CN108646034A - Rare cell interpretation method in cell mass - Google Patents
Rare cell interpretation method in cell mass Download PDFInfo
- Publication number
- CN108646034A CN108646034A CN201810718281.2A CN201810718281A CN108646034A CN 108646034 A CN108646034 A CN 108646034A CN 201810718281 A CN201810718281 A CN 201810718281A CN 108646034 A CN108646034 A CN 108646034A
- Authority
- CN
- China
- Prior art keywords
- cell
- protein
- rare
- antibody
- signal strength
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention relates to biometric analysis fields, specifically, the rare cell interpretation method being related in a kind of cell mass, the different expressions that the method passes through detection cell line target spot, calculate cell ratio R (R is the negative signal value F' of the positive signal value F/ cells of cell), with ROC curve analysis positive cell group ratio and negative group cell ratio, fractional threshold Rt is obtained.Calculate fluorescence threshold simultaneously(average value of the F of cell mass)+ns (s is the standard deviation of the F of cell mass), obtains fluorescence threshold Ft, according to the relationship and F of ratio R and Rt and the relationship of Ft can interpretation cell yin and yang attribute.Compared with prior art, this method is simple, quick, accuracy is high, consistency is good.
Description
Technical field
The present invention relates to biometric analysis fields, in particular to the rare cell interpretation side in a kind of cell mass
Method.
Background technology
Rare cell interpretation in cell mass is biology or the common purpose of medical domain；Wherein common application is for example from blood
Circulating tumor cell (Circulating tumor cell, CTC) is found in liquid cell.
Circulating tumor cell is that primary tumors tissue site sheds into sanguimotor tumour cell, many experiments card
The detection of real CTC contributes to monitoring, the judgement of patient's prognosis and the guidance of patient's aftertreatment that tumor recurrence shifts[1,2]。
The determination method of CTCs is more, and common technology includes that flow cytometry, genetic chip, reverse transcription-polymerase chain are anti-
It answers, immunofluorescence technique etc.[3].Currently, commercialized product mainly has the Cellsearch of Johson ＆ Johnson to CTCs Immunofluorescence tests
Detecting system, the CTC-Biopsy detecting systems etc. of Wuhan You Zhiyou medical science and technologies limited liability company.
Cellsearch detecting systems are generally the criterion of tumour cell：1) have typical cells form, 2)
With karyomorphism, 3) cellular morphology is had any different in WBC Appearance, 4) CK dyeing is the positive, 5) CD45 dyeing is feminine gender,
6) form ratio is more than 4 × 4 μm, 7) CK dyeing is compared at least 3 times or more of background colour, 8) cell membrane CK pigmented sections at least 50% with
On, 9) cell membrane CK dyeing is more uniform, and non-point-like dyes[4].Although the analysis system has had been incorporated into computer analysis side
Method can quickly judge tumour cell through computer auxiliary, but still analyst needed to check result, and give
The not useful number of the Judging index gone out is quantified, and still relies heavily on the subjective impression of analyst, and based on letter
Single area of computer aided be easy to cause the loss of information, and result is caused to be judged by accident.Also research is based on cellsearch detecting systems,
Formulate HER2 positive cells interpretation method, the interpretation method with relative ratio (cell HER2 fluorescent stainings value/cell area)/
(background fluorescence intensity value/cell area)[5].Although this method directly carries out HER2 positive cells with relatively simple numerical value
Definition, but this method is easy to be influenced by background stainings, and result is susceptible to false positive.
CTC-Biopsy detecting systems are the pathological states based on cell to carry out the judgement of CTC, although the system is collected
The CTC cellular morphology standards that a large amount of cell pathology experts propose at present are summarized, while also thin with more of whole nation Grade A hospital
Born of the same parents' pathologist has carried out a large amount of immunohistochemistry verification work together, but requirement of the system for analyst is harsh, it is desirable that
Analyst must be by screwing up discipline and the pathology knowledge frequently with certain level completes the interpretation of result, in practical operation
In, same a result is generally required to refer to the stringent analysis result of at least two analysts, this is not for clinical examination
Small challenge[6].As clinically as the immunohistochemistry of goldstandard, interpretation method is also based on a large amount of cell pathologies at present
Knowledge is established, but clinically, extremely harsh for the requirement for sentencing reader, and different analyst's sentencing for same a result
Reading has a certain difference.
Therefore, it is particularly important to establish simple, quick, accurate immunofluorescence positive cell interpretation method.
Bibliography
[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 for
cancer screening,patient stratification and monitoring.Translational Cancer
Research 4(3).
[3]Zhang Z,Ramnath N and Nagrath S.(2015)Current status of CTCs as
liquid biopsy in lung cancer and future directions.Front Oncol.5:209.
[4]KC Andree,G Van Dalum,LWMM Terstappen.(2016)Challenges in
circulating tumor cells detection by the cellsearch system.Molecular Oncology
10(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] Lee is huge.(2015) optimization of improvement ISET (CTC-Biopsy) technical system and its detection tumor patient cycle
The verification of tumour cell effect.University Of Ji'nan, master thesis.
Invention content
The present invention relates to the rare cell interpretation methods in a kind of cell mass, include the following steps：
The marker of rare cell to be detected is referred to as X protein, the marker of cell mass is referred to as Y albumen；It uses
The anti-X protein antibody of first signal strength tracer-labelling uses the anti-Y protein antibodies of second signal intensity tracer-labelling；
X protein positive cell and negative cells are respectively added in the cell mass, with the anti-X protein antibody and institute
It states anti-Y protein antibodies and detects X protein positive cell and negative cells respectively, the first signal for counting every cell picture respectively is strong
Indicator signal strength values F and second signal intensity indicator signal strength values F' is spent, and calculates the R=F/ of every cell picture
F'；Calculate the average value of all FWith standard deviation s, fluorescence threshold is obtained
When the fluorescence thresholdWhen taking 95% confidence interval, n=2；When taking 99% confidence interval, n=3；
When taking 99.9% confidence interval, n=4；When taking 99.99% confidence interval, n=5；
Each group R values are analyzed with ROC curve, determine the letter for distinguishing the positive cell and the negative cells
Number intensity rate threshold value Rt；
For certain cell of the cell mass, if its F ＞ Ft, and R ＞ Rt, then it is rare thin with X protein feature
Born of the same parents.
Compared with prior art, this method is simple, quick, accuracy is high, consistency is good.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is that HER2 detects antibody in the embodiment of the present invention and Herceptin antibody is determined using concentration；
Fig. 2 is that HER2 detects antibody and Herceptin antibody test cell line HER2 expressions in the embodiment of the present invention；
Intensity refers to cell FITC fluorescent values, represents HER2 expressions；
Ratio refers to CY5 fluorescent value of the FITC fluorescent values than cell picture of cell picture；
Fig. 3 is cell dyeing and ratio relation in the embodiment of the present invention；
BT474, SKBR3 cell are HER2 high-expression cell lines, and FITC detections are positive, and CY5 detections are negative；A431、MDA-
MB-231, MCF7 are HER2 low expression cell lines, and FITC detections are negative, and CY5 detections are negative；WBC is HER2 low expressions, CD45 tables
Up to cell, FITC detections are negative, and CY5 detections are positive.
Fig. 4 is the ratio Distribution value of different cells in the embodiment of the present invention；
A：Different cell ratio distributions, ratio are cloudy in 3.0~5.0, HER2 positive cells group (SKBR3, BT474) and HER2
There are more apparent differences for property groups of cells (MCF7, A431, MDA-MB-231, WBC)；B：Ratio Analysis HER2 positive cell groups
(SKBR3, BT474) and HER2 negative cells group (MCF7, A431, MDA-MB-231, WBC)；
Fig. 5 is that difference fractional threshold sentence read result of the embodiment of the present invention compares；
A：3.5 analysis Healthy Peoples, benign lesion, breast cancer sample, B：Ratio 4.0 analyzes Healthy People, benign lesion, mammary gland
Cancer sample, C：Ratio 4.5 analyzes Healthy People, benign lesion, breast cancer sample；
Fig. 6 compares for difference interpretation method of the embodiment of the present invention；
A：Ratio interpretation method analyzes Healthy People, benign lesion, breast cancer sample；B：Statistics interpretation method analysis health
People, benign lesion, breast cancer sample.
Specific implementation mode
The present invention relates to the rare cell interpretation methods in a kind of cell mass, include the following steps：
The marker of rare cell to be detected is referred to as X protein, the marker of cell mass is referred to as Y albumen；It uses
The anti-X protein antibody of first signal strength tracer-labelling uses the anti-Y protein antibodies of second signal intensity tracer-labelling；
X protein positive cell and negative cells are respectively added in the cell mass, with the anti-X protein antibody and institute
It states anti-Y protein antibodies and detects X protein positive cell and negative cells respectively, the first signal for counting every cell picture respectively is strong
Indicator signal strength values F and second signal intensity indicator signal strength values F' is spent, and calculates the R=F/ of every cell picture
F'；Calculate the average value of all FWith standard deviation s, fluorescence threshold is obtained
When the fluorescence thresholdWhen taking 95% confidence interval, n=2；When taking 99% confidence interval, n=3；
When taking 99.9% confidence interval, n=4；When taking 99.99% confidence interval, n=5；
Each group R values are analyzed with ROC curve, determine the letter for distinguishing the positive cell and the negative cells
Number intensity rate threshold value Rt；
For certain cell of the cell mass, if its F ＞ Ft, and R ＞ Rt, then it is rare thin with X protein characteristic
Born of the same parents.
The present invention marks cell mass using anti-Y protein antibodies；It can be very well residing for simulation cell mass and rare cell
Detection environment.
In some embodiments, the anti-X protein antibody is one or more；
It is respectively added to cell mass when the anti-X protein antibody is a variety of, then by X protein positive cell and negative cells
In before further include：
With anti-X protein antibody A b1, anti-X protein antibody A b2, the anti-X protein antibody A bn of anti-X protein antibody A b3 ... and institute
It states anti-Y protein antibodies and detects X protein positive cell and negative cells respectively, and calculate separately the first signal strength indicator
The ratio R 1 of signal strength values and the second signal intensity indicator signal strength values, R2, R3 ... Rn.
The data consistency of R1, R2, R3 ... Rn are analyzed, if various antibody consistency are good, subsequent step chooses it
In an antibody verified；If consistency is poor, separately verified by packet authentication similar in consistency or each antibody.
Data consistency can be verified by statistical analysis technique well known in the art, (the example when antibody levels are few
As 2 or 3 kind of antibody), then can be analyzed by comparing each group R values and the R values after leucocyte is verified by those skilled in the art
Whether it is enough to distinguish negative cells and positive cell, to determine whether to divide unified threshold value standard.
Different antibodies refer generally to identify the monoclonal antibody of X protein difference epitope.
In some embodiments, the first signal strength indicator and second signal intensity indicator are selected from fluorescence
Matter, quantum dot, digoxin labelled probe, biotin, radioactive isotope, electron dense substances, colloidal gold or enzyme.
Theoretically speaking the first signal strength indicator and second signal intensity indicator can be measured selected from different
The signal of change, as long as the two can distinguish.Such as first signal strength indicator be selected from Alexa 647, and second letter
Number intensity indicator is selected from radioactive isotope.
In some embodiments, the fluorescent material includes Alexa 350, Alexa 405, Alexa 430, Alexa
488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY 630/650, BODIPY 650/665, BODIPY-
FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, 5-
Carboxyl -2 ', 4 ', 5 ', 7 '-tetrachlorofluoresceins, 5-carboxyfluorescein, 5- carboxyrhodamines, 6- carboxyrhodamines, 6- carboxyl tetramethyls
Base rhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,6-FAM, dansyl Cl, FITC (fluorescein isothiocynate),
HEX, 6-JOE, NBD (7- nitro benzo -2- oxa-s -1,3- diazole), Oregon Green 488, Oregon Green 500,
The solid purple, cresyl blue of Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), M-phthalic acid, cresols
Purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluoresceins, rare earth gold
Belong to cryptate, three pairs of pyridyl group diamines europiums, europium cryptate or chelate, diamines, dicyanin, La Jolla indigo plants dye
Material, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamines, algae red green white, phycoerythrin R,
REG, rhodamine be green, rhodamine isothiocyanates, rhodamine are red, ROX, TAMRA, TET, TRIT (different sulphur of tetramethylrhodamine
Alcohol), it is one or more in tetramethylrhodamine and texas Red.
In some embodiments, the radioactive isotope includes110In、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 and83Any one of Sr.
In some embodiments, the enzyme includes in horseradish peroxidase, alkaline phosphatase and glucose oxidase
It is any.
In some embodiments, the X protein include HER2, NapsinA, TTF-1, ER α, ER β, PR, Bcl-2,
Any one of EGFR, ERCC1, RRM1, CEA, AFP, ER α, CA242, CA199, CA724.
In some embodiments, the cell mass is leucocyte, and the rare cell is circulating tumor cell, in cycle
Any one of chrotoplast, circulating endothelial cells, stem cell, residual disease cell；
Or, the cell mass is the tissue reached maturity, the rare cell is stem cell.
In some embodiments, when cell mass be leucocyte when, the Y albumen include CD45, CD3, CD50, CD66B,
It is one or more in CD69, CD84；
In some embodiments, the signal strength threshold for distinguishing the positive cell and the negative cells is being determined
It is worth after range, the method further includes：
The threshold range is verified with the blood sample containing the positive cell or the negative cells.
In some embodiments, when the rare cell is circulating tumor cell, the X protein includes HER2, ER
One kind in α, ER β, PR, Bcl-2, EGFR, ERCC1, RRM1, CEA, AFP, CA242, CA199, CA724, PDL1, PDL2 or
It is a variety of；
When rare cell be cycle epithelial cell when, the X protein include Epcam, N- cadherin, FSP1, CK,
It is one or more in hMAM, MUC1, PMSA；
When rare cell is circulating endothelial cells, the X protein includes in CD34, P1H12, CD31, CD62, CD102
It is one or more；
When rare cell is stem cell, the X protein includes one kind in CD44, CD133, CD90, ALDH1, ABCG2
Or it is a variety of.
In some embodiments, the X protein is HER2；
The anti-X protein antibody is that Herceptin antibody and HER2 detect antibody (the beautiful standing grain diagnostic products technology in Zhuhai are limited
Company, CBS006)；
In some embodiments, the first signal strength indicator and second signal intensity indicator are selected from fluorescence
Substance, and for two kinds of exciting lights and emit the different fluorescent material of light；
In some embodiments, the first signal strength indicator is FITC, the second signal intensity indicator
For CY5.
In some embodiments, HER2 positive cells must meet：
Positive signal fluorescence intensity>19, R values>3.5, and dyed with intact cell core and cell membrane.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment
The present embodiment is by taking the judgement of the positive cell of HER2 as an example, to illustrate technical scheme of the present invention.
1.HER2 detects antibody optimal reaction concentration and determines
Herceptin antibody and HER2 detection antibody are diluted to 16ug/ml with combination buffer, carry out 2 times of ladders respectively
Degree dilution, is detected with SKBR3 cells.The result shows that Herceptin antibody and HER2 detection antibody use a concentration of 2ug/
Ml~4ug/ml, cell-bound activity reaches saturation, and when a concentration of 4ug/ml of use, fluorescence intensity level is more stable.
2. Immunofluorescence test breast cancer cell line HER2 expressions
According to existing document report, SKBR3, BT474 cell line are that HER2 high expresses breast cancer cell line, in immune group
It is HER2 3+ defined in change, FISH detects ErBb2 gene magnifications；MCF7, MDA-MB-231 cell line are HER2 low expression mammary gland
Cancerous cell line is 0~1+ of HER2 defined in immunohistochemistry, and FISH detection ErBb2 genes do not expand；A431 is the low tables of HER2
Up to skin cancer cell system；WBC is HER2 low expression human leukocytes.With Herceptin antibody test cell lines SKBR3,
The HER2 expressions of BT474, MCF7, MDA-MB-231, A431, the results showed that SKBR3, BT474 cell are expressed for HER2 high
Cell, FITC fluorescence intensity levels 19~254.98；A431, MCF7, MDA-MB-231, WBC are HER2 low expression cells, and FITC is glimmering
Light intensity value 5.29~25.22.Similarly, with HER2 detection antibody test cell lines SKBR3, BT474, MCF7, MDA-MB-
231, the HER2 expressions of A431, the results showed that the FITC fluorescence intensity levels 22.29~254.8 of SKBR3, BT474 cell,
The FITC fluorescence intensity levels 4.38~29.63 of MCF7, MDA-MB-231, A431.Therefore, SKBR3, BT474 cell are classified as
HER2 positive cell groups, FITC fluorescence intensity levels 19.00~254.98, feminine gender group fluorescence intensity level 4.86~29.63 (table 1, table
2, Fig. 2).
Table 1Herceptin antibody test results
Table 2HER2 detects antibody test result
3.ROC analyzes the HER2 positives and negative cells group ratio
100 SKBR3, BT474, MCF7, MDA-MB-231, A431 cells are added to 1.25 × 10 respectively7It is a white thin
In born of the same parents, cell line capture is carried out with hybrid acquisition antibody and magnetic bead respectively, antibody test cell line is detected with HER2, uses CD45-
APC detects antibody test leucocyte.Leukocyte population (CY5 fluorescent values are determined according to cell CD45-APC fluorescent staining values>
7.2) cell that nucleus, is more than to leukocyte cell core according to cellular morphology is classified as cell line, and according to FITC fluorescence
Cell is sorted out (HER2 positives group (SKBR3, BT474) FITC fluorescent values 21.64~228.8, HER2 feminine gender groups by value
The FITC fluorescent stainings value 5.5~19.3 of (MDA-MB-231, MCF7, A431)).The FITC for counting every cell picture respectively is glimmering
Light intensity value and CY5 fluorescence intensity levels calculate the ratio (FITC fluorescence intensity levels/CY5 fluorescence intensity levels) of every cell picture
(table 3).Data analysis is carried out with Graphpad softwares, for ratio in 3.0~5.0 ranges, HER2 positive group cells are cloudy with HER2
There are larger difference (Fig. 4) for property group cell.It is analyzed with ROC curve, when ratio 3.5~4.0, sensitivity is 99.63~
100%, specificity is 99.00~99.88%, p<0.0001 (table 4, table 5).
3 cell ratio of table is distributed
Table 4ROC tracing analysis fractional thresholds
The different fractional threshold sensitivity and specificity analyses of table 5
4. ratio Analysis Healthy People, benign lesion, breast cancer HER2 positive cell number differences
Use ratio 3.5,4.0,4.5 as threshold value, 72 Healthy Peoples of analysis, 35 benign lesions, 71 breast cancer respectively
Sample HER2 positive cell numbers, according to HER2 positive cell number statistical sample positive rates.The result shows that when ratio is 4.0,
There are bigger difference (Fig. 5) for Healthy People, benign lesion, breast cancer sample.Therefore, select ratio 4.0 as ratio interpretation method
Critical value carries out human assistance interpretation in 3.5~4.0 cell for ratio, integrality is dyed according to nucleus and cell membrane
Carry out interpretation.
5. ratio interpretation method is established
According to the FITC fluorescence intensity levels of HER2 positive cells group and HER2 negative cells groups, Therefore, HER2 positive cells must expire
Foot：FITC fluorescence intensities>19, ratio>3.5, and dyed with intact cell core and cell membrane.
6. ratio interpretation method compares other interpretation methods
10 parts of Immunofluorescence test samples are randomly selected, are sentenced respectively with ratio interpretation method, statistics by two analysts
Reading method is analyzed, and the time-consuming of two methods, accuracy rate, consistency are compared.The result shows that averagely being consumed with ratio interpretation method
When 10 minute/part samples, rate of accuracy reached 99%, two analyst's analysis result consistency are up to 100% (table 6).Meanwhile with this two
Kind method analyzes 72 Healthy Peoples, 35 benign lesions, 71 breast cancer samples respectively, the results showed that uses ratio approach interpretation
When, there are larger difference (Fig. 6) between Healthy People, benign lesion, breast cancer sample three.
6 interpretation method of table compares
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but it will be understood by those of ordinary skill in the art that：Its
It still can be with technical scheme described in the above embodiments is modified, either to which part or all technical features
Carry out equivalent replacement；And these modifications or replacements, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. the rare cell interpretation method in a kind of cell mass, which is characterized in that include the following steps：
The marker of rare cell to be detected is referred to as X protein, the marker of cell mass is referred to as Y albumen；Use first
The anti-X protein antibody of signal strength tracer-labelling uses the anti-Y protein antibodies of second signal intensity tracer-labelling；
X protein positive cell and negative cells are respectively added in the cell mass, with the anti-X protein antibody and described anti-
Y protein antibodies detect X protein positive cell and negative cells respectively, and the first signal strength for counting every cell picture respectively refers to
Show agent signal strength values F and second signal intensity indicator signal strength values F', and calculates the R=F/F' of every cell picture；
Calculate the average value of all FWith standard deviation s, fluorescence threshold is obtained
When the fluorescence thresholdWhen taking 95% confidence interval, n=2；When taking 99% confidence interval, n=3；It takes
When 99.9% confidence interval, n=4；When taking 99.99% confidence interval, n=5；
Each group R values are analyzed with ROC curve, determination is strong for distinguishing the signal of the positive cell and the negative cells
Spend fractional threshold Rt；
For certain cell of the cell mass, if its F ＞ Ft, and R ＞ Rt, then it is the rare cell with X protein feature.
2. according to the method described in claim 1, it is characterized in that, the anti-X protein antibody is one or more；
It is respectively added to it in cell mass when the anti-X protein antibody is a variety of, then by X protein positive cell and negative cells
Before further include：
With anti-X protein antibody A b1, anti-X protein antibody A b2, anti-X protein antibody A b3 ... the anti-X protein antibody A bn and anti-Y
Protein antibodies detect X protein positive cell and negative cells respectively, and calculate separately the first signal strength indicator signal
The ratio R 1 of intensity value and the second signal intensity indicator signal strength values, R2, R3 ... Rn.
3. according to the method described in claim 1, it is characterized in that, the first signal strength indicator and second signal intensity
Indicator is selected from fluorescent material, quantum dot, digoxin labelled probe, biotin, radioactive isotope, electron dense substances, glue
Body gold or enzyme.
4. according to the method described in claim 3, it is characterized in that, the fluorescent material include Alexa 350, Alexa 405,
Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY 630/650, BODIPY
650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-two
Methoxyl group fluorescein, 5- carboxyls -2 ', 4 ', 5 ', 7 '-tetrachlorofluoresceins, 5-carboxyfluorescein, 5- carboxyrhodamines, 6- carboxyl sieve
Red bright, 6- carboxyls tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,6-FAM, dansyl Cl, FITC (different sulphur
Cyanic acid fluorescein), HEX, 6-JOE, NBD (7- nitro benzo -2- oxa-s -1,3- diazole), Oregon Green 488, Oregon
Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), M-phthalic acid, cresols
Gu purple, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl are glimmering
Light element, rare earth metal cryptate, three pairs of pyridyl group diamines europiums, europium cryptate or chelate, diamines, dicyanin,
La Jolla indigo plants dyestuff, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamines, algae red green white,
Phycoerythrin R, REG, rhodamine be green, rhodamine isothiocyanates, rhodamine are red, ROX, TAMRA, TET, TRIT (tetramethyl sieve
Red bright different mercaptan), it is one or more in tetramethylrhodamine and texas Red.
5. according to the method described in claim 3, it is characterized in that, the radioactive isotope includes110In、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 and83Any in Sr
Kind.
6. according to the method described in claim 3, it is characterized in that, the enzyme include horseradish peroxidase, alkaline phosphatase and
Any one of glucose oxidase.
7. according to claim 1~6 any one of them method, which is characterized in that the cell mass is leucocyte, described rare
Cell is any one of circulating tumor cell, cycle epithelial cell, circulating endothelial cells, stem cell, residual disease cell；
Or, the cell mass is the tissue reached maturity, the rare cell is stem cell.
8. the method according to the description of claim 7 is characterized in that when cell mass is leucocyte, the Y albumen includes
It is one or more in CD45, CD3, CD50, CD66B, CD69, CD84；
Preferably, after determining the signal strength threshold range for distinguishing the positive cell and the negative cells, institute
The method of stating further includes：
The threshold range is verified with the blood sample containing the positive cell or the negative cells.
9. method according to claim 7 or 8, which is characterized in that when the rare cell is circulating tumor cell, institute
State X protein include HER2, ER α, ER β, PR, Bcl-2, EGFR, ERCC1, RRM1, CEA, AFP, CA242, CA199, CA724,
It is one or more in PDL1, PDL2；
When rare cell be cycle epithelial cell when, the X protein include Epcam, N- cadherin, FSP1, CK, hMAM,
It is one or more in MUC1, PMSA；
When rare cell is circulating endothelial cells, the X protein includes one in CD34, P1H12, CD31, CD62, CD102
Kind is a variety of；
When rare cell is stem cell, the X protein includes one kind or more in CD44, CD133, CD90, ALDH1, ABCG2
Kind.
10. according to the method described in claim 9, it is characterized in that, the X protein is HER2；
The anti-X protein antibody is that Herceptin antibody and HER2 detect antibody；
Preferably, the first signal strength indicator and second signal intensity indicator are selected from fluorescent material, and are two kinds
The exciting light fluorescent material different with transmitting light.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810718281.2A CN108646034B (en) | 2018-07-03 | 2018-07-03 | Method for determining rare cells in cell population |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810718281.2A CN108646034B (en) | 2018-07-03 | 2018-07-03 | Method for determining rare cells in cell population |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108646034A true CN108646034A (en) | 2018-10-12 |
| CN108646034B CN108646034B (en) | 2020-10-23 |
Family
ID=63750192
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810718281.2A Active CN108646034B (en) | 2018-07-03 | 2018-07-03 | Method for determining rare cells in cell population |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108646034B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110458835A (en) * | 2019-08-16 | 2019-11-15 | 腾讯科技（深圳）有限公司 | A kind of image processing method, device, equipment and medium |
| WO2021213298A1 (en) * | 2020-04-20 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting ca199 expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
| CN114085911A (en) * | 2022-01-19 | 2022-02-25 | 珠海圣美生物诊断技术有限公司 | Method for detecting tumor cells or tumor cell fragments |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007069233A2 (en) * | 2005-12-13 | 2007-06-21 | Applied Spectral Imaging Ltd. | Automatic characterization of pathological specimen |
| WO2007089911A2 (en) * | 2006-01-30 | 2007-08-09 | The Scripps Research Institute | Methods for detection of circulating tumor cells and methods of diagnosis of cancer in a mammalian subject |
| CN102782498A (en) * | 2009-10-21 | 2012-11-14 | 斯克里普斯研究所 | Method of using non-rare cells to detect rare cells |
| CN104634720A (en) * | 2015-03-13 | 2015-05-20 | 南京医科大学第一附属医院 | Method for detecting weak antigen expression level based on geometric mean fluorescence index |
| CN106190945A (en) * | 2015-05-05 | 2016-12-07 | 深圳华大基因研究院 | Automatically the method and system of rare cell are identified |
-
2018
- 2018-07-03 CN CN201810718281.2A patent/CN108646034B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007069233A2 (en) * | 2005-12-13 | 2007-06-21 | Applied Spectral Imaging Ltd. | Automatic characterization of pathological specimen |
| WO2007089911A2 (en) * | 2006-01-30 | 2007-08-09 | The Scripps Research Institute | Methods for detection of circulating tumor cells and methods of diagnosis of cancer in a mammalian subject |
| CN102782498A (en) * | 2009-10-21 | 2012-11-14 | 斯克里普斯研究所 | Method of using non-rare cells to detect rare cells |
| CN104634720A (en) * | 2015-03-13 | 2015-05-20 | 南京医科大学第一附属医院 | Method for detecting weak antigen expression level based on geometric mean fluorescence index |
| CN106190945A (en) * | 2015-05-05 | 2016-12-07 | 深圳华大基因研究院 | Automatically the method and system of rare cell are identified |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110458835A (en) * | 2019-08-16 | 2019-11-15 | 腾讯科技（深圳）有限公司 | A kind of image processing method, device, equipment and medium |
| WO2021213298A1 (en) * | 2020-04-20 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting ca199 expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
| CN114085911A (en) * | 2022-01-19 | 2022-02-25 | 珠海圣美生物诊断技术有限公司 | Method for detecting tumor cells or tumor cell fragments |
| CN114085911B (en) * | 2022-01-19 | 2022-05-17 | 珠海圣美生物诊断技术有限公司 | Method for detecting tumor cells or tumor cell fragments |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108646034B (en) | 2020-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104007257B (en) | Method for detecting non-humoral rare karyotes, and kit thereof | |
| Marrinucci et al. | Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers | |
| CA2778328C (en) | Method of using non-rare cells to detect rare cells | |
| CN108646034A (en) | Rare cell interpretation method in cell mass | |
| US20130295580A1 (en) | Oral cancer point of care diagnostics | |
| CN107402296A (en) | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell | |
| US11988658B2 (en) | Methods of assessing cellular breast samples and compositions for use in practicing the same | |
| JP2017207516A (en) | Malignancy detection method and kit of peripheral circulating tumor cell unit | |
| Lei et al. | Combined detection of aneuploid circulating tumor‐derived endothelial cells and circulating tumor cells may improve diagnosis of early stage non‐small‐cell lung cancer | |
| WO2011002649A1 (en) | Analysis of circulating tumor-related microparticles | |
| CN107850587A (en) | Application of the circulating tumor cell mitotic index in cancer is layered and is diagnosed | |
| CN112313497A (en) | Methods of diagnosing disease using microflow cytometry | |
| Chudasama et al. | Inertia based microfluidic capture and characterisation of circulating tumour cells for the diagnosis of lung cancer | |
| CN115128285A (en) | Kit and system for identifying and evaluating thyroid follicular tumor by protein combination | |
| Sapir et al. | Improved automated localization and quantification of protein multiplexes via multispectral fluorescence imaging in heterogenous biopsy samples | |
| WO2022000041A1 (en) | Biomarker combinations for determining aggressive prostate cancer | |
| US20150004633A1 (en) | Assays and methods for the diagnosis of ovarian cancer | |
| CN107121551A (en) | Biomarker combinations, detection kit and the application of nasopharyngeal carcinoma | |
| CN109799347B (en) | Leukemia biomarker IGFBP3 and application thereof | |
| Lo et al. | A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer | |
| Futia | Circulating Tumor Cells: Mathematical Theory of Detectability with Simulations and Experimental Results in a Model System | |
| CN118505594A (en) | Colorectal cancer peripheral blood circulation tumor cell epithelium combined interstitial marker dual morphology parting model and application thereof | |
| Lei et al. | Aneuploid circulating tumor endothelial cells (CTECs) affect the detection of CTC, but they are fundamentally different | |
| Heussner et al. | Quantitative image analysis pipeline for detecting circulating hybrid cells in immunofluorescence images with human-level accuracy | |
| CN118501467A (en) | Bladder cancer prognosis marker, prognosis evaluation system and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 519000 3 3 level 3, No. 266 Tong Hang Road, Xiangzhou District, Zhuhai, Guangdong. Applicant after: Zhuhai Sheng Mei biological diagnostic technology Co., Ltd. Address before: 519000 3 3 level 3, No. 266 Tong Hang Road, Xiangzhou District, Zhuhai, Guangdong. Applicant before: Zhuhai Lizhu wie Medical Diagnostic Technology Co Ltd |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |