EP0855030A1

EP0855030A1 - Method and installations for separating magnetic particles in a fluid for biological analysis, and application of said method

Info

Publication number: EP0855030A1
Application number: EP97923133A
Authority: EP
Inventors: Jean-Claude Bisconte De Saint Julien
Original assignee: Biocom SA
Current assignee: Biocom SA
Priority date: 1996-05-07
Filing date: 1997-05-05
Publication date: 1998-07-29
Also published as: US6143577A; FR2748569B1; WO1997042503A1; JP2000500871A; FR2748569A1

Abstract

The invention discloses a magnetic immunoseparation method of cells, particularly of bacteria, foetal cells, bone marrow stem cells and systemic cancerous cells, consisting in fixing target cells on paramagnetic balls and causing a magnetic field to act on a sample containing the fixed cells, the free cells and the supernumerary paramagnetic balls, to isolate the paramagnetic balls. The sample is circulated in a tube (2), the cross-section of which is much less than the length on which the magnetic field is applied.

Description

METHOD AND PLANTS FOR SEPARATING MAGNETIC PARTICLES IN A FLUID FOR BIOLOGICAL ANALYSIS, AND APPLICATION OF SAID METHOD. The present invention relates to a method and an installation for separating magnetic particles in a fluid for the biological analysis of rare events.

The invention more particularly finds its application in the fields of medical diagnosis and quality control, in particular in the food industry, but also in the therapeutic field for detecting and taking from samples a cell type with a low occurrence. As examples of medical applications of the invention, there may be mentioned:

Separation of fetal cells present in maternal blood. In this case, it involves selectively collecting cells present at the rate of approximately 1 fetal cell per 10 million non-fetal cells. The implementation of the present invention should make it possible to replace the risk samples of amniotic cells.

- The preparation of stem cells from the bone marrow making it possible to re-seed marrow destroyed following anticancer treatment by chemotherapy or radiotherapy. As in the case of fetal cells, the number of bone marrow cells in the peripheral blood is much lower than that of other cells, of the order of 1 per 200,000.

The early detection of circulating cancer cells or micro metastasis, which is of the greatest interest in determining exploratory and therapeutic strategies. Again, the goal is to isolate and identify 1 cell for about 5 million nucleated cells.

In the field of biological control of food or the environment, the objective is to obtain a rapid result without going through the traditional phase of multiplication in culture. The expected sensitivities are of the order of 1 cell (bacteria, yeast or mold) for 1 gram or even 10 grams of product. In the prior art, there are many devices making it possible to achieve this goal. Among these are:

Physical methods based on differences in size, density or electrical charge (De Duve, 1971; Zeiller, 1972; Pretlow and Pretlow, 1982), but these methods lack specificity.

- The immuno-affinity methods consisting in fixing an antibody on a support, which antibody reacts vis-à-vis an antigenic motif present on the surface of the cells sought (Forsgren and Sjoquist, 1986; Langone, 1982). Different methods deriving from immunoaffinity methods, such as affinity chromatography (Hunt et al., 1982), have been proposed.

- Cell separation associated with fluorescence detection under the so-called method

"FACS" for the English expression "Fluorescence Activated Cell Sorting". This widely described method uses sophisticated equipment comprising a liquid flow through which the cells pass. A laser beam excites the fluorescence and thus triggers a signal which allows the cell to be deflected electrically in a container. This method is very effective and achieves an enrichment of almost 100% but it is not suitable for sorting large populations. Magnetic separation methods designated "MACS" for the English expression "Magnetic Affinity Cell Sorting", which are based on the use of magnetic particles ingested by cells (Melville et al., 1975) or selectively attached to cells through antibodies (Molday et al., 1977).

FACS and MACS methods are well known today and have already given rise to industrial implementation. In general, the results obtained with each of these methods differ little in terms of effectiveness and allow between 70 and 100% recovery. However, they are more or less suitable for the various applications envisaged.

The present invention is also based on the use of magnetic particles on the surface of which is immobilized a substance capable of specifically binding to a complementary substance which is free or present on the surface of cells. The analysis is carried out in a liquid medium by subjecting the biological medium into which said magnetic particles have been introduced to a magnetic field.

The magnetic particles more particularly envisaged within the framework of the invention are microbeads of the type of those sold by the companies Dynal, Rhône Poulenc or Sigma.

These paramagnetic microbeads of micron size can have on their surface different ligands depending on the application which is envisaged. These ligands are fixed to the surface of the microbeads by the techniques described in the prior art, these ligands can be:

Poly or monoclonal antibodies specific for antigens present on the surface of certain cell types, such as the proteins CD2, CD4, CD8 expressed on the surface of T lymphocytes. antibodies are attached to the surface of microbeads, for example through immunoglobulins.

- Oligonuleotides, for example oligo (dT) or oligo (dA) of variable sizes, biotinylated and therefore fixed to the surface of the microbeads by means of streptavidin. These oligonucleotides make it possible, by hybridization, to purify or extract RNA or DNA from samples previously treated so as to render the nucleic acids which it contains capable of hybridizing. The use of these oligonucleotides attached to the surface of the paramagnetic beads can also constitute a preparatory phase for an amplification protocol by PCR or a cloning after PCR, or even be used for sequencing in solid phase. The beads thus loaded with a ligand are brought into contact with the sample which may have undergone a preliminary treatment, in order to achieve the segregation of the event sought by the action of a magnetic field. However, the use of this technique has the drawback of requiring multiple manipulations and leads to a set of beads more or less aggregated by the action of the magnet, among which are some rare cells. The disproportion of the number of beads compared to the number of cells, of the order of 1 to 10, then makes the identification and collection of these cells impossible. Various solutions have been proposed in the prior art to eliminate these drawbacks, such as the action of a detergent which releases the cell nuclei but subsequently prohibits the use or the visual identification or by an antibody of the separated cells, or even the use of detachment products which act in a variable way. However, in certain cases, the vital character of the separated cells is essential, either because we envisage a therapeutic re-use of the cells, case of hematopoietic stem cells, or a recultivation of cell culture in vitro to amplify a signal, case of cells fetal. The conventional techniques using microbeads described above can then have an injurious action on the cells and prevent the use of separating columns imposing on the cells a complicated and traumatic course.

The processes of the state of the art are therefore not entirely satisfactory. In particular, artifacts reduce theoretically foreseeable performances. In fact, excess paramagnetic beads sometimes tend to form a gangue that completely traps a target cell. Given the very low occurrence of target cells, this phenomenon is particularly harmful. In addition, the separation operations require careful observance of a tedious and repetitive protocol, which is a source of error.

The object of the present invention is therefore to offer a method eliminating the steps of rinsing and elimination of cells and liquids not sought and jointly the automatic selection of the sought elements and the segregation with free paramagnetic beads.

Another object of the present invention is to improve the efficiency of the magnetic methods by avoiding the artefacts noted in the methods of the prior art and by simplifying the implementation protocol.

Its purpose is to facilitate separation for the purpose of counting, analysis, or processing, biological material present at very low concentrations in a sample. The sensitivities sought in the abovementioned fields can be of the order of 1 cell for 1 or even 10 grams of sample.

The European patent EP206077 is also known in the state of the art.

This patent discloses the general principle of magnetic immunoseparation by circulation in a tubular segment placed in a uniform magnetic field. It has appeared in certain use cases that the excess particles could be attracted too quickly at the start of the tube, and that agglomerates were created blocking the circulation of the labeled or unmarked cells. Given the scarcity of target cells, this situation is very troublesome. The invention aims to remedy this drawback by proposing to create an inhomogeneous magnetic field. This characteristic prevents the formation of agglomerates of unbound particles in the upstream part of the tubular segment. The application of an inhomogeneous magnetic field associated with the flow produced by the flow of the sample in the tube of small cross section with respect to the length leads to a better distribution of the particles and facilitates the release of the agglomerates by the effect of entrainment of free or fixed cells, of larger dimensions. These cells exert a slight pressure on the agglomerates of magnetic particles, which are released in the weak field zones to come back to be isolated in isolation and no longer agglomerated in the next strong field zone.

These aims are achieved by a method and an installation combining, after the contact of the sample and the balls, both the flux and the magnetic separation. To this end, the invention firstly relates to a method of magnetic separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and of circulating cancerous cells, of the type consisting in fixing the target cells. on paramagnetic beads and causing a magnetic field to act on a sample containing the fixed cells, the free cells and the excess paramagnetic beads, to isolate the paramagnetic beads, characterized in that the sample is circulated in a tube whose the section is much less than the length over which the magnetic field is applied.

The sample preferably flows at a speed of a few centimeters per second. The excess beads, due to their lower density than that of marked or unlabeled cells, or the clusters of paramagnetic beads, due to their high sensitivity to the magnetic field, are quickly attracted against the wall of the tube and are immobilized in the part proximal of the tube.

Unfixed cells are insensitive to the magnetic field and pass through the tube. The cells fixed on paramagnetic beads are entrained by the flow inside the tube, before being immobilized against the wall of the tube, in the distal part of the tube.

One thus proceeds to a geometric separation of the components of the sample, which allows recovery of the target cells by various means simple to implement.

According to a preferred embodiment, the sample is made to circulate in a spirally wound tube placed in a magnetic field whose lines of flows are substantially perpendicular to the plane of the spiral.

Advantageously, the section of the tube is between 0.5 and 3 millimeters and the length of the tube is greater than 10 centimeters.

Preferably, the tube is placed in a non-homogeneous magnetic field. This field can be produced by a permanent magnet or by an electromagnet. According to an advantageous variant, the preparation is introduced into a reservoir connected to the separation tube, said reservoir being raised relative to the tube to ensure circulation of the sample by gravity.

The invention also relates to an installation for the magnetic immuno-separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and of circulating cancerous cells, characterized in that it consists of a tube whose cross section is much less than the length, said tube being wound to form a spiral, and by at least one magnet arranged to create a magnetic field substantially perpendicular to the plane of the spiral formed by said tube. According to a particular embodiment, it comprises a tube having two sections of increasing section, the second section being at least 10 times greater than the first section.

The invention also relates to applications of this method for:

• immunofiltration of blood in the extracorporeal circulation

• prenatal diagnosis

• laboratory biological analysis. The invention will be better understood on reading the following, referring to the accompanying drawings relating to nonlimiting exemplary embodiments, where: - Figure 1 shows a front view of a device according to the invention;

- Figure 1 'shows a front view of a device according to the invention;

- Figure 2 shows a sectional view of the tubing; Figure 3 shows a view of an alternative embodiment.

- Figure 4 shows a cross-sectional view of an alternative embodiment, Figure 5 shows a view of a second alternative embodiment using a peristatic pump, Figures 6 shows a view of another alternative embodiment in operates a peristatic pump; FIGS. 6 'represents a view of another alternative embodiment using a peristatic pump;

- Figures 6 "shows a view of another alternative embodiment using a peristatic pump;

- Figure 7 shows a sectional view of a filtration module for the separation of magnetic particles. FIG. 1 represents a front view of an exemplary embodiment of a device according to the invention. It consists of a receptacle (1) which can contain approximately 10 to 50 milliliters, connected to a tube (2) wound in a spiral and ending in an outlet tube (3) provided with a valve (4). A set of permanent magnets (5) is arranged radially to create a non-homogeneous magnetic field. The arrangement of the magnets is indicated by way of example, a different arrangement, for example in the form of parallel magnets, also being applicable. The magnets (5) consist of bars made of samarium-cobalt. These magnetic bars can optionally be mounted on a rotating support disc. This variant makes it possible to drive the paramagnetic balls fixed towards the outlet of the tubing at one stage of the process. For example, they rotate alternately ± 20 °. The maximum drive speed is a few millimeters per second, so as to optimize the efficiency on paramagnetic particles.

We can provide different magnet drive modes:

If the movement is made continuously in the direction of flow, it will facilitate the evacuation of cells and particles;

If the movement is made continuously in the opposite direction to the flow, the evacuation of cells and particles will be delayed;

If the movement is reciprocating, the particles will be mixed to facilitate rinsing and the elimination of cells and particles.

The cross-section of the tubing (2) is approximately 0.5 millimeters. It is made of a material such as TEFLON (registered trademark). The length is about 50 centimeters.

The use of the device is as follows: a) first of all a PBS (commercial name) type rinsing liquid is introduced into a 20 milliliter pipette; b) the blood sample (7) which has been previously incubated with paramagnetic particles is aspirated. 9 milliliters of physiological saline are added (8). c) the receptacle (1) is connected to the spiral tube (2), scrupulously avoiding the formation of bubbles; d) the receptacle (1) is raised by about 30 centimeters relative to the spiral e) the valve (6) provided between the receptacle (1) and the tube (2) is opened. The liquids then flow continuously from the receptacle (1) to the tubing (2) in a spiral under the effect of hydrostatic pressure. f) the flow is stopped after allowing half of the 20 milliliters of rinse aid to pass. During this phase, you can increase the efficiency of rinsing by rotating the magnets

(5) alternatively, for example by repeating 5 times a movement cycle of 20 ° in the counterclockwise direction, then return to the initial position, then 20 ° in the anti-trigonometric direction.

The next step consists in recovering the rosettes or nuclei from the cells sought. 1) Recovery of rosettes

The spiral (2) is moved away from the influence of the magnetic bars (5), and the flow of the rest of the rinsing liquid is resumed. This one will entrain all the paramagnetic particles and the cells carrying paramagnetic particles. The desired particles can thus be recovered by filtering, using a filter allowing the paramagnetic particles to pass, but not the cells.

2) Recovery of the nuclei of the cells sought. To recover the nuclei of the cells, the magnetic influence will be maintained and a new reagent will be activated which will have the property of lysing the fixed cells by releasing the hardened and colored nuclei. By way of example, such a reagent consists of the following mixture:

- Detergent, for example CETRIMIDE (Trademark)

- Fixer, for example formaldehyde - Nuclear dye, for example propidium iodide.

This mixture is introduced into the receptacle (1) so that the volume does not exceed that of the spiral (2). The coloring makes it easier to visualize the progression of the liquid. This mixture is left in contact for 10 minutes with the spiral. The magnets are actuated in alternating movements to facilitate the release of the nuclei. We can then rinse to recover the pure nuclei. The magnets (5) are always present, no magnetic particles will contaminate the preparation. On the other hand, the cells being destroyed, the identification of the nuclei implies the use of molecular hybridization reagents. These make it possible to detect genetic anomalies such as the presence of a supernumerary centromere (trisomy 21) or amplified expressions of oncogenes, or even expressions of particular genes type P53. Furthermore, the stoichiometrically colored pure nuclei make it possible to define a level of ploid which characterizes the stages of tumor cells.

Figure 1 'shows an alternative embodiment of a device according to the invention. The device comprises three containers (31 to 33) of reagents and samples connected to a separator pipe (35) spirally wound. Automatic taps or valves open or close the flow of each of the containers (31 to 33) to the separator pipe (35). This separator pipe is placed in a magnetic field gradient generated by permanent magnets (36) arranged in a star, in alternating radial direction. The downstream end of the separator pipe (35) opens on the one hand into a collection pipe (37) and on the other hand into a discharge pipe (38). Valves or taps are provided on each of these pipes to control the flow from under to a rejection container (39), under to analysis means formed by a collection system on filter (40), by a collection container (41) or by a particle flow detector (42). Figure 2 shows a sectional view of a segment of tubing. Under the effect of Bernoulli's forces, the particles tend to concentrate in the center of the flux, where speed is most important. Under the effect of magnetic fluxes, the paramagnetic beads (10) lighter and less bulky than the unmarked particles (11) than the marked cells (12) are very quickly attracted to the wall (13) of the tubing.

The trajectory of the unmarked cells (11) is not disturbed by the magnetic fluxes and these therefore remain in the center of the flux where they are quickly drawn towards the exit.

The marked cells (12) are found in the last part of the tubing (2). Optionally, the tubing can be made by two segments of increasing section, the first serving to retain the excess beads, and the second to retain the marked cells. Figures 3 and 4 show views respectively in median section and in cross section of an alternative embodiment.

The tubing (2) is formed by a groove (14) made in a plastic plate (15). It opens on the one hand on one of the side edges to allow connection to a connecting pipe with the receptacle (1) supply, and on the other hand with a transverse orifice (16) passing through the plate (15) allowing the evacuation of fluids. The device is formed of two symmetrical plates (15, 15 ') and are connected so as to define between them the liquid circulation pipe. They have housings for the magnets (17) on the exterior surfaces.

FIG. 5 represents a view of a second alternative embodiment using a peristatic pump. In this variant, the progression of the liquids is not conditioned by gravity, but by a peristatic pump.

The device consists of a tubular element (22) forming a separating loop. One end sucks the treated sample as in the previous examples. The tubular element (22) is arranged in the field of permanent magnets (24 to 28) oriented perpendicular to the direction of movement of the liquid, in alternating directions. The tubular element (22) is disposable, which avoids any contamination of its interior walls. This tubular element (22) is connected to the suction tube of a peristaltic pump (29) of known type. The waste is collected in a test tube (30) at the outlet of the pump (29). The circulation of the fluid inside the tube is preferably continuous, with a speed of one order of 1 cm per second. The continuous circulation prevents the formation of cellular adhesions on the wall of the separation tube.

Figures 6, 6 and 6 "show a partial view of an alternative embodiment. The magnets form a squirrel cage (32) formed by a plurality of magnetic bars (33 to 40) magnetized radially in alternating directions. This structure allows one or more tubular elements (22) to be positioned and several samples to be processed in parallel. The use of such a device is as follows:

- The free end (23) of the separator pipe (22) is immersed in the sample which has previously received the paramagnetic particles. - It activates the pump (29) which will pass the magnetic particles and cells in the separator loop arranged in the magnetic field. The magnetic particles and the rosettes formed by the marked cells will stop in this loop while the other cells will evacuate. To do this in a cell purge, in particular for a therapeutic purpose, consisting in eliminating the marked cells in order to collect only the unmarked cells, it is preferable to replace the peristaltic pump by another means of aspiration, for example a syringe suction or a source of depression, to avoid crushing of unmarked cells.

This embodiment has several advantages:

- it allows a large number of samples to be processed in parallel, it automates the management of steps, it can process very large volumes,

- it avoids dead volumes upstream of the tubular element treatment area. Indeed, after the passage of the sample, the tube can be eliminated and the reagents pass from uncontaminated containers. The risk of recovering non-specific cells is very low. Furthermore, it is then possible to immerse all the pipes in the same reactive container, which simplifies handling.

The separation of the labeled cells and of the non-fixed magnetic particles after the implementation of a treatment system according to one of the preceding variants is advantageously carried out by means of a filtration device, an exemplary embodiment of which is described in the figure. 7.

The search for events as rare as fetal cells or micrometastases implies extreme qualities and efficiency of all stages of the process. First of all, the antibodies must be very effective in ensuring good recovery of the targeted cells. Cell loss should be avoided during rinsing and collection procedures. It is also necessary to minimize false positives resulting from non-specific cells, but also from dye crystals and various residues which in fluorescence can be confused with cells.

Image analysis is able to resolve certain discriminations. However, in the case of rare elements of the order of 1 to 50 cells per sample, the total number of artifacts must remain below these values, which requires high performance of the chain of separation of marked cells and unlabeled cells, and high quality of cell recovery. The usual slide collection and staining procedures generate "noise" which is not compatible with such requirements.

The filtration device according to the invention, on the other hand, makes it possible to meet these requirements. It consists of a piece (41) of molded silicone, for single use. This part (41) has a thickness of the order of 2 millimeters and has twelve protrusions (51) with a diameter of 6 millimeters and a height of 10 millimeters. These protrusions (51), normally closed, can be perforated by a hollow needle (48) provided at the end of the separator tube (42).

The silicone part (41) is positioned in a rigid perforated plate (43). The perforations are arranged in a manner complementary to the protuberances (51) of the part (41). This plate (43) can be tightly connected to a support (45) and held in position by claws (44). An O-ring completes the sealing of this assembly. The support (45) also has perforations arranged opposite the perforations of the plate (43). The support is connected to a vacuum source. A microporous filtration membrane (47) is disposed between the plate (43) and the support (45). It is advantageously a microporous polycarbonate membrane whose cells have a section of 2 microns.

The operation of this filtration module is as follows:

The part (41) is delivered with a protective film placed under the underside. One or more protrusions can be opened. The plate (41) is placed on the support (43) after positioning the membrane microporous, and the assembly is locked using the claws (44).

The separation tube (2) is delivered with a protective cap which prevents any contamination. This cap is removed and one of the protuberances of the part (41) is perforated.

When collecting the rosettes from the separator, the isolated paramagnetic particles pass through the membrane while the rosettes remain on the surface. The porosity of the microporous membrane allows the passage of paramagnetic particles whose cross-section is of the order of a micron, but not the rosettes, whose cross-section is of the order of 5 microns.

For the collection of nuclei, in the absence of beads, the porosity can be 1 to 2 microns.

The coloring can be done before filtration by fluorescent dyes of the IP or BET type (trade names) or even by coloring on the filter after the latter has been extracted from the containment system.

The combination of a separator device formed by a tube of section much less than the length placed in a magnetic field gradient, and a filtration system as described above, has many advantages. Such an association first of all facilitates the automation of biological analysis. It also makes it possible to carry out analyzes and uses for therapeutic purposes requiring a high degree of sterility. The paramagnetic beads allow sorting of the cells, and in particular a separation of the sought cells and the other cells. The filter removes certain excess cells, such as platelets, as well as excess paramagnetic beads. The filter also has the function of cell concentration rare sought on a reduced surface facilitating the analysis by imagery. This filter also makes it possible, for therapeutic applications in particular, to carry out a culture of the cells sought. For this purpose, the filter serves as a cell support and can be supplied with a nutritive liquid capable of promoting cell multiplication.

The invention will be better understood in the foregoing by way of nonlimiting example.

Claims

1 - Method for magnetic immuno-separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and circulating cancer cells, of the type consisting in fixing the target cells on paramagnetic beads and causing a field to act magnetic on a sample containing the fixed cells, the free cells and the excess paramagnetic beads, to isolate the paramagnetic beads, characterized in that the sample is circulated in a tube (2) whose cross section is much less than the length over which the inhomogeneous magnetic field applies. 2 A magnetic immuno-separation method according to claim 1 characterized in that the sample is circulated in a tube (2) wound in a spiral placed in a magnetic field gradient.

3 - A method of magnetic immuno-separation according to claim 1 or 2 characterized in that the section of the tube is between 0.5 and 3 millimeters.

4 - A method of magnetic immunoassay according to any one of claims 1 to 3 characterized in that the length of the tube is greater than 10 centimeters.

5 - Magnetic immuno-separation method according to any one of the preceding claims, characterized in that the preparation is introduced into a reservoir connected to the separation tube, said reservoir being raised relative to the tube to ensure circulation of the sample by gravity .

6 - Magnetic immunoseparation method according to claim 5 characterized in that one introduces without mixing and without interface layer in the reservoir a first volume of the sample and a second volume of a rinsing liquid.

7 - A method of magnetic immunoseparation according to any one of claims 1 to 4 characterized in that the preparation is aspirated in the tube placed in the gradient of magnetic fields, the downstream end of said tube being connected to a peristaltic pump.

8 - Method of magnetic immuno-separation according to any one of the preceding claims, characterized in that the magnets are movable relative to the tube, preferably in an alternating movement.

9 - Method of magnetic immuno-separation according to any one of the preceding claims, characterized in that the reagents and the samples flow continuously in the magnetic separation tube, at a speed of between 0.1 cm / s and 10 cm / s. 10 - A method of magnetic immuno-separation according to the preceding claim characterized in that one proceeds to a subsequent filtration step using a microporous membrane filter whose porosity is greater than the diameter of the paramagnetic beads and less than the diameter fixed cells.

11 - A method of magnetic immuno-separation according to the preceding claim characterized in that one proceeds to a subsequent filtration step using a microporous membrane filter whose porosity is greater than the diameter of the paramagnetic beads and some of the excess cells, and less than the diameter of fixed cells. 12 - filtration device for the implementation of the magnetic immuno-separation process according to claim 10 or 11 characterized in that it is constituted by a microporous membrane filter whose porosity is greater than the diameter of the paramagnetic beads and some of the excess cells, and less than the diameter of fixed cells.

13 - Installation for one magnetic immuno-separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and circulating cancerous cells characterized in that it consists of a tube whose section is very less than the length and by at least one magnet arranged to create a magnetic field in the space traversed by said tube.

14 - Installation for one magnetic immuno-separation of cells according to claim 13 characterized in that it further comprises a reservoir disposed upstream of the tube.

15 - Installation for one magnetic immuno-separation of cells according to claim 13 or 14 characterized in that it comprises a tube having two sections of increasing section, the second section being at least 10 times greater than the first section.

16 - Installation for one magnetic immuno-separation of cells according to claim 13 characterized in that it comprises two symmetrical plates (15, 15 ') having a groove forming a spiral, said plates supporting magnets.

17 - Installation for magnetic immuno-separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and circulating cancer cells, characterized in that it consists of a tube whose section is much less than the length, said tube having a first end for the aspiration of the sample and a second end connected to a peristaltic pump, the tube being placed in a gradient of magnetic fields.

18 - Installation for one magnetic immuno-separation of cells, in particular bacteria, fetal cells, stem cells of the bone marrow and those circulating cancer according to claim 17 characterized in that the alternating magnetic fields are produced by permanent magnets arranged in squirrel cage.

19 - filtration device for the implementation of the separation process according to claim 11 characterized in that it is constituted by a support tightly connected to a source of vacuum, and by an additional cover having means for connection from the downstream end of the cell separation tube, the interchangeable filtration membrane being disposed between the support and the cover.

20 - A filtration device according to claim 19 characterized in that the cover is constituted by a perforated plate and by a molded part having perforable areas by the end of one or more cell separation tubes.

21 - Process for the separation of fetal cells present in maternal blood, characterized in that the target cells are fixed on paramagnetic microbeads using a ligand capable of specifically binding to a free complementary substance or present in the cell surface and in that the sample is circulated through a tube having a length much greater than the section, said tube passing through a non-homogeneous magnetic field. 22 - Method for the early detection of circulating cancer cells or micro metastasis characterized in that the target cells are fixed on paramagnetic microbeads using a ligand capable of specifically binding to a free complementary substance or present in the cell surface and in that the sample is circulated through a tube having a length much greater than the section, said tube passing through a non-homogeneous magnetic field.

23 - Process for the preparation of cells, in particular of bone marrow strains making it possible to re-seed marrow destroyed following anticancer treatment by chemotherapy or radiotherapy, characterized in that the target cells are fixed on paramagnetic microbeads at 1 using a ligand capable of binding specifically to a free or present complementary substance on the surface of cells and in that the sample is circulated through a tube having a length much greater than the section, said tube crossing a non-homogeneous magnetic field.

24 - Process for the preparation of cells, in particular of bone marrow strains making it possible to re-seed marrow destroyed following anticancer treatment by chemotherapy or radiotherapy according to the preceding claim characterized in that the cells are filtered emerging from the tubing placed in the magnetic field, using a membrane filter having a porosity greater than the section of the paramagnetic beads, and less than the desired cells fixed.