CN101813694A - Method for determining and/or classifying the circulative macrophage and analyzing arrangement thereof - Google Patents
Method for determining and/or classifying the circulative macrophage and analyzing arrangement thereof Download PDFInfo
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- CN101813694A CN101813694A CN200910208853A CN200910208853A CN101813694A CN 101813694 A CN101813694 A CN 101813694A CN 200910208853 A CN200910208853 A CN 200910208853A CN 200910208853 A CN200910208853 A CN 200910208853A CN 101813694 A CN101813694 A CN 101813694A
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- Tropical Medicine & Parasitology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
This invention relates to a method for determining and/or classifying the circulative macrophage and analyzing arrangement thereof. Firstly, separate themacrophage from the taken whole blood by means of acentrical motion in grads. Then , perforate the macrophage and apply a staining process in a cell with at least a selected antibody. Thirdly, perform a flow cytometry analysis of a pretreated cell to give a statistics evaluation of the followed cellular content. Usually, PSA, cytokeratin and/or epithelium antigen are chosen as antibody.
Description
It is 03816702.6 that the application of this division is based on application number, the applying date is on June 2nd, 2003, and denomination of invention is divided an application for the original Chinese patent application of " character that is used for the cyclicity macrophage is determined and/or the method for classification and the analysis arrangement of implementing this method ".
The present invention relates to the method for and/or classification definite by the character that is used for the cyclicity macrophage of acellular autoantigen, and the analysis arrangement of implementing such method.
From Sinha, Wilson, Gleason:Immunoelectron microscopiclocation of prostatic-specific antigen in human prostate by theprotein A-gold complex, Cancer, 1987,60, known among the 1288-91, from the enforcement of the electron microscopy of the cell of prostata tissue, wherein the PSA antibody of prostatic normal structure, prostate cancer tissue and hyperplastic prostate tissue and golden mark is carried out incubation according to the document of being quoted.Inspection shows that gold grain is present in tenuigenin, cell endoparticle, RES and the lysosome.Along with the differentiation of end eventually of the tumour that increases, more gold grain appears in the membrane structure.This thinks the sign of following situation, and promptly PSA (prostate specific antigen) retains in the membrane structure along with the differentiation of end eventually of the tumour cell that increases.Another aspect of this inspection is for detecting gold grain equally in granulocyte and macrophage.
In flow cytometry is checked, in circulation blood, find the PSA positive cells.But in known before inspection, have only the surface of macrophage to be caught color because of PSA.
This fact of mRNA of not finding the PSA molecule in macrophage causes so single conclusion, has promptly only absorbed the PSA molecule, and has not related to the elimination micrometastasis.Referring to Brandt, Griwatz, Brinkmann:Circulating prostate-specific antigen/CD14-double-positive cells; Abiomarker indicating low risk for hematogeneousmetastasis of prostate cancer, J.Natl.Cancer Inst.1997; 89,174.
As everyone knows, accumulating in tissue in the more orderly or more unordered cell mass forms the pernicious variation of cell and is called tumour.These tumour cells are ignored the arrangement of tissue, and unrestrictedly growth and the increase by size and being impregnated in other the surrounding tissue, organ expanded, and perhaps cross over organizational boundaries and in intravasation system or the lymphatic system.
In case tumour arrives vascular system or lymphatic system, individual cells so herein or cell mass can be held by these systems are free, and are metastasis and adhering at other positions of health as the edema during pregnancy knurl.Have such danger at this, i.e. metastasis continued growth is also robbed the energy of health, knocks down until the health decline with by its disease.
During the formation of this tumour, tumour cell produces the material that is used to promote its growth.In addition, the material that can be used as the tumor growth indicant can be released.The latter is called tumor marker.But these marks are not special for tumour, and have only the quantity of concentration measured in the blood, because healthy cell also can discharge these materials.Therefore, tumor marker can not be used to detect tumour, and can only be used for the monitoring of disease or therapeutic process.A special marking of tumour is prostate specific antigen (PSA), and the finite concentration from blood begins it and just shows that prostate cancer is arranged.But prostatic optimum increase also can cause the rising of PSA value in the blood.
So far, tumor disease is mainly diagnosed by the method that provides image, for example ultrasound wave or computerized tomography, mammary gland maskaperture mask or the like., have only according to the tissue sample that contains tumour and just can make final determining, and definite therapeutic process.
Tumor disease causes the immune antagonism of human body self.This immune system is made up of the different cell types of a series of execution different tasks.Wherein, macrophage has such task, promptly discerns some sick sample material, engulfs it, and is broken down into its component.Subsequently, the fragment of the cell that will be engulfed is presented on the surface of other immunocytes, thus this make it possible to discern should attack structure.
The character that exists urgent demand to implement the cyclicity macrophage with stage in early days determines, and need not carry out the direct inspection of human body.
Task of the present invention is to provide the character that makes it possible to implement cyclicity macrophage (PBMC) to determine and/or the method and the analysis arrangement of classification.
According to the present invention,, therefore can implement direct and special lesion detection owing to can in the cyclicity macrophage, detect the antigen or the fragment of the tumour cell of being engulfed.
According to the present invention, extract whole blood sample, carry out gradient centrifugation subsequently to separate macrophage.Then with macrophage perforation, and implement the cell inner dyeing that uses at least a selected antibody.
Subsequently, the known flow cytometry of use itself is to write down cellularity on single level.
Flow cytometry makes it possible to carry out the physics and the molecular property of cell count and analysis of cells in flow of liquid.Particularly, on single level, measure the character and the record of cell or cell colony by means of the sample of fluorochrome label such as antibody.
At this, the antigen-antibody reaction of using the antibody of fluorochrome label is the basis.In order to analyze, the cell of single suspending liquid to be focused on (hydrodynamischeFokussierung) guiding by hydrodynamic force have the laser beam of suitable wavelength.By monochromatic laser beam correspondingly during the electronics of fluorescence excitation dyestuff, these electronic transitions are to higher energy level.After laser pulse, electronics returns its initial level, and the form with photon gives off energy simultaneously.Proportional with the described photon density of launching under the light detector recording with the antibody quantity that each cell institute combines.In addition, can further obtain about cell size and inner structure by optical diffraction and scattering is the information of cytoplasmic graininess and nucleus size.
Use prostate specific antigen, cytokeratin antibody and/or epithelial membrane antigen as selected antibody.
According to the present invention, just can determine by in macrophage, carrying out the PSA antibody staining whether the material of being engulfed is relevant with prostate then.
The antibody that the analysis arrangement that is used to implement this method comprises reagent, the gradient centrifugation machine that is used to separate macrophage, the reagent that is used for cell perforation that are used for the blood that heparinize extracts, be used to use fluorochrome label is to carrying out the device of cell inner dyeing and being used to measure each self-separation and through the cell inner structure of pretreated cell and have the fluidic cell metering instrument of the evaluation unit that computing machine supports, this analysis arrangement is used for the purpose of early detection tumour through pretreated cell.
To rely on embodiment to explain the present invention in more detail hereinafter.
In the step of blood draw and dyeing, begin to carry out heparinize from for example 6ml whole blood.By means of gradient centrifugation separating monocytic cell, macrophage and lymphocyte.
In next step, carry out the formaldehyde fixed and the saponarin of cell and handle to reach the purpose of perforation.
Be to use selected antibody in the following table for example to carry out the step of cell inner dyeing subsequently.
PSA antibody A b-1 (Clone ER-PRS)
Full cytokeratin-FITC
Epithelial membrane antigen (Clone E 29)
Homotype contrast IgG1 (Clone DAK-GO1)
Second antibody FITC goat anti-mouse (DAKO)
Then, will check in more detail by flow cytometry up to analyzing just fixing once more cell accordingly.Gating monocyte and macrophage, promptly only some measurement result is used for estimating, and carries out preselected.
Then, estimate homotype contrast and dyeing by histogram analysis, and provide the positive cell quantity of for example representing with number percent.
It shows, for the patient who suffers from the diffused prostate tumor, when carrying out the dyeing of macrophage with cytokeratin, finds histiocytic composition structure division in corresponding people's circulation immunity cell.Because these constituents are not the original contents of immunocyte, they must be included into by phagocytosis.Because the curve process of including in of cytokeratin is different with homotype obviously, so can get rid of non-specific effect.
PSA dyeing proof in the macrophage, the material through engulfing is certain relevant with prostata tissue, because this specific marker also is detectable.
In a word, by using described method and attached analysis arrangement, can access the novel method that the character that is used for the cyclicity macrophage is definite and classify, wherein classification makes it possible to point out possible prostate related content thing.
Claims (6)
1. be used for the method that the monocytic character of cyclicity macrophage and/or peripheral blood is determined and/or classified, it has the following step:
-extract whole blood and carry out gradient centrifugation with the separation macrophage,
-with the macrophage perforation,
-carry out the cell inner dyeing of described cell with at least a selected antibody, and
-carry out described fluidic cell quantitative analysis through pretreated cell, and carry out statistical evaluation for more cell subsequently,
2. according to the method for claim 1, it is characterized in that, use prostate specific antigen (PSA), cytokeratin and/or epithelial membrane antigen as described selected antibody.
3. according to the method for claim 1 or 2, it is characterized in that, after implementing flow cytometry, carry out the histogram analysis of homotype contrast and dyeing.
4. according to any one described method in the aforementioned claim, it is used for detecting the histocyte part of the diffused prostate tumor of including in by phagocytosis outside human body.
5. according to the method for claim 4, it is characterized in that, determine by the described PSA dyeing in the described macrophage whether the material of being engulfed is relevant with prostate.
6. be used for implementing analysis arrangement according to aforementioned any one described method of claim, the antibody that it comprises reagent, the gradient centrifugation machine that is used to separate macrophage, the reagent that is used for cell perforation that are used for the blood that heparinize extracts, be used to use fluorochrome label carries out the device of cell inner dyeing and is used to measure each self-separation and through the cell inner structure of pretreated cell and have the fluidic cell metering instrument of the evaluation unit that computing machine supports, this analysis arrangement is used for the purpose of early detection tumour through pretreated cell described.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10228548 | 2002-06-26 | ||
| DE10228548.9 | 2002-06-26 | ||
| DE10230893A DE10230893A1 (en) | 2002-06-26 | 2002-07-09 | Method for determining the properties and / or classification of circulating macrophages and / or peripheral blood mononuclear cells and analysis arrangement for carrying out the method |
| DE10230893.4 | 2002-07-09 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA038167026A Division CN1668922A (en) | 2002-06-26 | 2003-06-02 | Method for the determination of characteristics and/or the classification of circulating macrophages, and analysis arrangement for carrying out said method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101813694A true CN101813694A (en) | 2010-08-25 |
Family
ID=29723471
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910208853A Pending CN101813694A (en) | 2002-06-26 | 2003-06-02 | Method for determining and/or classifying the circulative macrophage and analyzing arrangement thereof |
Country Status (4)
| Country | Link |
|---|---|
| CN (1) | CN101813694A (en) |
| AT (1) | ATE479093T1 (en) |
| DE (2) | DE10230893A1 (en) |
| ES (1) | ES2351481T3 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6165460A (en) * | 1995-07-10 | 2000-12-26 | Therion Biologics Corporation | Generation of immune responses to prostate-specific antigen (PSA) |
| DE19814915A1 (en) * | 1998-04-03 | 1999-10-07 | Roche Diagnostics Gmbh | Immunological method for the determination of PSA |
-
2002
- 2002-07-09 DE DE10230893A patent/DE10230893A1/en not_active Withdrawn
-
2003
- 2003-06-02 AT AT03735519T patent/ATE479093T1/en active
- 2003-06-02 ES ES03735519T patent/ES2351481T3/en not_active Expired - Lifetime
- 2003-06-02 CN CN200910208853A patent/CN101813694A/en active Pending
- 2003-06-02 DE DE50313016T patent/DE50313016D1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE10230893A1 (en) | 2004-01-15 |
| DE50313016D1 (en) | 2010-10-07 |
| ES2351481T3 (en) | 2011-02-07 |
| ATE479093T1 (en) | 2010-09-15 |
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| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100825 |