-Positive Circulating Tumor Cells in Breast Cancer.PLoS ONE 6(1):e15624.
[6] Is plum blossom. (2015) Optimization of an improved ISET (CTC-Biopsy) technical system and verification of the effect of detecting circulating tumor cells of a tumor patient. University of Jinan, Master academic thesis.
Disclosure of Invention
The invention relates to a rare cell interpretation method in a cell population, which comprises the following steps:
the marker of the rare cell to be detected is called X protein, and the marker of the cell group is called Y protein; labeling the anti-X protein antibody with a first signal strength indicator and the anti-Y protein antibody with a second signal strength indicator;
adding X protein positive cells and negative cells into the cell population respectively, detecting the X protein positive cells and the negative cells by using the anti-X protein antibody and the anti-Y protein antibody respectively, counting a first signal intensity indicator signal intensity value F and a second signal intensity indicator signal intensity value F 'of each cell picture respectively, and calculating R ═ F/F' of each cell picture; calculate the average of all F
And standard deviation s, obtaining the fluorescence threshold
When the fluorescence threshold value
When a 95% confidence interval is taken, n is 2; when a 99% confidence interval is taken, n is 3; when a confidence interval of 99.9% is taken, n is 4; when a confidence interval of 99.99% is taken, n is 5;
analyzing each group of R values by using an ROC curve to determine a signal intensity ratio threshold value Rt for distinguishing the positive cells from the negative cells;
for a cell of the population, if it is F > Ft and R > Rt, it is a rare cell with the characteristics of the X protein.
Compared with the prior art, the method is simple, fast, high in accuracy and good in consistency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the concentration of HER2 detection antibody and Herceptin antibody used in the examples of the present invention;
FIG. 2 shows the expression level of HER2 in HER2 detection antibody and Herceptin antibody detection cell line in the example of the present invention;
intensity refers to the cellular FITC fluorescence value, representing HER2 expression level;
ratio refers to the FITC fluorescence value of the cell picture compared with the CY5 fluorescence value of the cell picture;
FIG. 3 is a graph showing the relationship between cell staining and ratio in examples of the present invention;
BT474 and SKBR3 cells are HER2 high expression cell lines, FITC detection is positive, and CY5 detection is negative; a431, MDA-MB-231 and MCF7 are HER2 low expression cell lines, FITC detection is negative, and CY5 detection is negative; WBC is HER2 low expression and CD45 expression cell, FITC detection is negative, and CY5 detection is positive.
FIG. 4 is a graph showing the ratio distribution of different cells in the examples of the present invention;
a: the cell ratio distribution is different, the ratio is 3.0-5.0, and the HER2 positive cell group (SKBR3, BT474) and the HER2 negative cell group (MCF7, A431, MDA-MB-231 and WBC) are obviously different; b: ratiometric analysis of the HER2 positive cell group (SKBR3, BT474) and HER2 negative cell group (MCF7, A431, MDA-MB-231, WBC);
FIG. 5 is a comparison of different ratio threshold interpretation results according to an embodiment of the present invention;
a: 3.5 analysis of healthy human, benign lesions, breast cancer samples, B: ratio 4.0 healthy, benign lesions, breast cancer samples were analyzed, C: analyzing healthy people, benign lesions and breast cancer samples according to the ratio of 4.5;
FIG. 6 is a comparison of different interpretation methods according to embodiments of the present invention;
a: analyzing healthy people, benign lesions and breast cancer samples by a ratio interpretation method; b: the statistical interpretation method analyzes healthy persons, benign lesions, breast cancer samples.
Detailed Description
The invention relates to a rare cell interpretation method in a cell population, which comprises the following steps:
the marker of the rare cell to be detected is called X protein, and the marker of the cell group is called Y protein; labeling the anti-X protein antibody with a first signal strength indicator and the anti-Y protein antibody with a second signal strength indicator;
adding X protein positive cells and negative cells into the cell population respectively, detecting the X protein positive cells and the negative cells by using the anti-X protein antibody and the anti-Y protein antibody respectively, counting a first signal intensity indicator signal intensity value F and a second signal intensity indicator signal intensity value F 'of each cell picture respectively, and calculating R ═ F/F' of each cell picture; calculate the average of all F
And standard deviation s, obtaining the fluorescence threshold
When the fluorescence threshold value
When a 95% confidence interval is taken, n is 2; when a 99% confidence interval is taken, n is 3; when a confidence interval of 99.9% is taken, n is 4; when a confidence interval of 99.99% is taken, n is 5;
analyzing each group of R values by using an ROC curve to determine a signal intensity ratio threshold value Rt for distinguishing the positive cells from the negative cells;
for a cell of the cell population, it is a rare cell with the property of an X protein if its F > Ft and R > Rt.
The invention adopts anti-Y protein antibody to mark cell group; can well simulate the detection environment of cell groups and rare cells.
In some embodiments, the anti-protein X antibody is one or more;
when the anti-protein X antibody is multiple, before adding the protein X positive cells and the protein X negative cells to the cell population respectively, the method further comprises the following steps:
detecting X protein positive and negative cells with an anti-X protein antibody Ab1, an anti-X protein antibody Ab2, an anti-X protein antibody Ab3 … …, an anti-X protein antibody Abn, and the anti-Y protein antibody, respectively, and calculating ratios R1, R2, R3 … … Rn of the first signal intensity indicator signal intensity value to the second signal intensity indicator signal intensity value, respectively.
Analyzing the data consistency of R1, R2 and R3 … … Rn, and if the consistency of each antibody is good, selecting one antibody in the subsequent steps for verification; if the consistency is poor, the verification is carried out according to groups with similar consistency, or each antibody is verified respectively.
Data consistency can be verified by statistical analysis methods known in the art, and when the number of antibodies is not large (e.g., 2 or 3 antibodies), one skilled in the art can analyze whether it is sufficient to distinguish between negative and positive cells by comparing the R values of each group with the R values after leukocyte verification to determine whether to partition a uniform threshold criterion.
Different antibodies generally refer to monoclonal antibodies that recognize different epitopes of protein X.
In some embodiments, the first and second signal intensity indicators are selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, electron-dense substances, colloidal gold, or enzymes.
In theory, the first signal strength indicator and the second signal strength indicator may be selected from different quantifiable signals, as long as the two are distinguishable. For example, the first signal intensity indicator is selected from Alexa 647 and the second signal intensity indicator is selected from a radioisotope.
In some embodiments, the fluorescent species include Alexa 350, Alexa 405, Alexa 430, Alexa488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5 ', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, FITC (fluorescein isothiocyanate), HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate, diamine, bispyanin, La Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and texas red.
In some embodiments, the radioactivity isIsotopes of the formulae110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82mRb and83sr.
In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
In some embodiments, the X protein comprises any one of HER2, napsin a, TTF-1, era, ER β, PR, Bcl-2, EGFR, ERCC1, RRM1, CEA, AFP, era, CA242, CA199, CA 724.
In some embodiments, the population of cells is leukocytes and the rare cell is any one of a circulating tumor cell, a circulating epithelial cell, a circulating endothelial cell, a stem cell, a residual diseased cell;
or, the cell population is a mature tissue and the rare cell is a stem cell.
In some embodiments, when the cell population is leukocytes, the Y protein comprises one or more of CD45, CD3, CD50, CD66B, CD69, CD 84;
in some embodiments, after determining a threshold range of signal intensity for distinguishing between the positive cells and the negative cells, the method further comprises:
validating the threshold range with a blood sample containing the positive cells or the negative cells.
In some embodiments, when the rare cell is a circulating tumor cell, the X protein comprises one or more of HER2, era, ER β, PR, Bcl-2, EGFR, ERCC1, RRM1, CEA, AFP, CA242, CA199, CA724, PDL1, PDL 2;
when the rare cell is a circulating epithelial cell, the X protein comprises one or more of Epcam, N-cadherin, FSP1, CK, hMAM, MUC1, PMSA;
when the rare cell is a circulating endothelial cell, the X protein comprises one or more of CD34, P1H12, CD31, CD62, CD 102;
when the rare cell is a stem cell, the X protein comprises one or more of CD44, CD133, CD90, ALDH1, ABCG 2.
In some embodiments, the protein X is HER 2;
the anti-X protein antibody is a Herceptin antibody and a HER2 detection antibody (Zhuhai grass diagnostic product technology Co., Ltd., CBS 006);
in some embodiments, the first signal intensity indicator and the second signal intensity indicator are each selected from fluorescent species and are fluorescent species that differ in both excitation and emission light;
in some embodiments, the first signal strength indicator is FITC and the second signal strength indicator is CY 5.
In some embodiments, a HER2 positive cell must satisfy:
the fluorescence intensity of the positive signal is >19, the R value is >3.5, and the staining of intact cell nucleus and cell membrane is realized.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Examples
In this example, HER2 positive cells were used as an example to illustrate the technical solution of the present invention.
Determination of the optimal reaction concentration of HER2 test antibody
The Herceptin antibody and HER2 detection antibody were diluted to 16ug/ml with binding buffer, and each diluted in 2-fold gradient for detection on SKBR3 cells. The results show that the concentration of the Herceptin antibody and the HER2 detection antibody is 2ug/ml to 4ug/ml, the cell binding activity is saturated, and the fluorescence intensity value is more stable when the concentration is 4 ug/ml.
2. Immunofluorescence detection of HER2 expression level of breast cancer cell line
According to the existing literature reports, SKBR3 and BT474 cell lines are HER2 high-expression breast cancer cell lines, are defined as HER 23 + in immunohistochemistry, and FISH detects ErBb2 gene amplification; MCF7 and MDA-MB-231 cell lines are HER2 low-expression breast cancer cell lines which are defined as HER 20-1 + in immunohistochemistry, and FISH detection ErBb2 gene is not amplified; a431 is HER2 low expression skin cancer cell line; WBCs are HER2 low expressing human leukocytes. Detecting the HER2 expression level of cell lines SKBR3, BT474, MCF7, MDA-MB-231 and A431 by using a Herceptin antibody, wherein the result shows that SKBR3 and BT474 cells are HER2 high-expression cells and FITC fluorescence intensity values are 19-254.98; a431, MCF7, MDA-MB-231 and WBC are HER2 low-expression cells, and FITC fluorescence intensity values are 5.29-25.22. Similarly, the HER2 detection antibody is used for detecting the expression level of HER2 of cell lines SKBR3, BT474, MCF7, MDA-MB-231 and A431, and the results show that the FITC fluorescence intensity values of SKBR3 and BT474 cells are 22.29-254.8, and the FITC fluorescence intensity values of MCF7, MDA-MB-231 and A431 are 4.38-29.63. Therefore, SKBR3 and BT474 cells are classified into a HER2 positive cell group, FITC fluorescence intensity values of 19.00-254.98 and negative group fluorescence intensity values of 4.86-29.63 (Table 1, Table 2 and FIG. 2).
TABLE 1Herceptin antibody test results
Table 2HER2 test antibody test results
ROC analysis of HER2 Positive to negative cell group ratio
100 SKBR3, BT474, MCF7, MDA-MB-231, A431 cells were added to 1.25X 10 cells, respectively7Among the leukocytes, cell line capture was performed using mixed capture antibody and magnetic beads, respectively, cell line detection was performed using HER2 detection antibody, and leukocytes were detected using CD45-APC detection antibody. Determination of leukocyte populations based on fluorescence of cellular CD45-APC staining (CY5 fluorescence value)>7.2), classifying cells with nuclei larger than leukocyte nuclei into cell lines according to cell morphology, and classifying the cells according to FITC fluorescence values (FITC fluorescence values of HER2 positive group (SKBR3, BT474) 21.64-228.8, and HER2 negative group (MDA-MB-231, MCF7, A431) 5.5-19.3). The FITC fluorescence intensity value and CY5 fluorescence intensity value of each cell image were counted, and the ratio (FITC fluorescence intensity value/CY 5 fluorescence intensity value) of each cell image was calculated (table 3). Data analysis is carried out by Graphpad software, the ratio is in the range of 3.0-5.0, and the HER2 positive group cells and the HER2 negative group cells have larger difference (figure 4). When the ratio is 3.5-4.0, the sensitivity is 99.63-100%, the specificity is 99.00-99.88%, and p is measured by ROC curve analysis<0.0001 (Table 4, Table 5).
TABLE 3 cell fraction distribution
TABLE 4ROC Curve analysis ratio thresholds
TABLE 5 different ratio threshold sensitivity and specificity analysis
4. Ratio analysis of the difference in the number of HER2 positive cells in healthy persons, benign lesions and breast cancer
The ratios of 3.5, 4.0 and 4.5 are respectively used as threshold values to analyze the numbers of HER2 positive cells of 72 healthy people, 35 benign lesions and 71 breast cancer samples, and the sample positive rate is counted according to the number of HER2 positive cells. The results show that the ratio is 4.0, and there is a large difference among healthy people, benign lesions, and breast cancer samples (fig. 5). Therefore, the ratio of 4.0 is selected as the critical value of the ratio interpretation method, the cells with the ratio of 3.5-4.0 are manually assisted and interpreted according to the dyeing integrity of cell nuclei and cell membranes.
5. Establishment of ratio interpretation method
According to the FITC fluorescence intensity values of a HER2 positive cell group and a HER2 negative cell group,
therefore, HER2 positive cells have to satisfy: FITC fluorescence intensity>19, ratio of>3.5, and has intact nuclear and cell membrane staining.
6. Comparison of ratio interpretation method with other interpretation methods
Randomly selecting 10 immunofluorescence detection samples, and analyzing by two analysts respectively by using a ratio interpretation method and a statistical interpretation method to compare the time consumption, the accuracy and the consistency of the two methods. The results show that the specific value interpretation method takes 10 minutes per sample on average, the accuracy reaches 99%, and the consistency of the analysis results of two analysts reaches 100% (Table 6). Meanwhile, the two methods are used for analyzing 72 healthy persons, 35 benign lesions and 71 breast cancer samples respectively, and the results show that when the ratio method is used for judging, the healthy persons, the benign lesions and the breast cancer samples have large differences (figure 6).
TABLE 6 comparison of interpretation methods
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (11)
1. A method for identifying rare cells in a cell population, comprising the steps of:
the marker of the rare cell to be detected is called X protein, and the marker of the cell group is called Y protein; labeling the anti-X protein antibody with a first signal strength indicator and the anti-Y protein antibody with a second signal strength indicator;
adding X protein positive cells and negative cells to the cell population respectively, detecting the X protein positive cells and the negative cells by using the anti-X protein antibody and the anti-Y protein antibody respectively, counting a first signal intensity indicator signal intensity value F and a second signal intensity indicator signal intensity value F 'of each cell picture respectively, and calculating R = F/F' of each cell picture; calculating the average value 'x and the standard deviation s of all F to obtain a threshold value Ft =' x + ns;
when the threshold Ft =' x + ns takes a 95% confidence interval, n = 2; when a 99% confidence interval is taken, n = 3; when a 99.9% confidence interval is taken, n = 4; when a confidence interval of 99.99% is taken, n = 5;
analyzing each group of R values by using an ROC curve to determine a signal intensity ratio threshold value Rt for distinguishing the positive cells from the negative cells;
for a cell of the population, if it is F > Ft and R > Rt, it is a rare cell with the characteristics of the X protein.
2. The method of claim 1, wherein the anti-protein X antibody is one or more;
when the anti-protein X antibody is multiple, before adding the protein X positive cells and the protein X negative cells to the cell population respectively, the method further comprises the following steps:
detecting X protein positive and negative cells with an anti-X protein antibody Ab1, an anti-X protein antibody Ab2, an anti-X protein antibody Ab3 … …, an anti-X protein antibody Abn, and the anti-Y protein antibody, respectively, and calculating ratios R1, R2, R3 … … Rn of the first signal intensity indicator signal intensity value to the second signal intensity indicator signal intensity value, respectively.
3. The method of claim 1, wherein the first and second signal intensity indicators are selected from the group consisting of fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, electron-dense substances, colloidal gold, and enzymes.
4. The method according to claim 3, wherein the fluorescent substance comprises Alexa 350, Alexa 405, Alexa 430, Alexa488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5 ', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, FITC (fluorescein isothiocyanate), HEX, 6-JOE, D (7-nitrobenzo-2-oxa-1, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate, diamine, bispyanin, La Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and texas red.
5. The method of claim 3, wherein the radioisotope comprises110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82mRb and83sr.
6. The method of claim 3, wherein the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
7. The method according to any one of claims 1 to 6, wherein the cell population is leukocytes, and the rare cells are any one of circulating tumor cells, circulating epithelial cells, circulating endothelial cells, stem cells, and residual diseased cells;
or, the cell population is a mature tissue and the rare cell is a stem cell.
8. The method of claim 7, wherein the Y protein comprises one or more of CD45, CD3, CD50, CD66B, CD69, CD84 when the cell population is leukocytes.
9. The method of claim 8, wherein after determining a threshold range of signal intensities for distinguishing the positive cells from the negative cells, the method further comprises:
validating the threshold range with a blood sample containing the positive cells or the negative cells.
10. The method of claim 7, wherein when the rare cell is a circulating tumor cell, the X protein comprises one or more of HER2, ER α, ER β, PR, Bcl-2, EGFR, ERCC1, RRM1, CEA, AFP, CA242, CA199, CA724, PDL1, PDL 2;
when the rare cell is a circulating epithelial cell, the X protein comprises one or more of Epcam, N-cadherin, FSP1, CK, hMAM, MUC1, PMSA;
when the rare cell is a circulating endothelial cell, the X protein comprises one or more of CD34, P1H12, CD31, CD62, CD 102;
when the rare cell is a stem cell, the X protein comprises one or more of CD44, CD133, CD90, ALDH1, ABCG 2.
11. The method of claim 10, wherein the protein X is HER 2;
the anti-X protein antibody is a Herceptin antibody and a HER2 detection antibody;
the first signal intensity indicator and the second signal intensity indicator are both selected from fluorescent substances, and are fluorescent substances different in both excitation light and emission light.
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