Compositions and methods for isolating, detecting and analyzing fetal cells

Abstract

To provide compositions, kits and methods for isolating, detecting and analyzing fetal cells.SOLUTION: A method of detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting a sample comprising a plurality of cells with a first antibody; (b) isolating cells bound to the first antibody to produce an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying cells bound to the second antibody as fetal cells, wherein the first antibody or the second antibody is (i) an antibody that binds to a trigger receptor-like 2 (TREML2) protein expressed on myeloid cells or (ii) comprises an antigen-binding fragment that binds to a TREML2 protein. Also provided are methods of preparing a fetal cell sample and methods of performing fetal genetic testing.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 874,306, filed July 15, 2019, the disclosure of which is incorporated by reference in its entirety. [Background technology]

[0002] For the past 40 years, researchers have attempted to isolate fetal cells from pregnant women in order to develop prenatal diagnostic tools. Amniocentesis was first developed in the early 1970s, followed by chorionic villus sampling (CVS) in the 1980s. Amniocentesis and chorionic villus sampling (CVS) are two invasive methods used in routine clinical practice to diagnose chromosomal abnormalities, such as common fetal aneuploidies (extra copies of chromosomes), such as trisomy 13, 18, and 21 (resulting in Down syndrome).

[0003] The ability to isolate fetal cells and fetal DNA from maternal blood during pregnancy has opened up remarkable opportunities for improving noninvasive prenatal testing. Recently, cell-free DNA-based screening (cfDNA), known as noninvasive prenatal testing (NIPT), has been introduced into prenatal screening and has been shown to be highly predictive of trisomy 21. Nevertheless, the performance of the screening is lower than that of invasive diagnostic tools, and confirmatory testing is still required. Furthermore, according to professional associations, NIPT does not predict copy number variations (CNVs) or microdeletions / microduplications (Practice bulletin n163 Obstet Gynecol. 2016; 127(5) 979-981). Therefore, current cell-free NIPT is not yet sufficient to detect subchromosomal deletions and duplications with high specificity, sensitivity, and positive predictive value.

[0004] Direct analysis of fetal cells from the maternal circulation has been difficult due to the scarcity of fetal cells in maternal blood. Numerous different enrichment methods have been tested, including filters, density gradients, fluorescence-activated cell sorting (FACS), microfluidics, and immunomagnetic beads. Although circulating fetal cells can be recovered, these methods lack consistency and reproducibility. The reason for this is the very low number of circulating fetal cells (0.1–10 cells per milliliter of maternal blood, which contains approximately 1–5 million cells), which has thus far prevented the establishment of a reproducible protocol. The challenge is to remove all contaminating nucleated blood cells without losing the very few circulating fetal cells in the first trimester of pregnancy. Summary of the Invention [Problem to be solved by the invention]

[0005] Given these limitations, and the fact that amniocentesis and chorionic villus sampling (CVS) are procedures with an associated risk of miscarriage, there is a need to develop new cell-based NIPD (non-invasive prenatal diagnostic) methods to select fetal cells from the maternal blood of pregnant women to screen for birth defects and genetic disorders.

[0006] Although fetal nucleated red blood cells (nRBCs) and trophoblast cells are known to be present in the maternal circulation, developing a reliable, cytogenetic, cell-based NIPT has been challenging. Recently, the possibility of developing a cell-based NIPT capable of detecting abnormalities with accuracy similar to that currently achieved by amniocentesis and CVS has been proposed (Amy M. Breman et al., Prenatal Diagnosis, 2016, 36(11):1009-1019).

[0007] Disclosed herein are fetal cell markers and agents that bind to the fetal cell markers. Also disclosed herein are compositions, kits, and methods for isolating, detecting, and analyzing fetal cells based on the fetal cell markers. [Means for solving the problem]

[0008] Disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising the steps of: (a) contacting a sample containing a plurality of cells with a first antibody; (b) isolating cells that bind to the first antibody to produce an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying cells that bind to the second antibody as fetal cells, wherein the first antibody or the second antibody is (i) an antibody that binds to Triggering Receptor Expressed on Myeloid Cells Like 2 (TREML2) protein, or (ii) comprises an antigen-binding fragment that binds to the TREML2 protein.

[0009] In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are trophoblast cells.

[0010] In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0011] In some embodiments, step (a) comprises adding a second EAEF comprising the other member of the specific binding pair to induce aggregation of the magnetic particles.

[0012] In some embodiments, step (b) comprises exposing the sample to a magnetic field.

[0013] In some embodiments, step (b) comprises adding a member of a specific binding pair to the enriched sample to reverse aggregation of magnetic particles in the enriched sample.

[0014] In some embodiments, the method further comprises, prior to step (a), adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0015] In some embodiments, the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 2-5.

[0016] In some embodiments, the method further comprises isolating a single fetal cell prior to step (d).

[0017] In some embodiments, the step of isolating a single fetal cell is carried out by isolating a single fetal cell bound to a second antibody.

[0018] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0019] In some embodiments, the step of isolating single fetal cells is based on immunofluorescence techniques. In some embodiments, the step of isolating single fetal cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the step of isolating single fetal cells is performed by DEPArray.

[0020] In some embodiments, step (d) comprises performing sequence analysis. In some embodiments, the sequence analysis comprises short tandem repeat (STR) analysis.

[0021] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0022] In some embodiments, the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 2-5.

[0023] In some embodiments, an antibody that binds to a TREML2 protein or an antigen-binding fragment that binds to a TREML2 protein comprises one or more CDRs selected from (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (iii) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (v) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, an antibody that binds to a TREML2 protein or an antigen-binding fragment that binds to a TREML2 protein comprises two, three, four, five, or six CDRs selected from (i) to (vi).

[0024] In some embodiments, the antibody that binds to the TREML2 protein is an anti-TREML2 antibody, hi some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0025] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising the steps of: (a) contacting a sample containing a plurality of cells with a magnetic reagent comprising magnetic particles conjugated to a first antibody that binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or an antigen-binding fragment thereof; and (c) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0026] In some embodiments, the method further comprises, prior to step (c), isolating cells that bind to the first antibody. In some embodiments, the isolating cells comprises exposing the sample to a magnetic field to enrich the sample for cells that bind to the first antibody.

[0027] In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic colloid particles are less than 200 nm. In some embodiments, the magnetic colloid particles are between about 80-200 nm. In some embodiments, the magnetic colloid particles are between about 90-150 nm. In some embodiments, the magnetic colloid particles have at least 50% magnetic mass. In some embodiments, the magnetic colloid particles have at least 60% magnetic mass. In some embodiments, the magnetic colloid particles have between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particles comprise a superparamagnetic crystalline core surrounded by coating molecules.

[0028] In some embodiments, the magnetic particles are further coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0029] In some embodiments, the method includes adding, during step (a), a second EAEF that comprises the other member of the specific binding pair to enhance particle aggregation.

[0030] In some embodiments, the method further comprises adding a member of the specific binding pair (a third EAEF) to the enriched sample to reverse the aggregation of the magnetic reagent in the sample, thereby facilitating identification of the cells.

[0031] In some embodiments, the method further comprises, prior to step (a), adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0032] In some embodiments, the method further comprises isolating the cells using an anti-TREML2 antibody or a second antibody prior to step (c), in some embodiments, the second antibody is selected from an anti-cytokeratin antibody and an anti-HLAG antibody.

[0033] In some embodiments, the anti-TREML2 antibody or the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0034] In some embodiments, the step of isolating the cells is based on immunofluorescence techniques. In some embodiments, the step of isolating the cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the step of isolating the cells is performed by DEPArray.

[0035] In some embodiments, identifying the cells comprises performing sequence analysis.

[0036] In some embodiments, the sequence analysis comprises short tandem repeat (STR) analysis.

[0037] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0038] In some embodiments, the fetal cells are fetal erythroblasts or fetal trophoblast cells.

[0039] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) selected from (i) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (iii) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (v) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0040] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0041] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample containing a plurality of cells with a first antibody (an anti-TREML2 antibody), wherein the first antibody binds to triggering receptor-like 2 (TREML2) protein expressed on myeloid cells (the anti-TREML2 antibody) or an antigen-binding fragment thereof; and (b) identifying cells that bind to the first antibody as fetal cells.

[0042] In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs).

[0043] In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic colloid particles are less than 200 nm. In some embodiments, the magnetic colloid particles are between about 80-200 nm. In some embodiments, the magnetic colloid particles are between about 90-150 nm. In some embodiments, the magnetic colloid particles have at least 50% magnetic mass. In some embodiments, the magnetic colloid particles have at least 60% magnetic mass. In some embodiments, the magnetic colloid particles have between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particles comprise a superparamagnetic crystalline core surrounded by coating molecules.

[0044] In some embodiments, the method further comprises exposing the sample to a magnetic field.

[0045] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0046] In some embodiments, the method further comprises, prior to step (b), adding a second EAEF comprising the other member of the specific binding pair to enhance aggregation of the magnetic particles.

[0047] In some embodiments, the method further comprises isolating cells that bound to the first antibody to generate an enriched sample.

[0048] In some embodiments, the method further comprises adding to the enriched sample a third EAEF capable of binding to the first EAEF or the second EAEF to reverse the aggregation of magnetic particles in the enriched sample, in some embodiments, the third EAEF is a member of the specific binding pair.

[0049] In some embodiments, the method further comprises, prior to step (a), adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0050] In some embodiments, the first antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0051] In some embodiments, the method further comprises, prior to step (b), isolating cells bound to the first antibody based on immunofluorescence techniques. In some embodiments, the step of isolating cells bound to the first antibody is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the step of isolating cells bound to the first antibody is performed by DEPArray.

[0052] In some embodiments, step (b) comprises performing sequence analysis. In some embodiments, the sequence analysis comprises short tandem repeat (STR) analysis. In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0053] In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of, one or more CDRs selected from (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two, three, four, five, or six CDRs selected from (a)-(f).

[0054] In some embodiments, the first antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0055] Further disclosed herein is a cell-based fetal genetic testing method comprising the steps of: (a) contacting a sample comprising a plurality of cells obtained from a pregnant subject with an anti-TREML2 antibody or antigen-binding fragment thereof; (b) isolating the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a probability of the fetus having one or more genetic abnormalities.

[0056] In some embodiments, the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof are fetal cells.

[0057] In some embodiments, the fetal cells are fetal erythroblasts. In some embodiments, the fetal cells are fetal trophoblast cells.

[0058] In some embodiments, analyzing one or more nucleic acid molecules comprises performing a karyotype analysis.

[0059] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing sequence analysis. In some embodiments, sequence analysis comprises short tandem repeat (STR) analysis.

[0060] In some embodiments, the one or more genetic abnormalities are selected from trisomy, sex chromosome abnormality, and structural abnormality. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21, and trisomy 22. In some embodiments, the sex chromosome abnormality is selected from monosomy X, triple X syndrome, and Klinefelter syndrome. In some embodiments, the structural abnormality is a copy number variation (CNV). In some embodiments, the structural abnormality is a CNV deletion or a CNV duplication.

[0061] In some embodiments, the anti-TREML2 antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a magnetic colloid particle. In some embodiments, the magnetic colloid particle is a ferrofluid magnetic particle.

[0062] In some embodiments, step (b) comprises exposing the sample to a magnetic field.

[0063] In some embodiments, the method further comprises, prior to step (a), contacting the sample with a first antibody that binds to a protein selected from EpCAM, CD105, and CD71.

[0064] In some embodiments, the method further comprises, prior to step (a), isolating cells that bind to the first antibody.

[0065] In some embodiments, the first antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a magnetic colloid particle. In some embodiments, the magnetic colloid particle is a ferrofluid magnetic particle. In some embodiments, the magnetic colloid particle is less than 200 nm. In some embodiments, the magnetic colloid particle is between about 80-200 nm. In some embodiments, the magnetic colloid particle is between about 90-150 nm. In some embodiments, the magnetic colloid particle has at least 50% magnetic mass. In some embodiments, the magnetic colloid particle has at least 60% magnetic mass. In some embodiments, the magnetic colloid particle has between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

[0066] In some embodiments, isolating cells that bind to the first antibody comprises exposing the sample to a magnetic field. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0067] In some embodiments, isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is based on immunofluorescence techniques. In some embodiments, isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed by DEPArray.

[0068] In some embodiments, the method further comprises contacting the cells bound to the anti-TREML2 antibody, or antigen-binding fragment thereof, with a second antibody, or antigen-binding fragment thereof.

[0069] In some embodiments, the second antibody is an anti-TREML2 antibody or an antigen-binding fragment thereof. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0070] In some embodiments, the method further comprises isolating cells bound to the second antibody or antigen-binding fragment thereof. In some embodiments, the isolating cells bound to the second antibody or antigen-binding fragment thereof is based on immunofluorescence techniques. In some embodiments, the isolating cells bound to the second antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolating cells bound to the second antibody or antigen-binding fragment thereof is performed by DEPArray.

[0071] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0072] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of, one or more CDRs selected from (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two, three, four, five, or six CDRs selected from (i) to (vi).

[0073] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting a maternal sample comprising fetal and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody and (ii) a magnetic colloid particle, wherein the first antibody is conjugated to the magnetic colloid particle; and (b) exposing the maternal sample to a magnetic field to isolate cells that bind to the first antibody conjugate, thereby preparing a fetal cell sample.

[0074] In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic colloid particles are less than 200 nm. In some embodiments, the magnetic colloid particles are between about 80-200 nm. In some embodiments, the magnetic colloid particles are between about 90-150 nm. In some embodiments, the magnetic colloid particles have at least 50% magnetic mass. In some embodiments, the magnetic colloid particles have at least 60% magnetic mass. In some embodiments, the magnetic colloid particles have between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particles comprise a superparamagnetic crystalline core surrounded by coating molecules.

[0075] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0076] In some embodiments, the method further comprises adding a second EAEF, which comprises the other member of the specific binding pair, to the maternal sample.

[0077] In some embodiments, the first antibody is an anti-TREML2 antibody.

[0078] In some embodiments, the first antibody is an anti-CD71 antibody.

[0079] In some embodiments, the first antibody binds to a protein selected from EpCAM and CD105.

[0080] In some embodiments, the step of preparing the fetal cell sample further comprises contacting the cells isolated from the maternal sample with a second antibody.

[0081] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0082] In some embodiments, the step of preparing the fetal cell sample further comprises isolating cells that bind to the second antibody.

[0083] In some embodiments, isolating cells bound to the second antibody is based on immunofluorescence techniques. In some embodiments, isolating cells bound to the second antibody is performed by fluorescence activated cell sorting (FACS). In some embodiments, isolating cells bound to the second antibody is performed by DEPArray.

[0084] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample containing a plurality of cells with a first antibody conjugate comprising a first antibody conjugated to magnetic colloid particles; (b) isolating cells that bind to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody that binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells.

[0085] In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are fetal trophoblast cells.

[0086] In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic colloid particles are less than 200 nm. In some embodiments, the magnetic colloid particles are between about 80-200 nm. In some embodiments, the magnetic colloid particles are between about 90-150 nm. In some embodiments, the magnetic colloid particles have at least 50% magnetic mass. In some embodiments, the magnetic colloid particles have at least 60% magnetic mass. In some embodiments, the magnetic colloid particles have between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particles comprise a superparamagnetic crystalline core surrounded by coating molecules.

[0087] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0088] In some embodiments, step (a) comprises adding a second EAEF comprising the other member of the specific binding pair to enhance aggregation of the magnetic particles.

[0089] In some embodiments, step (b) comprises adding a member of a specific binding pair to the enriched sample to reverse aggregation of magnetic particles in the enriched sample.

[0090] In some embodiments, the method further comprises, prior to step (a), adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0091] In some embodiments, the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0092] In some embodiments, the method further comprises isolating a single fetal cell prior to step (d). In some embodiments, the step of isolating a single fetal cell is carried out by isolating a single fetal cell bound to a second antibody.

[0093] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0094] In some embodiments, the step of isolating single fetal cells is based on immunofluorescence techniques. In some embodiments, the step of isolating single fetal cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the step of isolating single cells is performed by DEPArray.

[0095] In some embodiments, step (d) comprises performing sequence analysis. In some embodiments, the sequence analysis comprises short tandem repeat (STR) analysis.

[0096] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0097] In some embodiments, the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0098] In some embodiments, an antibody that binds to a TREML2 protein or an antigen-binding fragment that binds to a TREML2 protein comprises, or consists essentially of, one or more CDRs selected from (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR)1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (iii) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (v) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, an antibody that binds to a TREML2 protein or an antigen-binding fragment that binds to a TREML2 protein comprises two, three, four, five, or six of the CDRs selected from (i) to (vi). In some embodiments, any of SEQ ID NOs: 6 to 11 independently comprise one or more amino acid substitutions, additions, or deletions.

[0099] In some embodiments, the antibody that binds to a TREML2 protein is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0100] Further disclosed herein are anti-TREML2 antibodies. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, or consists essentially of, one or more CDRs selected from (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a) to (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a) to (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a) to (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more CDRs selected from (a) to (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs of (a) to (f).

[0101] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0102] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a magnetic colloid particle. In some embodiments, the magnetic colloid particle is a ferrofluid magnetic particle. In some embodiments, the magnetic colloid particle is less than 200 nm. In some embodiments, the magnetic colloid particle is between about 80-200 nm. In some embodiments, the magnetic colloid particle is between about 90-150 nm. In some embodiments, the magnetic colloid particle has at least 50% magnetic mass. In some embodiments, the magnetic colloid particle has at least 60% magnetic mass. In some embodiments, the magnetic colloid particle has between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

[0103] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0104] Disclosed herein is an anti-TREML2 antibody conjugate comprising: (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic particle conjugated to the anti-TREML2 antibody.

[0105] In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic colloid particles are less than 200 nm. In some embodiments, the magnetic colloid particles are between about 80-200 nm. In some embodiments, the magnetic colloid particles are between about 90-150 nm. In some embodiments, the magnetic colloid particles have at least 50% magnetic mass. In some embodiments, the magnetic colloid particles have at least 60% magnetic mass. In some embodiments, the magnetic colloid particles have between 70%-90% magnetic mass. In some embodiments, the magnetic colloid particles comprise a superparamagnetic crystalline core surrounded by coating molecules.

[0106] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0107] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of, one or more CDRs selected from (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of three or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of four or more CDRs selected from (a) through (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of five or more CDRs selected from (a) through (f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of all of the CDRs of (a) through (f).

[0108] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0109] Also disclosed herein are kits for isolating, detecting, and / or analyzing fetal cells. In some embodiments, the kits comprise, consist of, or consist essentially of (a) an antibody (anti-TREML2 antibody) or antigen-binding fragment thereof that binds to triggering receptor-like 2 (TREML2) protein expressed on myeloid cells, and (b) a magnetic reagent comprising magnetic colloid particles.

[0110] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0111] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists essentially of, one or more complementarity-determining regions (CDRs) selected from (a) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0112] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label to generate a conjugated antibody, hi some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0113] In some embodiments, the magnetic colloid particles have a dimension of less than 200 nm. In some embodiments, the magnetic colloid particles are ferrofluid particles.

[0114] In some embodiments, the magnetic colloid particles are conjugated to antibodies or antigen-binding fragments thereof.

[0115] In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of, one or more CDRs selected from (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two, three, four, five, or six CDRs selected from (i) to (vi).

[0116] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0117] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitor selected from the group consisting of a reducing agent, an immunoconjugate, a chelating agent, and diaminobutane. In some embodiments, the kit further comprises, consists of, or consists essentially of a chelating agent. In some embodiments, the chelating agent is EDTA.

[0118] In some embodiments, the kit further comprises, consists of, or consists essentially of an exogenous aggregation enhancing factor (EAEF), in some embodiments, the EAEF comprises, consists of, or consists essentially of one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0119] Further disclosed herein is a kit comprising (a) a first antibody bound to magnetic colloid particles capable of binding to a protein expressed on the surface of fetal cells, and (b) an anti-TREML2 antibody or an antigen-binding fragment thereof.

[0120] In some embodiments, the first antibody binds to a protein selected from EpCAM, CD105, and CD71.

[0121] In some embodiments, the magnetic colloid particles are ferrofluid particles.

[0122] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of, one or more CDRs selected from (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7, (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8, (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0123] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[0124] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitor selected from the group consisting of a reducing agent, an immunoconjugate, a chelating agent, and diaminobutane. In some embodiments, the chelating agent is EDTA.

[0125] In some embodiments, the kit further comprises, consists of, or consists essentially of one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin; or comprises, consists of, or consists essentially of an exogenous aggregation enhancing factor (EAEF). [Brief explanation of the drawings]

[0126] [Figure 1] FIG. 1 shows an exemplary method for isolating, detecting, and analyzing rare cells. [Figure 2] FIG. 2 shows an exemplary magnetic fluid structure. [Figure 3] FIG. 3 shows an exemplary method for detecting rare cells. [Figure 4] FIG. 4 shows an exemplary method for isolating and detecting rare cells. [Figure 5] Figures 5A-E show the gating of cells using a FACS instrument. [Figure 6] FIG. 6 shows an exemplary method for isolating, detecting, and analyzing rare cells. [Figure 7] FIG. 7 shows a schematic diagram of ferrofluid aggregation by controlled aggregation. [Figure 8] Figure 8 is a schematic of the fetal cell enrichment workflow. Fetal cells are enriched and stained from whole maternal blood. Pure single cells are isolated using DEPArray™ for whole genome amplification and analysis. [Figure 9]Figure 9 shows the gating strategy for erythroblasts isolated from fetal blood samples: (1) FSC-A / SSC-A to gate the major cell population, (2) gate Sytox Green-negative live cells, (3) FSC-H / W to exclude doublet cells, (4) gate double-positive GPA / Hoechst, (5) gate CD71-positive / CD45-negative, and (6) gate TLS1 / TREML2 and overlay with isotype control to determine the % of TREML2-positive cells. [Figure 10] Figure 10 shows the gating strategy for erythroblasts isolated from bone marrow samples: (1) FSC-A / SSC-A to gate the major cell population, (2) gate Sytox Green-negative live cells, (3) FSC-H / W to exclude doublet cells, (4) gate double-positive GPA / Hoechst, (5) gate CD71-positive / CD45-negative, and (6) gate TLS1 / TREML2 and overlay with isotype control to determine the % of TREML2-positive cells. [Figure 11] 11A-11J show TLS1 / TREML2 expression on fetal erythroblasts isolated from different fetal blood (FB) samples from different clones. [Figure 12] 12A-12L show TLS1 / TREML2 expression on adult erythroblasts isolated from different bone marrow (BM) samples from different clones. [Figure 13] FIG. 13 shows a scatter plot analysis of TLS1 / TREML-2 positive trophoblast cells identified by DEPArray™ after spiking and enrichment with CD105-FF and EpCAM-FF. [Figure 14] Figure 14 shows the CellBrowser® image gallery. Trophoblast cells show positive staining for the TREML-2-PE antibody, CK-APC and nuclear stain. [Figure 15]Figure 15A shows a scatter plot analysis of Draq5 / Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with CD71-FF. Figure 15B shows a CellBrowser® image gallery. Erythroblast cells show positive staining for CD71-PE antibody, Draq5, and Hoechst nuclear stain, and negative staining for CD45-FITC antibody. [Figure 16] Figure 16A shows a scatter plot analysis of Draq5 / Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with TLS1 / TREML-2-FF. Figure 16B shows a CellBrowser® image gallery. Erythroblast cells show positive staining for CD71-PE antibody, Draq5, and Hoechst nuclear stain, and negative staining for CD45-FITC antibody. [Figure 17] FIG. 17 shows STR analysis from a single fetal cell isolated from maternal blood. [Figure 18] FIG. 18 shows the results of CNV analysis of single fetal cells. [Figure 19] FIG. 19 shows the results of single-cell CNV analysis of healthy donors. [Figure 20] FIG. 20 shows an exemplary method for isolating and detecting rare cells. DETAILED DESCRIPTION OF THE INVENTION

[0127] Disclosed herein are compositions, kits, and methods for isolating, detecting, and / or analyzing rare cells in a sample. Generally, the compositions, kits, and methods disclosed herein include an agent that binds to the triggering receptor-like 2 (TREML2) protein expressed on myeloid cells (this protein is also referred to as TLS1 throughout this application). Alternatively or additionally, the compositions, kits, and methods disclosed herein include an antibody conjugate. The antibody conjugate includes an antibody conjugated to a magnetic colloid particle. The rare cell can be a fetal cell. The sample can be from a pregnant subject.

[0128] Methods for isolating, detecting and / or characterizing rare cells Disclosed herein are methods for isolating, detecting, and / or characterizing rare cells. In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are trophoblast cells. Generally, the methods include using an anti-TREML2 antibody or an antigen-binding fragment thereof to identify cells as fetal cells. Alternatively or additionally, the methods include using an antibody conjugated to a magnetic colloid particle to isolate the fetal cells.

[0129] Disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising the steps of (a) contacting a sample containing a plurality of cells with an anti-TREML2 antibody or an antigen-binding fragment thereof, and (b) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0130] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising the steps of: (a) contacting a sample containing a plurality of cells with a first antibody or antigen-binding fragment thereof; (b) isolating cells that bind to the first antibody or antigen-binding fragment thereof to produce an enriched sample; (c) contacting the enriched sample with a second antibody or antigen-binding fragment thereof; and (d) identifying cells that bind to the second antibody as fetal cells, wherein the first antibody or the second antibody is an antibody that binds to triggering receptor-like 2 (TREML2) protein expressed on myeloid cells.

[0131] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting a sample containing a plurality of cells with a magnetic reagent comprising magnetic particles conjugated to a first antibody or antigen-binding fragment thereof that binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or antigen-binding fragment thereof; and (c) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0132] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample containing a plurality of cells with a magnetic reagent comprising magnetic colloid particles conjugated to a first antibody or antigen-binding fragment thereof that binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying cells that bind to the second antibody as fetal cells.

[0133] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting a sample comprising a plurality of cells with a magnetic reagent and a second exogenous aggregation enhancing factor (EAEF), wherein the magnetic reagent comprises magnetic colloid particles conjugated to a first antibody or antigen-binding fragment thereof, the magnetic colloid particles being conjugated to the first EAEF, and the first antibody or antigen-binding fragment thereof binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying cells bound to the second antibody as fetal cells. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, the specific binding pair being selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0134] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample containing a plurality of cells with a first antibody conjugate comprising a first antibody or antigen-binding fragment thereof conjugated to magnetic colloid particles; (b) isolating cells that bind to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody or antigen-binding fragment thereof that binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells.

[0135] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting a maternal sample comprising fetal and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof, and (ii) a magnetic colloid particle, wherein the first antibody is conjugated to the magnetic colloid particle; and (b) exposing the maternal sample to a magnetic field to isolate cells that bind to the first antibody conjugate, thereby preparing a fetal cell sample.

[0136] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample containing fetal and maternal cells with a first antibody conjugate and a second exogenous aggregation enhancing factor (EAEF), wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof, (ii) magnetic colloid particles, and (iii) a first EAEF, wherein the first antibody is conjugated to the magnetic colloid particles, and the first EAEF is conjugated to the magnetic colloid particles; and (b) exposing the maternal sample to a magnetic field to isolate cells bound to the first antibody conjugate, thereby preparing the fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, the specific binding pair being selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0137] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting a maternal sample containing fetal and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof and (ii) a magnetic colloid particle, wherein the first antibody is conjugated to the magnetic colloid particle, and the first antibody is an anti-TREML2 antibody; and (b) exposing the maternal sample to a magnetic field to isolate cells bound to the first antibody conjugate, thereby preparing a fetal cell sample.

[0138] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample containing fetal and maternal cells with a first antibody conjugate and a second exogenous aggregation enhancing factor (EAEF), wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof and (ii) a magnetic colloid particle, wherein the first antibody is conjugated to the magnetic colloid particle, and the magnetic colloid particle is conjugated to the first EAEF; and (b) exposing the maternal sample to a magnetic field to isolate cells bound to the first antibody conjugate, thereby preparing a fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, the specific binding pair being selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0139] In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are erythroblasts. In some embodiments, the fetal cells are trophoblast cells.

[0140] In some embodiments, any of the methods disclosed herein further comprise the step of isolating cells that bind to the anti-TREML2 antibody or to the first antibody prior to identifying the cells.

[0141] In some embodiments, any of the methods disclosed herein includes the use of a first antibody. In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic particles are ferrofluid magnetic particles.

[0142] In some embodiments, any of the methods disclosed herein comprises isolating cells that bind to the first antibody or antigen-binding fragment thereof, hi some embodiments, isolating the cells comprises exposing the sample to a magnetic field.

[0143] In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) that comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0144] In some embodiments, any of the methods disclosed herein includes adding a second EAEF that contains the other member of the specific binding pair to induce aggregation of the magnetic particles.

[0145] In some embodiments, the step of isolating cells bound to the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of adding a member of the specific binding pair to the enriched sample to reverse aggregation of magnetic particles in the enriched sample.

[0146] In some embodiments, any method disclosed herein includes adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA).

[0147] In some embodiments, any of the methods disclosed herein uses a second antibody, which in some embodiments is, consists of, or consists essentially of an antibody that binds to the TREML2 protein, or an antigen-binding fragment that binds to the TREML2 protein.

[0148] In some embodiments, any of the methods disclosed herein includes isolating a single fetal cell. In some embodiments, isolating a single fetal cell is carried out by isolating a single fetal cell bound to a second antibody.

[0149] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the step of isolating single fetal cells is based on immunofluorescence techniques. In some embodiments, the step of isolating single fetal cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the step of isolating single fetal cells is performed by DEPArray.

[0150] In some embodiments, any of the methods disclosed herein comprises performing sequence analysis on one or more nucleic acid molecules isolated from the fetal cells. In some embodiments, the sequence analysis comprises short tandem repeat (STR) analysis.

[0151] In some embodiments, any of the methods disclosed herein includes analyzing the fetal cells. In some embodiments, analyzing the fetal cells includes performing genomic or genetic analysis. In some embodiments, performing genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0152] In some embodiments, the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0153] In some embodiments, an antibody that binds to TREML2 protein or an antigen-binding fragment that binds to TREML2 protein comprises one, two, three, four, five, or six CDRs selected from (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6, (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7, (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8, (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9, (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10, and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6-11 independently comprises one or more substitutions, additions, or deletions.

[0154] In some embodiments, the anti-TREML2 antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, isolating the cells comprises placing the sample in a magnetic separator. In some embodiments, isolating the cells comprises exposing the sample to a magnetic field.

[0155] In some embodiments, the anti-TREML2 antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the step of isolating the cells comprises flow cytometry. In some embodiments, the flow cytometry is fluorescence-activated cell sorting (FACS).

[0156] In some embodiments, isolating the cells comprises performing a DEPArray.

[0157] In some embodiments, identifying the cells comprises performing a sequencing reaction.

[0158] In some embodiments, the sample is enriched for fetal cells prior to contacting with the anti-TREML2 antibody, hi some embodiments, the sample is enriched for fetal cells by contacting the sample with a ferrofluid reagent, the ferrofluid comprising an antibody coupled to the ferrofluid.

[0159] In some embodiments, the antibody binds to a protein selected from EpCAM, CD105, and CD71.

[0160] In some embodiments, the methods disclosed herein further comprise isolating cells bound by the ferrofluid-coupled antibody, thereby producing a sample enriched for fetal cells.

[0161] In some embodiments, any of the methods disclosed herein further comprises performing sequencing analysis. In some embodiments, the sequencing analysis comprises short tandem repeat (STR) analysis.

[0162] In some embodiments, any method disclosed herein further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises detecting the presence or absence of one or more fetal abnormalities. In some embodiments, analyzing the fetal cells comprises performing genomic analysis. In some embodiments, analyzing the fetal cells comprises performing genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells. In some embodiments, performing genetic analysis comprises detecting the presence or absence of a chromosomal abnormality in the fetal cells. In some embodiments, the chromosomal abnormality is trisomy 21, trisomy 18, or trisomy 13.

[0163] In some embodiments, any method disclosed herein further comprises performing a genetic test on the fetal cells. In some embodiments, performing a genetic test on the fetal cells comprises detecting the presence or absence of one or more fetal abnormalities. In some embodiments, performing a genetic test on the fetal cells comprises performing a genomic analysis. In some embodiments, performing a genetic test on the fetal cells comprises performing a genetic analysis. In some embodiments, performing a genetic analysis comprises detecting the presence or absence of a chromosomal abnormality in the fetal cells. In some embodiments, the chromosomal abnormality is trisomy 21, trisomy 18, or trisomy 13.

[0164] In some embodiments, any of the methods disclosed herein further comprise providing a treatment recommendation based on the results of the analysis of the fetal cells. In some embodiments, any of the methods disclosed herein further comprise providing a treatment recommendation based on the results of the genetic testing on the fetal cells.

[0165] In some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the analysis of the fetal cells, hi some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the genetic testing on the fetal cells.

[0166] In some embodiments, any of the methods disclosed herein further comprises the step of recommending additional monitoring of the subject or fetus based on the results of the analysis of the fetal cells, hi some embodiments, any of the methods disclosed herein further comprises the step of recommending additional monitoring of the subject or fetus based on the results of the genetic testing on the fetal cells.

[0167]

[0013] Figure 1 illustrates an exemplary method for isolating, detecting, and / or analyzing rare cells. The methods disclosed herein may comprise, consist of, or consist essentially of one or more steps depicted in Figure 1. In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101), and (b) isolating rare cells (110). In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101), (b) isolating rare cells (110), and (c) analyzing the rare cells (120). In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101), (b) isolating rare cells (110), (c) analyzing the rare cells (120), and (d) generating one or more reports based on the analysis of the rare cells (106).

[0168] As shown in FIG. 1 , in some embodiments, a method comprises, or consists essentially of, (a) obtaining a sample from a subject (101) comprising a plurality of cells; (b) isolating rare cells (110) by (i) depleting non-rare cells from the sample to generate an enriched rare cell sample (102) and (ii) isolating rare cells from the enriched rare cell sample (103); (c) analyzing the rare cells (120) by (i) purifying nucleic acid molecules from the rare cells (104) and (ii) sequencing one or more nucleic acid molecules (105); and (f) generating one or more reports (106). In some embodiments, the rare cells are fetal cells. In some embodiments, enriching the rare cells (102) comprises, or consists essentially of, contacting the sample with a ferrofluid comprising an antibody coupled to magnetic particles that binds to a marker on the rare cells. In some embodiments, the marker on the rare cells is any of the markers disclosed herein. In some embodiments, the marker on the rare cells is TREML2 protein. In some embodiments, the antibody is any of the antibodies disclosed herein. In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the antibody is any of the anti-TREML2 antibodies disclosed herein. In some embodiments, the ferrofluid comprises, consists of, or consists essentially of the ferrofluid structure shown in FIG. 2. In some embodiments, enriching for rare cells (102) further comprises, consists of, or consists essentially of applying an external gradient magnetic separator to the sample to remove cells not bound to the ferrofluid. In some embodiments, the method further comprises, consists of, or consists essentially of contacting the rare cells with one or more additional antibodies conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, prior to isolating the rare cells (103), the rare cells are contacted with one or more additional antibodies. In some embodiments, isolating the rare cells (103) comprises, consists essentially of, or consists of selecting single cells bound by an antibody that binds to a marker on the rare cells. In some embodiments, the antibody is an anti-TREML2 antibody.In some embodiments, the anti-TREML2 antibody is any of the anti-TREML2 antibodies disclosed herein. In some embodiments, the anti-TREML2 antibody comprises (a) a heavy chain variable region (HCVR) complementarity determining region (CDR)1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6, (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7, (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8, (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9, (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10, and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the step (103) of isolating rare cells comprises, consists of, or consists essentially of sorting rare cells from the enriched cell sample. In some embodiments, the step of isolating rare cells (103) comprises, consists of, or consists essentially of performing fluorescence activated cell sorting (FACS). In some embodiments, the step of isolating rare cells (103) comprises, consists of, or consists essentially of performing DEPArray. In some embodiments, the step of purifying nucleic acid molecules from rare cells (104) comprises, consists of, or consists essentially of performing nucleic acid amplification. In some embodiments, the step of purifying nucleic acid molecules from rare cells (104) comprises, consists of, or consists essentially of generating a nucleic acid library.

[0169] 3 illustrates an exemplary method for detecting rare cells (e.g., fetal cells). In some embodiments, the method comprises, consists of, or consists essentially of (a) contacting a sample (301) containing a plurality of cells (302, 303) with a first antibody or antigen-binding fragment thereof (304), and (b) identifying the cells (303) to which the first antibody or antigen-binding fragment thereof (304) binds as fetal cells. In some embodiments, the first antibody (304) is an antibody that binds to the TREML2 protein. In some embodiments, the antigen-binding fragment (304) binds to the TREML2 protein. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of any one of the anti-TREML2 antibodies disclosed herein. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or essentially consists of: (a) a heavy chain variable region (HCVR) complementarity-determining region (CDR)1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:11. The first antibody or antigen-binding fragment thereof may be coupled to magnetic particles. For example, the first antibody or antigen-binding fragment thereof may be in the form of a ferrofluid. Alternatively, the first antibody or antigen-binding fragment may be conjugated to a label. The label can be any of the labels disclosed herein. For example, the first antibody or antigen-binding fragment can be conjugated to a fluorescent label. The cells can be identified by any of the identification techniques disclosed herein.In some embodiments, identifying cells bound to the first antibody or antigen-binding fragment thereof comprises isolating cells bound to the first antibody or antigen-binding fragment thereof. Isolating cells may include any cell isolation technique disclosed herein. In some embodiments, isolating cells includes magnetic separation. In some embodiments, identifying cells may include the use of a microscope. Identifying cells may include fluorescence microscopy. In some embodiments, identifying cells includes or is based on FACS. Alternatively or additionally, identifying cells includes or is based on a DEPArray.

[0170] 4 illustrates an exemplary method for isolating and detecting rare cells. In some embodiments, the method for detecting rare cells comprises, or consists essentially of, (a) contacting a sample (401) comprising a plurality of cells (402, 403) with an antibody conjugate (406) comprising a first antibody or antigen-binding fragment (404) coupled to magnetic particles (405), (b) enriching the rare cells (403) by exposing the sample to a magnetic field (407) and removing cells (402) that do not bind to the antibody conjugate, thereby producing an enriched rare cell sample (411), (c) contacting the enriched rare cell sample (411) with an antibody conjugate (410) comprising a second antibody or antigen-binding fragment (408) conjugated to a label (409), and (d) identifying cells that bind to the antibody conjugate as rare cells (403). In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the enriched rare cell sample (411) comprises rare cells (403) bound to an antibody conjugate (406) comprising a first antibody (404) or an antigen-binding fragment thereof (404) conjugated to a magnetic particle (405). Alternatively or additionally, the enriched rare cell sample (411) is further processed to separate the rare cells (403) from the antibody conjugate (406). In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, both the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment bind to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment, or (b) the second antibody or antigen-binding fragment, binds to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71, and (b) a second antibody or antigen-binding fragment binds to a TREML2 protein.In some embodiments, (a) a first antibody or antigen-binding fragment binds to EpCAM, and (b) a second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to CD105, and (b) a second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to CD71, and (b) a second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to TREML2 protein is any anti-TREML2 antibody or antigen-binding fragment disclosed herein. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity-determining region (CDR)1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:11. In some embodiments, the second antibody is an antibody that binds to the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any one of the labels disclosed herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the magnetic particles are further conjugated to a first exogenous aggregation enhancing factor (EAEF).In some embodiments, the method further comprises contacting the sample (401) with a second EAEF capable of binding to the first EAEF during step (A). In some embodiments, the addition of the second EAEF induces aggregation of the antibody conjugate (406). In some embodiments, the method further comprises adding a third EAEF capable of binding to the first or second exogenous aggregation enhancing factor. In some embodiments, the addition of the third EAEF reverses the aggregation of the first EAEF. In some embodiments, the method further comprises adding an aggregation inhibitor to the sample prior to step (a). Cells may be identified by any identification technique disclosed herein. In some embodiments, the step of identifying cells may include the use of a microscope. The step of identifying cells may include fluorescence microscopy. In some embodiments, the step of identifying cells includes or is based on FACS. Alternatively or additionally, the step of identifying cells includes or is based on DEPArray.

[0171] FIG. 20 shows another exemplary method for isolating and detecting rare cells. As shown in Figure 20, in some embodiments, a method for detecting rare cells comprises, or essentially comprises, step (A1): contacting a sample (2001) comprising a plurality of cells (2002, 2003) with a first antibody conjugate (2006) and a second exogenous aggregation enhancing factor (EAEF) (2011), wherein the first antibody conjugate (2006) comprises a first antibody or antigen-binding fragment (2004) coupled to a magnetic particle (2005), and the magnetic particle (2005) is further conjugated to a first EAEF (2012); and step (B1): exposing the sample to a magnetic field (2007) to remove cells (2002) that are not bound to the antibody conjugate (2006)-second EAEF (2011) complex, thereby enriching the rare cells (2003), thereby producing an enriched rare cell sample (2011). As shown in step (A2), the addition of the second EAEF (2011) induces aggregation of the first antibody conjugate (2006). In some embodiments, the method further comprises step (B2) adding a third EAEF (2013) to the enriched rare cell sample (2011). As shown in step (B3), the addition of the third EAEF (2013) reverses the aggregation of the first antibody conjugate (2006). In some embodiments, the method further comprises step (C) contacting the enriched rare cell sample (2011) with a second antibody conjugate (2010) comprising a second antibody or antigen-binding fragment (2008) conjugated to a label (2009). In some embodiments, the method further comprises step (D) identifying cells bound to the first antibody conjugate (2006) as rare cells (2003). In some embodiments, the method further comprises step (D) identifying cells that bind to the second antibody conjugate (2010) as rare cells (2003). In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs).In some embodiments, the enriched rare cell sample (2011) comprises rare cells (2003) bound to a first antibody conjugate (2006) comprising a first antibody (2004) or an antigen-binding fragment thereof (2004) conjugated to a magnetic particle (2005), the magnetic particle (2005) being further conjugated to a first EAEF (2012). Alternatively or additionally, the enriched rare cell sample (2011) is further treated to separate the rare cells (2003) from the antibody conjugate (2006). In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, both the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment bind to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment, or (b) a second antibody or antigen-binding fragment, binds to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71, and (b) a second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to EpCAM, and (b) a second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to CD105, and (b) a second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to CD71, and (b) a second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to the TREML2 protein is any anti-TREML2 antibody or antigen-binding fragment disclosed herein.In some embodiments, the TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity-determining region (CDR)1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:11. In some embodiments, the second antibody is an antibody that binds to the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any one of the labels disclosed herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, the magnetic colloid particles are ferrofluid magnetic particles. In some embodiments, the method further comprises adding an aggregation inhibitor to the sample prior to step (A1). In some embodiments, the first EAEF (2012) is desthiobiotin. In some embodiments, the second EAEF (2011) is streptavidin. In some embodiments, the third EAEF (2013) is biotin. Cells may be identified by any identification technique disclosed herein. In some embodiments, identifying cells may include the use of a microscope. Identifying cells may include fluorescent microscopy. In some embodiments, identifying cells includes or is based on FACS. Alternatively or additionally, identifying cells includes or is based on a DEPArray. In some embodiments, identifying the cells comprises or is based on an immune-based assay.

[0172] Although the methods disclosed herein may recite the use of an anti-TREML2 antibody or an antigen-binding fragment thereof, or an antibody conjugate comprising an anti-TREML2 antibody, any of these methods may be carried out using any agent capable of binding to the TREML2 protein, or a conjugate comprising an agent capable of binding to the TREML2 protein. Thus, the methods disclosed herein are not limited to the use of an anti-TREML2 antibody or an antigen-binding fragment thereof, or an antibody conjugate comprising an anti-TREML2 antibody.

[0173] Cell-based fetal genetic testing methods The identification of novel fetal cell markers, such as TREML2, allows for the isolation and / or detection of fetal cells and subsequent analysis of such cells. Accordingly, disclosed herein are cell-based fetal genetic testing methods. In some embodiments, the method comprises (a) using an anti-TREML2 antibody to isolate fetal cells from a sample from a pregnant subject, and (b) analyzing one or more nucleic acid molecules from the fetal cells to determine the likelihood that the fetus has one or more genetic abnormalities. Alternatively, the method comprises isolating fetal cells using any of the methods for isolating or detecting fetal cells disclosed herein, and analyzing one or more nucleic acid molecules from the isolated or detected fetal cells to determine the likelihood that the fetus has one or more genetic abnormalities. In some embodiments, the method comprises analyzing fetal cells isolated and / or detected by any of the methods disclosed herein. In some embodiments, the method comprises analyzing fetal cells prepared by any of the methods disclosed herein.

[0174] Disclosed herein is a cell-based fetal genetic testing method comprising the steps of: (a) contacting a sample containing a plurality of cells obtained from a pregnant subject with an anti-TREML2 antibody or antigen-binding fragment thereof; (b) isolating cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood of the fetus having one or more genetic abnormalities.

[0175] Disclosed herein is a cell-based fetal genetic testing method comprising the steps of: (a) contacting a sample containing a plurality of cells obtained from a pregnant subject with a first antibody or antigen-binding fragment thereof conjugated to magnetic colloid particles and binding to a marker on fetal cells; (b) isolating the cells that bind to the first antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the first antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood of the fetus having one or more genetic abnormalities.

[0176] Disclosed herein is a cell-based fetal genetic testing method comprising the steps of: (a) contacting a sample comprising a plurality of cells obtained from a pregnant subject with a first antibody or antigen-binding fragment thereof and a second exogenous aggregation enhancing factor (EAEF), wherein the first antibody or antigen-binding fragment is conjugated to magnetic colloid particles that are conjugated to the first EAEF and binds to a marker on fetal cells; (b) isolating the cells that bind to the first antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the first antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood of the fetus having one or more genetic abnormalities. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, the specific binding pair being selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0177] In some embodiments, the first antibody is an anti-TREML2 antibody. In some embodiments, the first antibody is an anti-CD71 antibody. In some embodiments, the first antibody is an anti-EpCAM antibody. In some embodiments, the first antibody is an anti-CD105 antibody. In some embodiments, when the first antibody is an anti-EpCAM antibody or an anti-CD105 antibody, the method further comprises contacting the isolated cells with a second antibody or antigen-binding fragment thereof that binds to a marker on fetal cells. In some embodiments, the second antibody is an anti-TREML2 antibody. In some embodiments, the second antibody is an anti-CD71 antibody. In some embodiments, the second antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the method further comprises isolating cells bound to the second antibody. In some embodiments, the method further comprises analyzing nucleic acid molecules from cells bound to the second antibody or antigen-binding fragment thereof.

[0178] In some embodiments, the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof are fetal cells. In some embodiments, the fetal cells are fetal erythroblasts. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are fetal trophoblast cells.

[0179] In some embodiments, analyzing one or more nucleic acid molecules comprises performing karyotyping. Karyotyping may be performed using any technique known in the art.

[0180] In some embodiments, analyzing one or more nucleic acid molecules comprises performing sequence analysis. Sequence analysis can be performed using any technique known in the art. In some embodiments, sequence analysis comprises short tandem repeat (STR) analysis.

[0181] In some embodiments, analyzing one or more nucleic acid molecules comprises performing one or more amplification reactions. Nucleic acid amplification can be performed by any technique known in the art. In some embodiments, nucleic acid amplification is performed by polymerase chain reaction (PCR).

[0182] In some embodiments, the one or more genetic abnormalities are selected from a trisomy, a sex chromosome abnormality, and a structural abnormality. In some embodiments, the genetic abnormality is a trisomy. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21, and trisomy 22. In some embodiments, the genetic abnormality is a sex chromosome abnormality. In some embodiments, the sex chromosome abnormality is selected from monosomy X, triple X syndrome, and Klinefelter syndrome. In some embodiments, the genetic abnormality is a structural abnormality. In some embodiments, the structural abnormality is a copy number variation (CNV). In some embodiments, the structural abnormality is a CNV deletion or a CNV duplication.

[0183] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle, hi some embodiments, the magnetic particle is a magnetic colloid particle.

[0184] In some embodiments, isolating cells that bind to the anti-TREML2 antibody, or antigen-binding fragment thereof, comprises exposing the sample to a magnetic field.

[0185] In some embodiments, the methods disclosed herein further comprise contacting the sample with a first antibody that binds to a protein selected from EpCAM, CD105, and CD71 before contacting the sample with the anti-TREML2 antibody. In some embodiments, the methods disclosed herein further comprise isolating cells bound to the first antibody before contacting the sample with the anti-TREML2 antibody. In some embodiments, the first antibody is conjugated to magnetic particles. In some embodiments, the magnetic particles are magnetic colloid particles. In some embodiments, isolating cells bound to the first antibody comprises exposing the sample to a magnetic field.

[0186] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the step of isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is based on immunofluorescence techniques. In some embodiments, the step of isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the step of isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed by DEPArray.

[0187] In some embodiments, the methods disclosed herein further comprise contacting the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof with a second antibody or antigen-binding fragment thereof. In some embodiments, the second antibody is an anti-TREML2 antibody or antigen-binding fragment thereof. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the methods disclosed herein further comprise isolating the cells bound to the second antibody or antigen-binding fragment thereof. In some embodiments, the isolating the cells bound to the second antibody or antigen-binding fragment thereof is based on immunofluorescence techniques. In some embodiments, the isolating the cells bound to the second antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolating the cells bound to the second antibody or antigen-binding fragment thereof is performed by DEPArray.

[0188] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661. Alternatively or additionally, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6, (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7, (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8, (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9, (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10, and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, any one of SEQ ID NOs: 6-11 independently contains two or more amino acid substitutions, additions, or deletions.

[0189] In some embodiments, any of the methods disclosed herein further comprise providing a treatment recommendation based on the results of the genetic testing on the fetal cells.

[0190] In some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the genetic testing on the fetal cells.

[0191] In some embodiments, any of the methods disclosed herein further comprise the step of recommending additional monitoring of the subject or fetus based on the results of the genetic testing on the fetal cells.

[0192] Although the methods disclosed herein may recite the use of an anti-TREML2 antibody or an antigen-binding fragment thereof, or an antibody conjugate comprising an anti-TREML2 antibody, any of the methods may be carried out using any agent capable of binding to the TREML2 protein, or a conjugate comprising an agent capable of binding to the TREML2 protein. Thus, the methods disclosed herein are not limited to the use of an anti-TREML2 antibody or an antigen-binding fragment thereof, or an antibody conjugate comprising an anti-TREML2 antibody.

[0193] Drugs that bind to rare cell markers Disclosed herein are agents that bind to rare cell markers. As used herein, a "rare cell marker" is a marker (e.g., a cell surface protein) on rare cells (e.g., fetal cells). A rare cell marker can be a cell surface protein that is expressed at a higher level on rare cells than on other cell types in a sample. A rare cell marker can be a triggering receptor-like 2 (TREML2) protein expressed on myeloid cells. A rare cell marker can be a human TREML2 protein. The human TREML2 protein can have the amino acid sequence of SEQ ID NO: 1. Alternatively, a rare cell marker can be CD71. In some embodiments, a rare cell marker is not CD71.

[0194] As used herein, the terms "TREML2" and "TLS1" refer to the same protein and are used interchangeably. TLS1 and TREML2 refer to the same marker, which has the same amino acid sequence as SEQ ID NO: 1 and includes domains and fragments having the amino acid sequences of SEQ ID NOs: 2 to 5.

[0195] As used herein, "rare cells" refer to cells present in a sample from a subject at a concentration of less than 10% of the total cell population, where the sample is a non-purified or non-enriched sample. In some embodiments, the rare cells are present in the sample at a concentration of less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cell population. In some embodiments, the rare cells are present in the sample at a concentration of less than 1% of the total cell population. In some embodiments, the rare cells are fetal cells, and the sample is from a pregnant subject.

[0196] As used herein, the terms "non-purified sample" or "non-enriched sample" can be used interchangeably and refer to a sample obtained from a subject that has not been treated to remove or isolate cells from the sample. Alternatively or additionally, a non-purified or non-enriched sample refers to a sample obtained from a subject that has not been depleted of one or more cells. Alternatively or additionally, a non-purified or non-enriched sample refers to a sample obtained from a subject that contains multiple different cell types.

[0197] In some embodiments, the agent that binds to the rare cell marker is selected from an antibody, an antibody fragment, a receptor, and a ligand. In some embodiments, the antibody fragment comprises an antigen-binding domain of an antibody. In some embodiments, the antibody fragment is selected from a monovalent antigen-binding fragment (Fab or Fab'), a bivalent antigen-binding fragment ((Fab)2 or (Fab')2), a variable fragment (Fv), a single-chain variable fragment (scFv), a bivalent diabody, a triabody, a tetrabody, a minibody, and a bispecific scFv (bis-scFv).

[0198] Generally, a monovalent Fab fragment has one antigen-binding site, while a bivalent (Fab)2 fragment has two antigen-binding regions linked by a disulfide bond. Fab fragments contain the variable heavy (V) chain of an antibody. H ) region and the light chain variable (V L ) region and heavy chain 1 constant (C H 1 ) region and light chain 1 (C L1 ) constant region. The Fv fragment consists of the heavy chain variable (V H ) region and the light chain variable (V L ) region of the Fab, but the antigen-binding site is composed of the Fab constant region (C H 1 and C L ) area is missing. V H and V L are linked in the Fv fragment by non-covalent interactions. Fab can be dimeric (Fab2) or trimeric (Fab3), allowing binding of two or three different antigens, respectively.

[0199] The orientation of the V domains and the length of the linker can be varied to create different types of Fv molecules. Generally, scFv fragments are predominantly monomeric when the linker is at least 12 residues long. Linkers of 3 to 11 residues long result in scFv molecules that are unable to fold into a functional Fv domain. These molecules associate with a second scFv molecule to create a bivalent diabody. Triabodies or tetrabodies can be formed when the linker length is less than 3 residues. Minibodies are scFv-C fragments that assemble into bivalent dimers. H Bis-scFv fragments are fusion proteins consisting of scFv fragments with two different variable domains and can simultaneously bind to two different epitopes.

[0200] The antibody may be a polyclonal antibody. Alternatively or additionally, the antibody may be a monoclonal antibody. The antibody may be an immunoglobulin gamma (IgG) antibody. The IgG antibody may be an IgG1 antibody. The IgG antibody may be an IgG2 antibody. The IgG antibody may be an IgG3 antibody. The IgG antibody may be an IgG4 antibody. The antibody may be an immunoglobulin mu (IgM) antibody. The antibody may be an immunoglobulin epsilon (IgE) antibody. The antibody may be an immunoglobulin delta (IgD) antibody. The antibody may be an immunoglobulin alpha (IgA) antibody. The IgGA antibody may be an IgGA1 antibody. Alternatively, the IgG antibody may be an IgGA2 antibody.

[0201] In some embodiments, the agent is an antibody or antibody fragment that binds to the TREML2 protein. In some embodiments, the antibody or antibody fragment binds to the extracellular domain of the TREML2 protein. In some embodiments, the extracellular domain has the amino acid sequence of SEQ ID NO: 2. Alternatively, the antibody or antibody fragment may bind to a fragment of the extracellular domain of TREML2. The fragment of the extracellular domain has the amino acid sequence of SEQ ID NO: 3-4. The antibody or antibody fragment may bind to the N-terminal domain of the TREML2 protein.

[0202] In some embodiments, the anti-TREML2 antibody is a polyclonal antibody, such as sc-109096 (Santa Cruz Biotechnology, Inc.), ARP49877_P050 (Aviva Systems Biology), OACA04996 (Aviva Systems Biology), AF3259 (R&D Systems), PA5-47471 (Thermo Fisher), ABIN634968 (Antibodies-online.com), ABIN928294 (Antibodies-online.com), 30-552 (ProSci), ABIN2463297 (antibodies-online.com), ABIN749888 (antibodies-online.com), bs-2737r (Bioss), ABIN1999045 (antibodies-online.com), 11655-rp02 (Sino The antibody may be selected from anti-TREML2 antibodies selected from Novus Biologicals), ABIN293207 (antibodies-online.com), ABIN2387613 (antibodies-online.com), t8282-40 (USBio), ABIN4249314 (antibodies-online.com), and nbp1-70737-20ul (Novus Biologicals).

[0203] The anti-TREML2 antibody may be a monoclonal antibody selected from MA5-30973 (Thermo Fisher), ABIN19999041 (antibodies-online.com), 11655-r001 (Sino Biological), and BD563661 (Fisher Scientific).

[0204] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0205] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, or three CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; and (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8.

[0206] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one, two, or three CDRs selected from: (a) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (b) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (c) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0207] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or essentially consists of: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0208] In some embodiments, any one of SEQ ID NOs: 6-11 independently contains one, two, or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 6 contains one, two, or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 7 contains one, two, or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 8 contains one, two, or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 9 contains one, two, or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 10 contains one amino acid substitution, addition, or deletion. In some embodiments, SEQ ID NO: 11 contains one, two, or more amino acid substitutions, additions, or deletions.

[0209] In some embodiments, the anti-TREML2 antibody is conjugated to a label to generate a conjugated antibody. In some embodiments, the label is selected from a fluorescent label, a radionuclide, an enzyme label, a chemiluminescent label, and a hapten. In some embodiments, the detectable label is a hapten. In some embodiments, the hapten is selected from DCC, biotin, nitropyrazole, thiazole sulfonamide, benzofurazan, and 2-hydroxyquinoxaline. In some embodiments, the detectable label is biotin. In some embodiments, the label is a fluorescent molecule. In some embodiments, the fluorescent molecule is selected from a fluorophore, a cyanine dye, and a near-infrared (NIR) dye. In some embodiments, the fluorescent molecule is fluorescein. In some embodiments, the fluorescent molecule is fluorescein isothiocyanate (FITC). In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin. In some embodiments, the conjugated antibody is selected from ABIN6070559 (antibodies-online.com), abx307664 (Abbexa, polyclonal), ABIN6070561 (antibodies-online.com), abx307665 (Abbexa, polyclonal), ABIN2662892 (antibodies-online.com), bld-351203 (BioLegend), ABIN2662891 (antibodies-online.com), bld-351204 (BioLegend), ABIN2662890 (antibodies-online.com, monoclonal), and bld-351104 (BioLegend).

[0210] magnetic particles The methods, compositions, and kits disclosed herein may include or use magnetic particles. For example, any antibody (or more generally, any agent that binds to a rare cell marker) disclosed herein may be conjugated to a magnetic particle. In some embodiments, an agent that binds to a rare cell marker (e.g., TREML2) is conjugated to a magnetic particle. The magnetic particle may be a magnetic colloid particle. The magnetic colloid particle may be a ferrofluid.

[0211] As used herein, the term "magnetic particle" refers to a particle that can be manipulated using a magnetic field. Magnetic particles include metals. Examples of metals include, but are not limited to, iron, nickel, cobalt, and copper.

[0212] As used herein, the term "magnetic colloidal particles" refers to magnetic particles coated with a non-magnetic material. An example of a non-magnetic particle is bovine serum albumin (BSA).

[0213] As used herein, the term "ferrofluid magnetic particles" refers to magnetic colloidal particles that contain iron.

[0214] In some embodiments, the magnetic particles are characterized by their submicron particle size. In some embodiments, the particle diameter is generally less than about 300 nanometers (nm), 275 nm, 250 nm, 225 nm, 200 nm, 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, or 100 nm. In some embodiments, the particle diameter is generally at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or 120 nm or more. In some embodiments, the particle diameter is between about 40 nm and 250 nm, 40 nm and 200 nm, 50 nm and 200 nm, 50 nm and 190 nm, 50 nm and 180 nm, 50 nm and 170 nm, 60 nm and 200 nm, 70 nm and 200 nm, 80 nm and 200 nm, 90 nm and 200 nm, 90 nm and 175 nm, or 90 nm and 150 nm.

[0215] In some embodiments, the particles have a magnetic mass of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% or more, hi some embodiments, the particles have a magnetic mass of between about 40%-95%, 45%-95%, 50%-90%, 55%-90%, 60%-90%, or 70%-90%.

[0216] In some embodiments, particles in the range of 90-150 nm and having between 70-90% magnetic mass may be used.

[0217] In some embodiments, particles are characterized by their resistance to gravitational separation from a solution. The particles may be resistant to gravitational separation for an extended period of time. The particles may be resistant to gravitational separation for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes or more. The particles may be resistant to gravitational separation for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 75 hours, 90 hours, 105 hours, or 120 hours or more. The particles may be resistant to gravity separation for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 75 days, 90 days, 105 days, or 120 days or more.

[0218] In some embodiments, magnetic particles are composed of a superparamagnetic crystalline core surrounded by coating molecules that are bound to the magnetic core, e.g., by physical adsorption or covalent bonding, to impart stabilizing colloidal properties. The coating material can be applied in an amount effective to prevent nonspecific interactions between the magnetic core and biopolymers found in the sample. Such biopolymers can include sialic acid residues on the surface of non-target cells, lectins, glycoproteins, and other membrane components. Furthermore, the coating material can contain as high a magnetic mass-to-nanoparticle ratio as possible. The dimensions of the magnetic crystals comprising the core are small enough so as not to contain a complete magnetic domain. The dimensions of the nanoparticles are such that their Brownian energy dominates their magnetic moment. Therefore, north-south pole alignment and subsequent mutual attraction / repulsion of these magnetic colloidal particles are unlikely to occur, even in moderately strong magnetic fields, contributing to their solution stability.

[0219] Magnetic particles can be separated in a high magnetic gradient external field separator, a property that facilitates sample manipulation and offers economic advantages over more complex internal gradient columns loaded with ferromagnetic beads or steel wool.

[0220] Magnetic particles can be prepared by modification of the substrates described in EP0842042, which is incorporated by reference in its entirety.

[0221] The magnetic particles can be coated with Abs (or more generally any agent) that can recognize differentially expressed proteins that correspond to the top candidates identified in Example 1. In some embodiments, the magnetic particles can be coated with an agent that binds to a rare cell marker (e.g., TREML2). The magnetic particles can be coated with any antibody or agent disclosed herein.

[0222] Coating of the magnetic particles can be done by any method known in the art, for example, the magnetic particles can be coated with antibodies as described in US 6,365,362 B1, which is incorporated by reference in its entirety.

[0223] FIG. 2 illustrates an exemplary ferrofluid magnetic particle structure. The ferrofluid magnetic particles disclosed herein can comprise, consist of, or consist essentially of the ferrofluid magnetic particle structure shown in FIG. 2. In some embodiments, the ferrofluid magnetic particles disclosed herein have the ferrofluid magnetic particle structure shown in FIG. 2. As shown in FIG. 2, the exemplary ferrofluid magnetic particle structure comprises, consists of, or consists essentially of an iron atom surrounded by bovine serum albumin (BSA). The BSA is attached to streptavidin (SA) which is attached to biotin (BT). The BT may be attached to another BSA which is attached to an exogenous aggregation enhancing factor (e.g., desthiobiotin (Dt-BT)). The BT may also be attached to an antibody (Y) that binds to a marker on rare cells. In some embodiments, the rare cells are fetal cells. In some embodiments, the marker is TREML2. Alternatively, the marker is EpCAM, CD105, or CD71.

[0224] FIG. 7 shows a schematic diagram of magnetic particle aggregation by controlled aggregation. As shown in FIG. 7, magnetic particles, such as the ferrofluid magnetic particles of FIG. 2, are coupled to an exogenous aggregation enhancing factor (EAEF, e.g., desthiobiotin (Dt-BT)). Addition of a second EAEF (e.g., streptavidin (SA)) capable of binding to the first EAEF promotes aggregation of the antibody-magnetic particle conjugates. In some embodiments, aggregation of the antibody-magnetic particle conjugates is reversed by addition of a third EAEF capable of binding to the first or second EAEF. In some embodiments, the third EAEF is the same as the first EAEF. Alternatively, the third EAEF is the same as the second EAEF. In another embodiment, the third EAEF is a binding partner (e.g., biotin) of the first or second EAEF.

[0225] Compositions and Kits Disclosed herein are compositions and kits comprising any of the anti-TREML2 antibodies or antigen-binding fragments thereof disclosed herein, wherein the composition or kit further comprises one or more components selected from a magnetic reagent, one or more additional antibodies or antibody conjugates, an aggregation inhibitor, and an aggregation factor.

[0226] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) a magnetic reagent.

[0227] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) magnetic colloid particles.

[0228] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) one or more additional antibodies or antigen-binding fragments thereof.

[0229] Further disclosed herein is a kit comprising (a) an anti-TREML2 antibody or an antigen-binding fragment thereof, and (b) a second antibody or an antigen-binding fragment thereof that binds to a protein expressed on the surface of fetal nucleated red blood cells (fnRBCs).

[0230] Further disclosed herein is a kit comprising (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) a second antibody or antigen-binding fragment thereof conjugated to a label.

[0231] Further disclosed herein is a kit comprising (a) a first anti-TREML2 antibody or antigen-binding fragment thereof conjugated to a magnetic particle, and (b) a second anti-TREML2 antibody or antigen-binding fragment thereof conjugated to a label.

[0232] In some embodiments, a composition or kit comprises an anti-TREML2 antibody or antigen-binding fragment thereof, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of (i) a complementarity-determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6, (ii) a CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7, and (iii) a CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8, and (b) a light chain variable region (LCVR) comprising, consisting of, or consisting essentially of (i) a CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9, (ii) a CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0233] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) a buffer comprising an aggregation inhibitor.

[0234] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof, and (b) an exogenous aggregation enhancing agent.

[0235] Any of the compositions, kits, or methods disclosed herein may include one or more magnetic reagents. The magnetic reagents may include one or more magnetic particles. The magnetic reagents may include ferromagnetic particles, superparamagnetic particles, or ferrofluid reagents.

[0236] As used herein, the term "ferromagnetic particles" refers to particles that are permanently magnetizable.

[0237] The magnetic reagent may include superparamagnetic particles. As used herein, the term "superparamagnetic particles" may refer to particles that are magnetically responsive particles. Superparamagnetic particles are particles that exhibit magnetic behavior only when exposed to a magnetic field. In some embodiments, the magnetic colloid particles are superparamagnetic particles.

[0238] In some embodiments, the magnetic reagent comprises magnetic particles. In some embodiments, the magnetic particles have dimensions of about 1.5 to about 50 microns, 0.7 to 1.5 microns, or less than 200 nm. In some embodiments, the magnetic particles have dimensions of less than 200 nm. In some embodiments, the magnetic reagent comprises magnetic particles conjugated to an antibody. In some embodiments, the antibody conjugated to the magnetic particle is an antibody that binds to a protein selected from epithelial cell adhesion molecule (EpCAM) and endoglin (CD105). Alternatively, the antibody conjugated to the magnetic particle binds to CD147. In a further embodiment, the antibody conjugated to the magnetic particle binds to CD45. In another embodiment, the antibody conjugated to the magnetic particle binds to a protein expressed on the surface of fetal cells.

[0239] In some embodiments, the magnetic reagent comprises a ferrofluid reagent. As used herein, the term "ferrofluid reagent" refers to a liquid suspension comprising magnetic particles. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to an anti-TREML2 antibody. Alternatively, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to one or more antibodies disclosed herein. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to an anti-EPCAM antibody. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to an anti-CD105 antibody. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to an antibody that binds to a protein expressed on the surface of fetal cells. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising magnetic particles conjugated to an anti-CD147 antibody.

[0240] In some embodiments, the kit further comprises one or more staining reagents. In some embodiments, the one or more staining reagents comprise one or more antibody conjugates. In some embodiments, the antibody conjugate in the one or more antibody conjugates is an antibody conjugated to a label. In some embodiments, the antibody binds to a protein selected from CD71, glycophorin A (GPA), and CD45.

[0241] In some embodiments, any of the antibodies disclosed herein (e.g., the anti-TREML2 antibody or one or more additional antibodies) further comprises a label. In some embodiments, the label is conjugated to the antibody. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0242] Any kit disclosed herein may include one or more antibodies or fragments thereof. The one or more antibodies may bind to a protein expressed on the surface of fetal cells. Alternatively or additionally, the one or more antibodies may bind to a protein expressed on the surface of maternal cells. The one or more antibodies may bind to a protein selected from EpCAM, CD105, CD147, CD15, CD71, GPA, and CD45. The one or more antibodies may bind to a protein selected from CD15, CD71, GPA, and CD45.

[0243] Any of the kits disclosed herein may include one or more antibodies or fragments thereof that bind to proteins expressed on the surface of fetal nucleated red blood cells (fnRBCs) or trophoblast cells. The antibodies may bind to a protein selected from EpCAM, CD105, CD71, and CD147.

[0244] Any of the kits disclosed herein may include one or more aggregation inhibitors. The kits disclosed herein may include one, two, three, four, or five or more aggregation inhibitors. The aggregation inhibitor may inhibit endogenous ferrofluid aggregation factors. In some embodiments, the aggregation inhibitor is selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane. The reducing agent may be mercaptoethanesulfonic acid. The aggregation inhibitor may be bovine serum albumin (BSA). The chelating agent may be EDTA.

[0245] The aggregation inhibitor may comprise an antibody or fragment thereof of the same isotype as the anti-TREML2 antibody. The antibody may be a non-specific antibody. In some embodiments, the antibody is a murine antibody.

[0246] Any of the kits disclosed herein may include an anti-TREML2 antibody that may be coupled to a ferrofluid. Any of the kits disclosed herein may include an anti-TREML2 antibody conjugated to a magnetic particle. The magnetic particle may be a magnetic colloid particle. The magnetic particle may be a ferrofluid magnetic particle.

[0247] Any of the kits disclosed herein can include an exogenous aggregation enhancing factor (EAEF). In some embodiments, the kits disclosed herein include one, two, three, four, or more EAEFs. In some embodiments, the magnetic particles disclosed herein are coupled to one or more EAEFs. In some embodiments, the EAEF includes one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0248] In some embodiments, the kits disclosed herein comprise two or more EAEFs, where a first EAEF comprises one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin, and a second EAEF comprises the other member of the specific binding pair.

[0249] In some embodiments, the kit disclosed herein further comprises a third EAEF. In some embodiments, the third EAEF is the same as the first EAEF. Alternatively, the third EAEF is the same as the second EAEF. In some embodiments, the third EAEF is capable of interacting with the first EAEF. In another embodiment, the third EAEF is capable of interacting with the second EAEF. When having a third EAEF that is the same as or capable of interacting with the first or second EAEF, adding the third EAEF results in reversal of aggregation of magnetic particles.

[0250] In some embodiments, the kits disclosed herein further comprise one or more aggregation inhibitors. In some embodiments, the aggregation inhibitor is selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA). The present invention encompasses, for example, the following embodiments: [1] A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody; (b) isolating cells that bind to the first antibody to generate an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying cells that bind to the second antibody as fetal cells; The first antibody or the second antibody is (i) an antibody that binds to the triggering receptor-like 2 (TREML2) protein expressed on myeloid cells, or (ii) an antigen-binding fragment that binds to the TREML2 protein. [2] The method according to [1], wherein the fetal cells are fetal nucleated red blood cells (fnRBCs). [3] The method according to [1] or [2], wherein the first antibody is conjugated to one or more magnetic particles. [4] The method according to [3], wherein the magnetic particles are magnetic colloidal particles. [5] The method according to [4], wherein the magnetic colloid particles are magnetic fluid magnetic particles. [6] The method according to any one of [3] to [5], wherein step (b) comprises exposing the sample to a magnetic field. [7] The method of [6], wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. [8] The method according to [7], wherein step (a) comprises adding a second EAEF containing the other member of the specific binding pair to induce aggregation of the magnetic particles. [9] The method according to [8], wherein step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse the aggregation of magnetic particles in the enriched sample.

[10] The method according to any one of [1] to [9], further comprising, before step (a), a step of adding at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample.

[11] The method according to

[10] , wherein the aggregation inhibitor is a chelating agent that is EDTA.

[12] The method according to any one of [1] to

[11] , wherein the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[13] The method according to

[12] , further comprising the step of isolating a single fetal cell prior to step (d).

[14] The method according to

[13] , wherein the step of isolating single fetal cells is carried out by isolating single fetal cells bound to a second antibody.

[15] The method of

[14] , wherein the second antibody is conjugated to a label.

[16] The method according to

[15] , wherein the label is a fluorescent label.

[17] The method according to

[16] , wherein the step of isolating single fetal cells is based on immunofluorescence techniques.

[18] The method according to

[17] , wherein the step of isolating single fetal cells is performed by fluorescence-activated cell sorting (FACS).

[19] The method according to

[17] , wherein the step of isolating a single fetal cell is carried out using DEPArray.

[20] The method according to any one of [1] to

[19] , wherein step (d) comprises a step of performing sequence analysis.

[21] The method according to

[20] , wherein the sequence analysis includes short tandem repeat (STR) analysis.

[22] The method according to any one of [1] to

[21] , further comprising the step of analyzing fetal cells.

[23] The method according to

[22] , wherein the analysis of fetal cells includes a step of performing genomic analysis or genetic analysis.

[24] The method according to

[23] , wherein the step of performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in fetal cells.

[25] The method according to any one of [1] to

[24] , wherein the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[26] An antibody that binds to the TREML2 protein or an antigen-binding fragment that binds to the TREML2 protein, (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 The method according to any one of [1] to

[25] , comprising one or more CDRs selected from:

[27] A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a magnetic reagent comprising magnetic particles conjugated to a first antibody that binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or antigen-binding fragment thereof; and (c) identifying the cells that bind to the anti-TREML2 antibody as fetal cells. A method comprising:

[28] The method according to

[27] , further comprising, before step (c), a step of isolating cells that bind to the first antibody.

[29] The method according to

[28] , wherein the step of isolating the cells comprises the step of exposing the sample to a magnetic field to concentrate the sample containing cells that are bound to the first antibody.

[30] The method according to any one of

[27] to

[29] , wherein the magnetic particles are magnetic colloidal particles.

[31] The method according to

[30] , wherein the magnetic colloid particles are ferrofluid magnetic particles.

[32] The method of any of

[27] to

[31] , wherein the magnetic particles are further coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[33] The method according to

[32] , comprising, during step (a), adding a second EAEF containing the other member of the specific binding pair to induce particle aggregation.

[34] The method of

[33] , further comprising the step of adding a member of a specific binding pair to the enriched sample to reverse agglutination of the sample, thereby facilitating identification of the cells.

[35] The method according to any one of

[27] to

[34] , further comprising, before step (a), a step of adding at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample.

[36] The method according to

[35] , wherein the aggregation inhibitor is a chelating agent.

[37] The method according to

[36] , wherein the chelating agent is EDTA.

[38] The method according to any one of

[27] to

[37] , further comprising, before step (c), a step of isolating cells using an anti-TREML2 antibody or a second antibody.

[39] The method according to

[38] , wherein the second antibody is selected from an anti-cytokeratin antibody and an anti-HLAG antibody.

[40] The method according to

[38] or

[39] , wherein the anti-TREML2 antibody or the second antibody is conjugated to a label.

[41] The method according to

[40] , wherein the label is a fluorescent label.

[42] The method according to

[41] , wherein the step of isolating cells is based on immunofluorescence techniques.

[43] The method according to

[42] , wherein the step of isolating cells is carried out by fluorescence-activated cell sorting (FACS).

[44] The method according to

[41] , wherein the step of isolating cells is carried out using DEPArray.

[45] The method according to any one of

[27] to

[44] , wherein the step of identifying the cells includes a step of performing sequence analysis.

[46] The method according to

[45] , wherein the sequence analysis includes short tandem repeat (STR) analysis.

[47] The method according to any one of

[27] to

[46] , further comprising the step of analyzing fetal cells.

[48] ​​The method according to

[47] , wherein the step of analyzing fetal cells includes a step of performing genomic analysis or genetic analysis.

[49] The method according to

[48] , wherein the step of performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in fetal cells.

[50] The method according to any one of

[27] to

[49] , wherein the fetal cells are fetal erythroblasts or fetal trophoblast cells.

[51] The anti-TREML2 antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 The method according to any one of

[27] to

[50] , comprising one or more complementarity determining regions (CDRs) selected from:

[52] The method according to

[51] , wherein any one of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[53] The method according to any one of

[27] to

[50] , wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[54] A kit comprising: (a) an antibody (anti-TREML2 antibody) or an antigen-binding fragment thereof that binds to triggering receptor-like 2 (TREML2) protein expressed on myeloid cells; and (b) a magnetic reagent containing magnetic colloid particles.

[55] The kit according to

[54] , wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

[56] The anti-TREML2 antibody or antigen-binding fragment thereof is (a) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 The kit according to

[54] , comprising one or more complementarity determining regions (CDRs) selected from:

[57] The kit according to

[56] , wherein any one of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[58] The kit according to any one of

[54] to

[57] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label to generate a binding antibody.

[59] The kit according to

[58] , wherein the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[60] The kit according to any one of

[54] to

[59] , wherein the dimensions of the magnetic colloidal particles are less than 200 nm.

[61] The kit according to any one of

[54] to

[60] , wherein the magnetic colloid particles are magnetic fluid particles.

[62] The kit according to any one of

[54] to

[61] , wherein the magnetic colloid particles are conjugated to an antibody or an antigen-binding fragment thereof.

[63] The kit according to

[62] , wherein the antibody is an anti-TREML2 antibody.

[64] The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 The kit according to

[63] , comprising one or more CDRs selected from:

[65] The kit according to

[65] , wherein any one of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[66] The kit according to any one of

[54] to

[65] , further comprising an inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

[67] The kit according to

[66] , wherein the chelating agent is EDTA.

[68] The kit according to any one of

[54] to

[67] , further comprising an exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[69] A kit comprising: (a) a first antibody bound to magnetic colloid particles, the first antibody being capable of binding to a protein expressed on the surface of fetal cells; and (b) an anti-TREML2 antibody or antigen-binding fragment thereof.

[70] The kit according to

[65] , wherein the first antibody binds to a protein selected from EpCAM, CD105, and CD71.

[71] The kit according to

[69] or

[27] , wherein the magnetic colloid particles are magnetic fluid particles.

[72] The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 The kit according to any one of

[69] to

[71] , comprising one or more CDRs selected from:

[73] The kit according to

[72] , wherein any one of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions, or deletions.

[74] The kit according to any one of

[69] to

[73] , further comprising an inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

[75] The kit according to

[74] , wherein the chelating agent is EDTA.

[76] The kit according to any one of

[69] to

[75] , further comprising an exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[77] A cell-based fetal genetic testing method, comprising: (a) contacting a sample obtained from a pregnant subject, the sample comprising a plurality of cells, with an anti-TREML2 antibody or antigen-binding fragment thereof; (b) isolating cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, the report providing a probability of the fetus having one or more genetic abnormalities. A method comprising:

[78] The method according to

[77] , wherein the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are fetal cells.

[79] The method according to

[78] , wherein the fetal cells are fetal erythroblasts.

[80] The method of

[78] , wherein the fetal cells are fetal trophoblast cells.

[81] The method according to any one of

[77] to

[80] , wherein the step of analyzing one or more nucleic acid molecules comprises a step of performing karyotyping.

[82] The method according to any one of

[77] to

[80] , wherein the step of analyzing one or more nucleic acid molecules comprises a step of performing sequence analysis.

[83] The method according to

[82] , wherein the sequence analysis comprises short tandem repeat (STR) analysis.

[84] The method according to any one of

[77] to

[83] , wherein the one or more genetic abnormalities are selected from trisomy, sex chromosome abnormality and structural abnormality.

[85] The method according to

[84] , wherein the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21 and trisomy 22.

[86] The method according to

[85] , wherein the sex chromosome abnormality is selected from monosomy X, triple X syndrome, and Klinefelter syndrome.

[87] The method according to

[86] , wherein the structural abnormality is copy number variation (CNV).

[88] The method according to

[87] , wherein the structural abnormality is a CNV deletion or a CNV duplication.

[89] The method according to any one of

[77] to

[88] , wherein the anti-TREML2 antibody is conjugated to a magnetic particle.

[90] The method of

[89] , wherein the magnetic particles are magnetic colloidal particles.

[91] The method of

[90] , wherein the magnetic colloid particles are ferrofluid magnetic particles.

[92] The method of any of

[89] to

[91] , wherein step (b) comprises exposing the sample to a magnetic field.

[93] The method according to any one of

[77] to

[88] , further comprising, before step (a), a step of contacting the sample with a first antibody that binds to a protein selected from EpCAM, CD105, and CD71.

[94] The method according to

[93] , further comprising, before step (a), a step of isolating cells that bind to the first antibody.

[95] The method according to

[93] or

[94] , wherein the first antibody is conjugated to a magnetic particle.

[96] The method of

[95] , wherein the magnetic particles are magnetic colloidal particles.

[97] The method of

[96] , wherein the magnetic colloid particles are ferrofluid magnetic particles.

[98] The method according to any one of

[95] to

[97] , wherein the step of isolating cells bound to the first antibody comprises the step of exposing the sample to a magnetic field.

[99] The method of any one of

[77] to

[88] and claims 93 to 98, wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label.

[0100] The method according to

[99] , wherein the label is a fluorescent label.

[0101] The method according to

[0100] , wherein the step of isolating cells bound to the anti-TREML2 antibody or its antigen-binding fragment is based on immunofluorescence techniques.

[0102] The method described in

[0101] , wherein the step of isolating cells bound to the anti-TREML2 antibody or its antigen-binding fragment is carried out by fluorescence-activated cell sorting (FACS).

[0103] The method described in

[0101] , in which the step of isolating cells bound to the anti-TREML2 antibody or its antigen-binding fragment is carried out using DEPArray.

[0104] A method according to any one of

[77] to

[92] , further comprising a step of contacting cells bound to the anti-TREML2 antibody or its antigen-binding fragment with a second antibody or its antigen-binding fragment.

[0105] The method described in

[0104] , wherein the second antibody is an anti-TREML2 antibody or an antigen-binding fragment thereof.

[0106] A method according to

[0104] or

[0105] , wherein the second antibody is conjugated to a label.

[0107] The method described in

[0106] , wherein the label is a fluorescent label.

[0108] The method described in

[0107] , further comprising the step of isolating cells that bind to the second antibody or its antigen-binding fragment.

[0109] The method according to

[0108] , wherein the step of isolating cells bound to the second antibody or its antigen-binding fragment is based on immunofluorescence techniques.

[0110] The method described in

[0109] , wherein the step of isolating cells bound to the second antibody or its antigen-binding fragment is carried out by fluorescence-activated cell sorting (FACS).

[0111] The method described in

[0109] , wherein the step of isolating cells bound to the second antibody or its antigen-binding fragment is carried out using DEPArray.

[0112] A method described in any of

[77] to

[0111] , wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0113] The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 A method according to any one of

[77] to

[0111] , comprising one or more CDRs selected from:

[0114] The method described in

[0113] , wherein any one of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody, wherein the first antibody binds to a triggering receptor-like 2 (TREML2) protein expressed on myeloid cells (an anti-TREML2 antibody) or an antigen-binding fragment thereof; and (b) identifying cells that bind to the first antibody as fetal cells; A method comprising:

[0116] The method described in

[0115] , wherein the fetal cells are fetal nucleated red blood cells (fnRBCs).

[0117] A method according to

[0115] or

[0116] , wherein the first antibody is conjugated to one or more magnetic particles.

[0118] The method described in

[0117] , wherein the magnetic particles are magnetic colloid particles.

[0119] The method described in

[0118] , wherein the magnetic colloid particles are magnetic fluid magnetic particles.

[0120] A method according to any one of

[0117] to

[0119] , further comprising a step of exposing the sample to a magnetic field.

[0121] A method according to any one of

[0117] to

[0120] , wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0122] The method described in

[0121] , further comprising, before step (b), a step of adding a second EAEF containing the other member of the specific binding pair to induce aggregation of the magnetic particles.

[0123] The method of

[0122] , further comprising the step of isolating cells that bind to the first antibody to produce an enriched sample.

[0124] The method of

[0123] , further comprising the step of adding a third EAEF capable of binding to the first EAEF or the second EAEF to the enriched sample in order to reverse the aggregation of magnetic particles in the enriched sample.

[0125] The method of

[0124] , wherein the third EAEF is a member of the specific binding pair.

[0126] A method described in any of

[0115] to

[0125] , further comprising a step of adding at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample before step (a).

[0127] The method described in

[0126] , wherein the aggregation inhibitor is a chelating agent.

[0128] The method described in

[0127] , wherein the chelating agent is EDTA.

[0129] A method according to

[0115] or

[0116] , wherein the first antibody is conjugated to a label.

[0130] The method described in

[0129] , wherein the label is a fluorescent label.

[0131] The method according to

[0130] , further comprising, before step (b), a step of isolating cells bound to the first antibody based on an immunofluorescence technique.

[0132] The method according to

[0131] , wherein the step of isolating cells bound to the first antibody is carried out by fluorescence-activated cell sorting (FACS).

[0133] The method described in

[0131] , wherein the step of isolating cells bound to the first antibody is carried out using DEPArray.

[0134] A method according to any one of

[0115] to

[0133] , wherein step (b) includes a step of performing sequence analysis.

[0135] The method described in

[0134] , wherein the sequence analysis includes short tandem repeat (STR) analysis.

[0136] A method according to any one of

[0115] to

[0135] , further comprising a step of analyzing fetal cells.

[0137] The method described in

[0136] , wherein the step of analyzing fetal cells includes a step of performing genomic analysis or genetic analysis.

[0138] The method described in

[0137] , wherein the step of performing genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in fetal cells.

[0139] The first antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 A method according to any one of

[0115] to

[0138] , comprising one or more CDRs selected from:

[0140] The method described in

[0139] , wherein any of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[0141] A method described in any of

[0115] to

[0138] , wherein the first antibody is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 An anti-TREML2 antibody or an antigen-binding fragment thereof, comprising one or more CDRs selected from:

[0143] An anti-TREML2 antibody described in

[0142] , wherein any of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[0144] An anti-TREML2 antibody described in

[0142] or

[0143] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a) to (f).

[0145] An anti-TREML2 antibody described in

[0142] or

[0143] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a) to (f).

[0146] An anti-TREML2 antibody described in

[0142] or

[0143] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a) to (f).

[0147] An anti-TREML2 antibody described in

[0142] or

[0143] , wherein the anti-TREML2 antibody or its antigen-binding fragment comprises five or more CDRs selected from (a) to (f).

[0148] An anti-TREML2 antibody according to

[0142] or

[0143] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs (a) to (f).

[0149] An anti-TREML2 antibody described in any of

[0142] to

[0148] , wherein the anti-TREML2 antibody or its antigen-binding fragment is conjugated to a label.

[0150] An anti-TREML2 antibody described in

[0149] , wherein the label is a fluorescent label.

[0151] An anti-TREML2 antibody described in any of

[0142] to

[0148] , wherein the anti-TREML2 antibody or its antigen-binding fragment is conjugated to a magnetic particle.

[0152] An anti-TREML2 antibody described in

[0151] , wherein the magnetic particles are magnetic colloid particles.

[0153] An anti-TREML2 antibody described in

[0152] , wherein the magnetic colloid particles are magnetic fluid magnetic particles.

[0154] An anti-TREML2 antibody described in

[0152] or

[0153] , wherein the magnetic colloid particles are less than 200 nm.

[0155] An anti-TREML2 antibody described in

[0152] or

[0153] , wherein the magnetic colloid particles are between approximately 80 and 200 nm.

[0156] An anti-TREML2 antibody described in

[0152] or

[0153] , wherein the magnetic colloid particles are between approximately 90 and 150 nm.

[0157] An anti-TREML2 antibody described in any of

[0152] to

[0156] , wherein the magnetic colloid particles have a magnetic mass of at least 50%.

[0158] An anti-TREML2 antibody described in

[0157] , wherein the magnetic colloid particles have a magnetic mass of at least 60%.

[0159] An anti-TREML2 antibody described in

[0157] , wherein the magnetic colloid particles have a magnetic mass of between 70% and 90%.

[0160] An anti-TREML2 antibody described in any of

[0152] to

[0159] , wherein the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

[0161] An anti-TREML2 antibody described in any of

[0151] to

[0160] , wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0162] An anti-TREML2 antibody conjugate comprising: (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic particle conjugated to the anti-TREML2 antibody.

[0163] An anti-TREML2 antibody conjugate described in

[0155] , wherein the magnetic particles are magnetic colloid particles.

[0164] An anti-TREML2 antibody conjugate described in

[0156] , wherein the magnetic colloid particles are magnetic fluid magnetic particles.

[0165] An anti-TREML2 antibody conjugate described in

[0163] or

[0164] , wherein the magnetic colloid particles are less than 200 nm.

[0166] An anti-TREML2 antibody conjugate described in

[0163] or

[0164] , wherein the magnetic colloid particles are between approximately 80 and 200 nm.

[0167] An anti-TREML2 antibody conjugate described in

[0163] or

[0164] , wherein the magnetic colloid particles are between approximately 90 and 150 nm.

[0168] An anti-TREML2 antibody conjugate described in any of

[0163] to

[0167] , wherein the magnetic colloid particle has a magnetic mass of at least 50%.

[0169] An anti-TREML2 antibody conjugate described in

[0168] , wherein the magnetic colloid particles have a magnetic mass of at least 60%.

[0170] An anti-TREML2 antibody conjugate described in

[0169] , wherein the magnetic colloid particles have a magnetic mass of between 70% and 90%.

[0171] An anti-TREML2 antibody conjugate described in any of

[0163] to

[0170] , wherein the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

[0172] An anti-TREML2 antibody conjugate described in any of

[0162] to

[0171] , wherein the magnetic particle is coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0173] The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 An anti-TREML2 antibody conjugate according to any one of

[0162] to

[0171] , comprising one or more CDRs selected from:

[0174] An anti-TREML2 antibody conjugate described in

[0173] , wherein any of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[0175] An anti-TREML2 antibody conjugate described in

[0173] or

[0174] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a) to (f).

[0176] An anti-TREML2 antibody conjugate described in

[0173] or

[0174] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a) to (f).

[0177] An anti-TREML2 antibody conjugate described in

[0173] or

[0174] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a) to (f).

[0178] An anti-TREML2 antibody conjugate described in

[0173] or

[0174] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more CDRs selected from (a) to (f).

[0179] An anti-TREML2 antibody conjugate according to

[0173] or

[0174] , wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs (a) to (f).

[0180] Anti-TREML2 antibodies include sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, and ABIN7498. An anti-TREML2 antibody conjugate described in any of

[0162] to

[0171] , wherein the anti-TREML2 antibody conjugate is selected from 88, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. A method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, comprising: (a) contacting a maternal sample containing fetal cells and maternal cells with a first antibody conjugate, the first antibody conjugate comprising (i) a first antibody and (ii) magnetic colloid particles, wherein the first antibody is conjugated to the magnetic colloid particles; and (b) isolating cells that bind to the first antibody conjugate by exposing the maternal sample to a magnetic field, thereby preparing a fetal cell sample. A method comprising:

[0182] The method described in

[0181] , wherein the magnetic colloid particles are less than 200 nm.

[0183] The method according to

[0181] , wherein the magnetic colloid particles are between about 80 and 200 nm.

[0184] The method described in

[0181] , wherein the magnetic colloid particles are between about 90 and 150 nm.

[0185] A method according to any one of

[0181] to

[0184] , wherein the magnetic colloid particles have a magnetic mass of at least 50%.

[0186] The method of

[0185] , wherein the magnetic colloid particles have a magnetic mass of at least 60%.

[0187] The method according to

[0185] , wherein the magnetic colloid particles have a magnetic mass between 70% and 90%.

[0188] A method according to any one of

[0181] to

[0187] , wherein the magnetic colloidal particles comprise a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0189] A method according to any one of

[0181] to

[0188] , wherein the magnetic colloid particles are magnetic fluid magnetic particles.

[0190] A method according to any of

[0181] to

[0189] , wherein the magnetic colloid particles are further conjugated to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0191] The method according to

[0190] , further comprising the step of adding to the maternal sample a second EAEF comprising the other member of the specific binding pair.

[0192] A method according to any one of

[0181] to

[0191] , wherein the first antibody is an anti-TREML2 antibody.

[0193] A method described in any one of

[0181] to

[0191] , wherein the first antibody is an anti-CD71 antibody.

[0194] A method according to any one of

[0181] to

[0191] , wherein the first antibody binds to a protein selected from EpCAM and CD105.

[0195] The method of

[0194] , wherein the step of preparing a fetal cell sample further comprises contacting cells isolated from the maternal sample with a second antibody.

[0196] The method described in

[0195] , wherein the second antibody is conjugated to a label.

[0197] The method described in

[0196] , wherein the label is a fluorescent label.

[0198] The method described in

[0197] , wherein the step of preparing a fetal cell sample further comprises the step of isolating cells that bind to the second antibody.

[0199] The method described in

[0198] , wherein the step of isolating cells bound to the second antibody is based on immunofluorescence techniques.

[0200] The method described in

[0199] , wherein the step of isolating cells bound to the second antibody is carried out by fluorescence-activated cell sorting (FACS).

[0201] A method according to

[0199] , in which the step of isolating cells bound to the second antibody is carried out using DEPArray.

[0202] The method described in

[26] , wherein the antibody that binds to the TREML2 protein or the antigen-binding fragment that binds to the TREML2 protein comprises two, three, four, five or six CDRs selected from (i) to (vi).

[0203] The method according to

[26] , wherein any of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[0204] The method according to any one of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles are less than 200 nm.

[0205] The method according to any one of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles are between about 80 and 200 nm.

[0206] The method of any of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles are between about 90 and 150 nm.

[0207] The method of any of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles have at least 50% magnetic mass.

[0208] The method of any of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles have a magnetic mass of at least 60%.

[0209] The method according to any one of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles have a magnetic mass between 70% and 90%.

[0210] The method according to any one of [4], [5],

[30] ,

[31] ,

[90] ,

[91] ,

[96] ,

[97] ,

[0118] and

[0119] , wherein the magnetic colloidal particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0211] A method described in any of [1] to

[25] , wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody conjugate comprising a first antibody bound to magnetic colloid particles; (b) isolating cells that bind to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody that binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells. A method comprising:

[0213] The method described in

[0212] , wherein the fetal cells are fetal nucleated red blood cells (fnRBCs).

[0214] A method according to

[0212] or

[0213] , wherein the magnetic colloid particles are magnetic fluid magnetic particles.

[0215] A method according to any one of

[0212] to

[0214] , wherein the magnetic colloidal particles are less than 200 nm.

[0216] A method according to any one of

[0212] to

[0214] , wherein the magnetic colloidal particles are between about 80 and 200 nm.

[0217] A method according to any one of

[0212] to

[0214] , wherein the magnetic colloidal particles are between about 90 and 150 nm.

[0218] A method according to any one of

[0212] to

[0217] , wherein the magnetic colloid particles have a magnetic mass of at least 50%.

[0219] A method according to any one of

[0212] to

[0217] , wherein the magnetic colloid particles have a magnetic mass of at least 60%.

[0220] A method according to any one of

[0212] to

[0217] , wherein the magnetic colloidal particles have a magnetic mass between 70% and 90%.

[0221] A method according to any one of

[0212] to

[0220] , wherein the magnetic colloidal particles comprise a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0222] A method according to any of

[0212] to

[0221] , wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group including biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0223] The method described in

[0222] , wherein step (a) comprises adding a second EAEF containing the other member of the specific binding pair to induce aggregation of magnetic particles.

[0224] The method according to

[0223] , wherein step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse the aggregation of magnetic particles in the enriched sample.

[0225] A method described in any of

[0212] to

[0224] , further comprising a step of adding at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample before step (a).

[0226] The method described in

[0225] , wherein the aggregation inhibitor is a chelating agent.

[0227] The method described in

[0226] , wherein the chelating agent is EDTA.

[0228] A method according to any one of

[0212] to

[0227] , wherein the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0229] The method described in

[0228] , further comprising a step of isolating a single fetal cell prior to step (d).

[0230] The method described in

[0229] , wherein the step of isolating single fetal cells is carried out by isolating single fetal cells bound to a second antibody.

[0231] The method described in

[0230] , wherein the second antibody is conjugated to a label.

[0232] The method described in

[0231] , wherein the label is a fluorescent label.

[0233] The method described in

[0232] , wherein the step of isolating single fetal cells is based on immunofluorescence techniques.

[0234] The method described in

[0233] , wherein the step of isolating single fetal cells is carried out by fluorescence-activated cell sorting (FACS).

[0235] The method described in

[0233] , wherein the step of isolating single fetal cells is performed using DEPArray.

[0236] A method according to any one of

[0212] to

[0233] , wherein step (d) includes a step of performing sequence analysis.

[0237] The method described in

[0236] , wherein the sequence analysis includes short tandem repeat (STR) analysis.

[0238] A method according to any one of

[0212] to

[0237] , further comprising a step of analyzing fetal cells.

[0239] The method described in

[0238] , wherein the step of analyzing fetal cells includes a step of performing genomic analysis or genetic analysis.

[0240] The method described in

[0239] , wherein the step of performing genetic analysis includes a step of detecting the presence or absence of one or more genetic abnormalities in fetal cells.

[0241] A method according to any one of

[0212] to

[0240] , wherein the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0242] An antibody that binds to the TREML2 protein, or an antigen-binding fragment that binds to the TREML2 protein, (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 A method according to any one of claims 241 to 228, comprising one or more CDRs selected from:

[0243] A method described in

[0242] , wherein the antibody that binds to TREML2 protein or the antigen-binding fragment that binds to TREML2 protein comprises two, three, four, five or six CDRs selected from (i) to (vi).

[0244] A method described in

[0242] or

[0243] , wherein any of SEQ ID NOs: 6 to 11 independently contains one or more amino acid substitutions, additions or deletions.

[0245] A method described in any one of claims 228 to 235 and

[0241] , wherein the antibody that binds to the TREML2 protein is selected from sc-109096, ARP49877#P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0251] [Example] [Example 1] Identification of novel markers for fetal cells This example describes the identification of novel markers for fetal cells.

[0252] Preparation of nucleated red blood cells (nRBCs) Using ultrasound-guided techniques, fetal whole blood (n=5) was administered to pregnant women (10 years of pregnancy) scheduled for surgical termination of pregnancy. +0 15 +6 week).

[0253] Twenty milliliters of peripheral blood was collected from pregnant women at the time of delivery (n=2) or before surgical termination of pregnancy (n=1).

[0254] After collection, fetal and maternal blood samples were diluted with an equal volume of phosphate-buffered saline (PBS) and gently layered onto the Percoll gradient. Samples were centrifuged at 1800 rpm for 10 minutes at room temperature. The interface layer containing fetal or adult erythroblasts was collected and washed twice with PBS.

[0255] For maternal blood, a depletion step of CD45 / CD15 positive cells was performed using an LD column (Miltenyi Biotec) by labeling the cells with anti-CD45 microbeads and anti-CD15 microbeads (Miltenyi Biotec).

[0256] For maternal blood, a microfluidic device was also used to remove contaminating RBC cells and enrich for adult erythroblasts.

[0257] Flow cytometric enrichment and cell sorting To prepare samples for FACS sorting, enriched cells from fetal and maternal blood were stained with anti-CD71 antibody (Miltenyi Biotec), anti-GPA antibody (BD Bioscience), anti-CD45 antibody (Miltenyi Biotec), Hoechst (nuclear stain), and Sytox Green dye (live / dead cell stain) for 30 minutes at room temperature.

[0258] FACS sorting Erythroid cells were gated and sorted as shown in Figures 5A-5E. Figure 5B: FSC-H / W gate to exclude doublet cells. Figure 5C: Sytox Green-negative live cells were gated. Figure 5D: Double-positive GPA / Hoechst cells were gated. Figure 5E: CD71-positive / CD45-negative cells were gated.

[0259] For fetal blood samples, fewer than 200,000 target erythroblasts were sorted.

[0260] For maternal blood samples, maternal erythroblast counts never exceeded 1,000.

[0261] RNA extraction using sorted populations Treated and sorted cells were used for total RNA extraction.

[0262] Total RNA was extracted from sorted cells using the Picopure RNA isolation kit (Applied Biosystems), quantified by the Quant-iT RiboGreen RNA assay kit (Thermo Fisher), and analyzed on an Agilent 2100 bioanalyzer using the RNA 6000 Pico kit for quality control.

[0263] RNAseq and library preparation cDNA libraries were prepared based on Illumina sequencing (Appendix A; RNA-seq protocol). Sequencing was performed using a HiSeq 2000 with 20 million reads per sample.

[0264] Sequencing Reads obtained from next-generation sequencing (Illumina) were first quality checked using standard procedures using the FastQC protocol, then mapped to the reference genome and subsequently quantified using STAR software version 2.5. The dimensionality of the generated read count matrix (i.e., a table of read counts for all features—coding or non-coding RNAs—detected in each sample) was reduced by a data reduction step, retaining only genes with at least one single count in a single sample.

[0265] Data analysis using bioinformatics tools The resulting data were further processed using the DESeq2 R / Bioconductor package for differential expression to find genes whose expression was significantly higher or lower between the two samples being compared.

[0266] A DESeq2 default differential expression analysis was performed, consisting of the following steps: for each sample, estimate a size coefficient using the "median ratio method" (Anders and Huber, 2010); for each gene, estimate the found variance using a fitting method that optimizes the variance of negative binomial distributed data; finally, the resulting size coefficient and variance estimates were used to test for significance of the coefficient of the fitted distribution.

[0267] Finally, the result table from the DESeq2 analysis was extracted to obtain the base mean, log2 fold change, standard error, test statistic, p-value, and adjusted p-value across samples for 20,205 features (genes) with non-zero total read counts. Differentially expressed genes were filtered according to an adjusted p-value (Benjamini-Hochberg / FDR method) cutoff of 0.01 and a cutoff of 2-fold change in expression. These parameters selected 3,233 genes as differentially expressed, the majority of which (2,961) were up-regulated in fetal blood (regardless of the fold change cutoff).

[0268] The resulting gene table was annotated and functionally described using the Ensembl database. Genes associated with the annotation "plasma membrane" according to the Gene Ontology term GO:0005886 were flagged and further tagged with "transmembrane" using data from the Uniprot database. Among them, 366 plasma membrane genes were differentially expressed, and the majority of them (336) were upregulated in fetal cells (regardless of the fold-change cutoff).

[0269] In parallel with the differential expression analysis, read counts were normalized and transformed using DESeq2 to allow selection using more stringent expression criteria. The first selection started with a list of genes expressed only in fetal samples, i.e., genes with zero reads in all three maternal samples (12,187 genes, list "ALL DATA"), and was performed according to the following criteria: 1) selection of genes detected (i.e., expressed) in all fetal samples, 2) selection of genes with a mean / standard deviation ratio of expression >1, and 3) selection of genes associated with the plasma membrane GO tag and tagged as "predicted transmembrane" by the Uniport database. See selection scheme below. The resulting 77 genes were then ranked according to their reduced mean fetal expression (list "RANKED"). The final manual curated selection took into account known biological function, stability of expression levels across samples, crude estimates of absolute expression levels (reads relative to gene length), antibody availability, and other biological considerations (16 genes, list "Selected").

[0270] Selection Scheme Below is the selection scheme used to identify potential novel markers for fetal cells. 1) Genes not expressed in any maternal samples: 12,187 2) Genes expressed exclusively in all fetal samples: 2079 2.1) Genes annotated as associated with GO plasma membrane terms: 213 2.2) Genes predicted to be transmembrane by UniProt: 305 3) GO plasma membrane-associated and predicted transmembrane genes: 89 4) Genes with mean / standard deviation ratios > 1: 77

[0271] Ranking differentially expressed genes from gene lists Further manual curation of the final selection and ranking method took into account transcript length, number of reads, and other biologically relevant criteria, resulting in the top candidates:

[0272] [Table 1]

[0273] Identification of antibodies specific for selected target molecules Testing Ab candidates against erythroblasts by FACS analysis To determine whether differential expression at the RNA level was reflected at the level of the respective proteins, immunostaining was performed using commercially available antibodies (n=13) for flow cytometry and DEPArray analysis.

[0274] As a negative control, an isotype-matched Ab conjugated with the same fluorochrome as the commercial antibody was used at the same concentration.

[0275] All 13 antibodies shown in Table 2 were first tested on frozen fetal blood.

[0276] Only antibodies that were clearly expressed on fetal erythroblasts were tested against frozen maternal blood samples.

[0277] In addition to the specific antibody or isotype control to be tested, the antibodies used for staining included CD71 Ab (Miltenyi Biotec), GPA Ab (BD Bioscience), CD45 Ab (Miltenyi Biotec), and Hoechst for the identification of erythroblasts. Briefly, cells (2.5–5 × 10 5 ) were incubated with Ab in the presence of FcR blocking reagent (Miltenyi Biotec) for 30 min at room temperature. After washing off unbound Ab, the cell pellet was resuspended in AutoMACS running buffer (Miltenyi) containing Sytox Green dye.

[0278] [Table 2]

[0279] Ab1, TLT2, was used against TREML2 (hereafter also referred to as TLS-1)

[0280] [Example 2] Magnetic fluid technology for cell capture and selection In this example, selected antibodies expressed exclusively in fetal cells were used for ferrofluid conjugation: TREML2-FF (also called TLS1-FF) refers to a ferrofluid-conjugated antibody capable of binding to a protein expressed by the TLS1 gene.

[0281] The size of the FF-Ab was determined using a NanoBrook Zeta Plus particle size analyzer and the concentration was determined using a spectrophotometer.

[0282] JPEG2026032067000004.jpg64146JPEG2026032067000005.jpg9147

[0283] Controlled Concentration To inhibit endogenous ferrofluid agglutination factors, the blood sample is preincubated with a buffer containing one or more inhibitors before adding the ferrofluid to the blood (as described in EP 1311820, incorporated by reference in its entirety). One inhibitor can be a reducing agent, such as 100 mM mercaptoethanesulfonic acid, which inhibits IgM-induced agglutination without affecting the ligand used to label the cells. The reducing agent can be added to the blood as a single reagent. A second inhibitor can be bovine serum albumin, which can be included in the buffer at 10 mg / ml to neutralize any HAMAA. A third inhibitor can be a nonspecific mouse antibody, particularly one of an appropriate isotype matching the antibody in the ferrofluid. This third inhibitor can be included in the buffer at a concentration of 0.5-5 mg / ml to neutralize even the most troublesome HAMAA. A fourth inhibitor can be streptavidin, which can be included in the buffer if necessary to neutralize any anti-streptavidin antibodies present in the plasma. Pretreatment of blood with the above buffer and reducing agent can take 15 to 30 minutes to neutralize all endogenous agglutination factors. After neutralization, exogenous ferrofluid agglutination factors are added to the sample, followed by the addition of ferrofluid. The ferrofluid is coupled to a target-specific antibody as well as to another ligand specific for the exogenous agglutination factor. After optimally labeling the target cells with ferrofluid and inducing ferrofluid agglutination with the exogenous agglutination factor, the sample is subjected to magnetic separation to enrich the target.

[0284] The sample is placed in a magnetic separator (Immunicon catalog number QS-012) for 10 minutes. The sample is removed from the magnet, vortexed, and placed back in the magnetic separator for 10 minutes to collect the magnetically labeled cells. The uncollected sample is aspirated, and the magnetically collected cells are resuspended in 0.75 ml of wash dilution buffer and re-separated in the magnetic separator for 10 minutes. The uncollected sample is discarded, and the collected cells are resuspended after removing the tube from the magnetic separator. After removing all non-targets, the magnetically labeled targets and free ferrofluid are resuspended in buffer. In some cases, exogenous-mediated ferrofluid aggregation should be reversed. This is accomplished by resuspending the final sample in a buffer containing a disaggregation factor that binds to the exogenous aggregation factor. The disaggregation factor disaggregates all ferrofluid aggregates, making the cells more accessible for further analysis.

[0285] [Example 3] Fetal Cell Detection and Analysis This example describes the isolation and analysis of single fetal cells. DEPArray may be performed as described in EP2152859, which is incorporated by reference in its entirety.

[0286] Pregnant women and healthy volunteers Peripheral blood samples were collected by venipuncture into 10 mL CellSave storage tubes (Menarini Silicon Biosystems, Huntingdon Valley, PA, USA) from 14 pregnant women aged 12–17+2 weeks. For the spiking experiment, peripheral blood was collected from healthy donors. All donors provided written consent, and the study protocol was approved by the Medical Ethics Committee of San Gerardo Hospital, Monza, Italy. All samples were processed 1–4 days later.

[0287] To enrich fetal trophoblasts from contaminating cells, we used antibodies against epithelial cell adhesion antigen (EpCAM), vascular endothelial marker (CD105), and / or TREML2 coupled to magnetic fluid. The enriched cells were labeled with phycoerythrin (PE)-conjugated anti-TREML2 monoclonal antibody (mAb). To recognize leukocytes, the enriched cells were also fluorescently labeled with allophycocyanin (APC)-conjugated anti-cytokeratin mAb C11, APC-conjugated anti-HLA-G mAb, and fluorescein isothiocyanate (FITC)-conjugated anti-CD45 mAb.

[0288] This enrichment procedure for target cells (e.g., trophoblast cells) is necessary because their frequency in maternal blood is known to be very low, at only 1-10 cells per ml of whole blood (containing over 1 billion cells).

[0289] CVS and cord blood for spiking Whole blood from healthy volunteers was spiked with fetal trophoblast cells derived from chorionic villus sampling (CVS) or fetal erythroblast cells derived from umbilical cord blood.

[0290] CVS cultures were selected for their CD105 / EpCAM expression. Cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco), and L-glutamine (Gibco) at 37°C and 5% CO2. Before spiking, cells were detached from the flask, resuspended in 10 ml of PBS (Gibco), and placed in CellSave storage tubes for at least 1 day.

[0291] Cord blood samples obtained from San Gerardo Hospital were collected in CellSave storage tubes.

[0292] Spiking experiments were performed to demonstrate the specificity of the selection procedure when using ferrofluid-conjugated antibodies to capture fetal cells.

[0293] Fetal blood and bone marrow samples Using ultrasound-guided techniques, fetal whole blood (n=3) was collected from pregnant women (10 years of pregnancy) scheduled for surgical termination of pregnancy. +0 15 +6 week).

[0294] All donors provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of KK Maternal and Child Health Centre (Singapore).

[0295] After collection, fetal blood was diluted with an equal volume of PBS and gently layered onto the Percoll gradient. The sample was centrifuged at 1800 rpm for 10 minutes at room temperature. The interface layer containing fetal erythroblasts was collected and washed twice with PBS.

[0296] Cryopreserved bone marrow mononuclear cells containing adult erythroblasts were purchased (Lonza, catalog 2M-125C). Cells were thawed, treated with DNase I, and washed according to the manufacturer's instructions. Cells were then rested for 1 hour at 37°C in RPMI medium supplemented with 10% FBS, penicillin / streptomycin, and L-glutamine and used as a negative control (three different donors were tested).

[0297] Four clones of commercially available TREML2 antibodies were tested in the samples using flow cytometry.

[0298] Isotype-matched Abs conjugated with the same fluorescent dye as the commercially available TREML2 Ab were used at the same concentration. In addition to the TREML2 Ab or isotype control, Abs used for staining included CD71 Ab (Miltenyi Biotec), GPA Ab (BD Bioscience), CD45 Ab (Miltenyi Biotec), and Hoechst. Briefly, cells (2.5–5 × 10 5 ) were incubated with Ab in the presence of FcR blocking reagent (Miltenyi Biotec) for 30 min at room temperature. After washing off unbound Ab, the cell pellet was resuspended in running buffer containing Sytox Green dye for gating live cells in FACS analysis.

[0299] Preparation of desthiobiotin ferrofluid antibodies for controlled aggregation In some embodiments, the magnetic fluids used in the practice of the present invention are particles that behave like colloids. Such particles are characterized by submicron particle sizes, generally less than 200 nanometers (nm), and long-term resistance to gravitational separation from solution. Particles in the 90-150 nm range and between 70-90% magnetic mass are used. Suitable magnetic particles consist of a superparamagnetic crystalline core surrounded by coating molecules that are bound to the magnetic core by, for example, physical adsorption or covalent bonding, imparting stabilizing colloidal properties. Preferably, the coating material is applied in an amount effective to prevent nonspecific interactions between the magnetic core and biopolymers found in the sample. Such biopolymers may include sialic acid residues on the surface of non-target cells, lectins, glycoproteins, and other membrane components. Furthermore, the coating material contains as high a magnetic mass-to-nanoparticle ratio as possible. The dimensions of the magnetic crystals comprising the core are small enough so as not to contain complete magnetic domains. The dimensions of the nanoparticles are such that their Brownian dynamics outweigh their magnetic moment. Therefore, north-south pole alignment of these magnetic colloidal particles and the subsequent mutual attraction / repulsion are unlikely to occur even in moderately strong magnetic fields, contributing to the stability of the solution. Finally, magnetic particles can be separated in an external high-gradient magnetic separator, a property that facilitates sample manipulation and offers economic advantages over more complex internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles with the above properties can be prepared by modification of the substrate described in EP 0 842 042. In a preferred embodiment of the present invention, magnetic particles coated with anti-CD105 antibodies are prepared as described in US Pat. No. 6,365,362 B1, the entire contents of which are incorporated by reference.

[0300] Recombinant human antibody against the CD105 antigen was obtained from hybridoma number 166707 (R&D Systems) and conjugated to the substrate using standard coupling chemistry as described in U.S. Patent Application No. 09 / 248,388. The CD105 Ab ferrofluid was then resuspended in 20 mM HEPES, pH 7.5, for conjugation to desthiobiotin using N-hydroxysuccinimide-DL-desthiobiotin (NHS-desthiobiotin) (Sigma, Cat. No. H-2134). A stock solution of NHS-desthiobiotin was made at 1 mg / ml in DMSO. Five mg of NHS-desthiobiotin was added to 1 mg of CD105 Ab ferrofluid and incubated at room temperature for 2 hours. Unreacted NHS-desthiobiotin was removed using a high-gradient magnet by three washes with 20 mM HEPES, pH 7.5, containing 1 mg / ml BSA and 0.05% Proclin 300. After the final wash, the desthiobiotin / CD105 Ab ferrofluid was resuspended in water / BSA / Proclin 300 and filtered through a 0.2 μm syringe filter. The iron concentration of the CD105 Ab ferrofluid was determined using a spectrophotometric assay and adjusted to 0.22 mg / ml. Particle size measurements were performed using a particle size analyzer, NanoBrook 90Plus (Brookhaven Instruments Corporation).

[0301] Anti-CD71, anti-TREML2, and anti-EpCAM antibodies were conjugated to ferrofluid using the same method.

[0302] Blood processing A 7.5 ml aliquot of blood was diluted with 6.5 ml of dilution buffer (Menarini Silicon Biosystems).

[0303] To inhibit endogenous ferrofluid aggregating factors, blood samples (7.5 ml aliquots) are preincubated with 6.5 ml of dilution buffer (Menarini Silicon Biosystems) containing one or more inhibitors (described in EP 1311820, incorporated by reference in its entirety) before adding ferrofluid to the blood. One inhibitor can be a reducing agent, such as 100 mM mercaptoethanesulfonic acid, which inhibits IgM-induced agglutination without affecting the ligand used to label the cells. The reducing agent can be added to the blood as a single reagent. A second inhibitor can be bovine serum albumin, which can be included in the buffer at 10 mg / ml to neutralize any HAMAA. A third inhibitor can be a nonspecific mouse antibody, particularly one of an appropriate isotype matching the antibody in the ferrofluid. This third inhibitor can be included in the buffer at a concentration of 0.5–5 mg / ml to neutralize even the most troublesome HAMAA. A fourth inhibitor can be streptavidin, which can be included in the buffer if necessary to neutralize any anti-streptavidin antibodies present in the plasma. Pretreatment of the blood with the above buffer and reducing agent can last from 15 to 30 minutes to neutralize all endogenous coagulation factors. During this incubation period, the diluted blood is centrifuged at 800 g for 10 minutes at room temperature without braking to remove the plasma.

[0304] After neutralizing all endogenous agglutinants, an exogenous ferrofluid agglutinant (streptavidin) is added to the sample followed by the addition of ferrofluid, which is coupled to a target-specific antibody and to another ligand specific for the exogenous agglutinant, e.g., desthiobiotin (binding pair desthiobiotin-streptavidin).

[0305] For enrichment of fetal trophoblast cells, anti-CD105 ferrofluid, anti-EpCAM ferrofluid, and / or anti-TREML2 ferrofluid were used. For enrichment of fetal erythroblasts, anti-CD71 ferrofluid and / or anti-TREML2 ferrofluid were used. After optimally labeling target cells with ferrofluid and inducing ferrofluid aggregation with exogenous agglutination factors, the sample was subjected to magnetic separation to enrich for the target.

[0306] The sample was placed in a magnetic separator (Immunicon catalog number QS-012) for 10 minutes. The sample was removed from the magnet, mixed by vortexing, and placed back in the magnetic separator for an additional 10 minutes. The sample was removed from the magnet, mixed again, and placed back in the magnetic separator for an additional 20 minutes to capture the magnetically labeled cells. The uncollected sample was aspirated, and the magnetically captured cells were resuspended in 3 ml of wash dilution buffer and re-separated in the magnetic separator for 10 minutes. The uncollected sample was discarded, and the captured cells were resuspended after removing the tube from the magnetic separator. After removing all non-targets, the magnetically labeled targets and free ferrofluid were resuspended in buffer. In some cases, exogenous ferrofluid aggregation is reversed. Reversal of aggregation is achieved by resuspending the final sample in a buffer containing a disaggregating agent that binds to the exogenous aggregation agent (if the binding pair is desthiobiotin-streptavidin, the exogenous agent that breaks aggregation can be biotin). Without wishing to be bound by theory, the disaggregating agent disaggregates any ferrofluid aggregates, making the cells available for further analysis.

[0307] For trophoblasts, the enriched cells were fluorescently labeled with phycoerythrin (PE)-conjugated anti-TREML2 monoclonal antibody (mAb), a nucleic acid dye (Hoechst 33342) for DNA staining, and allophycocyanin (APC)-conjugated anti-cytokeratin mAb C11, APC-conjugated anti-HLA-G mAb, and / or fluorescein isothiocyanate (FITC)-conjugated anti-CD45 mAb for leukocyte recognition.

[0308] For erythroblasts, the enriched cells were fluorescently labeled with phycoerythrin (PE)-conjugated anti-TREML2 monoclonal antibody (mAb), and for erythroblasts, the enriched cells were further fluorescently labeled with a nucleic acid dye (Hoechst 33342) for DNA staining, phycoerythrin (PE)-conjugated anti-CD71 monoclonal antibody (mAb), and / or fluorescein isothiocyanate (FITC)-conjugated anti-CD45 mAb.

[0309] Stained cells were fixed with 2% paraformaldehyde (PFA) for 20 minutes at room temperature, then washed and resuspended in the appropriate buffer (and volume) for DEPArray™ NxT system (Menarini Silicon Biosystems) or FACS analysis.

[0310] DEPArray analysis DEPArray™ NxT is a semiconductor-based technology for the precise isolation of pure single cells. Comprised of a DEPArray™ control unit and disposable cartridge, the DEPArray™ NxT combines state-of-the-art microfluidic and silicon biochip technologies to gently manipulate each single target cell in an enriched sample.

[0311] The phenomenon that allows cells to be manipulated inside the chip is called "dielectrophoresis" and is based on the ability to polarize particles within a liquid suspension medium through the action of an electric field. Polarization creates a field of force that can be used to trap each individual particle in a series of potential wells, thus allowing the particle's position to be controlled. Each potential well can be controlled by changing the chip's programming to move one or more particles from their initial position to their final destination for collection.

[0312] DEPArray™ enables the selection and isolation of rare cells with very high resolution (down to single cells) and very high purity. Cells are selected via multiparameter analysis of fluorescent signals and morphological properties obtained by processing brightfield or fluorescent images.

[0313] This technique has already been used to isolate and select single circulating tumor cells in the blood of tumor patients (as described in EP 1311820, which is incorporated by reference in its entirety).

[0314] Whole blood samples from healthy volunteers were spiked with villous cultures containing fetal trophoblast cells with trisomy 21. Samples were concentrated and stained as previously described.

[0315] Trophoblast cells were analyzed using the DEPArray™ NxT system. Trophoblast cells showed positive staining for TREML2. Cells showing positive staining for pan-cytokeratin (CK), undetectable CD45 labeling, and positive nuclear staining were classified as fetal trophoblasts and isolated as single cells.

[0316] Whole blood samples from healthy volunteers were spiked with umbilical cord blood containing fetal erythroblasts prelabeled with Draq5 nuclear dye. Samples were enriched and stained using CD71-Ab ferrofluid and TREML2-Ab ferrofluid as previously described. Erythroblast-enriched cells were analyzed using a DEPArray™ NxT system. Cells showing positive staining for CD71, undetectable CD45 labeling, and positive nuclear staining for Hoechst / Draq5 were classified as fetal erythroblasts.

[0317] Demonstration of fetal cell origin by short tandem repeat (STR) analysis The isolated cells were lysed using the DEPArray™ LysePrep kit (MSB, Italy) according to the manufacturer's instructions.

[0318] DNA from single cells was PCR amplified using the PowerPlex Fusion 6c Human DNA Amplification Kit (Promega TMD045), which consists of a multiplex primer set targeting 27 loci spanning the human genome.

[0319] Genomic DNA was also isolated from 200 μl of maternal whole blood using a QIAgen DSP Blood Mini Kit (QIAgen) and served as a control. When available, fetal genomic DNA obtained from either direct or cultured CVS tissue or amniotic fluid was also analyzed.

[0320] STR was performed according to the manufacturer's recommendations, and fragment analysis was performed using a ThermoFisher Scientific 3500 Genetic Analyzer (POP-4 and 36 cm capillary array). Subsequent software analysis was performed using GeneMapper® ID-X v1.4. The allele patterns of isolated single cells were then compared with fetal and parental genomic DNA patterns to assess allele dropout and potential inheritance patterns.

[0321] POC: Clinical study of 20 first trimester pregnant women Twenty milliliters of peripheral blood samples were collected by venipuncture into 10-mL CellSave storage tubes (Menarini Silicon Biosystems, Huntingdon Valley, PA, USA) from 14 pregnant women aged 12 to 17+2 weeks. All samples were processed 1 to 4 days later. Fetal trophoblast cells were successfully isolated from the 14 pregnant women (Table X). An average of 1.4 fetal trophoblast cells were isolated from the 14 positive pregnant women.

[0322] Copy number variation analysis (CNV) Whole blood samples from healthy volunteers were spiked with villous cultures containing fetal trophoblast cells, and the samples were concentrated and stained as previously described.

[0323] Whole genome amplification (Ampli1 WGA, Menarini Silicon Biosystems) was performed using single fetal trophoblast cells recovered from the DEPArray™ NxT.

[0324] Five microliters of the Ampli1™ WGA product was purified using 1.8× SPRIselect beads (Beckman Coulter) according to the manufacturer's instructions and eluted in 12.5 μl of TE buffer using the Ampli1™ Low pass kit (Menarini Silicon Biosystems) for library preparation.

[0325] FASTQ files from 13 Ampli1™ LowPass libraries were aligned to the hg19 reference genome using BWA. Copy number profiles were calculated using Control-FREEC with GC normalization without control samples. Copy number plots were generated using custom Python scripts.

[0326] result Figure 8 shows a schematic diagram of the workflow for fetal cell enrichment. As shown in Figure 8, the workflow consists of three separate steps: 1. Sample collection and capture of target cells using ferrofluid-coupled antibodies that specifically select the target cells. 2. Target cells are labeled with the selected antibody and loaded into a DEPArray cartridge for screening and selection. The selected single cells are then sorted using the DEPArray instrument. 3. The sorted single cells are analyzed by STR (short tandem repeat) technology to verify their fetal cell origin.

[0327] In this example, fetal cells are enriched and stained from whole blood of a pregnant woman. Pure single cells are isolated using DEPArray™ for whole genome amplification and analysis.

[0328] Figures 9-10 demonstrate the specificity of the novel TREML2 antibody, as shown by flow cytometry analysis of TREML2 (i.e., TLS) expression on erythroblasts isolated from fetal blood (FB) (Figure 9) and bone marrow (BM) samples (Figure 10). As shown in Figures 9-10, erythroblasts isolated from fetal blood or bone marrow samples were gated as follows: (1) FSC-A / SSC-A to gate the major cell population, (2) gate Sytox Green-negative live cells, (3) FSC-H / W to exclude doublet cells, (4) gate double-positive GPA / Hoechst, (5) gate CD71-positive / CD45-negative, and (6) gate TLS and overlay with isotype controls to determine the % of TREML2-positive cells.

[0329] Figures 11A-11J show TLS expression on erythroblasts isolated from various fetal blood (FB) samples from various clones. Figures 12A-12L show TLS expression on erythroblasts isolated from various bone marrow (BM) samples from various clones. As Figures 11A-11J show, fetal erythroblasts from fetal blood showed positive staining with the TREML2 antibody, whereas no expression is detectable in adult erythroblasts isolated from bone marrow (BM) (Figures 12A-12L).

[0330] [Table 3]

[0331] [Table 4]

[0332] To demonstrate the specificity of CD105-FF and EpCAM-FF capture and enrichment, trophoblast cells from CVS cultures were used.

[0333] Figure 13 shows a scatter plot analysis of TREML2-positive trophoblast cells identified by DEPArray™ after spiking and enrichment with CD105-FF and EpCAM-FF. Figure 14 shows a CellBrowser® image gallery. Trophoblast cells show positive staining for the TREML-2-PE antibody, CK-APC, and nuclear stain.

[0334] To demonstrate the specificity of CD71 or TREML-2 capture and enrichment, umbilical cord blood-derived erythroblasts were used.

[0335] Figure 15A shows a scatter plot analysis of Draq5 / Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with CD71-FF. Figure 15B shows a CellBrowser® image gallery. Erythroblast cells show positive staining for CD71-PE antibody, Draq5, and Hoechst nuclear stain, and negative staining for CD45-FITC antibody.

[0336] Figure 16A shows a scatter plot analysis of Draq5 / Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with TREML-2-FF. Figure 16B shows a CellBrowser® image gallery. Erythroblast cells show positive staining for CD71-PE antibody, Draq5, and Hoechst nuclear stain, and negative staining for CD45-FITC antibody.

[0337] The sorted single cells were analyzed by STR (short tandem repeat) technology to verify their fetal cell origin (by comparison with maternal DNA and fetal DNA analysis from amniocentesis). The same locus profile was detected. Figure 17 shows the STR analysis from a single fetal cell.

[0338] In a preliminary clinical trial, 14 pregnant women at various stages of gestation were enrolled, and STR analysis showed that fetal cells were positively detected in blood samples from the pregnant women.

[0339] [Table 5]

[0340] To demonstrate that sampling of trisomy chr21 can be detected from single-cell recovery of fetal cells derived from chorionic villus sampling (VK), we performed copy number variation (CNV) analysis.

[0341] Figure 18 shows the results of CNV analysis of fetal cells. As Figure 18 shows, single cell recoveries from the DEPArray (e.g., from recovery 1 (R1), recovery 3 (R3), and recovery 6 (R6)) confirm the presence of trisomy chr21 on the library from chorionic villus sampling (VK).

[0342] Figure 19 shows the results of CNV analysis of healthy donors. As Figure 19 shows, healthy donors (HD) exhibited flat copy number profiles, similar to the profiles obtained with PBMC single cells isolated from DEPArray.

[0343] [Example 4] Workflow sequence for selecting nRBCs from maternal blood This example describes one method for selecting nucleated red blood cells (nRBCs) from a blood sample from a pregnant subject. As shown in FIG. 6, a blood sample is collected from a pregnant subject (601). The blood sample is collected into a CellSave tube (601). The nRBCs can be concentrated by magnetic separation (602, e.g., ferrofluid concentration). Alternatively or additionally, the sample can be processed using a CELLTRACKS® AUTOPREP® system (603). Single cells can be visualized and isolated using DEPArray™ NxT control unit image-based technology. Once the cells are isolated, nucleic acids are purified from the isolated cells (605). Genomic and / or genetic analysis is performed (606). For example, nucleic acid molecules are sequenced to detect chromosomal abnormalities.

[0344] [Example 5] RNA Sequencing Protocol Nucleic acid molecules, such as RNA, can be isolated from rare cells (e.g., fetal cells). This example provides an exemplary method for sequencing RNA from fetal cells.

[0345] SMART-Seq V2 For RT-PCR, Smartseq version 2 was adapted with some modifications.

[0346] For fetal erythroblasts (EBs), 2 ng of total RNA input was used for reverse transcription. For maternal EBs, total RNA was concentrated and used for reverse transcription due to the limited number of maternal EBs available for sorting.

[0347] (1) Reverse transcription 1 μl of oligo dT 30VN primer (10 uM) and 1 ul of dNTP mix (10 mM each) are added to the sample tube.

[0348] Samples are incubated at 72°C for 3 minutes and immediately placed on ice.

[0349] Reverse transcription mix is ​​prepared on ice as follows and 5.7 ul is added to each sample.

[0350] JPEG2026032067000009.jpg84157

[0351] The reaction is incubated in a thermocycler as follows.

[0352] JPEG2026032067000010.jpg47157

[0353] (2) PCR preamplification Prepare PCR mix on ice as follows and add 15 ul to each sample.

[0354] JPEG2026032067000011.jpg41157

[0355] The samples are placed in a thermocycler and the following program is run:

[0356] JPEG2026032067000012.jpg54157

[0357] The number of PCR cycles depends on the cell type and can be increased (cells with low RNA content) or decreased (cells with higher amounts of RNA).

[0358] (3) PCR purification The amplified cDNA product is purified twice using 0.5x reaction volume of AMPure XP beads (Beckman Coulter). The purified cDNA is quantified on an Agilent 2100 Bioanalyzer using a high-sensitivity DNA kit.

[0359] (4) Illumina Nextera XT DNA sample preparation To prepare the libraries, the Illumina NEXTERA XT DNA kit is used with modifications (cDNA sample, reagent volumes and reaction volumes were optimized to 1 / 4 of the volumes in the manufacturer's instructions).

[0360] Dilute the cDNA accordingly to obtain 300 pg.

[0361] 1.25 ul (300 pg) of cDNA was dispensed into 0.2 PCR tubes.

[0362] Add 2.5 ul of tagment DNA buffer and 1.25 ul of amplicon tagment mix.

[0363] The tagmentation reaction is incubated in a thermocycler at 55 degrees for 5 minutes.

[0364] Immediately add 1.25 ul of NT and incubate at room temperature for 5 minutes.

[0365] Add 1.25ul of Index1, 1.25ul of Index2 and 3.75ul of Nextera PCR Master Mix (NPM) to the tagmented DNA.

[0366] Amplification is carried out using the following program:

[0367] JPEG2026032067000013.jpg61157

[0368] (5) Library DNA cleanup (purification): AMPure XP beads (0.6x the reaction volume) are added to the library DNA.

[0369] Discard the beads and save the supernatant for the first cleanup.

[0370] In the second cleanup, AMPure XP beads (0.7x the reaction volume) are added.

[0371] The beads are set aside and the DNA fragments are eluted.

[0372] Successful libraries (average 400 bp) are quantified on an Agilent 2100 bioanalyzer using a high sensitivity DNA kit.

[0373] To pool libraries, adjust each of the library samples to 10 nM and pool by volume.

[0374] (6) Library DNA sequencing: The library was submitted to a sequencing facility and paired-end sequenced (2 x 101 bp) using an Illumina HiSeq™ High Output v3 system.

[0375] [Example 6] Detection of trophoblast cells This example describes one method for detecting trophoblast cells, using FerroFluid technology for cell capture and selection, and DEPArray technology for cell sorting.

[0376] Trophoblast cells are captured using FerroFluid technology and controlled aggregation. A blood sample from a pregnant subject is contacted with a magnetic fluid containing magnetic colloid particles conjugated to an anti-EpCAM antibody (EpCAM-FF) or a magnetic fluid containing magnetic colloid particles conjugated to an anti-CD105 antibody (CD105-FF). The blood sample contains multiple cells (fetal cells and maternal cells). A first exogenous aggregation enhancer, such as desthiobiotin, is conjugated to the magnetic colloid particles. A second exogenous aggregation enhancer, such as streptavidin, is added to the sample. Without wishing to be bound by theory, the addition of the second exogenous aggregation enhancer induces aggregation of the magnetic colloid particles, thereby facilitating the isolation of fetal cells and reducing contamination with non-fetal cells. The sample is then placed on a magnetic separator to isolate cells bound to EpCAM-FF or CD105-FF.

[0377] To facilitate further analysis of cells bound with EpCAM-FF or CD105-FF, a third exogenous aggregation-enhancing factor, such as biotin, is added to the isolated cells. Without wishing to be bound by theory, the addition of the third exogenous aggregation-enhancing factor reverses the aggregation of magnetic colloid particles, making single cells easier to analyze.

[0378] DEPArray technology for cell sorting: TLS1 (i.e., TREML2) is used as a candidate stain for trophoblast cells. Isolated cells are stained with fluorescently labeled anti-TLS antibody (i.e., anti-TREML2 antibody), anti-HLA-G antibody, and cytokeratin. The isolated and stained cells are loaded onto a DEPArray cartridge and analyzed using a DEPArray instrument. Cells are identified as trophoblast cells if they stain positive for TLS, HLA-G, and cytokeratin staining.

[0379] [Example 7] Diagnosis of fetal abnormalities Fetal cells isolated or identified by any of the methods disclosed herein can be further analyzed to diagnose fetal abnormalities. A karyotype test is performed on the fetal cells to detect chromosomal abnormalities. If a chromosomal abnormality is detected, the fetus is diagnosed with the corresponding disorder. For example, if three copies of chromosome 21 are detected, the fetus is diagnosed with Down syndrome. In another example, if three copies of chromosome 18 are detected, the fetus is diagnosed with Edwards syndrome.

[0380] JPEG2026032067000014.jpg88170JPEG2026032067000015.jpg127170

[0381] SEQUENCE LISTING <110> A. MENARINI BIOMARKERS SINGAPORE PTE LTD. <120> COMPOSITIONS AND METHODS FOR ISOLATING, DETECTING, AND ANALYZING FETAL CELLS <130> PA25-503 <141> 2020-07-14 <150> US 62 / 874,306 <151> 2019-07-15 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 321 <212> PRT <213> Homo sapiens <400> 1 Met Ala Pro Ala Phe Leu Leu Leu Leu Leu Leu Trp Pro Gln Gly Cys 1 5 10 15 Val Ser Gly Pro Ser Ala Asp Ser Val Tyr Thr Lys Val Arg Leu Leu 20 25 30 Glu Gly Glu Thr Leu Ser Val Gln Cys Ser Tyr Lys Gly Tyr Lys Asn 35 40 45 Arg Val Glu Gly Lys Val Trp Cys Lys Ile Arg Lys Lys Lys Cys Glu 50 55 60 Pro Gly Phe Ala Arg Val Trp Val Lys Gly Pro Arg Tyr Leu Leu Gln 65 70 75 80 Asp Asp Ala Gln Ala Lys Val Val Asn Ile Thr Met Val Ala Leu Lys 85 90 95 Leu Gln Asp Ser Gly Arg Tyr Trp Cys Met Arg Asn Thr Ser Gly Ile 100 105 110 Leu Tyr Pro Leu Met Gly Phe Gln Leu Asp Val Ser Pro Ala Pro Gln 115 120 125 Thr Glu Arg Asn Ile Pro Phe Thr His Leu Asp Asn Ile Leu Lys Ser 130 135 140 Gly Thr Val Thr Thr Gly Gln Ala Pro Thr Ser Gly Pro Asp Ala Pro 145 150 155 160 Phe Thr Thr Gly Val Met Val Phe Thr Pro Gly Leu Ile Thr Leu Pro 165 170 175 Arg Leu Leu Ala Ser Thr Arg Pro Ala Ser Lys Thr Gly Tyr Ser Phe 180 185 190 Thr Ala Thr Ser Thr Thr Ser Gln Gly Pro Arg Arg Thr Met Gly Ser 195 200 205 Gln Thr Val Thr Ala Ser Pro Ser Asn Ala Arg Asp Ser Ser Ala Gly 210 215 220 Pro Glu Ser Ile Ser Thr Lys Ser Gly Asp Leu Ser Thr Arg Ser Pro 225 230 235 240 Thr Thr Gly Leu Cys Leu Thr Ser Arg Ser Leu Leu Asn Arg Leu Pro 245 250 255 Ser Met Pro Ser Ile Arg His Gln Asp Val Tyr Ser Thr Val Leu Gly 260 265 270 Val Val Leu Thr Leu Leu Val Leu Met Leu Ile Met Val Tyr Gly Phe 275 280 285 Trp Lys Lys Arg His Met Ala Ser Tyr Ser Met Cys Ser Asp Pro Ser 290 295 300 Thr Arg Asp Pro Pro Gly Arg Pro Glu Pro Tyr Val Glu Val Tyr Leu 305 310 315 320 Ile <210> 2 <211> 250 <212> PRT <213> Homo sapiens <400> 2 Gly Pro Ser Ala Asp Ser Val Tyr Thr Lys Val Arg Leu Leu Glu Gly 1 5 10 15 Glu Thr Leu Ser Val Gln Cys Ser Tyr Lys Gly Tyr Lys Asn Arg Val 20 25 30 Glu Gly Lys Val Trp Cys Lys Ile Arg Lys Lys Lys Cys Glu Pro Gly 35 40 45 Phe Ala Arg Val Trp Val Lys Gly Pro Arg Tyr Leu Leu Gln Asp Asp 50 55 60 Ala Gln Ala Lys Val Val Asn Ile Thr Met Val Ala Leu Lys Leu Gln 65 70 75 80 Asp Ser Gly Arg Tyr Trp Cys Met Arg Asn Thr Ser Gly Ile Leu Tyr 85 90 95 Pro Leu Met Gly Phe Gln Leu Asp Val Ser Pro Ala Pro Gln Thr Glu 100 105 110 Arg Asn Ile Pro Phe Thr His Leu Asp Asn Ile Leu Lys Ser Gly Thr 115 120 125 Val Thr Thr Gly Gln Ala Pro Thr Ser Gly Pro Asp Ala Pro Phe Thr 130 135 140 Thr Gly Val Met Val Phe Thr Pro Gly Leu Ile Thr Leu Pro Arg Leu 145 150 155 160 Leu Ala Ser Thr Arg Pro Ala Ser Lys Thr Gly Tyr Ser Phe Thr Ala 165 170 175 Thr Ser Thr Thr Ser Gln Gly Pro Arg Arg Thr Met Gly Ser Gln Thr 180 185 190 Val Thr Ala Ser Pro Ser Asn Ala Arg Asp Ser Ser Ala Gly Pro Glu 195 200 205 Ser Ile Ser Thr Lys Ser Gly Asp Leu Ser Thr Arg Ser Pro Thr Thr 210 215 220 Gly Leu Cys Leu Thr Ser Arg Ser Leu Leu Asn Arg Leu Pro Ser Met 225 230 235 240 Pro Ser Ile Arg His Gln Asp Val Tyr Ser 245 250 <210> 3 <211> 63 <212> PRT <213> Homo sapiens <400> 3 Ser Ala Asp Ser Val Tyr Thr Lys Val Arg Leu Leu Glu Gly Glu Thr 1 5 10 15 Leu Ser Val Gln Cys Ser Tyr Lys Gly Tyr Lys Asn Arg Val Glu Gly 20 25 30 Lys Val Trp Cys Lys Ile Arg Lys Lys Lys Cys Glu Pro Gly Phe Ala 35 40 45 Arg Val Trp Val Lys Gly Pro Arg Tyr Leu Leu Gln Asp Asp Ala 50 55 60 <210> 4 <211> 50 <212> PRT <213> Homo sapiens <400> 4 Gly Arg Tyr Trp Cys Met Arg Asn Thr Ser Gly Ile Leu Tyr Pro Leu 1 5 10 15 Met Gly Phe Gln Leu Asp Val Ser Pro Ala Pro Gln Thr Glu Arg Asn 20 25 30 Ile Pro Phe Thr His Leu Asp Asn Ile Leu Lys Ser Gly Thr Val Thr 35 40 45 Thr Gly 50 <210> 5 <211> 50 <212> PRT <213> Homo sapiens <400> 5 Thr Gly Tyr Ser Phe Thr Ala Thr Ser Thr Thr Ser Gln Gly Pro Arg 1 5 10 15 Arg Thr Met Gly Ser Gln Thr Val Thr Ala Ser Pro Ser Asn Ala Arg 20 25 30 Asp Ser Ser Ala Gly Pro Glu Ser Ile Ser Thr Lys Ser Gly Asp Leu 35 40 45 Ser Thr 50 <210> 6 <211> 10 <212> PRT <213> Mus musculus <400> 6 Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 10 <210> 7 <211> 7 <212> PRT <213> Mus musculus <400> 7 Ile Trp Trp Tyr Asp Asp Lys 1 5 <210> 8 <211> 12 <212> PRT <213> Mus musculus <400> 8 Val Arg Ile Glu Ser Thr Met Ile Thr Gly Asp Tyr 1 5 10 <210> 9 <211> 10 <212> PRT <213> Mus musculus <400> 9 Gln Ser Val Asp Tyr Asp Gly Tyr Ser Tyr 1 5 10 <210> 10 <211> 3 <212> PRT <213> Mus musculus <400> 10 Ala Ala Ser 1 <210> 11 <211> 9 <212> PRT <213> Mus musculus <400> 11 Gln Gln Ser Ile Glu Asp Pro Trp Thr 1 5

Claims

1. 1. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody; (b) isolating cells that bind to the first antibody to generate an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying cells that bind to the second antibody as fetal cells; The first antibody or the second antibody is (i) an antibody that binds to the triggering receptor-like 2 (TREML2) protein expressed on myeloid cells, or (ii) an antigen-binding fragment that binds to the TREML2 protein.

2. 2. The method of claim 1, wherein the fetal cells are fetal nucleated red blood cells (fnRBCs).

3. 3. The method of claim 1 or 2, wherein the first antibody is conjugated to one or more magnetic particles.

4. The method of claim 3 , wherein the magnetic particles are magnetic colloidal particles.

5. 5. The method of claim 4, wherein the magnetic colloid particles are ferrofluid magnetic particles.

6. 6. The method of claim 3, wherein step (b) comprises exposing the sample to a magnetic field.

7. 7. The method of claim 6, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

8. The method of claim 7, wherein step (a) comprises adding a second EAEF containing the other member of the specific binding pair to induce aggregation of the magnetic particles.

9. 9. The method of claim 8, wherein step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse aggregation of magnetic particles in the enriched sample.

10. 10. The method of claim 1, further comprising, prior to step (a), adding to the sample at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

11. 11. The method of claim 10, wherein the aggregation inhibitor is a chelating agent that is EDTA.

12. 12. The method of claim 1, wherein the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

13. 13. The method of claim 12, further comprising the step of isolating a single fetal cell prior to step (d).

14. 14. The method of claim 13, wherein the step of isolating a single fetal cell is carried out by isolating a single fetal cell bound to a second antibody.

15. 15. The method of claim 14, wherein the second antibody is conjugated to a label.

16. 16. The method of claim 15, wherein the label is a fluorescent label.

17. 17. The method of claim 16, wherein the step of isolating single fetal cells is based on immunofluorescence techniques.

18. 18. The method of claim 17, wherein the step of isolating single fetal cells is performed by fluorescence-activated cell sorting (FACS).

19. 18. The method of claim 17, wherein the step of isolating a single fetal cell is performed by DEPArray.

20. 20. The method of any one of claims 1 to 19, wherein step (d) comprises performing sequence analysis.

21. 21. The method of claim 20, wherein the sequence analysis comprises short tandem repeat (STR) analysis.

22. 22. The method of any one of claims 1 to 21, further comprising the step of analyzing fetal cells.

23. 23. The method of claim 22, wherein analyzing the fetal cells comprises performing genomic or genetic analysis.

24. 24. The method of claim 23, wherein performing a genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

25. 25. The method of any one of claims 1 to 24, wherein the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

26. An antibody that binds to the TREML2 protein, or an antigen-binding fragment that binds to the TREML2 protein, (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 26. The method of any one of claims 1 to 25, comprising one or more CDRs selected from:

27. 1. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a magnetic reagent comprising magnetic particles conjugated to a first antibody that binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or antigen-binding fragment thereof; and (c) identifying the cells that bind to the anti-TREML2 antibody as fetal cells. A method comprising:

28. 28. The method of claim 27, further comprising, prior to step (c), isolating cells that bind to the first antibody.

29. 30. The method of claim 28, wherein isolating the cells comprises subjecting the sample to a magnetic field to enrich the sample for cells that are bound to the first antibody.

30. 30. The method of any one of claims 27 to 29, wherein the magnetic particles are magnetic colloidal particles.

31. 31. The method of claim 30, wherein the magnetic colloid particles are ferrofluid magnetic particles.

32. 32. The method of any one of claims 27 to 31, wherein the magnetic particles are further coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

33. 33. The method of claim 32, further comprising, during step (a), adding a second EAEF comprising the other member of the specific binding pair to induce particle agglutination.

34. 34. The method of claim 33, further comprising the step of adding a member of a specific binding pair to the enriched sample to reverse agglutination, thereby facilitating identification of the cells.

35. 35. The method of any one of claims 27 to 34, further comprising the step of adding to the sample, prior to step (a), at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

36. 36. The method of claim 35, wherein the aggregation inhibitor is a chelating agent.

37. 37. The method of claim 36, wherein the chelating agent is EDTA.

38. 38. The method of any one of claims 27 to 37, further comprising, prior to step (c), isolating the cells using an anti-TREML2 antibody or a second antibody.

39. 39. The method of claim 38, wherein the second antibody is selected from an anti-cytokeratin antibody and an anti-HLAG antibody.

40. 40. The method of claim 38 or 39, wherein the anti-TREML2 antibody or the second antibody is conjugated to a label.

41. 41. The method of claim 40, wherein the label is a fluorescent label.

42. 42. The method of claim 41, wherein the step of isolating the cells is based on immunofluorescence techniques.

43. 43. The method of claim 42, wherein the step of isolating the cells is performed by fluorescence-activated cell sorting (FACS).

44. The method of claim 41, wherein the step of isolating cells is performed by DEPArray.

45. 45. The method of any one of claims 27 to 44, wherein identifying the cells comprises performing sequence analysis.

46. 46. ​​The method of claim 45, wherein the sequence analysis comprises short tandem repeat (STR) analysis.

47. 47. The method of any one of claims 27 to 46, further comprising analyzing fetal cells.

48. 48. The method of claim 47, wherein analyzing the fetal cells comprises performing genomic or genetic analysis.

49. 49. The method of claim 48, wherein performing a genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

50. 50. The method of any one of claims 27 to 49, wherein the fetal cells are fetal erythroblasts or fetal trophoblast cells.

51. The anti-TREML2 antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 51. The method of any one of claims 27 to 50, comprising one or more complementarity determining regions (CDRs) selected from:

52. 52. The method of claim 51, wherein any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

53. 51. The method of any one of claims 27 to 50, wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

54. A kit comprising: (a) an antibody (anti-TREML2 antibody) or an antigen-binding fragment thereof that binds to the triggering receptor-like 2 (TREML2) protein expressed on myeloid cells; and (b) a magnetic reagent comprising magnetic colloid particles.

55. 55. The kit of claim 54, wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

56. The anti-TREML2 antibody or antigen-binding fragment thereof is (a) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 55. The kit of claim 54, comprising one or more complementarity determining regions (CDRs) selected from:

57. 57. The kit of claim 56, wherein any one of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions.

58. 58. The kit of any one of claims 54 to 57, wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label to generate a binding antibody.

59. 59. The kit of claim 58, wherein the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

60. 60. The kit of any one of claims 54 to 59, wherein the magnetic colloid particles have dimensions of less than 200 nm.

61. 61. The kit of claims 54 to 60, wherein the magnetic colloid particles are ferrofluid particles.

62. 62. The kit of any one of claims 54 to 61, wherein the magnetic colloid particles are conjugated to an antibody or antigen-binding fragment thereof.

63. 63. The kit of claim 62, wherein the antibody is an anti-TREML2 antibody.

64. The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 64. The kit of claim 63, comprising one or more CDRs selected from:

65. 66. The kit of claim 65, wherein any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

66. 66. The kit of any one of claims 54 to 65, further comprising an inhibitor selected from the group consisting of a reducing agent, an immunoconjugate, a chelating agent, and diaminobutane.

67. 67. The kit of claim 66, wherein the chelating agent is EDTA.

68. 68. The kit of any one of claims 54 to 67, further comprising an exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

69. A kit comprising: (a) a first antibody bound to magnetic colloid particles, capable of binding to a protein expressed on the surface of fetal cells; and (b) an anti-TREML2 antibody or antigen-binding fragment thereof.

70. 66. The kit of claim 65, wherein the first antibody binds to a protein selected from EpCAM, CD105, and CD71.

71. 69. The kit of claim 27, wherein the magnetic colloid particles are ferrofluid particles.

72. The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 72. The kit of any one of Claims 69 to 71, comprising one or more CDRs selected from:

73. 73. The kit of claim 72, wherein any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

74. 74. The kit of any one of claims 69 to 73, further comprising an inhibitor selected from the group consisting of a reducing agent, an immunoconjugate, a chelating agent, and diaminobutane.

75. 75. The kit of claim 74, wherein the chelating agent is EDTA.

76. 76. The kit of any one of claims 69 to 75, further comprising an exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

77. 1. A cell-based fetal genetic testing method comprising: (a) contacting a sample obtained from a pregnant subject, the sample comprising a plurality of cells, with an anti-TREML2 antibody or antigen-binding fragment thereof; (b) isolating cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, the report providing a probability of the fetus having one or more genetic abnormalities. A method comprising:

78. 78. The method of claim 77, wherein the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof are fetal cells.

79. 79. The method of claim 78, wherein the fetal cells are fetal erythroblasts.

80. 79. The method of claim 78, wherein the fetal cells are fetal trophoblast cells.

81. 81. The method of any one of Claims 77 to 80, wherein analyzing one or more nucleic acid molecules comprises performing karyotyping.

82. 81. The method of any one of Claims 77 to 80, wherein analyzing the one or more nucleic acid molecules comprises performing sequence analysis.

83. 83. The method of claim 82, wherein the sequence analysis comprises short tandem repeat (STR) analysis.

84. 84. The method of any one of claims 77 to 83, wherein the one or more genetic abnormalities are selected from trisomy, sex chromosome abnormalities and structural abnormalities.

85. 85. The method of claim 84, wherein the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21 and trisomy 22.

86. 86. The method of claim 85, wherein the sex chromosome abnormality is selected from monosomy X, triple X syndrome, and Klinefelter syndrome.

87. 87. The method of claim 86, wherein the structural abnormality is a copy number variation (CNV).

88. 88. The method of claim 87, wherein the structural abnormality is a CNV deletion or a CNV duplication.

89. 89. The method of any one of claims 77 to 88, wherein the anti-TREML2 antibody is conjugated to a magnetic particle.

90. 90. The method of claim 89, wherein the magnetic particles are magnetic colloidal particles.

91. 91. The method of claim 90, wherein the magnetic colloid particles are ferrofluid magnetic particles.

92. 92. The method of any one of claims 89 to 91, wherein step (b) comprises exposing the sample to a magnetic field.

93. 89. The method of any one of claims 77 to 88, further comprising, prior to step (a), contacting the sample with a first antibody that binds to a protein selected from EpCAM, CD105, and CD71.

94. 94. The method of claim 93, further comprising, prior to step (a), isolating cells that bind to the first antibody.

95. 95. The method of claim 93 or 94, wherein the first antibody is conjugated to a magnetic particle.

96. 96. The method of claim 95, wherein the magnetic particles are magnetic colloidal particles.

97. 97. The method of claim 96, wherein the magnetic colloid particles are ferrofluid magnetic particles.

98. 98. The method of any one of Claims 95 to 97, wherein isolating cells that bind to the first antibody comprises exposing the sample to a magnetic field.

99. 99. A method according to any one of claims 77 to 88 and claims 93 to 98, wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label.

100. 100. The method of claim 99, wherein the label is a fluorescent label.

101. The method of claim 100, wherein the step of isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is based on immunofluorescence techniques.

102. The method of claim 101, wherein the step of isolating cells bound to the anti-TREML2 antibody or its antigen-binding fragment is carried out by fluorescence-activated cell sorting (FACS).

103. The method of claim 101, wherein the step of isolating cells bound to the anti-TREML2 antibody or its antigen-binding fragment is carried out using DEPArray.

104. 93. The method of any one of claims 77 to 92, further comprising contacting the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof with a second antibody or antigen-binding fragment thereof.

105. The method of claim 104, wherein the second antibody is an anti-TREML2 antibody or an antigen-binding fragment thereof.

106. 106. The method of claim 104 or 105, wherein the second antibody is conjugated to a label.

107. 107. The method of claim 106, wherein the label is a fluorescent label.

108. 108. The method of claim 107, further comprising the step of isolating cells that bind to the second antibody or antigen-binding fragment thereof.

109. 109. The method of claim 108, wherein the step of isolating cells bound to the second antibody or antigen-binding fragment thereof is based on immunofluorescence techniques.

110. 110. The method of claim 109, wherein the step of isolating cells bound to the second antibody or antigen-binding fragment thereof is carried out by fluorescence-activated cell sorting (FACS).

111. The method of claim 109, wherein the step of isolating cells bound to the second antibody or its antigen-binding fragment is carried out by DEPArray.

112. 112. The method of any one of claims 77 to 111, wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

113. The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 112. The method of any one of claims 77 to 111, comprising one or more CDRs selected from:

114. 114. The method of claim 113, wherein any one of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions or deletions.

115. 1. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody, wherein the first antibody binds to a triggering receptor-like 2 (TREML2) protein expressed on myeloid cells (an anti-TREML2 antibody) or an antigen-binding fragment thereof; and (b) identifying cells that bind to the first antibody as fetal cells; A method comprising:

116. 116. The method of claim 115, wherein the fetal cells are fetal nucleated red blood cells (fnRBCs).

117. 117. The method of claim 115 or 116, wherein the first antibody is conjugated to one or more magnetic particles.

118. 118. The method of claim 117, wherein the magnetic particles are magnetic colloidal particles.

119. 119. The method of claim 118, wherein the magnetic colloid particles are ferrofluid magnetic particles.

120. 120. The method of any one of claims 117 to 119, further comprising exposing the sample to a magnetic field.

121. 121. The method of any one of claims 117 to 120, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

122. 122. The method of claim 121, further comprising, before step (b), adding a second EAEF containing the other member of the specific binding pair to induce aggregation of the magnetic particles.

123. 123. The method of claim 122, further comprising isolating cells that bind to the first antibody to produce an enriched sample.

124. The method of claim 123, further comprising adding a third EAEF capable of binding to the first EAEF or the second EAEF to the concentrated sample in order to reverse the aggregation of magnetic particles in the concentrated sample.

125. 125. The method of claim 124, wherein the third EAEF is a member of the specific binding pair.

126. 126. The method of any one of claims 115 to 125, further comprising the step of adding to the sample, prior to step (a), at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

127. 127. The method of claim 126, wherein the aggregation inhibitor is a chelating agent.

128. 128. The method of claim 127, wherein the chelating agent is EDTA.

129. 117. The method of claim 115 or 116, wherein the first antibody is conjugated to a label.

130. 130. The method of claim 129, wherein the label is a fluorescent label.

131. 131. The method of claim 130, further comprising, prior to step (b), isolating cells bound to the first antibody based on immunofluorescence techniques.

132. 132. The method of claim 131, wherein the step of isolating cells bound to the first antibody is performed by fluorescence-activated cell sorting (FACS).

133. The method of claim 131, wherein the step of isolating cells bound to the first antibody is performed by DEPArray.

134. 134. The method of any one of claims 115 to 133, wherein step (b) comprises performing sequence analysis.

135. 135. The method of claim 134, wherein the sequence analysis comprises short tandem repeat (STR) analysis.

136. 136. The method of any one of claims 115 to 135, further comprising analyzing fetal cells.

137. 137. The method of claim 136, wherein analyzing the fetal cells comprises performing genomic or genetic analysis.

138. 138. The method of claim 137, wherein performing the genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

139. the first antibody or antigen-binding fragment thereof (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 139. The method of any one of claims 115 to 138, comprising one or more CDRs selected from:

140. 140. The method of claim 139, wherein any of SEQ ID NOs: 6-11 independently comprises one or more amino acid substitutions, additions or deletions.

141. 139. The method of any one of claims 115 to 138, wherein the first antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

142. (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 An anti-TREML2 antibody or an antigen-binding fragment thereof, comprising one or more CDRs selected from:

143. 143. The anti-TREML2 antibody of claim 142, wherein any of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions or deletions.

144. The anti-TREML2 antibody of claim 142 or 143, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a) to (f).

145. The anti-TREML2 antibody of claim 142 or 143, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a) to (f).

146. The anti-TREML2 antibody of claim 142 or 143, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a) to (f).

147. The anti-TREML2 antibody of claim 142 or 143, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more CDRs selected from (a) to (f).

148. The anti-TREML2 antibody of claim 142 or 143, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs (a) to (f).

149. 149. The anti-TREML2 antibody of any one of claims 142 to 148, wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label.

150. The anti-TREML2 antibody of claim 149, wherein the label is a fluorescent label.

151. 149. The anti-TREML2 antibody of any one of claims 142 to 148, wherein the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle.

152. The anti-TREML2 antibody of claim 151, wherein the magnetic particles are magnetic colloid particles.

153. The anti-TREML2 antibody of claim 152, wherein the magnetic colloid particles are ferrofluid magnetic particles.

154. 154. The anti-TREML2 antibody of claim 152 or 153, wherein the magnetic colloid particles are less than 200 nm.

155. 154. The anti-TREML2 antibody of claim 152 or 153, wherein the magnetic colloid particles are between about 80 and 200 nm.

156. 154. The anti-TREML2 antibody of claim 152 or 153, wherein the magnetic colloid particles are between about 90 and 150 nm.

157. 157. An anti-TREML2 antibody according to any one of claims 152 to 156, wherein the magnetic colloid particles have a magnetic mass of at least 50%.

158. 158. The anti-TREML2 antibody of claim 157, wherein the magnetic colloid particle has a magnetic mass of at least 60%.

159. 158. The anti-TREML2 antibody of claim 157, wherein the magnetic colloid particle has a magnetic mass of between 70% and 90%.

160. 160. The anti-TREML2 antibody of any one of claims 152 to 159, wherein the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

161. 161. The anti-TREML2 antibody of any one of claims 151 to 160, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group comprising biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

162. An anti-TREML2 antibody conjugate comprising: (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic particle conjugated to the anti-TREML2 antibody.

163. 156. The anti-TREML2 antibody conjugate of claim 155, wherein the magnetic particles are magnetic colloid particles.

164. 157. The anti-TREML2 antibody conjugate of claim 156, wherein the magnetic colloid particles are ferrofluid magnetic particles.

165. 165. The anti-TREML2 antibody conjugate of claim 163 or 164, wherein the magnetic colloidal particles are less than 200 nm.

166. 165. The anti-TREML2 antibody conjugate of claim 163 or 164, wherein the magnetic colloid particle is between about 80 and 200 nm.

167. 165. The anti-TREML2 antibody conjugate of claim 163 or 164, wherein the magnetic colloid particle is between about 90 and 150 nm.

168. 168. An anti-TREML2 antibody conjugate according to any one of claims 163 to 167, wherein the magnetic colloid particle has a magnetic mass of at least 50%.

169. 169. The anti-TREML2 antibody conjugate of claim 168, wherein the magnetic colloid particle has a magnetic mass of at least 60%.

170. 170. The anti-TREML2 antibody conjugate of claim 169, wherein the magnetic colloid particle has a magnetic mass of between 70% and 90%.

171. 171. An anti-TREML2 antibody conjugate according to any one of claims 163 to 170, wherein the magnetic colloid particle comprises a superparamagnetic crystalline core surrounded by coating molecules.

172. 172. The anti-TREML2 antibody conjugate of any one of claims 162 to 171, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group comprising biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

173. The anti-TREML2 antibody or antigen-binding fragment thereof is (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 172. The anti-TREML2 antibody conjugate of any one of claims 162 to 171, comprising one or more CDRs selected from:

174. 174. The anti-TREML2 antibody conjugate of claim 173, wherein any of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions or deletions.

175. 175. The anti-TREML2 antibody conjugate of claim 173 or 174, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a) to (f).

176. 175. The anti-TREML2 antibody conjugate of claim 173 or 174, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a) to (f).

177. 175. The anti-TREML2 antibody conjugate of claim 173 or 174, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a) to (f).

178. 175. The anti-TREML2 antibody conjugate of claim 173 or 174, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more CDRs selected from (a) to (f).

179. 175. The anti-TREML2 antibody conjugate of claim 173 or 174, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs (a) to (f).

180. 172. The anti-TREML2 antibody conjugate of any one of claims 162 to 171, wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

181. 1. A method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, comprising: (a) contacting a maternal sample containing fetal cells and maternal cells with a first antibody conjugate, the first antibody conjugate comprising (i) a first antibody and (ii) magnetic colloid particles, wherein the first antibody is conjugated to the magnetic colloid particles; and (b) isolating cells that bind to the first antibody conjugate by exposing the maternal sample to a magnetic field, thereby preparing a fetal cell sample. A method comprising:

182. 182. The method of claim 181, wherein the magnetic colloid particles are less than 200 nm.

183. 182. The method of claim 181, wherein the magnetic colloid particles are between about 80 and 200 nm.

184. 182. The method of claim 181, wherein the magnetic colloid particles are between about 90 and 150 nm.

185. 185. The method of any one of claims 181 to 184, wherein the magnetic colloid particles have a magnetic mass of at least 50%.

186. 186. The method of claim 185, wherein the magnetic colloid particles have a magnetic mass of at least 60%.

187. 186. The method of claim 185, wherein the magnetic colloid particles have a magnetic mass between 70% and 90%.

188. 188. The method of any one of claims 181 to 187, wherein the magnetic colloidal particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

189. 189. The method of any one of claims 181 to 188, wherein the magnetic colloid particles are ferrofluid magnetic particles.

190. 190. The method of any one of claims 181 to 189, wherein the magnetic colloid particles are further conjugated to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group comprising biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

191. 191. The method of claim 190, further comprising adding to the maternal sample a second EAEF comprising the other member of the specific binding pair.

192. 192. The method of any one of claims 181 to 191, wherein the first antibody is an anti-TREML2 antibody.

193. 192. The method of any one of claims 181 to 191, wherein the first antibody is an anti-CD71 antibody.

194. 192. The method of any one of claims 181 to 191, wherein the first antibody binds to a protein selected from EpCAM and CD105.

195. 195. The method of Claim 194, wherein the step of preparing a fetal cell sample further comprises contacting cells isolated from the maternal sample with a second antibody.

196. 196. The method of claim 195, wherein the second antibody is conjugated to a label.

197. 197. The method of claim 196, wherein the label is a fluorescent label.

198. 198. The method of claim 197, wherein the step of preparing the fetal cell sample further comprises the step of isolating cells that bind to the second antibody.

199. 199. The method of claim 198, wherein the step of isolating cells bound to the second antibody is based on immunofluorescence techniques.

200. 200. The method of claim 199, wherein the step of isolating cells bound to the second antibody is performed by fluorescence-activated cell sorting (FACS).

201. The method of claim 199, wherein the step of isolating cells bound to the second antibody is performed by DEPArray.

202. The method of claim 26, wherein the antibody that binds to the TREML2 protein or the antigen-binding fragment that binds to the TREML2 protein comprises two, three, four, five, or six of the CDRs selected from (i) to (vi).

203. 27. The method of claim 26, wherein any of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions, or deletions.

204. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloidal particles are less than 200 nm.

205. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloid particles are between about 80 and 200 nm.

206. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloid particles are between about 90 and 150 nm.

207. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloid particles have at least 50% magnetic mass.

208. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloid particles have a magnetic mass of at least 60%.

209. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloid particles have a magnetic mass between 70% and 90%.

210. 120. The method of any one of claims 4, 5, 30, 31, 90, 91, 96, 97, 118 and 119, wherein the magnetic colloidal particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

211. 26. The method of any one of claims 1 to 25, wherein the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

212. 1. A method for detecting fetal cells in a sample from a pregnant subject, comprising: (a) contacting a sample comprising a plurality of cells with a first antibody conjugate comprising a first antibody bound to magnetic colloid particles; (b) isolating cells that bind to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody that binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells. A method comprising:

213. 213. The method of claim 212, wherein the fetal cells are fetal nucleated red blood cells (fnRBCs).

214. 214. The method of claim 212 or 213, wherein the magnetic colloid particles are ferrofluid magnetic particles.

215. 215. The method of any one of claims 212 to 214, wherein the magnetic colloid particles are less than 200 nm.

216. 215. The method of any one of claims 212 to 214, wherein the magnetic colloid particles are between about 80 and 200 nm.

217. 215. The method of any one of claims 212 to 214, wherein the magnetic colloid particles are between about 90 and 150 nm.

218. 218. The method of any one of claims 212 to 217, wherein the magnetic colloid particles have a magnetic mass of at least 50%.

219. 218. The method of any one of claims 212 to 217, wherein the magnetic colloid particles have a magnetic mass of at least 60%.

220. 218. A method according to any one of claims 212 to 217, wherein the magnetic colloidal particles have a magnetic mass between 70% and 90%.

221. 221. The method of any one of claims 212 to 220, wherein the magnetic colloidal particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

222. 222. The method of any one of claims 212 to 221, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

223. 223. The method of claim 222, wherein step (a) comprises adding a second EAEF comprising the other member of the specific binding pair to induce aggregation of the magnetic particles.

224. 224. The method of claim 223, wherein step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse aggregation of magnetic particles in the enriched sample.

225. 225. The method of any one of claims 212 to 224, further comprising the step of adding to the sample, prior to step (a), at least one aggregation inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane.

226. 226. The method of claim 225, wherein the aggregation inhibitor is a chelating agent.

227. The method of claim 226, wherein the chelating agent is EDTA.

228. 228. A method according to any one of claims 212 to 227, wherein the second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

229. 229. The method of claim 228, further comprising the step of isolating a single fetal cell prior to step (d).

230. 230. The method of claim 229, wherein isolating a single fetal cell is carried out by isolating a single fetal cell bound to a second antibody.

231. 231. The method of claim 230, wherein the second antibody is conjugated to a label.

232. 232. The method of claim 231, wherein the label is a fluorescent label.

233. 233. The method of claim 232, wherein the step of isolating single fetal cells is based on immunofluorescence techniques.

234. 234. The method of claim 233, wherein the step of isolating single fetal cells is performed by fluorescence-activated cell sorting (FACS).

235. The method of claim 233, wherein the step of isolating a single fetal cell is performed by DEPArray.

236. 234. The method of any one of claims 212 to 233, wherein step (d) comprises performing sequence analysis.

237. 237. The method of claim 236, wherein the sequence analysis comprises short tandem repeat (STR) analysis.

238. 238. The method of any one of claims 212 to 237, further comprising analyzing fetal cells.

239. 239. The method of claim 238, wherein analyzing the fetal cells comprises performing genomic or genetic analysis.

240. 240. The method of claim 239, wherein performing the genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

241. 241. The method of any one of claims 212 to 240, wherein the first antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

242. An antibody that binds to the TREML2 protein, or an antigen-binding fragment that binds to the TREML2 protein, (i) a heavy chain variable region (HCVR) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (vi) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11 242. The method of any one of claims 228 to 235 and claim 241, comprising one or more CDRs selected from:

243. The method of claim 242, wherein the antibody that binds to TREML2 protein or the antigen-binding fragment that binds to TREML2 protein comprises two, three, four, five, or six of the CDRs selected from (i) to (vi).

244. 244. The method of claim 242 or 243, wherein any of SEQ ID NOs: 6 to 11 independently comprises one or more amino acid substitutions, additions or deletions.

245. 242. The method of any one of claims 228 to 235 and claim 241, wherein the antibody that binds to the TREML2 protein is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul, and BD563661.

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