Method for identification, selection and analysis of tumour cells

A method, comprising the steps of:

• a) obtaining a sample of a biological fluid and preparing from the sample a suspension of cells of interest and contaminating cells in a fluid wherein the cells of interest are nucleated cells and the contaminating cells comprise nucleated cells, the cells of interest being circulating tumour cells and/or disseminating tumour cells;
• b) labeling the suspension with:
• a first antibody conjugated to a first fluorescent marker that excites at a first wavelength, the first antibody binding to the cells of interest, wherein the first antibody is an anti-cytokeratin antibody, an anti-EpCAM antibody, an anti-PSA antibody, an anti-TTF-1 antibody, or an anti-HER2/neu antibody,
• a second antibody conjugated to a second fluorescent marker that excites at a second wavelength, the second antibody binding to the contaminating cells and does not bind to the cells of interest, wherein the second antibody is an anti-CD45 antibody, and,
• a third fluorescent marker that binds to the cells of interest, which are nucleated cells, and to the nucleated contaminating cells and excites at a third wavelength;
• c) introducing the suspension into a chamber of a microfluidic device, wherein the chamber is bounded by a top surface adjacent to at least one electrode and a bottom surface that is adjacent to an array of electrodes arranged in the chamber to generate dielectrophoretic cages and immobilize the cells of interest and the contaminating cells in the chamber using dielectrophoretic cages;
• d) applying a first pattern of voltages to the array of electrodes to generate a first field of force acting on the cells of interest and the contaminating cells to individually trap the cells of interest and contaminating cells in the dielectrophoretic cages;
• e) scanning the suspension loaded in the chamber of the microfluidic device with a fluorescence microscope in three different fluorescence channels at first, second, and third wavelengths, respectively, to generate fluorescent images of the cells of interest and the contaminating cells of the suspension while the cells of interest and the contaminating cells are trapped in the dielectrophoretic cages, the fluorescent images showing morphology of the cells of interest and the contaminating cells, and a distribution of three fluorescence signals across the three channels within each of the dielectrophoretic cages;
• f) individually selecting single cells of interest in the suspension on the basis of identification in the fluorescent images of a distinct combination of the three signals and morphology compatible with tumour cells, while the single cells of interest are immobilized in the dielectrophoretic cages in the chamber of the microfluidic device, wherein the distinct combination of the three signals is positive to the first fluorescent marker, negative to the second fluorescent marker, and positive to the third fluorescent maker;
• and
• g) individually manipulating the selected cells of interest immobilized in the chamber of the microfluidic device into a purified specimen using dielectrophoretic isolation by applying a second pattern of voltages to the array of electrodes to generate a second field of force acting only on the selected cells of interest to shift the selected cells of interest from the dielectrophoretic cages into the purified specimen,
• wherein:
• the purified specimen has a purity of at least 90% as defined by the ratio of the number of the cells of interest to the total number of cells in the purified specimen.

A method, comprising the steps of:

• a) labeling a suspension comprising cells of interest and contaminating cells, the suspension being enriched in cells of interest and the cells of interest being circulating tumor cells and/or disseminating tumour cells, wherein the cells of interest are nucleated cells and the contaminating cells comprise nucleated cells, with:
• a first antibody conjugated to a first fluorescent marker that excites at a first wavelength, the first antibody binding to the cells of interest,
• a second antibody conjugated to a second fluorescent marker that excites at a second wavelength, the second antibody binding to the contaminating cells and does not bind to the cells of interest, wherein the second antibody is an anti-CD45 antibody or anti-vimentin antibody, and,
• a third fluorescent marker that binds to the cells of interest, which are nucleated cells, and nucleated contaminating cells and excites at a third wavelength;
• ;
• b) introducing the suspension into a chamber of a microfluidic device, wherein the chamber is bounded by a top surface adjacent to at least one electrode and a bottom surface that is adjacent to an array of electrodes arranged in the chamber to generate dielectrophoretic cages and immobilize the cells of interest and the contaminating cells in the chamber using dielectrophoretic cages;
• c) applying a first pattern of voltages to the array of electrodes to generate a first field of force acting on the cells of interest and the contaminating cells, to individually trap the cells of interest and the contaminating cells in the dielectrophoretic cages;
• d) scanning the suspension loaded in the chamber of the microfluidic device with a fluorescence microscope in three different fluorescence channels at first, second, and third wavelengths, respectively, to generate fluorescent images of the cells of interest and the contaminating cells while the cells of interest and the contaminating cells, are trapped in the dielectrophoretic cages, the fluorescent images showing morphology of the cells of interest and the contaminating cells, and a distribution of three fluorescence signals across the three channels within each of the dielectrophoretic cages;
• e) individually selecting single cells of interest in the suspension on the basis of identification in the fluorescent images of a distinct combination of the three signals and morphology compatible with tumour cells, while the single cells of interest are immobilized in the dielectrophoretic cages in the chamber of the microfluidic device, wherein the distinct combination of the three signals is positive to the first fluorescent marker, negative to the second fluorescent marker, and positive to the third fluorescent maker;
• and
• f) individually manipulating the selected cells of interest immobilized in the chamber of the microfluidic device into a purified specimen using dielectrophoretic isolation by applying a second pattern of voltages to the array of electrodes to generate a second field of force acting only on the selected cells of interest to shift the selected cells of interest from the dielectrophoretic cages into the purified specimen,
• wherein:
• the purified specimen has a purity of at least 90% as defined by the ratio of the number of the cells of interest to the total number of cells in the purified specimen.