Isolation of fetal cells using facs

Abstract

The present invention relates to automated methods for isolating fetal cells from a sample, such as a blood sample, derived from a pregnant woman. The isolated fetal cells can be used for identifying genetic abnormalities in the fetal DNA.

1 - 30 . (canceled) 
     
     
         31 . A method of isolating a fetal cell from maternal blood comprising the steps of:
 A. providing a blood sample from a woman carrying a fetus;   B. contacting cells comprised in said blood sample with at least one labelling agent directed against a fetal trophoblast marker;   C. enriching said sample for fetal trophoblasts with magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), or using antibodies immobilized onto a solid surface, to form enriched cells;   D. contacting the enriched cells with at least one labelling agent selected from each of the groups to form labelled cells:
 i. a labelling agent directed against the nucleus, 
 ii. a labelling agent directed against a maternal cell marker, and 
 iii. a labelling agent directed against a fetal trophoblast marker; 
   E. single cell sorting the labelled cells, wherein single cell sorted cells are sorted into single cell compartments based on:
 i. positive selection of said labelling agent directed against a fetal trophoblast marker, 
 ii. positive selection of said labelling agent directed against the nucleus, and 
 iii. negative selection of said labelling agent directed against a maternal cell marker; 
   F. identifying fetal trophoblasts among said single cell sorted cells.   
     
     
         32 . The method according to  claim 31 , wherein said labelling agent directed against a fetal trophoblast marker is a labelling agent directed against an endothelial or an epithelial marker. 
     
     
         33 . The method according to  claim 31 , wherein said labelling agent directed against a fetal trophoblast marker is a labelling agent directed against an endothelial marker. 
     
     
         34 . The method according to  claim 33 , wherein said endothelial marker is CD105, CD90 and/or CD141. 
     
     
         35 . The method according to  claim 31 , wherein said labelling agent directed against a fetal trophoblast marker is a labelling agent directed against an epithelial marker. 
     
     
         36 . The method according to  claim 35 , wherein said epithelial marker is a cytokeratin, Human Cytokeratin 7 CK7, Human Cytokeratin 8 CK8, Human Cytokeratin 18 CK18 and/or Human Cytokeratin 19 CK19. 
     
     
         37 . The method according to  claim 31 , wherein said labelling agents are antibodies. 
     
     
         38 . The method according to  claim 31 , wherein at least one of said labelling agents are indirectly and/or directly labelled by at least one fluorophore. 
     
     
         39 . The method according to  claim 38 , wherein said fluorophore is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 555, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), and BV421. 
     
     
         40 . The method according to  claim 31 , wherein said labelling agent directed against the nucleus is selected from any Hoechst dye, such as Hoechst 33342, DAPI, propidium iodide, 7-AAD, Vybrant DyeCycle Stains, SYTOX stains, or SYTO stains. 
     
     
         41 . The method according to  claim 31 , wherein said labelling agent directed against a maternal cell marker is a labelling agent directed against a leucocyte marker selected from CD45, CD3, CD14, CD15, CD16, and/or CD19. 
     
     
         42 . The method according to  claim 31 , wherein said fetal trophoblasts are identified by obtaining a genotype from said fetal trophoblasts by STR analysis or SNP analysis. 
     
     
         43 . The method according to  claim 31 , wherein said fetal cell is identified by detecting one or more markers associated with a genetic abnormality in the genome of the fetal cell. 
     
     
         44 . The method according to  claim 43 , wherein said fetal cell is identified by detecting one or more markers associated with a genetic abnormality in the genome of the fetal cell, wherein said genetic abnormality is detected by one or more methods selected from Microarray-based Comparative Genomic Hybridization (aCGH), Short Tandem Repeat analysis (STR analysis), whole genome amplification, whole genome scan, SNP array, Polony sequencing, Shotgun sequencing, Massively parallel signature sequencing (MPSS), Sanger Sequencing, PCR-based methods and Next-Generation Sequencing methods. 
     
     
         45 . The method according to  claim 43 , wherein said genetic abnormality is aneuploidy, monosomy, polysomy, trisomy, copy number variation (CNV), single nucleotide variation (SNV), or a monogenic disorder. 
     
     
         46 . The method according to  claim 31 , wherein said blood sample is between 5-50 mL such as 5-30 mL and 10-30 mL. 
     
     
         47 . The method according to  claim 31 , wherein said blood sample comprises a cellular fraction, optionally a cellular fraction separated from plasma of said blood sample optionally by centrifugation. 
     
     
         48 . The method according to  claim 47 , said method further comprising a step of fixating the cells in said blood sample subsequent to being separated from said plasma fraction, wherein fixation is optionally a paraformaldehyde fixation. 
     
     
         49 . The method according to  claim 47 , wherein said cellular fraction comprises both maternal cells and fetal cells optionally selected from the group consisting of red blood cells, white blood cells, fetal trophoblasts, fetal extravillous trophoblasts, and fetal endovascular trophoblasts. 
     
     
         50 . The method according to  claim 49 , said method further comprising a step of selectively lysing red blood cells of said cellular fraction using a detergent subsequent to separation of said cellular fraction from said plasma fraction, wherein said lysing also permeabilizes the remaining cells in said cellular fraction.