Method for separating rare cells from a population of cells
AssigneeApplied Imaging Corporation
InventorsAlexander M. Saunders, Michael A. M. Zarowitz, Patricia J. Baldwin
Life of this patent
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Abstract
A method for isolating and enriching rare cells, including fetal nucleated erythrocytes from a peripheral blood sample is described. The method includes two centrifugation steps, the first a bulk separation step to enrich erythrocytes from other blood components. The second centrifugation comprises a colloidal density gradient medium dispersed in a hypertonic meltable gel. In one aspect of the invention, maternal erythrocytes may be hemolyzed prior to the second density gradient centrifugation to provide additional enrichment for fetal nucleated erythrocytes.
Claims25
A method for obtaining fetal nucleated red blood cells from maternal blood in a blood sample, the method comprising:
- a) centrifuging the blood sample in a first centrifugation vessel to obtain a red blood cell fraction;
- b) transferring the red blood cell fraction to an upper portion of a second centrifugation vessel, the second centrifugation vessel containing a density gradient medium consisting of a colloid dispersed in a meltable gel, wherein the colloid is capable of maintaining the red blood cell fraction in a substantially unaggregated state;
- c) hemolysis of maternal red blood cells in the red blood cell fraction to obtain an enriched fetal red blood cell fraction, wherein the hemolysis occurs in a water soluble medium disposed on the upper portion of the density gradient medium;
- d) melting the gel;
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- e) centrifuging the enriched fetal red blood cell fraction through the density gradient medium to obtain a fraction enriched in fetal nucleated red blood cells.
A method for obtaining fetal nucleated red blood cells from maternal blood in a blood sample, the method comprising:
- a) centrifuging the blood sample in a first centrifugation vessel to obtain a red blood cell fraction;
- b) hemolyzing maternal red blood cells in the red blood cell fraction to obtain an enriched fetal red blood cell fraction;
- c) and centrifuging the enriched fetal red blood cell fraction through a density gradient medium consisting of a colloid dispersed in a sol state meltable gel, wherein the colloid is capable of maintaining the enriched fetal red blood cell fraction in a substantially unaggregated state, to obtain a fraction enriched in fetal nucleated red blood cells.
A method of isolating fetal nucleated red blood cells from maternal blood in a whole blood sample, the method comprising:
- (a) hemolyzing maternal red blood cells in the blood sample with a hemolyzing agent, wherein the hemolysis occurs in the presence of an inhibitor to slow hemolysis of fetal red blood cells;
- (b) terminating the hemolysis reaction;
- (c) concentrating all the remaining non-hemolysed red blood cells by centrifugation;
- (d) separating the remaining non-hemolysed red blood cells from material white blood cells by density in a density gradient medium comprising a colloid dispersed in a sol state meltable gel, wherein the colloid is capable of maintaining the non-hemolysed red blood cells in a substantially unaggregated state;
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- (e) recovering a fraction enriched in the fetal nucleated red blood cells.
A kit for the enrichment or isolation of fetal nucleated red blood cells from maternal red blood cells, the kit comprising a first plastic centrifuge vessel having a narrowed recovery portion and an outer support vessel, a second centrifuge vessel having an upper aperture and a base, the second centrifuge vessel containing a density gradient medium, the density gradient medium comprising a colloid dispersed in a meltable gel, wherein the colloid is capable of maintaining all red blood cells in a substantially unaggregated state.
A kit for the isolation of fetal nucleated red blood cells, the kit comprising:
- (a) a hemolyzing agent;
- (b) a hemolysis inhibitor;
- (c) a hemolysis termination reagent;
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- (d) a density gradient medium comprising a colloid dispersed in a meltable gel, wherein the colloid is capable of maintaining the cells in a substantially unaggregated state.
Dependencies are inferred from the claim text.