Labeled cells for use as an internal functional control in rare cell detection assays
OwnerLeon W.M.M. Terstappen, Galla Chandra Rao +2inventors
InventorsLeon W.M.M. Terstappen, Galla Chandra Rao, Herman Rutner, Paul A. Liberti
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Abstract
Stabilized control cells, methods for making the same and kits comprising the stabilized cells are disclosed. Also provided are improved methods for detecting rare cells in biological samples.
Claims54
A process for producing a stabilized cell for use as an internal control in methods for isolating and identifying rare cells, said stabilized control cell having determinants in common with said rare cells, said process comprising,
- a) redundantly labeling said control cell with at least two fluorescent labels having the same spectral properties;
- b) contacting said labeled cells with a cell fixative said fixative effecting stabilization of both cellular structure and antigenic moieties present on said control cells;
- c) subsequently removing the excess fixative to promote long-term storage of said control cells, said control cells being physically and biologically stable for a period up to at least six months.
A process for producing a stabilized cell for use as an internal control in methods for isolating and identifying rare cells, said stabilized control cell having determinants in common with said rare cells, said process comprising,
- a) redundantly membrane labeling said control cell with at least two fluorescent labels having the same spectral properties;
- b) contacting said labeled cells with a cell fixative said fixative effecting stabilization of both cellular structures and antigenic moieties present on said control cells;
- c) subsequently removing the excess fixative to promote long-term storage of said control cells, said control cells being physically and biologically stable for a period up to at least to six months, wherein said control cell expresses epithelial cell adhesion molecule (EpCam) on its surface and also expresses cytokeratin intracellularly.
A stabilized cell for use as an internal control in methods for isolating and identifying rare cells, said control cell having determinants in common with said rare cells, wherein said control cell is labeled redundantly with at least two fluorescent labels having the same spectral properties, and cellular components and antigenic moieties of said control cell have been stabilized for a period up to at least six months by exposure to fixative.
A stabilized cell for use as an internal control in methods for isolating and identifying rare cells, said stabilized control cell having determinants in common with said rare cells, and comprising a detectably labeled membrane, said cells further comprising stabilized cellular components and antigenic moieties, said stabilization being effected by exposure to a fixative, wherein said control cell is a tumor cell expressing EpCam on its surface and cytokeratin intracellularly.
The control cell of 16, wherein said membrane is redundantly labeled with at least two fluorescent labels having the same spectral properties.
A stabilized cell for use as an internal control in methods for isolating and identifying rare cells, said stabilized control cell having determinants in common with said rare cells, and comprising a redundantly labeled membrane, said membrane being labeled with at least two fluorescent labels having the same spectral properties, said cells further comprising stabilized cellular components and antigenic moieties, said stabilization being effected by exposure to a fixative, wherein said control cell is selected from the group consisting of tumor cells, bacterially infected cells, virally infected cells, myocardial cells, and endothelial cells in circulation, and fetal cells in maternal circulation.
A method for detecting and enumerating rare cells in a mixed cell population, the presence of said rare cells in said population being indicative of severity of a disease state, comprising:
- a) obtaining a blood sample from a test subject, said sample comprising a mixed cell population suspected of containing said rare cells;
- b) preparing an immunomagnetic sample wherein said biological specimen is mixed with magnetic particles coupled to a ligand which reacts specifically with a determinant of the rare cells, to the substantial exclusion of other sample components;
- c) contacting said immunomagnetic sample with at least one reagent which labels a determinant of said rare cells;
- and
- d) analyzing said labeled rare cells to determine the presence and number of any rare cells in said immunomagnetic sample, the greater the number of rare cells present in said sample, the greater the severity of said disease state, wherein the improvement comprises the addition of a stabilized cell for use as an internal control cell in said method, said control cell having determinants in common with said rare cells and wherein
- said membrane of said control cell is detectably labeled and cellular components and antigenic moieties of said control cell have been stabilized for a period up to at least six months by exposure to fixative.
An improved kit for screening a patient sample for the presence of circulating tumor cells, comprising:
- a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and anti-EpCAM coupled, directly or indirectly, to said base coating material;
- b) at least one antibody having binding specificity for a cancer cell determinant;
- c) cell specific dye for excluding sample components other than said tumor cells from analysis wherein the improvement comprises the addition of a container comprising stabilized cells for use as an internal control, said stabilized control cells having determinants in common with said rare cells, wherein said membrane of said control cell is detectably labeled, and cellular components and antigenic moieties of said control cell have been stabilized for a period up to at least six months by exposure to fixative, said stabilized control cells being suspended in a buoyant density medium.
Dependencies are inferred from the claim text.