Identification and analysis of fetal trophoblast cells in cervical mucus for prenatal diagnosis
A method of retrieving fetal cells from an endocervical sample by removing the mucus from the endocervical sample by disassociating fetal cells and maternal cells in the endocervical sample; and isolating disassociated fetal cells from other cells in the endocervical sample. Also provided is a method of retrieving fetal cells from an endocervical sample, by obtaining a mixture of disassociated cells prepared by the above method, treating the cells with a fetal-specific antibody, identifying cells that have bound to the fetal-specific antibody, and isolating the identified cells. The disassociated cell prepared by the above method can be analyzed and used for a variety of purposes including, but not limited to, the identification of fetal cells among cervical cells, determination of fetal cell density to predict high risk pregnancy, genetic analysis of fetal cells, and determination of growth factor or other biomarker expression to predict obstetrical disorders.
1. A method of retrieving fetal cells from an endocervical sample, comprising:
obtaining an endocervical sample;
placing the endocervical sample in a fixative;
removing the mucus from the endocervical sample, thereby disassociating and fixing fetal cells and maternal cells in the endocervical sample; and
isolating disassociated fetal cells en masse from maternal cells in the endocervical sample, wherein said isolating comprises labeling fetal cells in the sample to aid in isolating the fetal cells en masse, wherein said labeling step includes labeling the fetal cells using column-free immunomagnetic nanoparticles and recovering isolated dissociated fetal cells, wherein a majority of fetal cells present in the endocervical sample are recovered as isolated dissociated fetal cells, and wherein the isolated dissociated fetal cells are characterized by purity of 90% or greater.
2. The method according to claim 1 , wherein said obtaining step comprises obtaining the endocervical sample using a method selected from the group consisting of: intrauterine lavage, aspiration of cervical mucus, and removal of surface tissue from the endocervical canal.
3. The method according to claim 1 , wherein said obtaining step further comprises culturing the sample in a trophoblast culture medium.
4. The method according to claim 1 , further comprising identifying cells isolated in the sample for diagnostic purposes.
5. The method according to claim 4 , wherein said identifying step includes analyzing cells using a method selected from the group consisting of fluorescent in situ hybridization, polymerase chain reaction with reverse transcription, polymerase chain reaction without reverse transcription, and immunohistochemistry.
6. The method according to claim 4 , further comprising analyzing the identified cells using a method selected from the group consisting of: biochemical testing and whole genome amplification followed by genomic hybridization or sequencing.
7. The method of claim 1 , wherein the isolating disassociated fetal cells from maternal cells comprises:
contacting the cells of the endocervical sample with a fetal-specific antibody;
identifying contacted cells that have bound to the fetal-specific antibody; and
isolating the identified cells.
8. The method according to claim 1 , further comprising:
identifying fetal cells in the isolated dissociated fetal cells;
determining fetal cell density;
performing a genetic analysis of the fetal cells; or
identifying one or more biomarkers in the isolated dissociated fetal cells.
9. A method of retrieving fetal cells from an endocervical sample comprising:
obtaining an endocervical sample;
placing the endocervical sample in a fixative;
removing mucus from the endocervical sample;
immunomagnetically labeling fetal cells of the endocervical sample using column-free immunomagnetic nanoparticles;
disassociating fetal cells from maternal cells in the endocervical sample, and
retrieving disassociated fetal cells en masse from the sample, wherein a majority of fetal cells present in the endocervical sample are recovered as dissociated fetal cells, and wherein the dissociated fetal cells are characterized by purity of 90% or greater.
10. The method of claim 1 , wherein removing the mucus from the endocervical sample comprises contacting the mucus with acetic acid.
11. A method of retrieving fetal cells from an endocervical sample consisting of:
obtaining a fixed or an unfixed endocervical sample;
treating the sample by acidification or treating the sample with a preservative;
centrifuging the sample and resuspending pelleted cells, thereby washing the pelleted cells, producing a washed endocervical sample;
immunomagnetically labeling fetal cells of the washed endocervical sample using column-free immunomagnetic nanoparticles;
disassociating fetal cells from maternal cells in the washed endocervical sample; and
retrieving disassociated fetal cells en masse from the washed sample, wherein a majority of fetal cells present in the endocervical sample are recovered as dissociated fetal cells, and wherein the dissociated fetal cells are characterized by purity of 90% or greater.