CN111454906A - Composition and kit for rapidly extracting circulating non-blood related nucleated cells from peripheral blood and application of composition and kit - Google Patents
Composition and kit for rapidly extracting circulating non-blood related nucleated cells from peripheral blood and application of composition and kit Download PDFInfo
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Abstract
The invention combines a centrifugation method based on a special density separating agent with a means of removing a blood-related nucleated cell reagent, and on one hand, provides a composition which is used for separating circulating non-blood-related nucleated cells, such as circulating tumor epithelial cells, circulating vascular endothelial cells and the like from peripheral blood in a unique and efficient manner. The present invention relates to a composition of non-related nucleated rare cells for rapid acquisition of circulation from blood. The composition comprises the following components: buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-blood related nucleated cell surface marker antibodies, and a cell separation medium.
Description
Technical Field
The invention belongs to the field of medical biology, and relates to a composition containing nucleated cells and a kit.
Background
The great clinical significance of extracting and detecting circulating non-blood related nucleated cells (such as vascular endothelial cells, tumor epithelial cells) in peripheral blood has long been demonstrated. The current method for extracting circulating non-blood related nucleated cells is to combine antibodies with special markers on the surfaces of the cells so as to achieve the aim of direct capture. However, this direct capture technique has low sensitivity and poor reproducibility, and the surface marker has a great difference in expression level for different types of cells, so it cannot be used as a general means for extracting non-related nucleated cells from blood samples.
Recently, a great deal of scientific research and clinical experiments have proved that the enrichment and extraction of non-blood-related nucleated cells in blood by removing blood-related nucleated cells (such as leucocytes and the like) in blood is the most effective means. Although some single methods involved in this process have been reported, such as the immunomagnetic particle method for removing leukocytes, the glycophorin-A (glycophorin A) cross-linking method for removing erythrocytes, and the preliminary attempt of combining immunomagnetic particles with density centrifugation, which must be performed by means of a special cell separation apparatus, these methods are time-consuming, have low leukocyte removal rate, poor target cell recovery rate, and require special equipment, thereby causing inconvenience in application.
For the reasons, the invention combines the centrifugal method based on the composition or the kit using the special density separating agent with the means for removing the leukocyte reagent, and unexpected experimental results prove that the composition or the kit combined with the method can rapidly, efficiently and specifically remove most of blood-related nucleated cells, red blood cells and plasma proteins in a blood sample, thereby rapidly enriching and extracting circulating non-blood-related nucleated cells in peripheral blood, such as circulating tumor epithelial cells, circulating vascular endothelial cells and the like.
The invention does not need to use a special device magnetic frame, simplifies the step of reducing the leukocyte removal of the magnetic adsorption immunomagnetic beads, reduces the step of centrifugation and effectively shortens the experimental time by 60 minutes.
Disclosure of Invention
The invention combines a centrifugation method based on a special density separating agent with a means of removing a blood-related nucleated cell reagent, and on one hand, provides a composition which is used for separating circulating non-blood-related nucleated cells, such as circulating tumor epithelial cells, circulating vascular endothelial cells and the like from peripheral blood in a unique and efficient manner.
In another aspect, the invention provides a kit for uniquely and efficiently separating circulating non-limbed nucleated cells, such as circulating tumor epithelial cells and circulating vascular endothelial cells, from peripheral blood.
In a further aspect, the present invention also provides the use of the above-described composition or kit for biomedical experiments, or manual clinical testing operations, or for clinical testing operations with the aid of automated detection instrumentation.
In one aspect, the present invention provides a composition for rapidly obtaining circulating non-related nucleated rare cells from blood, comprising the following components: the kit comprises a buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-related nucleated cell surface marker antibodies and a cell separation medium, wherein the osmotic pressure of the buffer solution is 280-20 mOsm/kg H2O, pH is 6.8 to 7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07156 to 1.07738 g/ml, and the cell separation medium comprises one or more of the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate or derivatives thereof, a sugar-containing solution, a nonionic polymer polymerized from sucrose and epichlorohydrin.
Preferably, the specific gravity of the immune microspheres is higher than that of the cell separation medium, and the immune microspheres or immune particles are covalently or non-covalently bound with antibodies that specifically recognize one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA.
Preferably, the circulating non-limbic nucleated rare cells are selected from the group consisting of: any solid tumor cells that are circulating and have epithelial origin, or any tumor cells that are not circulating and have epithelial origin, circulating vascular endothelial cells, tumor stem cells, and immune cells.
Preferably, the osmotic pressure of the buffer solution is 280-320 mOsm/kgH2O, pH is 6.85-7.75, the cell separation medium comprises one or more of the following reagents: polyvinylpyrrolidone coated colloidal silica, polysaccharide and sodium diatrizoate or derivatives thereof, a sugar containing solution, a non-ionic polymer polymerized from sucrose and epichlorohydrin, said sugar containing solution being a solution of a sugar selected from the group consisting of: such as dextran, polysaccharide and sodium diatrizoate or derivatives thereof, sucrose and the like, and the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml.
Preferably, the proportion of the circulating non-blood related nucleated rare cells in the collected blood sample accounts for less than 0.1% of all nucleated cells, and the tumor cells are selected from all solid tumor cells of epithelial origin, such as lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer and the like; or tumor cells not of epithelial origin, such as melanoma and the like; circulating vascular endothelial cells, tumor stem cells, blood fetal cells, stem cells, and immune cells.
In another aspect, the present invention provides a kit for rapidly obtaining circulating non-limbed nucleated rare cells from blood, comprising:
a composition comprising the following ingredients: buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-blood related nucleated cell surface marker antibodies, and a cell separation medium; wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, pH is 6.8 to 7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07156 to 1.07738 g/ml, and the cell separation medium comprises one or more of the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate or derivatives thereof, a sugar-containing solution, a nonionic polymer polymerized from sucrose and epichlorohydrin; containerA container containing the composition and a blood collection tube with an anticoagulant; and instructions for use of the kit.
Preferably, the specific gravity of said immunomicrosphere or immunomer is higher than the specific gravity of said cell separation medium, said immunomicrosphere or immunomer having covalently or non-covalently bound thereto an antibody specifically recognizing one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA.
Preferably, the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, pH is 6.85-7.75, and the sugar-containing solution is a solution of sugar selected from the following: such as dextran, polysaccharides and sodium diatrizoate or derivatives thereof, or sucrose, etc.
Preferably, the circulating non-limbic nucleated rare cells are selected from the group consisting of: any solid tumor cells of epithelial origin that circulate, or any tumor cells that do not have epithelial origin, vascular endothelial cells that circulate, tumor stem cells, fetal cells in blood, stem cells, and immune cells.
Preferably, the proportion of the circulating non-blood related nucleated rare cells in the collected blood sample accounts for less than 0.1% of all nucleated cells, and the tumor cells are selected from all solid tumor cells of epithelial origin, such as lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer and the like; or tumor cells not of epithelial origin, such as melanoma and the like; circulating vascular endothelial cells, tumor stem cells, blood fetal cells, stem cells, and immune cells.
In a further aspect, the present invention also provides the use of the above-described composition or kit for biomedical experiments, or manual clinical testing operations, or for clinical testing operations with the aid of automated detection instrumentation.
Compared with the common commercial centrifugation medium on the market at present, the novel composition or the kit can greatly shorten the centrifugation time and stably improve the cell recovery rate from the original 60 +/-20% to more than 95%. The composition is combined with a leukocyte removal reagent and a means of hypotonic erythrocyte fusion, can effectively and rapidly enrich and extract circulating non-blood related nucleated rare cells in peripheral blood, such as tumor epithelial cells, vascular endothelial cells and the like, and has great practical clinical significance for early diagnosis of tumors and heart diseases. Moreover, the present invention provides the above-mentioned composition or kit for use in biomedical experiments, or manual clinical test operations or in clinical test operations with the aid of automated test equipment, which greatly simplifies the operation and improves the efficiency.
The main improvements of the present invention over the existing composition (CN101880650B) are in the following aspects:
(1) the composition of the present invention does not include a bleed cell lysis solution, which destroys fine surface proteins and affects downstream cell staining and identification.
(2) The invention replaces the original antibody with CD14, CD45RO and CD45RA, and can more comprehensively, effectively and specifically remove blood-borne leucocytes.
(3) The invention also avoids using a magnetic frame and adsorbing immune antibody magnetic beads by using the magnetic force of the magnetic frame, and avoids washing the supernatant after the magnetic beads are removed.
Through the modification, the invention shortens and simplifies the experimental steps, thereby reducing the loss of the enriched target cells in the experiment and obviously improving the cell recovery rate over the prior art.
Detailed Description
The present invention combines a centrifugation method based on a separating agent of a specific density with a means for removing a reagent for removing blood-related nucleated cells, and in one embodiment provides a unique and highly efficient composition for separating circulating non-blood-related nucleated cells, such as circulating tumor epithelial cells and circulating vascular endothelial cells, from peripheral blood.
In another embodiment, the invention also provides a kit for separating circulating non-limbed nucleated cells, such as circulating tumor epithelial cells and circulating vascular endothelial cells, from peripheral blood with unique and high efficiency.
In yet another embodiment, the present invention also provides the use of the above-described composition or kit for biomedical experiments, or manual clinical testing procedures, or for clinical testing procedures with the aid of automated detection instrumentation.
In one embodiment, the present invention provides a composition for rapidly obtaining circulating non-related nucleated rare cells from blood, comprising the following components: the kit comprises a buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-related nucleated cell surface marker antibodies and a cell separation medium, wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, pH is 6.8 to 7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07156 to 1.07738 g/ml, and the cell separation medium comprises one or more of the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate or derivatives thereof, a sugar-containing solution, a nonionic polymer polymerized from sucrose and epichlorohydrin.
In a preferred embodiment, the specific gravity of the immune microspheres or immune particles is higher than the specific gravity of the cell separation medium, and the immune microspheres or immune particles are covalently or non-covalently bound with antibodies that specifically recognize one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA.
In another preferred embodiment, the circulating non-limbed nucleated rare cell is selected from the group consisting of: circulating any solid tumor cell with epithelial origin, or any tumor cell without epithelial origin, circulating vascular endothelial cells, tumor stem cells, blood fetal cells, stem cells, and immune cells, etc.
In another preferred embodiment, the buffer has an osmotic pressure of 280-320 mOsm/kg H2O, pH is 6.85-7.75, and the sugar-containing solution is a solution of sugar selected from the following: such as dextran, polysaccharide and sodium diatrizoate or derivatives thereof, sucrose and the like, and the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml.
In another preferred embodiment, the proportion of the circulating non-blood related rare nucleated cells in the collected blood sample accounts for less than 0.1% of all nucleated cells, and the tumor cells are selected from all solid tumor cells of epithelial origin, such as lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer and the like; or tumor cells not of epithelial origin, such as melanoma and the like.
In another embodiment, the present invention provides a kit for rapidly obtaining circulating non-limbed nucleated rare cells from blood comprising:
a composition comprising the following ingredients: buffer solution, magnetic or non-magnetic immune microspheres coated with anti-blood related nucleated cell surface marker antibodies, and a cell separation medium; wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, pH is 6.8 to 7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07156 to 1.07738 g/ml, and the cell separation medium comprises one or more of the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharide, sodium diatrizoate or a derivative thereof, a sugar-containing solution, a nonionic polymer polymerized from sucrose and epichlorohydrin;
a container containing the composition and a blood collection tube with an anticoagulant; and
instructions for use of the kit.
In another preferred embodiment, the specific gravity of the immune microspheres or immune particles is higher than the specific gravity of the cell separation medium, and the immune microspheres or immune particles are covalently or non-covalently bound with antibodies that specifically recognize one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA. In another preferred embodiment, the buffer has an osmotic pressure of 280-320 mOsm/kg H2O, pH is 6.85-7.75, and the sugar-containing solution is a solution of sugar selected from the following: such as dextran, polysaccharides and sodium diatrizoate or derivatives thereof, or sucrose, etc.
In another preferred embodiment, the circulating non-limbed nucleated rare cell is selected from the group consisting of: any solid tumor cells that are circulating and have epithelial origin, or any solid tumor cells that are not circulating and have epithelial origin, circulating vascular endothelial cells, tumor stem cells, and immune cells.
In another preferred embodiment, the proportion of the circulating non-blood related rare nucleated cells in the collected blood sample accounts for less than 0.1% of all nucleated cells, and the tumor cells are selected from all solid tumor cells of epithelial origin, such as lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer and the like; or tumor cells not of epithelial origin, such as melanoma and the like.
In a specific embodiment of the present invention, the reagent for removing the blood-related nucleated cells is an immunoparticle obtained by covalently coupling one or more antibodies that recognize a leukocyte surface marker to a magnetic or non-magnetic solid particle or solid surface. The density of the centrifugal separation medium is 1.07156-1.07738 g/ml, and the specific gravity can be any osmotic pressure of 280-320 mOsm/kg H2O, pH 6.8.8 to 7.8, respectively. The separation medium can effectively separate non-blood-related nucleated cells from red blood cells and immune particles combined with blood-related nucleated cells.
In yet another embodiment, the present invention also provides the use of the above-described composition or kit for biomedical experiments, or manual clinical testing procedures or for clinical testing procedures with the aid of automated detection instrumentation.
Noun interpretation
Blood-related nucleated cells: refers to leukocytes, including lymphocytes (T cells, B cells, etc.); neutrophils; eosinophils; basophils, and the like.
Circulating non-limbed nucleated cells: mainly tumor cells with epithelial origin or without epithelial origin and circulating vascular endothelial cells circulating in the blood; rare cells such as tumor stem cells, fetal cells in blood, certain immune cells and the like.
The invention is mainly used for quickly removing plasma protein, red blood cells and blood-related nucleated cells from a blood sample, thereby achieving the purpose of enriching non-blood-related nucleated cells in the blood sample.
In the practice of the invention, blood is collected in any of the commercially available blood collection tubes containing an anticoagulant.
The main steps for enriching non-blood related nucleated cells in a blood sample using the composition of the present invention comprise:
removing plasma proteins;
immune microspheres or immune particles coated with antibodies against the surface markers of the blood-related nucleated cells (antibodies are covalently or non-covalently coupled to the solid surface);
adding a cell separation medium with the specific gravity of 1.07156-1.07738 g/ml, centrifuging the cell separation medium based on special density,
thereby enriching the blood for non-blood related nucleated cells.
In the composition, the specific gravity of the cell separation medium is 1.07156-1.07738 g/ml (gr/ml or gr/cm)3). The cell separation medium comprises any one or more than two of the following reagent components: polyvinylpyrrolidone coated colloidal silica (polyvinylpyrrolidone coated colloidal silica); a mixture of polysaccharide (polysucrose) and sodium diatrizoate (sodium diatrizoate) or a derivative thereof; a nonionic polymer polymerized from sucrose and epichlorohydrin; and optionally any protein solution. The specific gravity of the cell separation medium can be any osmotic pressure of 280-320 mOsm/kg H2O, pH 6.8.8 to 7.8, respectively.
In another preferred embodiment, the buffer has an osmotic pressure of 280-320 mOsm/kg H2O, pH is 6.85 to 7.75.
In another preferred embodiment, the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml.
Example 1
Rapid collection of non-limbed circulating nucleated cells from a human blood sample using the compositions of the present invention 6ml of human peripheral blood was collected in any blood collection tube (BD, New Jersey, USA) containing an anticoagulant (e.g., EDTA, heparin, ACD, etc.) and after centrifugation (1000 × g, 15 minutes), plasma proteins were removed.
After adding 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), the mixture was mixed by inversion 10 times and incubated at room temperature for 20 minutes.
Adding 6ml of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 ml of a spacer solution of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml, the cell separation medium contains polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer formed by polymerizing sucrose and epichlorohydrin), and centrifuging the sample again (900 × g, 6 minutes).
The supernatant was collected and centrifuged again at 1000 × g for 4 minutes.
And discarding the supernatant to obtain sedimentary cells, namely the enriched non-blood related nucleated cells.
The sedimented cells obtained after centrifugation were resuspended in phosphate buffer for further analysis.
The cell separation medium in the compositions of the invention in this example was prepared by: a mixture of 5.8% polysaccharide (polysucrose) and 9.1% sodium diatrizoate (Sigma, Missouri, UDA) was accurately adjusted to a density of 1.07260-1.07650 g/ml using Phosphate Buffered Saline (PBS) under monitoring at 20 ℃ using a high precision digital densitometer
Compared with the current commercial centrifugation medium on the market, the composition of the invention can greatly shorten the centrifugation time for obtaining the circulating non-blood-related nucleated cells from the human blood sample by the steps and stably improve the recovery rate of the circulating non-blood-related nucleated cells from the original 60 +/-20 percent to 96 percent.
Specifically, in the composition of the present invention, the immune microspheres or immune particles are formed by covalent or non-covalent coupling of antibodies specifically recognizing leukocyte markers to the surface of microspheres, which may or may not be chemically treated to be suitable for coupling to proteins, the diameter of the microspheres is 10 nm to 100 μm, and the immune microspheres or immune particles comprise or partially comprise any one of the following components: silica, dextran, agarose, or cross-linked dextran. The microspheres used to prepare the immune microspheres are magnetic or non-magnetic.
In the compositions of the present invention, the antibodies used to prepare the immunomicrosphere or immunomer specifically recognize one or more of the following, but not limited to, these leukocyte surface markers: CD14, CD45RO, CD45RA, and the like.
In the compositions of the invention, the immune microspheres or particles are prepared by covalent or non-covalent coupling of any chemically treated solid surface, such as silica particles, to a ligand or specific monoclonal or polyclonal antibody, including antibodies against leukocyte surface markers such as CD14, CD45RO, CD45RA, with or without a suitable binding protein.
In the composition of the present invention, the specific gravity of the unique cell separation medium at 20 ℃ is preferably in the range of 1.07260-1.07650 g/ml (gr/ml or gr/cm)3) A density within this particular range is suitable for separating almost all nucleated cells from erythrocytes and from immune microspheres or particles. The cell separation medium comprises any one or any two or more of the following reagent components: polyvinylpyrrolidone-coated colloidal silica; a polysaccharide; sodium diatrizoate or a derivative thereof; a nonionic polymer composed of sucrose and epichlorohydrin; or any sugar-containing solution, such as dextran or sucrose; iodinated small compounds (such as metrizamide); or any protein solution. The specific gravity of the cell separation medium can be any osmotic pressure between 285 and 315mOsm/kgH2O, pH 6.8.8-7.8 at 20 deg.C in 1.07260-1.07650 g/ml buffer solution. The specific gravity of the immune microspheres or immune particles is higher than that of the cell separation medium. Centrifugation based on the cell separation medium is performed in a common commercial centrifuge tube.
All technical approaches to detect circulating rare cells to date are based on immunofluorescence methods.
However, the major disadvantage of non-specific binding to target cells due to the self-negative charge of the fluorochrome is inevitable. This disadvantage brings about a great trouble to people in the process of distinguishing true and false positive staining signals. The composition or the kit provided by the invention can overcome non-specific staining caused by immunofluorescence, and enables detection of stained circulating rare cells to be carried out under a common optical microscope or a scanner based on a microscope principle. Has proved to be a technical means with high specificity, rapidness, simplicity and low cost, and no need of any fluorescent dye and expensive fluorescent microscope.
In the compositions of the invention, monoclonal or polyclonal antibodies that recognize keratin, such as keratin 8, 18, 19 or any one or two or more of a broad spectrum of keratin, are used to identify circulating tumor cells that have shed into the blood from any solid tumor of epithelial origin. Another monoclonal or polyclonal antibody recognizing the leukocyte surface marker CD45 was used for leukocyte staining to distinguish false positives.
It is also an object of the present invention to provide a kit for rapidly obtaining circulating non-related nucleated rare cells from blood, comprising:
a composition comprising the following ingredients: buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-blood related nucleated cell surface marker antibodies, and a cell separation medium; wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, pH is 6.8 to 7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07156 to 1.07738 g/ml, and the cell separation medium comprises one or two or more of the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharide, sodium diatrizoate or its derivative, a sugar-containing solution, and a nonionic polymer obtained by polymerizing sucrose and epichlorohydrin. The kit also includes a container for holding the composition and a blood collection tube optionally with an anticoagulant.
The kit also includes instructions for use of the kit.
In the kit of the present invention, a buffer solution, magnetic or non-magnetic immunomicrospheres or immunomicropalls coated with antibodies against the hematopoietic border nucleated cell surface markers, and a cell separation medium are separately contained in different containers.
The container may be an ampoule or a sealed bag. The blood collection tube with anticoagulant may contain any anticoagulant such as EDTA, heparin, or ACD.
In the kit of the present invention, the immune microspheres or immune particles are formed by covalent or non-covalent coupling of antibodies specifically recognizing leukocyte markers to the surface of microspheres or particles, which may or may not be chemically treated to be suitable for coupling to proteins, said microspheres or particles having a diameter of 10 nm to 100 μm, said microspheres or particles comprising or partially comprising any one of the following components: silica, dextran, agarose, or cross-linked dextran. The microspheres used to prepare the immune microspheres are magnetic or non-magnetic.
In the kit of the present invention, the antibody used to prepare the immunomicrosphere or immunomarticle specifically recognizes one or more of the following, but not limited to, these leukocyte surface markers: CD14, CD45RO, CD45RA, and the like.
In the kits of the invention, the immunomicrospheres or immunomicropalls are prepared by covalent or non-covalent coupling of any chemically treated solid surface such as silica microspheres, or dextran, agarose, or sephadex microspheres with ligands or specific monoclonal or polyclonal antibodies including antibodies against leukocyte surface markers such as CD45 and/or CD 50.
In the kit of the present invention, it is preferable that the specific gravity of the cell separation medium at 20 ℃ is preferably in the range of 1.07260 to 1.07650 g/ml (gr/ml or gr/cm)3) The density in this particular range is suitable for separating almost all nucleated cells from erythrocytes and immune microspheres.
The cell separation medium comprises any one or any two or more of the following reagent components: polyvinylpyrrolidone-coated colloidal silica; polysaccharide plus diatrizoic acid sodium or derivatives thereof; non-ionic polymerization of sucrose and epichlorohydrinAn agent; or any sugar-containing solution, such as dextran or sucrose; iodinated small compounds (such as metrizamide); or any protein solution. The specific gravity of the cell separation medium can be any osmotic pressure of 280-320 mOsm/kg H2O, pH 6.8.8-7.8 at 20 deg.C in 1.07260-1.07650 g/ml buffer solution. The specific gravity of the immune microspheres or immune particles is higher than that of the cell separation medium. Centrifugation based on the cell separation medium is performed in a commonly commercially available centrifuge tube.
Example 2
The kit of the present invention is used for rapidly obtaining non-blood related circulating nucleated cells from a human blood sample, 6ml of human peripheral blood is collected in any blood collection tube (BD, New Jersey, USA) containing an anticoagulant (such as EDTA, heparin, ACD, etc.), and plasma proteins are removed after centrifugation (1000 × g, 15 minutes).
After adding 50. mu.l of magnetic beads coated with monoclonal antibodies against CD14, CD45RO/CD45RA (Invitrogen, California, USA), the mixture was mixed by inversion 10 times and incubated at room temperature for 20 minutes.
Adding 6ml of cell buffer solution, gently transferring the sample to a centrifuge tube containing 3 ml of a spacer solution of cell separation medium (the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml, the cell separation medium contains polyvinylpyrrolidone-coated colloidal silica, polysaccharide and sodium diatrizoate; a sugar solution containing dextran; a nonionic polymer formed by polymerizing sucrose and epichlorohydrin), and centrifuging the sample again (900 × g, 6 minutes).
The supernatant was collected and centrifuged again at 1000 × g for 4 minutes.
And discarding the supernatant to obtain sedimentary cells, namely the enriched non-blood related nucleated cells.
The sedimented cells obtained after centrifugation were resuspended in phosphate buffer carried by the kit for further analysis.
Compared with the commercial centrifugation medium commonly used in the market at present, the kit of the invention can greatly shorten the centrifugation time for obtaining the circulating non-blood-related nucleated cells from the human blood sample by using the steps and improve the recovery rate of the circulating non-blood-related nucleated cells to 95 percent.
The composition or kit of the present invention can be used in biomedical experiments, or in manual clinical testing operations or in clinical testing operations with the aid of automated testing equipment, all of which are routine procedures well known to those skilled in the art and need not be described herein in detail.
Claims (10)
1. A composition for rapidly obtaining circulating non-blood related nucleated rare cells from blood comprising the following components: the buffer solution comprises magnetic or non-magnetic immune microspheres or immune particles coated with anti-related nucleated cell surface marker antibodies and a cell separation medium, wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, the pH value is 6.8-7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml, the specific gravity of the immune microspheres or immune particles is higher than that of the cell separation medium, and the cell separation medium comprises any one or any two or more than two of the following reagents: polyvinylpyrrolidone coated colloidal silica, polysaccharide and sodium diatrizoate or its derivatives methopamide, dextran, sucrose, nonionic polymer polymerized from sucrose and epichlorohydrin.
2. The composition of claim 1, wherein the immune microspheres or immune particles are covalently or non-covalently bound with an antibody that specifically recognizes one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA.
3. The composition of claim 1 or 2, wherein the circulating non-limbed nucleated rare cell is selected from the group consisting of: circulating any solid tumor cells of epithelial origin or any tumor cells not of epithelial origin, circulating vascular endothelial cells, stem cells, and immune cells.
4. The composition of claim 3, wherein the circulating non-limbed nucleated rare cells are selected from tumor stem cells.
5. The composition of claim 1 or 2, wherein the buffer has an osmotic pressure of 280 to 320mOsm/kgH2O and a PH of 6.85 to 7.75.
6. The composition of claim 3, wherein the proportion of circulating non-limbed nucleated rare cells selected from all solid tumor cells having epithelial origin, or any tumor cells not having epithelial origin, circulating vascular endothelial cells, fetal cells in blood, stem cells, and immune cells in the collected blood sample is less than 0.1% of all nucleated cells.
7. The composition of claim 6, wherein the proportion of circulating non-bloodborne nucleated rare cells selected from tumor stem cells in the collected blood sample is less than 0.1% of all nucleated cells.
8. The composition according to claim 6, wherein the solid tumor cells of epithelial origin are selected from the following cells: cells of lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer, all solid tumor cells; any tumor cell that is not of epithelial origin is a melanoma cell.
9. A kit for rapidly obtaining circulating non-limbed nucleated rare cells from blood comprising:
a composition comprising the following ingredients: buffer solution, magnetic or non-magnetic immune microspheres or immune particles coated with anti-blood related nucleated cell surface marker antibodies, and a cell separation medium; wherein the osmotic pressure of the buffer solution is 280-320 mOsm/kg H2O, the pH value is 6.8-7.8, the specific gravity of the cell separation medium at 20 ℃ is 1.07260-1.07650 g/ml, the specific gravity of the immune microspheres or immune particles is higher than that of the cell separation medium, and the cell separation medium comprises any one or any two or more components selected from the following reagents: polyvinylpyrrolidone-coated colloidal silica, polysaccharides and sodium diatrizoate or its derivatives methoxamide, dextran, sucrose, nonionic polymers polymerized from sucrose and epichlorohydrin;
a container containing the composition and a blood collection tube optionally with an anticoagulant; a slide and instructions for use of the kit.
10. The kit of claim 9, wherein the immune microspheres or immune particles are covalently or non-covalently bound with antibodies that specifically recognize one or more of the following leukocyte surface markers: CD14, CD45RO, CD45 RA;
wherein the circulating non-limbed nucleated rare cell is selected from the group consisting of: circulating any solid tumor cells of epithelial origin or any tumor cells not of epithelial origin, circulating vascular endothelial cells, fetal cells in blood, stem cells, and immune cells;
wherein the circulating non-limbed nucleated rare cells are selected from tumor stem cells;
wherein the proportion of circulating non-limbed rare nucleated cells in the collected blood sample is less than 0.1% of all nucleated cells, the circulating non-limbed rare nucleated cells being selected from the group consisting of all solid tumor cells having epithelial origin, or solid tumor cells not having epithelial origin, circulating vascular endothelial cells, fetal cells in blood, stem cells, and immune cells;
wherein the proportion of the circulating non-bloodborne nucleated rare cells in the collected blood sample accounts for less than 0.1 percent of all nucleated cells, and the circulating non-bloodborne nucleated rare cells are selected from tumor stem cells;
wherein the solid tumor cells of epithelial origin are selected from the following cells: cells of lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, gastric cancer and liver cancer; any tumor cell that is not of epithelial origin is a melanoma cell.
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