Analysis of circulating tumor cells, fragments, and debris
First Claim
1. A method for diagnosing disease in a test subject comprising:
- a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact rare cells and further comprising;
i. circulating cell fragments derived from rare cells, orii. circulating cellular debris derived from rare cells;
b. preparing a magnetically-labeled sample wherein said biological sample is mixed with colloidal magnetic nanoparticles, having sizes sufficiently small so as to have a magnetic moment that ensures complete separation from the specimen when exposed to an external magnet, coupled to a first biospecific ligand which reacts and specifically binds to a common specific binding site present in said intact rare cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components;
c. contacting said magnetically-labeled sample with at least one additional biospecific ligand conjugated to a label and specifically binding to another specific binding site common amongst said intact rare cells, and said cell fragments or said cellular debris which are bound to the first biospecific ligand in step b, to the substantial exclusion of other specimen components;
d. subjecting said magnetically-labeled sample to the externally-applied high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact rare cells, and said cell fragments or said cellular debris; and
e. analyzing said labeled rare cells, and said labeled cell fragments or said labeled cellular debris from step d, the presence of said labeled rare cells, said labeled cell fragments, and said labeled cellular debris indicating the presence of disease.
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Accused Products
Abstract
The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CELLSPOTTER® fluorescent microscopy imaging system. Analyzing damaged cells has shown to be important. However, there are two sources of damage: in vivo and in vitro. Damage in vivo occurs by apoptosis, necrosis, or immune response. Damage in vitro occurs during sample acquisition, handling, transport, processing, or analysis. It is therefore desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. Described herein are methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also provided are kits for assaying biological specimens using these methods.
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Citations
17 Claims
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1. A method for diagnosing disease in a test subject comprising:
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a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact rare cells and further comprising; i. circulating cell fragments derived from rare cells, or ii. circulating cellular debris derived from rare cells; b. preparing a magnetically-labeled sample wherein said biological sample is mixed with colloidal magnetic nanoparticles, having sizes sufficiently small so as to have a magnetic moment that ensures complete separation from the specimen when exposed to an external magnet, coupled to a first biospecific ligand which reacts and specifically binds to a common specific binding site present in said intact rare cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components; c. contacting said magnetically-labeled sample with at least one additional biospecific ligand conjugated to a label and specifically binding to another specific binding site common amongst said intact rare cells, and said cell fragments or said cellular debris which are bound to the first biospecific ligand in step b, to the substantial exclusion of other specimen components; d. subjecting said magnetically-labeled sample to the externally-applied high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact rare cells, and said cell fragments or said cellular debris; and e. analyzing said labeled rare cells, and said labeled cell fragments or said labeled cellular debris from step d, the presence of said labeled rare cells, said labeled cell fragments, and said labeled cellular debris indicating the presence of disease. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for diagnosing malignancy in a test subject comprising:
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a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact malignant cells and further comprising; i. circulating cell fragments derived from malignant cells, or ii. circulating cellular debris derived from malignant cells; b. preparing a magnetically-labeled sample wherein said biological sample is mixed with colloidal magnetic nanoparticles, having sizes sufficiently small so as to have a magnetic moment that ensures complete separation from the specimen when exposed to an external magnet coupled to a first biospecific ligand which reacts and specifically binds to a common specific binding site present in said intact malignant cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components; c. contacting said magnetically-labeled sample with at least one additional biospecific ligand conjugated to a label and specifically binding to another specific binding site common amongst said intact malignant cells, and said cell fragments or said cellular debris which are bound to the first biospecific ligand in step b, to the substantial exclusion of other specimen components; d. subjecting said magnetically-labeled sample to the externally-applied high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact malignant cells, and said cell fragments or said cellular debris; and e. analyzing said labeled malignant cells, and said labeled cell fragments or said labeled cellular debris from step d, the presence of said labeled malignant cells, said labeled cell fragments, and said labeled cellular debris indicating the presence of malignancy. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14)
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15. A kit for assaying a biological specimen for the presence of malignant cells, and cell fragments derived from malignant cells or cellular debris derived from malignant cells, comprising:
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a. coated colloidal magnetic nanoparticles comprising; i. a magnetic core material having sizes sufficiently small so as to have a magnetic moment that ensures complete separation of malignant cell bound thereto and cell fragment or cellular debris derived from malignant cell bound thereto when exposed to an externally applied magnet, ii. a protein base coating material, and iii. an antibody that binds specifically to a first characteristic determinant of said malignant cell, and said cell fragments or said cellular debris, wherein said antibody is coupled to said base coating material on the colloidal magnetic nanoparticle; b. at least one antibody having binding specificity for a second characteristic determinant of said malignant cell, and said cell fragments or said cellular debris; c. an agent capable of staining further features of said malignant cells, and said cell fragments or said cellular debris. - View Dependent Claims (16, 17)
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Specification