Background of the invention The examination of fetal cells for early detection of fetal diseases and genetic ab- normalities is carried out in connection with many pregnancies, particularly when the maternal age is high (35 years or above) or where there is a family history of genetic diseases. Fetal cells may be obtained by amniocentesis, the removal of amniotic fluid from the amniotic cavity within the amniotic sac or by chorion biopsy, where biopsies are taken from the placenta, a so-called invasive sampling.

During pregnancy a variety of cell types of fetal origin cross the placenta and circu- late within the maternal peripheral blood. The feasability of using fetal cells in the maternal circulation for diagnostic purposes, has been hindered by the fact that fetal cells are present in maternal blood in very limited numbers, reported ratios have been from 1: 105 to 1: 108 fetal cells per nucleated maternal cells. In addition most fetal cells cannot be distinguished from maternal cells on the basis of morphology alone, but rather must be identified based upon detection of fetal cell markers. Evi- dently it would be of great advantage to perform fetal diagnostics by a less invasive procedure, such as through a maternal blood sample.

One particular fetal cell type within maternal blood that has been demonstrated to be useful for detecting fetal DNA is the nucleated erythrocyte.

Also, fetal leukocytes have been reported to be present in maternal blood. Leuko- cytes are one subpopulation of white blood cells found in the blood. There are three types of subsets of leukocytes (which also are referred to as polymorphnuclear leu- kocytes): neutrophils, basophils and eosinophils. All leukocytes have a distinctive morphology characterized by the nucleus and cellular granules. Furthermore, fetal lymphocytes and trophoblasts may be present in the maternal blood.

Due to the very limited number of fetal cells in maternal blood concentration or en- richment of the maternal blood sample with respect to the fetal cells have been con- ducted by negative selection, i. e. removal of maternal cells. Enrichment of fetal cells by density gradient centrifugation or by removing maternal cells with an antibody to a cell surface antigen is described in for example US 5,858,649, US 5,731,156, US 5,766,843 and US 5,861,253.

Yet another method of removing maternal cells, in particular maternal erythrocytes, is by lysing, again optionally combined with immunologic methods for removing the maternal cells.

Another selection procedure is to mark the fetal cells for example by the use of CD71 antibodies. This is called positive selection as opposed to negative selection mentioned above where maternal cells are removed.

Immobilisation of a ligand by linking the ligand to a surface is a well-known prepara- tion for a separation technique. For example in WO 96/31557 a method of photo- chemical immobilisation of ligands using quinones is described.

In various systems attempts for increasing the efficacy has been performed. For example, US 5,759,793 describes a method for the affinity separation of human stem cells in a fluidised bed system, using a magnetically stabilised fluidised bed.

Another approach is described in US 4,710,472 wherein a magnetic separation de- vice suitable for the removal of magnetic bead-coated cells is described.

It is an object of the present invention to improve the separation of cells by a method using antibody binding, in particular with respect to samples having a very low con- centration of the target cells, such as fetal cells in maternal blood or cancer cells.

Summary of the invention Accordingly, the present invention provides a method for the isolation of at least one target cell population, in particular a mammalian cell population, from a liquid sam- ple of cells comprising:

-establishing a sample flow over a surface coated with antibodies specific for said target cell population, -allowing said cell sample and surface to be in physical contact providing for the cells from the at least one mammalian target cell population to bind to the anti- bodies on the surface, -removing the liquid sample comprising non-bound cells from the surface, and -identifying the target cells on the surface.

By the present method it is possible to flow a large volume of the sample over the surface, whereby a large number of the target cells may get in contact with the sur- face. The term"sample flow"is used in its normal meaning, whereby a sample hav- ing a volume larger than the volume of the chamber wherein the surface is arranged may be flowed over and thereby brought in contact with the surface. Merely stirring or agitating a sample in a chamber without replacing at least part of the sample con- stantly is not understood as flow.

Another object of the present invention relates to a system providing for isolating at least one cell population from a liquid sample of cells, comprising a surface sur- rounded by at least one wall, said surface being coated with antibodies specific for said target cell population, means for establishing a sample flow over said surface allowing said cell sample and surface to be in physical contact such that the cells from the at least one mammalian target cell population may bind to the antibodies on the surface, means for removing liquid sample comprising non-bound cells, and means for identifying target cells on the surface.

Yet another object relates to the use of the target cell population for diagnostic pur- poses.

Drawings Figure 1: depicts a model of the system for the separation of a cell target population.

Figure 2: depicts another embodiment of the system of Figure 1.

Figure 3: is a schematic drawing of a system according to the invention, wherein fig.

3a is a closure and fig. 3b shows the system comprising the surface.

Detailed description of the invention The present invention reveals a novel method and a system for the isolation of mammalian target cell populations. The present method has improved the separa- tion of target cells from other cells in a liquid sample in particular when the ratio of target cells to the other cells in the liquid sample is small. By the term target cell population is meant the cells desired to identify, which cells are distinguishable from the other cells in the sample by the use of species selective identifiers, such as anti- bodies. The cell sample of the present invention is passed or flowed over an anti- body-covered surface.

The method may be conducted by use of a system as defined below.

In a further embodiment the invention relates to a system providing for the recovery of the target cells from the surface, for example when the target cell population is in a blood sample.

In one embodiment of the present invention the liquid sample of cells is a blood sample. More particularly the present invention has proven successful in the isola- tion of fetal cells from maternal cells. Fetal cells may occur in the maternal blood stream at a ratio of 1: 105 to 1: 108 with respect to the maternal cells. In the light of this naturally occurring ratio the present invention presents a method wherein fetal cells are isolated from maternal blood without prior enrichment or concentration of the sample. This provides for an improved method of isolation by which the risk of loosing fetal cells due to enrichment or concentration procedures has been greatly reduced.

In one aspect of the present invention the cell sample is diluted prior to being passed over the surface. In a preferred embodiment the cell sample is diluted 2-10 times, more preferably 4-6 times by the addition of isotonic buffers, such as saline

solutions, phosphat buffered saline solutions, PBS, and/or suitable growth media, such as basal media, and tissues growth media. The dilution of the cell sample en- hances the probability that substantially all of the fetal cells will experience being in contact with the antibody-covered surface.

In one aspect of the invention it is desirable to obtain as large a cell sample as pos- sible, such as a maternal blood sample, in order to increase the total number of fetal cells. However, due to practical problems the size of the sample must be within certain limits. Accordingly, the size of the maternal blood sample is preferably in the range of 5 to 40 ml, such as from 10 to 30 ml.

As described above an important aspect of the present invention is the establish- ment of a sample flow over the surface. The flow may be established by any suitable means, such as by gravity, by use of a pump or by suction. It is however of impor- tance that the flow is controllable and ajustable to be adapted to the specific sam- ple and surface used. According to the invention the flow is preferably created by a pump that preferably is arranged downstream of the surface in order to avoid any cell damage from the pump prior to binding the cells.

The flow of the cell sample may be a continuous flow, so that the contact between the surface and the cell sample is at a constant rate. In a more preferred embodi- ment however the flow is discontinuous, by changing the flow rate during the flow, such as by alternating high and low flow rates during the passing of the sample over the surface. In another embodiment of the invention the flow is sporadically dis- rupted. One way of creating a disrupted flow according to the invention is by apply- ing a stepped stop flow. In one embodiment of the present invention the stop flow comprises three steps: a flow step, and a sedimentation step followed by another flow step respectively. The time frame for the length of the individual steps may be of any length. However, within the scope of the present invention the ratio for the preferred time frames for the individual steps is from 1: 5 to 2: 1, such as from 1: 5 to 1: 60, or from 1: 10 to 2: 1 for the flow step to the sedimentation step, more preferred from 1: 20 to 1: 40 or from 1: 5 to 1: 1. It is further preferred that at least two sedimen- tation steps are provided for each volume during its pass over the surface.

The flow rate is held within predetermined range to obtain an optimized surface- sample contact. Accordingly, the flow applied to the cell sample may have a flow rate from 0.2-5 ml/min, such as 0.2-3 ml/min or 1-5 ml/min., more preferably about 1 ml, or about 2.5 ml/min.

Furthermore, a turbulence in the flow of the cells may be created. The object of the present invention is to provide for improved isolation of a cell target population, and a movement of cells within the sample may increase the probability of physical con- tact between the cells and the surface. For this purpose turbulence may be created in the sample ensuring an optimal contact. By creating a turbulence in the flow of the cell sample the probability that the surface-cell interaction will occur increases, and thereby the probability of isolating a target cell population increases. Turbulence may be arranged by any means suitable for a turbulent flow over the surface avoid- ing that the cells are damaged due to the turbulence. The turbulence may be cre- ated by arranging a rotating magnet in the flow, such as adjacent the surface, but preferably not in contact with the surface. In another embodiment the magnet is lo- cated in the flow upstream the surface creating the turbulence before the flow over the surface.

Another means for creating the turbulence may be mechanical hindrance of a lami- nar flow over the surface.

Other means for attracting the cells towards the surface may be used either alone or in addition to creating a turbulent flow and/or changing the flow.

Accordingly, in one embodiment of the present invention a magnetic field is effecting cells in the sample having metal particles positioned intracellularly and/or extracel- lularly. The positioning of metal particles on or inside the cells provides for the means of attracting the cells to the surface by applying a magnetic field. Thus, it is preferred that the magnetic field is attracting the cells towards the surface. The cells comprising metal particles are preferably only the target cells, however, the method may also be applied in case substantially all cells contain the metal particles, in that only the target cells are bound, whereby removal of the magnetic field will cause unbound cells to pass on. By the term metal particles is meant any type of magnetic

particles, such as magnetic beads. In particular magnetic beads comprising iron, such as ferro fluidics are used.

According to the invention the magnetic field may be constant or it may be pulsating.

By the word"constant"is meant a magnetic field, which is applied to the cell sample at the same magnitude throughout the isolation procedure. In the context of the pre- sent invention the word"pulsating"means applying a magnetic field to the cell sam- ple in intervals. Pulsating magnetic field is preferred, so that unbound cells continue flowing.

Further means for attracting a target cell population to the surface according to the invention are electrical forces, such as a charge flow wherein the knowledge of the different charges of various cell types are employed as a tool of isolation of a target cell population.

The surface according to the invention is any type of surface capable of being coated with antibodies under the flow conditions described above. By the term coated is meant any form of having the antibodies attached to the surface.

In a preferred embodiment of the present invention the surface on which the target cell population will bind to is levelled in the meaning of being non-curved. The sur- face according to the invention may be positioned in any possible angle provided it is levelled.

For the purpose of the present invention the surface is levelled and even thereby allowing for the identification of the target cell population in a microscope.

In the system according to the invention the surface is surrounded by at least one wall. Said wall may be divided into several wall pieces, such as four wall pieces around a rectangular or square surface. In another embodiment the wall is con- structed by an O-ring or the like situated on or around said surface. Said wall may also be arranged by locating the surface in a chamber, and the flow is then created in the chamber.

According to the invention the surface may constitute at least one wall of the cham- ber, such as the bottom wall. The surface may be situated in a chamber to ensure the enclosure of the cell sample and to allow for the creation of turbulence in the cell sample.

In a preferred embodiment the chamber is a closed chamber apart from at least one inlet and at least one outlet, said closed chamber being provided by arranging a closure of the chamber, preferably the closure provides a fluid tight sealing of the chamber.

The closure of the chamber according to the invention may be even or uneven, such as grooved, on the side of the closure facing the sample in the chamber. In case of the latter the created turbulence of the flow of the cell sample may be further poten- tiated by the shape of the closure. Any one or all of the walls and closure may be made of any type of glass or plastic. In particular with respect to the method of iden- tification it is preferred that at least the surface part and the closure are transparent or at least partly transparent allowing the cells to be microscopically identified when the surface is in the chamber.

However a part of the surface as well as a part of the closure may be produced from metal providing for electrical conduction, in particular in relation to a charge flow as discussed above. Either the whole surface and/or closure is made from metal or metal wires may be incorporated in glass or plastic surface and/or closure.

The dimensions of the chamber provided are not crucial for the method to be carried out. In practice the surface area is often between 10 and 100 cm2, such as between 10 and 50 cm2 It is preferred that the height of the chamber is below 1 cm, prefera- bly from 0.1 to 2 mm, such as from 0.2 to 2.0 mm, or from 0.1 to 1.0 mm.

For carrying out several isolations simultaneously it is preferred that the system comprises several individual surfaces or chambers, each separated from each other being supplied individually with a flow. Such system may comprise 5 or 10 adjacent surfaces or chambers or even more.

The fluid tight sealing of the chamber may be arranged by providing a surrounding wall made of rubber, synthetic rubber or other flexible material. Also the wall may be made of Teflon, thereby reducing the friction and attachment of cells to the wall.

The antibodies on the surface are antibodies specifically directed to the target cells, having no or little affinity towards the other cells of the sample. Accordingly, the an- tibody is preferably specific for cell-surface situated epitopes expressed by the tar- get cells. However, antibodies directed towards intracellular antigens may be used if the cells are conditioned therefor, i. e. by a permeabilising treatment.

When isolating fetal cells the antibodies are preferably antibodies directed to EPO erythrin or CD71 antibodies or CD36 antibodies.

The identification of the target cells may be carried out by any suitable method, such as labelling of the target cells, for example fetal cells or cancer cells.

Fetal cells may be distinguished from maternal cells by the specific recognition of a fetal cell antigen or they may be distinguished from maternal cells by the specific recognition of RNA encoding a protein selectively produced by fetal cells.

According to one aspect of the present invention the selective labelling is based on an antigen-antibody reaction wherein the antibodies are specific for epitopes ex- pressed by the target cell population, such as a protein selectively produced by fetal cells. Such a protein may be selected from the group consisting of embryonic he- moglobin, such as E and zeta hemoglobin, and fetal hemoglobin, such as gamma and alpha hemoglobin.

In particular the labelling may be carried out by the use of an antibody selected from antibodies against various types of normal globin chains in human hemoglobin, for example anti epsilon (e) antibodies, anti zeta antibodies, anti gamma (y) anti- bodies, anti alpha (a) antibodies, and anti beta antibodies.

The antibodies may be monoclonal or polyclonal antibodies, however the mono- clonal antibodies are preferred.

Furthermore, the antibodies may be labelle or unlabelled antibodies. The labelled antibodies are preferably FITC labelled, biotin labelled or TRITC labelled antibodies, but other labellings may be used as well.

Thus, according to the present invention the labelling may be carried out using anti epsilon (s) monoclonal antibodies or anti zeta (4) monoclonal antibodies, more pref- erably anti epsilon (e) unlabelled monoclonal antibodies or anti zeta () unlabelled monoclonal antibodies, or anti epsilon (E) monoclonal biotin labelle antibodies or anti zeta (4) monoclonal biotin labelle antibodies.

In yet another embodiment of the invention the labelling is performed using anti gamma (y) FITC labelle antibodies.

Two or more selective labellings may be performed in order to enhance the prob- ability and selectivity of identifying the fetal cells bound to the surface on the back- ground of maternal cells. The combination of two or more labellings may be a com- bination of any of the labellings used for single labelling.

The enhanced selective labelling may also be performed by the use of two or more antibodies directed against the same protein or different proteins. In this embodi- ment the labelling with two or more labels may be carried out simultaneously.

Preferably, fetal-cell-specific RNA sequences are used as fetal cell markers. Such RNA is generally messenger RNA (mRNA). The presence of such RNA indicates that the gene for the fetal protein is being transcribed and expressed. Fetal cells contain distinct mRNAs or RNA species that do not occur in other cell types. The detection of these RNAs, whether as mRNA can serve to identify cells, or even sub- cellular fractions of cells fetal or embryonic in origin. According to the present inven- tion the m-RNA may be coding for a protein selected from the group consisting of embryonic hemoglobin, such as s and zeta hemoglobin and fetal hemoglobin, such as gamma and alpha hemoglobin.

Further, according to the present invention DNA probes (oligos) for the hybridisation are directed against embryonic cell RNA, such as for E and zeta hemoglobin, and for fetal hemoglobin, such as for gamma and alpha hemoglobin. A DNA probe may be

synthesised as an oligodeoxynucleotide using a commercial synthesiser. Probes may be comprised of the natural nucleotide bases or known analogues of the natu- ral nucleotide bases.

Also, a combined labelling may be carried out by the use of two or more different hybridisation probes, such as a combination of a DNA probe and a PNA probe for hybridisation with the same fetal RNA or more preferred with different RNAs. Fur- thermore, two or more different DNA probes (or PNA probes or LNA probes) may be used for hybridisation with different fetal RNAs.

In another embodiment a combination of an immunological labelling and a hybridi- sation labelling may be employed according to the present invention. In this em- bodiment the labelling is normally carried out sequentially by a first immunological labelling step, then identification of the labelled cells, and then a second step of hy- bridisation labelling for verification of the identification of the cells labelle by the first step. It is preferred in the first step to use antibodies against epsilon, gamma and/or zeta globin and in the second step to use hybridisation for epsilon and/or zeta globin mRNA to verify the fetal cells identified.

The nucleic acid of the fetal cells can also be amplified prior to detection using a known amplification technique, such as the polymerase chain reaction (in situ RT- PCR). Primers for in situ RT-PCR amplification are chosen to specifically amplify a mRNA of interest in the target cell, as mentioned above for hybridisation.

The identification step may further comprise a microscopy step, wherein the cells labelle, independent of the labelling method, are identified by microscopy directly on the surface.

Due to the large amount of cells to be examined to find the small amount of fetal cells in the blood sample an important factor for the detection equipment is the rate of cells identified per unit of time. For example very fast scanning microscopes may be used for the identification. Also, laser scanners could be used. Preferably the laser scanner is equipped with at least two lasers emitting light with different wave- length capable to excite the various labels on the cells or in one laser emitting sev- eral wavelengths.

Preferably, during or after identification of the fetal cells the position of detected la- belled cells on the surface is recorded. This provides for the later collection of the detected cells from the position which has been recorded. The position of the de- tected labelle cells on the supporting surface may be recorded by use of a scanner provided with detectors registering the light emitted from the labelle cells, such photo-multipliers, CCDs, or the like detectors. Thereby it is possible to identify and specifically isolate substantially only the selectively labelled fetal cells.

In a preferred embodiment the scanner is arranged for detecting selectively labelle fetal cells, and when detecting a fetal cell, carrying out a verification step by switch- ing to another wavelength to verify presence of for example staining of the nucleus.

It is of importance for the use of the method that a fast scanning system is used, for example a scanner capable of scanning in the range of from 0.1 m/sec to 10 m/sec or faster, such as appr. 1 m/sec.

Further studies and use of the target cells may be performed on the cells situated on the surface. However, depending on the purpose of isolating the target cells, it may be appropriate to recover the identified cells from the surface.

In one aspect of the present invention the target cells are recovered from the sur- face by micro-dissection. In the present context, micro-dissection is used to mean that each target-cell is identified and removed individually. Micro-dissection may be carried out by means of a laser apparatus, whereby a laser beam may liberate the target cells from the surface preserving the viability of the target cells, and thereby ensuring the further in vivo studies of the living target cells.

Another method of micro-dissection is micro-manipulation, optionally by means of minute canulas.

The cells recovered and collected according to any of the procedures may be used for diagnostic purposes, i. e. being subjected to further identification and/or investi- gation, such as microscopic and/or molecular identification and/or investigation. The cells may be subjected to investigations of analysing the presence of genetic dis-

eases. For example the nucleic acid of fetal cells may be analysed for diagnostic or other purposes. For instance the presence or absence of a particular nucleic acid may indicate the presence or absence of certain genes coding for diseases, such as cystic fibrosis. The nucleic acid may additionally be analysed for X or Y specificity.

Thus, the presence of a Y chromosome encoded genes or gene products is a qualitative distinguishing feature of the cells of a male fetus.

Also, viable cells recovered from the surface may be cultured for further investiga- tions.

Verification of the selective identification of fetal cells may be carried out by several methods. In a model system the method may be performed on maternal blood sam- ples from pregnant women carrying a male fetus. The cells isolated may then be analysed for the presence of a Y chromosome, indicative of cells being from the male fetus.

Another verification method, which is independent of the sex of the fetus, is verifica- tion by the use of identification of small tandem repeats (STR), or variable number tandem repeats (VNTR) to detect genetic input from the father, thereby verifying fetal cells, as the only cells in the sample comprising input from the father.

As mentioned above the present method may be carried out for the isolation of any kind of rare event target cells, and is particular interesting when used for rare event cells being present in very low concentrations, such as those for the fetal cells in maternal blood or for blood-borne cells in some cancer forms.

The system and method according to the invention is further described with relation to the drawings: The system 1 is depicted in Fig. 1, wherein a chamber 1 is arranged in the flow of the sample. A sample, optionally diluted, located in a container 3 provided with an outlet 4. The sample is preferably stirred, such as by means of a rotating magnet 5 in the container to obtain a homogenous slurry before entering into the flow. The sample is flowing from the container 3 through outlet 4 into the line 6 towards inlet 7 of the chamber 1. In chamber 1, the sample is passing over the surface 8 arranged

in the bottom of the chamber 1. In Fig. 1 the chamber 1 is defined by the walls 12 made of Teflon in the present case, and transparent closure 2.

Unbound cells and liquid is flowing further out of outlet 9 into line 10. In a preferred embodiment, the flow is created by means of pump 11 arranged in line 10. The liq- uid comprising unbound cells may then be discarded or used elsewhere. The liquid may also be reflowed to the system at least once in order to increase the number of target cells bound.

Turning to Fig. 2, it may be seen that a rotating magnet 12 is arranged in the cham- ber 1 for providing turbulence in the flow in the chamber 1. The magnet 12 is located adjacent the closure 2, not to be brought in contact with the antibody-coated surface 8, in order to avoid damaging the surface 8. Means for establishing a magnetic field in the chamber (not shown) may be provided outside the chamber.

In Fig. 3a the closure 2 is shown in greater detail. The inlet 7 and outlet 9 is led through the closure 2. In Fig. 3a the inlet 7 is divided into 3 inlet streams 13 during the flow through the closure. The division into several inlet streams may be carried out before the flow reaches the closure or during the flow through the closure 2. Cor- respondingly, the outlet 9 may collect flows from one to several outlet streams 14, in the depicted example 3 outlet streams 14 are shown. It is within the scope of the invention that from one to several inlet 13 and outlet 14 streams may be comprised in the flow. Variations in the location of the inlet and outlet streams is foreseen with the present invention, such as inlet streams perpendicular to the face of the closure facing the sample or oblique inlet streams, whereby the inlet is led obliquely through the closure towards the face of the closure facing the sample. In any embodiment it is the object to obtain a flow of the sample over substantially all of the surface coated with antibodies.

In Fig. 3b an example of the surface 8 surrounded by four wall pieces 12 is shown.

When covered by the closure of Fig. 3a a chamber is created having the surface 8 as one wall of the chamber. By combining Fig. 3a and 3b it may be seen that a sample may be flowing in line 6 through inlet 7 into the inlet streams 13 onto the surface 8. The liquid comprising unbound cells is removed from the surface through outlet streams 14 via outlet 9 into line 10.