WO2013043126A1

WO2013043126A1 - Method(s) for obtaining a foetal red blood cell

Info

Publication number: WO2013043126A1
Application number: PCT/SG2012/000345
Authority: WO
Inventors: Jae Hyeok Choi; John Christopher Love; Sherry Sze Yee HO; Mahesh Choolani
Original assignee: National University Of Singapore; Massachussetts Institute Of Technology
Priority date: 2011-09-20
Filing date: 2012-09-20
Publication date: 2013-03-28
Also published as: WO2013043126A8

Abstract

In one aspect, the invention is directed to a method of separating at least one foetal nucleated red blood cell (FNRBC) from a sample comprising: a) placing the sample on at least one support surface b) contacting the sample with at least one antibody or antigen binding fragment thereof that specifically binds to at least one white blood cell, thereby producing at least one labelled cell; and c) providing a means capable of detection and isolation of at least one cell in the sample on the support surface, the cell comprising the features of: (i) being an unlabelled cell; (ii) comprising nucleic acid; and (iii) being 5 to 25pm in size, wherein the detection of the features (i), (ii) and (iii) is done simultaneously and the cell comprising the features (i), (ii) and (iii) is the FNRBC.

Description

METHOD(S) FOR OBTAINING A FOETAL RED BLOOD CELL

FIELD OF THE INVENTION

The present invention relates to a method of identifying and/or isolating at least one foetal erythroblast. In particular, the invention relates to an automated method of identifying at least one foetal erythroblast in a sample on a substrate and/or using an automated method of isolating the foetal erythroblast from the sample.

BACKGROUND OF THE INVENTION

Prenatal diagnosis provides valuable information on the health of the unborn child and can include invasive and non-invasive methods. Current methods of prenatal diagnosis for common chromosomal and single gene disorders involve invasive procedures such as amniocentesis, chorion villus biopsy and foetal blood sampling to obtain foetal DNA for cytogenetic and/or molecular analysis. Such procedures carry a small but significant (0.13-2.2%) risk of procedural- related foetal miscarriage. Accordingly, non-invasive prenatal diagnosis (NIPD) methods are preferred to invasive methods. The development of non-invasive prenatal screening also improved the ability to detect cases of aneuploidies and limit amniocentesis to high-risk patients.

Circulating foetal cells and cell-free foetal DNA provide a non-invasive source of foetal DNA for genetic analysis. In particular, identification of cell-free DNA, mRNA and foetal cells in the maternal circulation made the possibility of NIPD for diagnosis of chromosomal anomalies and single gene defects of the foetus. While it has been shown that circulating foetal nucleic acid in maternal plasma is useful in the non-invasive prenatal diagnosis of chromosomal aneuploidies, monogenic disorders and foetal rhesus status in Rh-negative pregnancies, one limitation still remains. It cannot be readily segregated from the abundant noise of maternal DNA circulating in the same plasma. Accordingly, the foetal genetic materials obtained from the maternal circulation are rather inadequate to provide reliable information on chromosomal abnormalities. In particular, the cell-free DNA in maternal circulation is rather insufficient to provide complete chromosomal information such as aneuploidies for diagnosis and is also expensive. On the other hand, the foetal cells are promising candidates for detecting chromosomal abnormalities but their cell numbers are very few. In particular, the utilization of foetal cells circulating in the maternal blood is both promising for detection of aneuploidies as well as in providing complete genetic information of the foetus. Here again the major limitations are their scarcity in maternal circulation and lack of efficient separation techniques. Moreover, some of these cells might persist from previous pregnancy and may not be indicative of the current foetal status. Further, first and second trimester prenatal screening involve combinations of foetal nuchal translucency (NT), maternal serum free-^-human chorionic gonadotrophin (β-hCG), pregnancy- associated plasma protein-A (PAPP-A), unconjugated estriol, and inhibin-A, all of which can only identify -80-90% of foetuses with trisomy 21 and other major aneuploidies at a fixed 5% false- positive rate. Single gene disorders are not screened for, and invasive procedures are mandatory for a definite genetic diagnosis after high-risk pregnancies are identified.

Conventional foetal cell separation strategies include density gradient centrifugation, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), micromanipulation, selective lysis and galactose-lectin-based methods. Unfortunately, recovery of foetal cells is low, and the recovered foetal cell fraction is not pure due to non-specific expression of cell surface antigens on both foetal and adult RBCs.

Accordingly, there is a need for the development and/or offer of NIPD assays that are genetic- based to enhance detection and lower false-positive rates (<5%). This will subsequently improve the uptake of prenatal screening and diagnostic tests.

In view of the scarcity of foetal erythroblasts, to date no method has successfully identified and/or isolated foetal erythroblasts. Studies on foetal erythroblasts have relied only on heterogenous culture of cells, which may not provide accurate information in view of maternal cells or other impurities. Poor in vitro viability of foetal erythroblasts also severely limits the possibility of performing further analysis or studies on these cells.

There is thus a need in the art for a method for detecting, and/or isolating foetal erythroblasts obtained by non-invasive means that is efficient and effective for use.

SUMMARY OF THE INVENTION

The present invention is defined in the appended independent claims. Some optional features of the present invention are defined in the appended dependent claims.

The present invention is directed towards a method of identifying at least one foetal nucleated red blood cell (FNRBC) from a sample comprising:

a) placing the sample on at least one support surface

b) contacting the sample with at least one antibody or antigen binding fragment thereof that specifically binds to at least one white blood cell, thereby producing at least one labelled cell;

and

c) providing a means capable of detection and isolation of at least one cell in the sample on the support surface, the cell comprising the features of: (i) comprising nucleic acid;

(ii) being an unlabelled cell; and

(iii) being 5 to 25μιτι in size ,

wherein the detection of the features (i), (ii) and (iii) is done simultaneously or sequentially and the cell comprising the features (i), (ii) and (iii) is the FNRBC.

According to a further aspect, the present invention is directed to a device capable of performing the method according to any aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a study outline to explore cell micromanipulation with manual and automated methods, followed by downstream molecular genetic analysis.

Figure 2 is an image of immunocytochemistry of a mixed population of mononuclear cells and foetal nucleated red blood cells (FNRBCs).

Figure 3 are four images of four different sets of 10-cell FNRBCs.

Figure 4 is a schematic diagram of manual cell micromanipulation (Huang et al., 2011 )

Figure 5 is a schematic diagram of automated cell micromanipulation (Choi et al., 2010)

Figure 6 is a schematic diagram showing the procedure of a high-throughput automated cell micromanipulation.

Figure 7 are images showing the identification and separation of a target cell (i.e. FNRBC) outside a microwell using automated cell micromanipulation.

Figure 8 are images showing the identification and separation of a target cell (i.e. FNRBC) inside a microwell using automated cell micromanipulation.

Figure 9 images showing the identification of an FNRBC using Laser Capture Microdissection (LCM).

Figure 10 is a graph showing the results of real-time quantitative PCR of beta-globin post identification and separation of the FNRBC.

Figure 11 is a schematic diagram showing a high-throughput single cell assay.

Figure 12 are images of the results of automatic micromanipulation of FNRBC from a Pre- termination of pregnancy (Pre-TOP).

Figure 13 is an image showing distribution of more number of cells than that of microwells on PDMS stamp (FNRBC in the center).

Figure 14 is a graph showing the total amount of whole genome amplification for each FNRBC isolated.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference. Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" as used herein thus usually means "at least one".

The term "comprising" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Accordingly, the term "comprising" encompasses the more restrictive terms "consisting essentially of" and "consisting of."

The term "CD45 negative" as used herein refers to any cell that expresses no signal or is negative for native, recombinant or synthetic forms of the CD45 molecule/ marker. The presence of CD45 expression on a cell in a sample may be determined using any immunostaining method known in the art and using any anti-CD45 reagent. Any cells positively stained with anti-CD45 reagent may be excluded as these may include CD45 positive white blood cells.

The term "erythroblast" as used herein refers to a red blood cell having a nucleus. In particular, an erythroblast refers to a nucleated precursor cell from which a reticulocyte develops into an erythrocyte. "Erythroblast" may be used interchangeably with a "Normoblast" and refers to a nucleated red blood cell, the immediate precursor of an erythrocyte. For example, the erythroblast may be of mammalian origin. In particular, the erythroblast may be a primitive or human foetal erythroblast. "Erythrocytes" or "red blood cells" or "RBC" include non-nucleated adult and foetal red blood cells.

The term "mammalian" is herein defined as a mammalian individual, in particular, a primate for example a human being. For purposes of research, the subject may be a non-human. For example the subject may be an animal suitable for use in an animal model, e.g., a pig, horse, mouse, rat, cow, dog, cat, cattle, non-human primate (e.g. chimpanzee) and the like.

The phrase "morphology of a cell" as used herein refers in general to the form, structure and configuration of a cell and may include aspects of the cell appearance like shape, colour or pattern of internal or external part of a cell. Morphology of a cell may also include nucleation of a cell, whether the cell is nucleated or not nucleated, whether the cell is CD45 negative or positive and/or the cytoplasm to nuclear ratio of the cell. Other aspects that may fall within the definition of morphology of a cell includes the size of the cell, for example the diameter of the cell, the adherence ability of the cell, for example whether the cell adheres or does not adhere to a surface, malleability of a cell and the like. The phrase "form of a cell" may be used interchangeably with the phrase "shape of a cell" and as used herein refers to typical Cell forms like circular cells, elliptic cells, shmoo like cells, division forms like dumbbells, star-like cell forms, flat cells, scale-like cells, columnar cells, invaginated cells, cells with concavely formed walls, cells with convexly formed walls, the presence of prolongations, appendices or cilia, the presence of angles or corner etc. Typical morphologies or forms would be known to the person skilled in the art and can be derived.

The term "nucleated" as used herein refers to a cell that has a nucleus. Nucleated cells may be distinguished from red blood cells which are not nucleated based on any nuclear staining known in the art.

The term "prenatal disorder" as used herein refers to diseases or conditions in a foetus or embryo before it is born. The prenatal disorder may be selected from the non-limiting group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's disease, Charcot-Marie- Tooth disease, haemophilia, Duchenne Muscular Dystrophy, mitochondrial disorder, hereditary multiple exostoses, osteogenesis imperfecta disorder and the like.

As used herein, an agent that "specifically labels" or "specifically binds" refers to an agent that selectively labels or binds to (or has high affinity for) a target (e.g., White Blood Cells (WBCs)) in a sample to the exclusion of other molecules {e.g., other cells such as FNRBCs, ARBCs) in the sample (does not bind other molecules, or has a low affinity for other molecules, in the sample).

The term "sample" as used herein refers to a subset of tissues, cells or component parts (for example fluids) that may include, but are not limited to, maternal tissue, maternal blood, cord blood, amniocenteses, chorionic villus sample, foetal blood, and/or foetal tissue/fluids. In particular, foetal tissue may be trophoblast tissue, placental tissue or a combination thereof. The sample as used in the present invention may have been previously subjected to a density gradient purification which may comprise a polyvinyl-pyrrolidone coated silica, but not limited to, Ficoll gradient and Percoll™ gradient.

The present invention may be directed to a methodology where a manual or high-throughput automated micromanipulation-based system can selectively retrieve individual foetal cells from maternal blood cells based on parameters of morphology (size), staining of nucleus, and surface markers, which enhances the recovery of foetal cells from maternal blood. Also, the whole genome of each micromanipulated single foetal cell can be amplified for subsequent molecular analysis using, for example, single cell or multi-cell (>1 cell) PCR; each individual cell can be deposited into a PCR tube or a PCR well of a 96-well plate from any type of culture dish or dense arrays of subnanoliter microwell (PDMS) containers. This method allows for about 4 million cells to be scanned at one go and thus speeding up the process for detecting FNRBC which are rare.

According to one aspect, there is provided a method of separating at least one foetal nucleated red blood cell (FNRBC) from a sample comprising:

a) placing the sample on at least one support surface;

b) contacting the sample with at least one antibody or antigen binding fragment thereof that specifically binds to at least one white blood cell, thereby producing at least one labelled cell; and

c) providing a means capable of detection and isolation of at least one cell in the sample on the support surface, the cell comprising the features of:

(i) comprising nucleic acid

(ii) being an unlabelled cell;

and

(iii) being 5 to 25μιτι in size ,

The means capable of detection and/or isolation may be a manual and/or an automated method. In particular, the means may be a high-throughput automated method of detecting the FNRBC. In particular, the means may be a high-throughput automated micromanipulation-based system that can selectively retrieve individual foetal cells from maternal blood cells based on parameters of morphology (size), staining of nucleus, and surface markers, which enhances the recovery of foetal cells from maternal blood. In particular, the method may mostly automated and thus be reliably enriched and amplified from within maternal blood, and be accurately identified as foetal in origin. This may allow for foetal cells to be a richer and purer source of foetal DNA for chromosomal and genetic diagnosis.

The five main technical advantages of the method according to any aspect of the present invention are the;

(1 ) successful automatic micromanipulation of FNRBCs from maternal blood obtained before termination of pregnancy;

(2) reduced total amount of time spent for automatic micromanipulation;

(3) increased holding capacity of FNRBCs within one PDMS microwell stamp;

(4) sufficient amounts of DNA achieved by whole genome amplification from a single FNRBC that was retrieved by automated micromanipulation;

(5) establishment of whole genome sequencing strategy from single FNRBCs. Other methods for rare cell recovery focus on single parameters (e.g., EpCam+ for circulating tumor cells), and use flow devices (microfluidics) to separate cells, which cannot provide 100% pure FNRBCs as provided in the method of the present invention.

A variety of articles may comprise the support surface according to any aspect of the present invention and suitable articles will be evident to those of skill in the art. Such articles include cell culture vessels, such as slides (e.g., tissue slides, microscope slides, etc.), plates (e.g., culture plates or multi-well plates, including micro plates), flasks (e.g., stationary or spinner flasks), bottles (e.g., roller bottles), bioreactors, and the like. The support surface for use according to any aspect of the present invention may be made of a variety of materials, including natural polymers, synthetic polymers and inorganic composites. Natural polymers include, for example, collagen- and glycosaminoglycan (GAG)-based materials. Synthetic polymers include, for example, poly(a-hydroxy acids) such as polylactic acid (PLA), polyglycolic acid (PGA) and copolymers thereof (PLGA), poly(ortho ester), polyurethanes, and hydrogels, such as polyhydroxyethylmethacrylate (poly-HEMA) or polyethylene oxide-polypropylene oxide copolymer. Hybrid materials, containing naturally derived and synthetic polymer materials, may also be used. Non-limiting examples of such materials are disclosed in Chen et al. (Advanced Materials 12:455-457, 2000). Inorganic composites include, for example, calcium phosphate ceramics, bioglasses and bioactive glass-ceramics, in particular composites combining calcium hydroxyapatite and silicon stabilized tricalcium phosphate. In particular, support surfaces may be polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polytri- or tetra- fluoroethylene, polyhexafluoropropylene, polyvinyl chloride, polyvinylidine fluoride, polylactide, cellulose, glass, or a ceramic. In one example, the support surface may be a polystyrene tissue culture dish or multi-well plate. More in particular, the support surface may be an array which comprises a plurality of microwells. Each microwell may be about 30 μπι x about 30 m in size. The array may be made of a biocompatible, elastomeric rubber like material into which each microwell may be moulded. The material may be polydimethylsiloxane (PDMS) to reduce or prevent damage to the means for isolating the FNRBC.

In particular, as shown herein the whole genome of each micromanipulated single foetal cell can be amplified for subsequent molecular analysis using, for example, single cell or multi-cell (>1 cell) PCR; each individual cell can be deposited into a PCR tube or a PCR well of a 96-well plate from any type of culture dish or dense arrays of subnanoliter microwell (PDMS) containers.

In particular, this high-throughput PDMS microwell platform for foetal cells may be highly efficient and unique to separate cells and make them ready for micromanipulation. FNRBCs can be either outside of the subnanoliter microwell or inside of the microwell because the micromanipulator can pick either one. This separation between inside and outside of microwell helps to isolate each single FNRBC (or foetal cell) and prevent the contamination due to the retrieval of non- targeted cells during micromanipulation The sample may be contacted with an (one or more) agent (e.g., a first agent) that specifically labels white blood cells (WBCs), and may be detectable (either directly or indirectly), in order to distinguish the WBCs from the FNRBCs in the sample, thereby producing labelled cells. The sample may then be sorted for nucleated cells.

The isolation of the FNRBC may be done using at least one tip of a suitable tube. In particular, the isolation of the FNRBC may be done using at least one capillary tip, syringe needle, pipette tip and the like. A syringe needle of different ranges may be capable of being used for isolating FNRBCs. For example, the syringe needle may have a diameter of 0.5mm to 10mm. In particular, the syringe needle may have a diameter of 0.6, 0.8, 1 .0, 1 .5, 2.5, 5 mm, 9mm and the like. Even more in particular, the isolation of the FNRBC may be carried out using at least one capillary tip which may be made of glass. The aspiration rate of the capillary tip may programmatically controlled. "Aspiration rate" refers to the speed the cell and/or fluid in the sample is taken up and/or released from the tip. Use of the tip which may be programmatically controlled enables control of the aspiration rate to accurately increase or decrease the rate depending on the cell adhesion to the support surface. The aspiration rate may be optimised to minimise damage to the cell.

A variety of agents that specifically label WBCs are known in the art. In a particular aspect, the agent specifically binds to WBCs. For example, an agent that specifically labels (e.g., binds to) a cell surface marker (e.g., a cell surface polypeptide) of WBCs (a cell surface marker that identifies the cells as WBCs) can be used in the methods provided herein. As is also known in the art, there are a variety of cell surface markers that are specific to WBCs. Examples of such cell surface markers of WBCs include CD45, CD14, CD5, CD19, CD20, CD45, CD3, CD4, CD8, CD16, CD56 and the like.

In one aspect, the agent that specifically labels a WBC is an antibody or antigen binding fragment thereof. The antibody can be a polyclonal antibody, a nanobody or heavy chain antibody (Rothbauer, ., et al., Nature Methods, 3(11)887-889 (2006); Kirchhofer, A., et al., Nature Structural & Molecular Biology, 17(1 )r\ 33-139 (2010)), a monoclonal antibody, a chimeric antibody, a humanized antibody, a bi-specific antibody, a bi-functional antibody or human antibody and antigenic fragments thereof (a functional or biologically active fragment). As appreciated by those of skill in the art, antigenic fragments include Fab, Fab', Fab2, and Fab'2 fragments, single chain variable regions and variations of the same. In other aspects, the agent labels the WBC with a marker that can be detected by virtue of its ability to fluoresce at one or more wavelengths, a marker that can be detected magnetically such as a small magnetic bead, a quantum dot or combinations thereof. In particular, the antibody or antigen binding fragment thereof may be one that specifically binds to CD45. All the CD45 negative cells may be completely unlabelled. More in particular, only 100% unlabelled cells (i.e. a cell with absolutely no label) may be considered a CD45 negative cell. Even more in particular, about 100%, 99%, 98%, 97%, 96% or 95% unlabelled cells may be considered CD45 negative cells.

Cells that comprise nucleic acid can be detected using a variety of methods known to those of skill in the art. For example, cells that comprise nucleic acid are detected using phase contrast microscopy, colorimetric assays, autoradiography, Raman spectroscopy or combinations thereof. As known to those of skill in the art a variety of methods such as end-labelling (either at the 3' ends with DNA polymerase or at the 5' end using T4 polynucleotide kinase), nick translation using DNA polymerase and DNase I to form nicks that are filled in by polymerase, random primer labelling, and polymerase chain reaction (PCR) labelling can be used to label nucleic acid.

In other aspects, the method can further comprise contacting the sample with a second agent (third agent, fourth agent, etc.) that specifically labels nucleic acid in the sample and is also detectable (either directly or indirectly). As will be apparent to those of skill in the art, the agent that specifically labels nucleic acid is distinct, and separately detectable, from the agent that specifically labels WBCs. Agents, such as stains, dyes, radioactive labels (e.g., ³²P), chemically substituted nucleotides, or nanobodies that specifically label all or a portion of a nucleic acid in a cell are well known in the art. Examples of stains that specifically label nucleic acid include Hoechst stain, DAPI stain, acridine orange and the like.

Methods of detecting an agent that labels nucleic acid are also well known to those of skill in the art. For example, detection methods include fluorescence microscopy, fluorescent activated cells sorting (FACS), radioactive detection or a combination thereof. In particular, the sample may be sorted for nucleated cells using density gradient centrifugation.

FNRBCs are typically about 5 to about 25μπι in size. Thus, in the methods provided herein, cells that are about 5 to about 25μιη in size are also detected. In other aspects, cells that are about 10 to about 15μιτι in size are detected. In yet other aspects, cells that are about 10 to about 25μΓη in size are detected. Methods for detecting cells of a particular size are well known to those of skill in the art. In particular aspects, cells that are about 5 to about 25μηη, about 10 to about 15μηι, or about 1 0 to about 25μηη in size are detected using optical methods such as phase contrast microscopy, fluorescence microscopy and/or the like after staining the surface of a cell.

The means capable of detection and isolation according to any aspect of the present invention may be capable of separating the FNRBC from the sample on the support surface. In particular, the isolation of the mammalian nucleated foetal cell fronr the sample may be performed using, but not limited to, a micromanipulator or any system that allows individual picking of a foetal cell. The micromanipulator may have at least one micropipette. In one example, the micromanipulator may have two, three, four, five or more micropipettes, each capable of isolating an FNRBC separately/ independently. The micropipette may be customized with an internal diameter of between 20-25 μιη to suit the target cell size and a flat tip end to prevent physical damage on the cells.

In another example, since FNRBCs are specific for epsilon-globin, Laser Capture Microdissection (LCM) may be used to retrieve epsilon-globin positive FNRBCs enriched from digested trophoblasts to explore the possibility of genetic analysis using LCM cells (denoted ^* in Figure 1 ). However, epsilon-globin is a cytoplasmic protein and to identify epsilon-globin positive FNRBCs, cells need to be fixed before immunoc tochemistry (ICC).

The FNRBC may be a mammalian foetal erythroblast. More in particular, the FNRBC may be a primitive or human foetal erythroblast. FNRBCs are very elastic (pliable, flexible), so the use of microfluidics-based or size-based (use of sieves, flow cytometry, etc) platforms may not be suitable. Also, due to the rarity of FNRBCs, it is important to minimize or prevent the loss of targeted cells. The high-throughput microwell platform according to any aspect of the present invention reduces the loss of any FNRBC and can screen or micromanipulate again if it is needed.

In one aspect, the invention is directed to a method of separating foetal nucleated red blood cells (FNRBCs) from a sample. The method comprises contacting the sample with an agent that specifically labels a white blood cell, and maintaining the sample under conditions in which the agent labels white blood cells in the sample, thereby producing a sample comprising labelled cells. The sample with the labelled cells are loaded onto an array, wherein the array comprises a plurality of microwells and each microwell of the array contains about one cell from the sample. Each cell in a microwell of the array that (i) is not labelled with the agent, (ii) comprises nucleic acid and (iii) is about 5 to about 25μΓη in size is an FNRBC, thereby separating the FNRBCs from the sample.

In one example, the sample with the labelled cells may be loaded onto an array, wherein the array comprises a plurality of microwells and each microwell of the array contains about one cell from the sample. In one aspect, most or all of the microwells of the array contains (e.g., comprises, consists essentially of, consists of) a single cell from the sample.

As will be apparent to those of skill in the art, arrays are typically composed of biocompatible, elastomeric rubber-like material into which the plurality of microwells has been moulded. In one aspect, the biocompatible, elastomeric rubber-like material is polydimethylsiloxane (PDMS). In other aspects, the material is an off-stoichiometry thiol-ene (OSTE) (Carlborg, CF., et ai, Lab on a Chip (August 1 , 201 1 )), styrenic thermo elastomer (TPE), or thermoplastic elsatomer (Roy, E., et ai, Lab on a Chip (July 27, 201 1 )). In particular aspects, the overall dimension of the array may be about 25mm x about 60mm. In other aspects, the overall dimension of the array is about 2-fold, 3-fold, 4-fold or 5-fold larger than 25mm x 60mm. In yet other aspects, each microwell is about 5 ΐΌ - about 100μιη x about 5 Mm - about Ι ΟΟμιτι x about 5 μιη - about Ι ΟΟμιτι. Each microwell of the array may be typically of a size and dimension that accommodates at least one single cell.

Each cell in one or more of the microwells of the array that (i) is not labelled with the agent, (ii) comprises nucleic acid and (iii) is about 5 to about 25μιτι in size may be a FNRBC. In order to enhance the recovery of foetal cells from maternal blood, a manual and/or high-throughput automated micromanipulation-based system may be used to selectively retrieve foetal cells from maternal blood. In particular aspects, about 100% of the FNRBCs in the sample are separated from the sample. In other aspects, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the FNRBCs in the sample are separated from the sample. In other aspects, the one or more FNRBCs recovered from the one or more wells of the array are live FNRBCs. Proof-of- concept using manual micromanipulation showed that the retrieved cells can be successfully whole-genome amplified for subsequent PCR amplification and analysis. In order to screen and isolate foetal cells from a significant number of maternal cells in maternal blood, a high throughput system may be needed. Microfab cated systems are suited for this purpose. Highly concentrated arrays of microwells with microfluidic systems enable compartmentalization of 10⁴- 10⁶ cells in subnanoliter volumes for assay in a single assay.

As described herein, a first means according to any aspect of the present invention may be a commercially available instrument designed for colony picking that may be programmatically controlled. In particular, the first means may be used with a customised software module. This module may be able to translate data that may be captured from a dense array of microwells for high-throughput, single-cell screening by capturing secretion from individual cells. Instead of capturing secretion, the scanning module may be used to discriminate between target and background cells based on morphology (size), and cell and/or nucleus staining. This system may be able to list the exact locations from which to retrieve the desired individual cells, enabling 100% recovery of targeted cells.

Highly concentrated arrays of microwells with microfluidic systems enable compartmentalization of 10⁴— 10⁶ cells in subnanoliter volumes for characterization in a single assay. As described herein, a commercially available instrument designed for colony picking (Haupt et al., 2009) may be used with a customised software module. This module was able to translate data that was captured from a dense array of microwells for high-throughput, single-cell screening by capturing secretion from individual cells. Instead of capturing secretion, the scanning module is used to discriminate between target and background cells based on morphology (size), and cell staining. This system was able to list the exact locations from which to retrieve the desired individual cells, enabling 100% recovery of targeted cells.

A variety of detection methods for detecting a (one or more) cell that (i) is not labelled with the agent, (ii) comprises nucleic acid and/or (iii) is about 5 to about 25μηι in size are known to those of skill in the art.

In another aspect, the invention is directed to a method of separating FNRBCs from anucleated red blood cells (ARBCs) present in a sample. The method comprises contacting the sample with an agent that specifically labels a white blood cell, and maintaining the sample under conditions in which the agent labels white blood cells in the sample, thereby producing a sample comprising labelled cells. The sample with the labelled cells are loaded onto an array, wherein the array comprises a plurality of microwells and each microwell of the array contains about one cell from the sample. Each cell present in a microwell of the array that (i) is not labelled with the agent, (ii) comprises nucleic acid and (iii) is about 5 to about 25μιτι in size is a FNRBC, thereby separating the FNRBCs from the ARBCs in the sample.

In yet another aspect, the invention is directed to a method of detecting whether one or more FNRBCs are present in a sample (e.g., a blood sample). The method comprises contacting the sample with an agent that specifically labels a white blood cell, and maintaining the sample under conditions in which the agent labels white blood cells in the sample, thereby producing a sample comprising labelled cells. The sample with the labelled cells are loaded onto an array, wherein the array comprises a plurality of microwells and each microwell of the array contains about one cell from the sample. If one or more cells in one or more the microwells of the array that (i) is not labelled with the agent, (ii) comprises nucleic acid and (iii) is about 5 to about 25μηι in size is detected, then FNRBCs are detected in the sample.

The method according to any aspect of the present invention may comprise FNRBCs which may be recovered manually or automatically. The FNRBCs may be recovered using a manual micromanipulator or an automated micromanipulator.

The methods described herein overcome the risk of invasive diagnostic test; a small but significant (0.13-2.2%) risk of procedural-related foetal miscarriage. The development and offer of NIPD that are genetic-based enhances detection and lowers false-positive rates (<5%). The ease of peripheral blood collection without trained clinical expertise as compared with amniocentesis or chorion villus biopsy improves the uptake of prenatal screening and diagnostic tests. The methods described herein eliminate procedural-related risks of foetal miscarriages and lower the cost of patient management and provide for accurate non-invasive test of foetal genetic abnormalities by the use of foetal cells in maternal blood. In the methods of the invention any suitable sample, e.g., a biological sample can be used. Examples of such samples include biological fluids (e.g., blood, cord blood, plasma, urine and the like) and tissue (e.g., trophoblast tissue, liver tissue, and placenta). In particular aspects, the sample is a maternal sample (a sample obtained from a pregnant mother), and in other aspects, the sample is a foetal sample. In yet other aspects, the sample is obtained from a mother in a first trimester, a second trimester or a third trimester of a pregnancy. Typically, the sample comprises a variety of cell types in addition to FNRBCs such as anucleated red blood cells (ARBCs) and white blood cells (WBCs). In yet other aspects, the sample is from a mammal such as a primate (e.g. human), a canine, a feline, a bovine, a porcine, a murine and the like.

As described herein, the one or more FNRBCs recovered from the one or more wells of the array can be further analyzed. For example, the one or more FNRBCs can be further analyzed using polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), multiple-ligand dependent probe amplification (mlpa), short tendon repeat analysis, array competitive genomic hybridization (CGH), genotyping, single plex sequencing, massively parallel sequencing, next generation sequencing or a combination thereof. Array comparative genomic hybridization (aCGH) for the analysis of the complete genome can also be used in addition to real-time PCR and QF-PCR.

In particular aspects, the one or more FNRBCS are further tested for one or more prenatal disorders. Examples of such prenatal disorders include Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs Disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's Disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne muscular dystrophy, mitochondrial disorder, Hereditary multiple exostoses, osteogenesis imperfecta disorder or a combination thereof. In other aspects, the FNRBCs are tested for a foetus' gender.

The method according to any aspect of the present invention may thus be useful for diagnostics and especially for foetal cell micromanipulation. The method according to any aspect of the present invention, in particular with use of micromanipulation may be able to isolate and retrieve rare fetal cells (total 10 - 60 cells of FNRBCs after enrichment) without losing these rare cells. The majority of methods for separation of FNRBCs use magnetic beads to enrich populations of cells. These methods cannot produce pure FNRBCs as other non-target cells will still be present even after depletion.

For diagnostics, the image-based cytometry incorporated in the method according to any aspect of the present invention may be considered unique, especially in combination with the automated micromanipulation. Flow cytometry or other flow devices may not be proper for rare foetal cells, because of possible cell loss. Micromanipulated single cells on a polymerase chain reaction (PCR) plate or a specified plate enable direct whole genome amplification (WGA) / PCR for single cells or multi-cell (>1 cell). This may be considered the most simplified and time-efficient procedure for single cell WGA / PCR.

In addition, the method can further comprise additional steps to remove cells other than FNRBCs or further sort the cells in the sample. For example, prior to contacting the sample with an agent that specifically labels WBCs, the sample can be sorted for nucleated cells using, for example, density gradient centrifugation.

As will be apparent to those of skill in the art, the methods provided herein can further comprising contacting the sample with an agent that specifically labels FNRBCs. in one aspect, the method comprises contacting the sample with an (one or more) agent that labels CD147 (see U.S. Provisional Application No. 61/503,236 which is incorporated herein by reference in its entirety), MCT1 , CD164 or combinations thereof.

The methods provided herein can further comprise recovering one or more FNRBCs from the one or more microwells of the array either manually (e.g., using a manual micromanipulator) or automatically (e.g., using an automated micromanipulator) using a variety of known methods and/or devices such as integrated microfluidic systems (Huang, R., ei al., Prenatal Diagnosis, 2S.S92-899 (2008); Kim, J., ef al., Lab on a Chip (July 29, 201 1 )). In particular, the method according to any aspect of the present invention may not be restricted to a specific instrument used to date.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001 ).

The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

The study described herein is outlined in Figure 1 . Paired maternal peripheral blood and trophoblast samples were collected before termination of pregnancy (~9-1 1 gestational weeks). Nucleated cells were sorted using density gradient centrifugation. Magnetic-activated cell sorting (MACS) with specific antibodies was performed to enrich for target foetal cells. The nuclei of foetal nucleated red blood cells (FNRBCs) were labelled with fluorescent stains such as Hoechst, while mononuclear cells (MNCs) were labelled with specific surface antibodies (CD45) conjugated with fluorescent tags for identification. As cell morphology is not affected, Hoechst- positive/CD45-negative cells (i.e. FNRBCs) can be retrieved using manual and automated cell micromanipulation under microscope visualization. The cells were collected at varying numbers (1 , 3, 5, and 10) into PCR tubes and plates prior to whole genome amplification (WGA). Realtime PCR and quantitative fluorescence (QF)-PCR were performed on WGA DNA FNRBCs collected from paired digested trophoblasts wereblasts was included as controls for cell micromanipulation. Genomic DNA was isolated from CD45-positive fraction from the maternal blood (i.e. maternal DNA), and digested trophoblast cells (i.e. foetal DNA) as controls for downstream molecular genetic analysis.

Methods/Materials

Cell staining, visualization and identification

For cell micromanipulation, an example of live cell staining is described that enabled visual discrimination of target foetal cells based on cell morphology and fluorescence staining. For Laser Capture Microdissection (LCM), immunocytochemistry (ICC) was used.

Live cell staining

Cells were resuspended in RPMI-1640 media (Sigma-Aldrich, MO, USA) or 1 xPBS and stained with CD45 antibody. For the CD45-AF488 (Invitrogen, USA) staining, 5 μΙ of antibody was added to 1 ml of cell suspension followed by a 1 -hour incubation at 37°C. For CD45-FITC (Miltenyi Biotec, Germany) staining, 10 μΙ of antibody was added to 100 μΙ of cell suspension with up to 10⁷ cells followed by a 30-min incubation at room temperature. After CD45 staining, 1 μΙ of Hoechst dye (stock cone. 10 mg/ml, Invitrogen, USA) was added to 1 ml of cell suspension. Cells were incubated for 15 min at 37 °C and pelleted down at 3000 rpm for 5 min. The cell pellet was resuspended in RPMI-1640 media. CD45 and Hoechst stained cells were examined under the fluorescent microscope (Olympus IX70, USA) (Figures 2, 3). Figure 2 shows a mixed population of CD45-positive adult mononuclear cells (MNCs) and CD45-negative/Hoechst-positive FNRBCs. The presence of blue nuclear staining (i.e. Hoechst-positive) with green fluorescence (i.e. CD45- positive) were indicative of adult MNCs. These cells were excluded during the identification and selection for FNRBCs micromanipulation (Hoechst-positive/CD45-negative).

Figure 3 shows four 10-cell sets of Hoechst positive and CD45-negative foetal nucleated RBCs (FNRBCs) that were manually picked into PCR tubes. Only cells that showed Hoechst positive and CD45-negative fluorescence staining patterns were identified and selected as target FNRBCs for micromanipulation. Slide-making/ Immunocytochemistry (ICC)

The enriched cell fraction containing FNRBCs from digested trophoblast was cytospun onto polyethylene (PEN) membrane slides (Carl Zeiss, Munich, Germany). ICC using anti-s-globin antibody to identify epsilon-globin-positive FNRBCs was performed on Wright's stained slides. All slides were stored at -20 °C until LCM.

Manual cell micromanipulation

An efficient system was successfully developed using a Zeiss-Narashige micromanipulation system and 20 μπι bore micropipettes to pick up individual foetal cells into 0.2 ml PCR tubes for direct whole genome amplification (WGA) and subsequent molecular genetic analysis. In brief, the cells were resuspended in culture medium after staining with CD45 and Hoechst dye. 100 μΙ of culture medium was used for every 1 -3x10⁶ cells. Micromanipulator (Narishige, Japan) and inverted microscope (Olympus IX70, USA) were set up prior to the micromanipulation. 100 μΙ of cell suspension was loaded onto a 60 mm culture dish. 50 μΙ of culture medium and 50 μΙ of 1 x PBS were loaded onto the same dish in two separate droplets. A micropipette (Origio, USA) with internal diameter of 20 μιη was placed in the cell suspension droplet. FNRBCs were identified and picked up by checking the Hoechst dye (positive) and CD45 (negative) staining. Picked FNRBCs were immediately transferred to the medium droplet, followed by the PBS droplet to wash the cells. Picked FNRBCs were put into PCR tubes (size 200 μΙ) and stored at -20°C until analysis. There was no contamination of other cell types commonly present in the blood.

Figure 4 shows the setup of the manual cell micromanipulator, and the process of cell retrieval. A: A mixed population of cells in a large droplet of culture medium at the centre (arrow). Ai: Magnified phase contrast image of the mixed cell population before micromanipulation. B, C: Target cells were individually picked from the central droplet using a 20 μιη bore micropipette and Zeiss-Narashige micromanipulator under the microscope. The picked cells were transferred to 10 μΙ culture medium droplets under sterile mineral oil to obtain homogenous populations. Ci: Magnified phase contrast image of picked cells were individually monitored. D: Cross-sectional view of Petri dish showing a thin layer of equilibrated mineral oil covering droplets of culture medium containing picked cells.

Automated cell scanning/micromanipuiation

With the success of real-time PCR and QF-PCR amplifications of FNRBCs retrieved by manual cell micromanipulation, the use of automated cell micromanipulation was then explored. The automated platform consisted of dense arrays of subnanoliter microwell polydimethylsiloxane (PDMS) containers for high-throughput single-cell screening. Picking of single cell and/or colony was performed either by using in-house capillaries system or a commercially available instrument (CellCelector, AVISO GmbH, Germany). In addition, a customized software module for the automation of scanning and identification of target cell was included for automated cell retrieval {e.g., Choi, JH, et al., Biotechnol. Prog., 26:888-895 (2010)). The combination of automated screening and micromanipulation ensured consistency between scanning/retrieval time and cell quality, thus ensuring reproducibility regardless of environmental factors and level of operator experience.

As proof-of-concept, manual cell micromanipulation, in addition to automated cell micromanipulation, was used to pick foetal cells enriched from chorion villus biopsy. Although manual micromanipulation of foetal cells from maternal blood was successful, manual scanning and retrieving of targeted foetal cells among the randomly distributed cells on tissue culture plate were time-consuming and inefficient. Therefore, an automatic scanning and automated micromanipulation with high-throughput microfabricated platform was developed to enable rapid scanning and cell retrieving (one minute per cell) for millions of individual cells.

Figure 5 shows the instrument for transferring cells by automated micromanipulation, (a) Photograph of the CellCelector; (insert) enlarged photograph of the glass capillary, (b) Preaspiration of media into the glass capillary, (c) Aspiration (picking) of cell(s) from a targeted well; (insert) enlarged photograph of a glass capillary positioned in a well of the PDMS stamp, (d) Deposition of the retrieved cells into a 96-well plate filled with media.

Automated scanning and selective retrieval of foetal cells based on parameters of morphology (size), staining of nucleus, and surface markers were successful as shown in Figures 6, 7 and 8. In addition, for each micromanipulated single foetal cell, the whole genome can be amplified for subsequent molecular analysis using single cell or multi-cell (>1 cell) PCR; each individual cell can be deposited into a PCR tube or a PCR well of a 96-well plate from any type of culture dish or dense arrays of subnanoliter microwell (PDMS) containers. The low circulating foetal cell population in the maternal blood presented a challenge in the enrichment and isolation of foetal cells for genetic analysis (~1 .2 foetal cell per ml of maternal blood from 1 st trimester pregnancy). As described earlier, the current methodologies for foetal cell enrichment are FACS and MACS. However, as the surface antigens of the targeted cell are not entirely specific to foetal cells, adult cells are recovered together with the foetal cells, presenting a major source of contamination for downstream genetic analysis. In addition, cells are lost during density gradient centrifugation and MACS, leading to poor recovery. As the population of circulating foetal cell in maternal blood, recovery is of utmost importance and therefore, alternative methodologies that are specific and ensure high recovery of foetal cells need to be explored.

High-throughput single cell assay on a platform of PDMS stamp enabled the screening of over 200,000 wells (20-30 μηη). Every single foetal cell that was located on the PDMS was micromanipulated and retrieved with success (Figures 7 and 8). As all pictures and parameters (morphology, size, staining) were captured for every single cell that was retrieved, the documented pictures and parameters post-retrieval were reviewable when necessary. Thus, an advantage of the invention described herein is documentation afforded by the automated micromanipulation. This feature provides a metric for quality assurance and control e.g., for Federal Drug Adminstration (FDA) purposes. As shown herein, FNRBC were screened and micromanipulated using three criteria; (1 ) morphology (size: 10 ~ 15 μιη as compared to the adult red blood cells, aRBC) (2) positive staining: live cell staining of the nucleus (specific to cells containing nuclei and thus, excluding non-nucleated cells such as aRBC) and (3) negative staining: CD45 staining (specific to adult nucleated cells and not FNRBCs). The automated high-throughput single cell assay provided herein was able to check through customized criteria with automatic scanning and locate target single FNRBC automatically. That is, the assay can use defined criteria to automatically identify a single FNRBC. Figure 7 shows high-throughput morphology and fluorescence screening of individual cells of FNRBC on the PDMS platform and automated micromanipulation, (a) Captured image of FNRBC in the dotted circle line on the PDMS platform by using fluorescence (UV light source) with minimum light source on quick time, (b) nucleus staining by Hoechst (UV source only), (c) CD45 staining against maternal nucleus cell, (d) under the procedure of automated micromanipulation.

Figure 8 shows before and after high-throughput morphology and fluorescence screening of individual cells of FNRBC on the PDMS platform and automated micromanipulation, (a) Captured image of FNRBC in the dotted circle line on the PDMS platform by using fluorescence (UV light source) with minimum light source on a short exposure time, (b) Nucleus staining by Hoechst (UV source only), (c) Captured image by using fluorescence (UV light source) with minimum light source on a short exposure time right after automated micromanipulation, (d) Image from nucleus staining right after automated micromanipulation.

Laser Capture Microdissection (LCM)

LMPC was performed using a P.A.L.M. Robot-Microbeam System (Carl Zeiss) following the manufacturer's recommendations. Images were captured before and after microdissection to ensure that target epsilon-globin positive FNRBCs were successfully microdissected from the slide (Figure 9). Adhesive caps containing the laser-dissected and catapulted cells were stored at -20 °C before whole genome amplification (WGA).

Figure 9 shows (a) Epsilon-globin positive primitive foetal erythroblast before LCMP; (b) same picture showing the removal of the same cell after LCMP.

Whole Genome Amplification (WGA)

LCM cells were collected into PCR tubes by washing LCM caps with 7 μΙ Cell Extraction Buffer from the Picoplex™ WGA kit (Rubicon Genomics, Ml, USA). Manual- and automated- micromanipulated cells were collected into PCR tubes and 96-well plates respectively. DNA was extracted by 75 °C incubation for 10 min followed by 95 °C incubation for 4 min using the Extraction Cocktail provided in the kit. WGA was performed using the Picoplex™ WGA kit (Rubicon Genomics) according to manufacturer's recommendations with 16 cycles of amplification. The quality and quantity of all amplified DNA samples were assessed by gel electrophoresis and Picogreen quantitative assays before genetic analysis with quantitative fluorescence PCR (QF-PCR) and real-time PCR.

Quantitative measurement using real-time PCR

Quantitative real-time PCR of beta-globin (HBB), a chromosome 1 1 locus, was performed using Applied Biosystems 7000 Sequence Detector (Applied Biosystems, CA, US). Sex determining region Y (SRY) was included only in samples collected from women carrying male foetuses. Commercial male genomic DNA with known concentrations was serially diluted 5-fold to generate the standard curves for absolute quantitation. Each sample and standard was run in triplicate with both sample and standard running in parallel. Water blanks were included in triplicates for each PCR as amplification negative controls. Reactions were set up in a reaction volume of 25 μΙ using the TaqMan Universal PCR Master Mix (Applied Biosystems). Three microliters of WGA DNA (concentration unknown), and 30 ng genomic DNA were amplified. Thermal cycling for was initiated with a 2-min incubation at 50^°C, to allow the uracil N- glycosylase (UNG) to act, followed by a first denaturation step of 10 min at 95^*C and then 55 cycles of 95^°C for 15 s and 60 for 1 min.

Quantitative Fluorescence-PCR (QF-PCR)

PCR amplification was performed in a total reaction volume of 25 μΙ containing 30 ng DNA, 0.1 - 0.4 pmoles of each fluorescent-labelled and unlabeled primer (AITBiotech, Singapore) and 1 x PCR multiplex master mix (Qiagen, GmbH, Hilden, Germany). Following initial denaturation at 95 °C for 15 min, 28 cycles of denaturation at 94°C for 30 s, annealing for 90 s at 58 "C and extension at 72°C for 90 s. This was followed by a final extension step at 72°C for 10 min. Amplification was carried out in a Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). One microlitre of the amplified allelic fragments was mixed with 9.5 μΙ formamide and 0.5 μΙ Genescan-500 Rox (6-carboxy-X-rhodamine) (ABI) size standards in an optical 96-well reaction plate before denaturation at 95 °C for 2 min. This was followed by 4°C for 2 min to prevent re-annealing before capillary electrophoresis with an ABI Prism 310 Genetic Analyser (ABI). GeneScan Analysis Software version 3.1 (ABI) was used for data analysis.

Quantitative PCR Results

Real-time PCR of HBB and SRY were performed on WGA DNA samples from five LCM and five manually micromanipulated (MM) male FNRBCs to quantify the amounts of DNA, as well as compare differences in downstream PCR efficacies between LCM and manually picked cells, if any. The five FNRBCs were pooled together during LCM and MM before WGA as a whole. While HBB and SRY amplifications were successful in the MM DNA sample (HBB/SRY = 1 172.7/290.5 GE in 50 μΙ WGA eluate), PCR amplifications failed in the LCM DNA sample. To determine whether Wright's staining and ICC would affect the quality of the WGA DNA from LCM cells, WGA of varying cell numbers (1 , 3, 5, 10) from Wright's/ICC stained (labelled as "ICC LCM Cells") and unstained LCM cells (labelled as "Unstained LCM Cells") were performed. Similar numbers of MM cells stained with live cell nuclear staining (labelled as "Hoechst+ Live Cells"), and non-stained cells (labelled as "Unstained Live Cells") were also included. Real-time PCR of HBB was again used to quantify the amounts of DNA.

Figure 10 shows the quantity in genome equivalents (GE) of LCM and live FNRBCs from digested trophoblasts with varying cell numbers of 1 , 3, 5, and 10. PCR amplifications were directly proportional to the number of cells. A detrimental effect on PCR amplifications was also observed when LCM cells were Wright's stained followed by ICC. Unstained LCM cells showed better PCR amplifications, however, staining was included in a clinical sample. For optimal PCR amplifications, micromanipulated cells that are stained without affecting cell morphology (i.e. Hoechst) are preferred for downstream genetic analysis.

QF-PCR Results

The Table shows STR analysis of WGA DNA of 10 cells that were manually retrieved from MACS-sorted maternal blood (labelled as "10-cell from MB29"). Foetal DNA was isolated from chorion villus biopsy (labelled as "Villi DNA") while maternal DNA was isolated from CD45- positive cells that were MACS-sorted from maternal blood (labelled as "CD45+ DNA"). A total of 23 STRs throughout chromosomes 13, 18, 21 , X and Y were analyzed. Of these, nine STRs (highlighted in red) showed the presence of paternally-inherited foetal alleles in the amplified DNA of 10 cells from maternal blood. These alleles were present in villi DNA and absent in CD45-positive maternal cells.

DNA

(MB29)

CD+45 354 362 429 297 297 159 241 250 352 359

DNA

(MB29)

Sample D13S631 D21 S1412 D13S258 D21 S11 D21 S141 1 D21 S226

10-cell 195 199 403 407 -234 279 261 336 338 456

from

MB29

Villi 195 199 403 406 235 279 253 261 307 31 1 456

DNA

(MB29)

CD+45 199 212 395 406 234 275 253 261 307 311 456 459

DNA

(MB29)

Sample X22 XHPRT DXS6785 DXS6809 DXS6803 SRY SRY

10-cell 201 285 148 176 261 1 15 177 175 from

MB29

Villi 201 228 286 151 261 116 176 176

DNA

(MB29)

CD+45 217 228 286 151 261 1 16

DNA

(MB29)

Table 1. Quantitative Fluorescence (QF)-PCR results

Results

Successful automatic micromanipulation of FNRBCs from maternal blood obtained before termination of pregnancy

Pre - TOP matemal blood is the ideal sample for NIPD assay and the results showed that 10 - 20 FNRBCs from 20 ml matemal blood can be screened and automatically micromanipulated (Figure 12). These numbers corresponded with the expected numbers achieved from manual picking and other references (1 FNRBC/ ml maternal blood). Shortened total spending time for automatic micromanipulation

Automatic micromanipulation with scanning the whole sample took 1 - 2 hours whereas manual micromanipulation requires 4 - 5 hours by experts. Considering at least about 3000 amniocentesis per year in Singapore for high risk pregnancy, it needs 10 samples to do NIPD. Only automatic micromanipulation can perform this task with limiting the working hours with

Increased holding capacity of FNRBCs in one PDMS microwell stamp

One PDMS stamp could hold more than 4 x 10⁶ cells. This meant that only two PDMS stamps were needed for one sample. One PDMS stamp had about 0.1 x 10⁶ wells. This was because from the pre - enrichment procedure, the amount of nucleated cells reduced so in the PDMS stamp, well suspended nucleated cells were still far from each other, enough to micromanipulate one by one even if cells were densely packed (Figure 13). 20 x 20 or 30 x 30 pm microwells were used and both worked well for FNRBC screening.

Sufficient amounts of DNA achieved by whole genome amplification from a single FNRBC that was retrieved by automated micromanipulation

The amount, 7 pg of DNA from whole genome amplification for single cell was sufficient to perform downstream molecular genetic analysis (Figure 14).

Establishment of whole genome sequencing strategy from single FNRBCs

The FNRBCs contained a complete set of foetal genome and thus whole genome sequencing can be established as a diagnostic test. The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A method of separating at least one foetal nucleated red blood cell (FNRBC) from a sample comprising:

a) placing the sample on at least one support surface;

c) providing a means capable of detection and isolation of at least one cell in the sample comprising the features of:

(i) comprising nucleic acid

(ii) being an unlabelled cell; and

(iii) being 5 to 25μιη in size ,

wherein the detection of the features (i), (ii) and (iii) is done simultaneously and the cell comprising the features (i), (ii) and (iii) is the FNRBC.

2. A method of separating at least one foetal nucleated red blood cell (FNRBC) from a sample comprising:

a) placing the sample on at least one support surface;

(i) comprising nucleic acid;

(ii) being an unlabelled cell; and

(iii) being 5 to 25μιη in size ,

wherein the detection of the features (i), (ii) and (iii) is done sequentially and the cell comprising the features (i), (ii) and (iii) is the FNRBC.

3. The method according to either claim 1 or 2, wherein the support surface is selected from the group consisting of a tissue slide, a microscope slide, a plate, a multi-well plate, an array, a multi-well array, a two or three-dimensional scaffold, a membrane and a film.

4. The method according to any one of the preceding claims, wherein the support surface is an array which comprises a plurality of microwells.

5. The method according to claim 4, wherein the microwell is made of PDMS or glass.

6. The method according to either claim 4 or 5, wherein each microwell is about 30 μνη x about 30 μηη in size.

7. The method according to any one of the preceding claims, wherein the isolation of the FNRBC is done using at least one capillary tip.

8. The method according to claim 7, wherein the aspiration rate of the capillary tip is programmatically controlled.

9. The method according to any one of the preceding claims, wherein the detection is programmatically controlled.

10. The method according to any one of the preceding claims, wherein the isolation of the FNRBC is done using at least one micromanipulator.

1 1 . The method according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof specifically binds to CD45, CD14, CD5, CD19, CD20, CD3, CD4, CD8, CD1 6, CD56 or a combination thereof.

12. The method according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof specifically binds to CD45 and the CD45 negative cells are completely unlabelled.

13. The method according to any one of the preceding claims, wherein the presence of nucleic acid in the cell in step (b) is determined using at least one method selected from the group consisting of phase contrast microscopy, colorimetric assay, autoradiography, Raman spectroscopy and combination thereof.

14. The method according to any one of the preceding claims, wherein the size of cells is determined using at least one optical method.

15. The method according to any one of the preceding claims, wherein the sample is selected from the group consisting of maternal blood, maternal tissue, cord blood and a combination thereof.

16. The method according to claim 15, wherein the maternal tissue is selected from the group consisting of trophoblast tissue, liver tissue, placental tissue and a combination thereof.

17. The method according to any one of the preceding claims, wherein the isolated FNRBC is further analysed using at least one method selected from the group consisting of polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), multiple-ligand dependent probe amplification (mlpa), short tendon repeat analysis, array competitive genomic hybridization (CGH), genotyping, single plex sequencing, massively parallel sequencing, next generation sequencing and a combination thereof.

18. The method according to any one of the preceding claims, wherein the FNRBC is further tested for at least one prenatal disorder.

19. The method according to claim 18, wherein the prenatal disorder is selected from the group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs Disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's Disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne muscular dystrophy, mitochondrial disorder, Hereditary multiple exostoses, osteogenesis imperfecta disorder and a combination thereof.

20. The method according to any one of the preceding claims, wherein the FNRBC is tested for gender of a foetus.

21. The method according to any one of the preceding claims, wherein the sample is from a mother in a first trimester, a second trimester or a third trimester of pregnancy.

22. The method according to any one of the preceding claims, wherein the FNRBC is a human FNRBC.

23. The method according to any one of the preceding claims, wherein the sample comprises at least one anucleated red blood cell (ARBC).

24. A device capable of performing the method according to any one of the preceding claims.