FR2682481A1 - PROCESS FOR OBTAINING TROPHOBLASTIC CELLS FROM A FÓOETUS. - Google Patents

Abstract

Method of obtaining trophoblast cells from a foetus of a mammal, specifically from a sample of a the peripheral blood taken from the mother bearing the said foetus, involving the following sequence of steps: (a) separation of erythrocytes from the said sample; (b) separation of at least part of the leucocytes, marking at least part of the leucocytes with at least a first specific marker; (c) separation of the trophoblast cells, marking the latter with at least one second antibody substantially specific to the said cells; (d) positive selection of the trophoblast cells by flow cytometry. The method is characterized in that the first antibody of the pre-enrichement step is a common antileucocyte antibody, specifically marking 80 % of the leucocytes of the leucocyte fraction, to give a fraction pre-enriched with trophoblast cells, enabling the trophoblast cells marked only by the second antibody to be identified subsequently during flow cytometry.

Description

PROCESS FOR OBTAINING CELLS
TROPHOBLASTICS OF A FETUS
The present invention relates to obtaining trophoblastic cells from the fetus of a mammalian individual, from a blood sample taken from the peripheral venous circulation of the mother carrying said fetus, and therefore during her gestation.

 Without limitation, the present invention will be introduced, defined, described and discussed in relation to obtaining trophoblastic cells of the human race.

 By “trophoblastic cell”, within the meaning of the present invention, is meant any trophoblast cell, syncithiotrophoblast or cytotrophoblast, of villous or extravillous origin, whatever its type, anucleated, mononuclear, polynucleated, and whatever the period of their appearance in the maternal blood compared to the duration of gestation.

Different publications, among which we will cite
- COVONE, AE, MUTTON D., JOHNSON, PM, ADINOLFI,
M. (1984). Trophoblast cells in peripheral blood from
pregnant women. Lancet, II, 841-843.

- HERZENBERG, LA, BIANCHI, DW, SCHRÖDER, J., CANN,
HM, IVERSON, M. (1979). Fetal cells in the blood of
pregnant women: detection and enrichment by
fluorescence-activated cell sorting. Proc. Natl. Acad.

Sci. USA, 76.1453-1455
- HSI, BL-L, YEH, C.-JG (1986). Monoclonal antibodies
GB25 recognizes human villous trophoblasts, Am. J.

Reprod. Immunol. Microbiol., 10, 1-3
- HSI, BL-L, YEH, C.-JG, JOHNSON, PM, BERESFORD, N.,
STERN, PL (1987). Monoclonal antibody GB 17
recognizes human syncytiotrophoblast. J. Reprod.

Immunol., 12, 235-244
IKLE, Von-F, A. Dissemination von syncytiotrophoblastze
llen im mütterlichen Blut wàhrend der Graviditat.

 (1964). Bull. Schweiz. Akad. Med. Wiss., 20, 62-72.

- KOZMA, R., SPRING, J., JOHNSON, PM, ADINOLFI,
M. (1986). Detection of syncytiotrophoblast in
maternal peripheral and uterine veins using a
monoclonal antibody and flow-cytometry. Hmm. Reprod.,
1, 335-336
- KOZMA, R., CHAPMAN, M., LOKE, YW, JOHNSON, PM,
ADINOLFI, M. (1986). Detection of trophoblast-like
cells in maternal blood using specific monoclonal
antibodies. J. Reprod. Immunol., 11, 55-61
- YOUSSEF, M., SHULMAN, P., THARAPEL, AT, SIMPSON,
JL, ELIAS, E. (1990). Failure to document fetal cells
in maternal circulation using the Selypes-Lorencz
"air-culture" technical cytogenetic, Hum.Genet.,
85, 133-134.

have disclosed and proposed a process for obtaining trophoblastic cells or the like from a human fetus, as previously introduced, according to which, successively
- the erythrocytes are separated from a blood sample
taken from the peripheral venous circulation of the
mother, to get a fraction by negative sorting
leukocyte enriched with leukocytes
- an alleged fraction is separated by positive sorting
enriched in trophoblastic cells of the fraction
leukocyte (maternal blood minus erythrocytes),
labeling said cells with a biolabel
ie a substantially specific antibody
fique of said cells
- possibly, from the fraction enriched in
trophoblastic cells, we sort by cytometry of
flows a fraction presumably even richer in
trophoblastic cells.

 In general, apart from the antibodies selected below as biological markers, the term “marker” very generally means any substance, product, substituent or second compound, biological or not, capable of differentiating another substance or primary product, by incorporation or bonding of the second product to the first product, and this to subsequently recognize the first product, by any analysis process to which the second product specifically reacts.

 Within the meaning of the preceding definition, a marker can be a luminous substance (for example dye), chromophore (for example fluorescent), an enzyme, a hapten, an antigen, a radioactive isotope, etc.

 Such a method has been proposed subsequently by the document WO-A-9006509, according to which different antibodies called FD 0161 G, FD 066 Q and FD 0338 P have been isolated as more particularly specific for syncithiotrophoblastic and / or cytotrophoblastic cells, to mark these last during the positive sorting phase on the leukocyte fraction.

 In addition, these antibodies have been defined and identified by determining the protein sequences of the antigens respectively recognized by said antibodies.

 More recently, document EP-A-0 412 700 has described and proposed a process of the same type, possibly adding, between the step of separation of erythrocytes by negative sorting, and the step of obtaining by positive sorting the fraction enriched in trophoblastic cells, a step of separation of part of the leukocytes from the leukocyte fraction, to obtain by negative sorting a fraction pre-enriched in trophoblastic cells. This intermediate enrichment step by negative sorting consists in labeling the monocytes of the leukocyte fraction with a specific antibody, for example known as HLE-1.

 Furthermore, according to document EP-A-0 412 700, monoclonal antibodies have been isolated and produced, more specifically specific for cytotrophoblastic cells of the first trimester of gestation, and used for the separation step by positive sorting of the fraction presumably enriched in trophoblastic cells.

 The various methods described above have been presented by their authors or inventors, as making it possible to obtain fetal cells, by a "non-invasive" method, that is to say without taking an amniotic fluid from the mother, or biopsy from the embryo.

Obtaining these fetal cells, for its part, in a traditional manner known per se, the implementation of various tests or methods of prenatal diagnosis, for example by cytogenetics, such as sex determination, or congenital diseases like Down's syndrome (Mongolism).

 So far, the various methods described above have only identified the presence of trophoblastic cells in the final fraction obtained, including after flow cytometry, only by detecting, by biochemical genetic tests, the fetal DNA. occurrence that of chromosome Y of a male embryo in said final fraction. This means in particular that to date, on the one hand, trophoblastic cells have not been isolated in the final fraction of the process and recognized as such, in particular by a morphological examination, and on the other hand this fraction final cannot be described as substantially pure in trophoblastic cells.

 However, vis-à-vis the tests to be carried out only on fetal cells, in particular biochemical cells, for the purposes of prenatal diagnosis, obtaining a final fraction enriched in trophoblastic cells does not seem much more useful than the sample. initial whole blood from the mother, from which she came. What appears fundamental and essential in this respect is in fact the obtaining of a substantially pure fraction of trophoblastic cells, or even the obtaining of a sufficient number of duly isolated trophoblastic cells.

The isolation of a few trophoblastic cells or of a substantially pure fraction of said cells in fact allows a whole series of tests or prior verifications, essential for carrying out various reliable, sensitive, and repetitive prenatal diagnoses.
- it is first of all possible to recognize and identify
trust the trophoblastic cells, by all
traditional methods, such as morphology and
electron microscopy
- it is then possible to cultivate in a viable way
these trophoblastic cells, which opens the way
traditional cytogenetic tests only
on fetal cells
- to finish, and above all, all biochemical tests
traditional on DNA, or molecular genetics,
such as cytochemical hybridization, amplification
in vitro genomics (including PCR), can be put
implemented directly, that is to say without culture, without
risk an incorrect answer, or "parasitized" by
maternal cells; in this regard, if as stated
previously, we can consider a sex detection
of the fetus, by detection with a genetic marker
of the Y chromosome in a final fraction enriched in
trophoblastic cells it appears to date
impossible, always by biochi genetic pathway
to detect a chromosomal or gene abnormality
tick of the fetus, for example an autosomal abnormality
recessive, without having almost fetal cells
ment pure.

 The present invention therefore relates to a process as described above, making it possible to obtain a substantially pure fraction of trophoblastic cells, for example at 99%, having preserved their morphological and physiological integrity at the end of the various stages of positive sorting and / or negative, this process opens the way to all traditional biochemical tests, applied only to fetal DNA.

 The present invention begins with the discovery or observation that, during the stage of pre-enrichment in trophoblastic cells by positive sorting, the biological marker, for example an antibody specific for trophoblastic cells, can also mark the latter as maternal leukocytes having absorbed, ingested trophoblastic cells, or certain of their constituents after cell death, and having epitopes common with the latter.

 To easily distinguish, during the final separation step, for example by flow cytometry, the trophoblastic cells from the lymphocytes presenting with them a cross immunological reaction, the present invention has identified different operating parameters contributing separately or together to the desired result.

 Mainly, according to the present invention, it is first of all necessary, during the pre-enrichment step by negative sorting, to mark almost all of the leukocytes, including lymphocytes, polynuclear and monocytes, with an anti- common leukocyte (abbreviated AALC).

 The use of an AALC allows relatively complete negative sorting on leukocytes, with the advantage of not damaging the trophoblastic cells present in maternal blood. This advantage appears particularly significant with respect to the solutions which have retained a direct positive sorting of the trophoblastic cells on a sample of maternal blood, depleted of its erythrocytes.

 Different other parameters combine or complement the desired result.

 Thus, if the AALC is fixed on a solid support or phase, in the divided state, in the form of magnetized particles for example, in suspension in the leukocyte fraction, the latter is then brought into contact with the face of said support fixing 1 'TALC, and the leukocytes labeled with the latter can be easily and completely separated from the leukocyte fraction, for example by the action of a magnetic field.

 Furthermore, in the case of a support of the magnetic particle or magnetic bead type, it is preferable to operate with an excess of the number of AALCs, relative to the number of cells in the leukocyte fraction, and / or with several AALCs fixed on the same magnetized particle, so as to obtain, during the pre-enrichment step, a three-dimensional particle / leukocyte network, with a narrow mesh, unlikely to incorporate trophoblastic cells of relatively larger size. Still for magnetized particles, such as magnetic beads, by choosing between 0.5 and 1 the number of magnetized particles, relative to the number of cells in the leukocyte fraction, a too dense network is avoided, liable to damage the trophoblastic cells, during the action of the magnetic field on the particles and against the wall of the treatment container.

 According to one embodiment of the invention, the AALC of the negative sorting step and the marker of the positive sorting step, in this case an anti-trophoblastic cell antibody (abbreviated to AACT), carry respectively different chromophoric products, for example and respectively a fluorescent chemical group phycoerythrine, and a secondary anti-mouse antibody coupled to fluorescein isothiocyanate. Such an operating choice makes it possible precisely, for example by fluorescence flow cytometry, to distinguish not only the trophoblastic cells from the residual maternal lymphocytes, but also the trophoblastic cells from the maternal lymphocytes which have ingested the latter, which will carry the two markers.

 A representative process of the present invention for achieving relatively pure trophoblastic cells is described below in general.

 First, a sample of the peripheral venous blood from the mother carrying the fetus is taken. It follows from previous scientific work that the number of trophoblastic cells present in this sample is on average and about one cell per million.

 The erythrocytes are separated from this sample, by centrifugation under a density gradient, for example according to the FICOLL method, to obtain, by negative sorting, a leukocyte fraction, that is to say enriched in leukocytes. For example, centrifugation is carried out in a liquid with a density equal to 1.077, corresponding to that of lymphocytes. This gives a leukocyte fraction extracted from the centrifuged sample, containing lymphocytes, monocytes, and trophoblastic cells, while the centrifugation pellet contains red blood cells, granular polymorphonuclear cells, giant trophoblastic cells, as well as platelets. At the end of this separation phase, the leukocyte fraction obtained is already enriched in trophoblastic cells, by a factor of approximately 100, and consequently contains approximately one trophoblastic cell per 10,000.

 The leukocyte fraction is then subjected to a new enrichment in trophoblastic cells, by negative sorting, by labeling almost all of the leukocytes with a biological marker, in this case an AALC, to obtain two fractions, namely a pre-enriched fraction. into trophoblastic cells, containing only a few residual maternal leukocytes, and a fraction containing practically all leukocytes from the peripheral maternal blood, including lymphocytes, polynuclear cells and monocytes. To do this, and preferably, the retained AALC, for example the antibody known under the reference CD 45 RO, is fixed on a solid support in the divided state, consisting of magnetic particles or magnetic beads, capable of be separated from the suspension medium by the action of a magnetic field. The leukocyte fraction is then brought into contact with the face of the particulate support which has fixed the AALC.

 According to a preferred embodiment, the AALC is fixed on the magnetic particles, by means of a secondary antibody, for example a goat anti-mouse antibody, itself fixed on the solid support divided, by a covalent chemical bond in particular.

Advantageously, the anti-mouse goat antibody is fixed to the solid support by its Fc fragment, leaving free an Fab'2 fragment, affinity purified; this latter characteristic makes it possible to avoid the attachment of trophoblastic cells, which have receptors for the Fc fragment.

 Furthermore, still during the pre-enrichment step, the AALC carries a chromophore product, in particular a fluorescent chemical group, for example phycoerythrin. This will allow in later stages of the process to recognize residual leukocytes.

Preferably, the stage of pre-enrichment in trophoblastic cells is carried out with the following operating choices
- an excess of the number of antibody molecules, by
ratio to the estimated number of cell sites (i.e.
number of cells x by number of sites per cell)
- several AALCs on each magnetized particle or ball
magnetic
- a number of particles, related to the number of
cells in the leukocyte fraction, between
0.5 and 1.

 All of these operating parameters simultaneously make it possible to obtain a particle / white blood cell network with sufficiently narrow meshes so as not to trap the trophoblastic cells, while avoiding damaging them against the wall of the analysis container, during the action of the magnetic field.

 Next, the trophoblastic cells are separated from the pre-enriched fraction obtained previously, by labeling said cells with a biological marker, in this case an antibody substantially specific for trophoblastic cells, to thereby obtain a fraction substantially enriched in positive sorting. trophoblastic cells. Preferably, the AACT selected carries a chromophore product, for example a secondary anti-mouse antibody coupled to isothiocyanate-fluorescein. Advantageously, during this positive sorting phase, the AACT is an antibody substantially anti-syncythiotrophoblastic cells, for example the antibodies called GB 17, in the publications of Mr. HSI, identified in the preamble of this description.

 Finally, a substantially pure fraction of trophoblastic cells is obtained, by sorting by fluorescence flow cytometry the fraction enriched in trophoblastic cells previously obtained. Given the different chromophoric groups marking respectively the trophoblastic cells and the residual leukocytes, flow cytometry makes it possible to distinguish not only the residual leukocytes from the trophoblastic cells, but also the maternal white blood cells which have absorbed or ingested the latter, or certain of their constituents. after cell death, of these same trophoblastic cells.

 In total, and at the end of such a process, a substantially pure fraction of intact trophoblastic cells is obtained, allowing then the application of all traditional genetic tests, in particular biochemical tests on fetal DNA. For example, starting from 10 cm3 of maternal blood, such a process makes it possible to recover up to 3 x 10 3 trophoblastic cells, which then allows very high diagnostic accuracy; in particular, a large number of trophoblastic cells makes it possible to detect mosaic anomalies, that is to say those for which only part of the fetal cells is affected. Such anomalies are not easily detectable today by conventional cytogenetics, which only diagnoses a maximum of twenty cells.

 An example of obtaining trophoblastic cells according to the invention is described below.

 After a gestation time greater than or equal to 6 weeks, 10 cm3 of maternal blood are drawn from the vein of the fold of the mother's elbow, for example.

The sample taken contains 5.1010 red blood cells, 5.107 white blood cells (leukocytes, neutrophils, and eosinophils, basophils, lymphocytes, monocytes), 5.103 trophoblastic cells (anucleated, mononuclear, giant multinucleated), and 4.109 platelets.

 In a first step, with a discontinuous FICOLL gradient, in centrifugation in medium of density 1.077, approximately 99% of the red blood cells, 80% of the polynuclear cells, and 80% of the giant multinuclear trophoblastic cells are eliminated. After centrifugation, one can simply take the interface which contains all the mononuclear cells. Another sample is thus obtained containing approximately 5.102 red blood cells, 4.107 white blood cells, and 4.103 trophoblastic cells.

 In a second step, the white blood cells are eliminated to pre-enrich the sample with trophoblastic cells. To this end, a common anti-leukocyte antibody, for example CD45R0 coupled to phycoerythrin, which is an excitable fluorochrome at 488 nm, and whose emission can be collected at 575 nm more or less 20 nm, is used as described above. The white blood cells marked as above are placed in the presence of magnetic beads coated with an anti-mouse goat antibody, specific for immunoglobulins. Then we retain the complexes obtained, namely beads / goat anti-mouse antibody / CD45R0 / white blood cells, with a permanent magnet, to recover a supernatant. We thus obtain in the latter a residual population of cells, namely approximately 5.102 globules red, 4,105 white blood cells, and 4,103 trophoblastic cells, it being understood that the enrichment factor in trophoblastic cells can vary between 10 and 100 approximately.

In a third step, the trophoblastic cells are purified. To this end, the latter are coupled with any appropriate antibody, specific for one or more types of trophoblastic cells, and described in the various publications and patents identified in the preamble to this description; for example, the antibody known as GB 17 is used, as described by
Mr. Bae-Li HSI, et al, in an article entitled "Monoclonal antibody GB 17 recognizes human syncytiotrophoblasts", Journal of Reproductive Immunology, 12 (1987) 235-244.

 If the GB 17 antibody is used, the trophoblastic cell-GB 17 complex will be labeled with a goat anti-mouse antibody coupled to FITC (fluorescein-isothiocyanate), which is an excitable fluorochrome at 488 nm, emitting at 525 nm plus or minus 15 nm.

We can thus recognize later in flow cytometry the white blood cells marked by the CD45R0 antibody, the white blood cells having absorbed or ingested trophoblastic cells, and finally the latter.

 In a final step with a laser cell sorter, that is to say with a flow cytometer, for example an apparatus of the brand COULTER and of the reference ELITE, the fraction obtained at the end of the third step, on size / structure criteria to eliminate red blood cells and cell debris, including anucleated trophoblastic cells. From the fluorescence parameters retained, it is possible to discriminate white blood cells (fluorescence at 575 nm) from trophoblastic cells (fluorescence at 525 nm). On average, at the end of this sorting phase, 3.103 trophoblastic cells are obtained, with more or less 2.102 cells, on 12 samples. These purified cells are collected on microscope slides, for subsequent cytochemical hybridization (100 trophoblastic cells per slide ), and in 1.5 ml micro-tubes, for use of in vitro genomic amplification (PCR).

Finally, the identification of the labeled trophoblastic cells can be done under a fluorescence and phase contrast microscope. These cells can be counted under the microscope at the cell of
MALASSEZ, with a count of 400 cells.

 Subsequently, and for diagnostic purposes, various in situ hybridization probes, for example biotinylated by "nick translation", are used. These probes can be revealed, after in situ hybridization, by application of avidin-FITC, and of a biotinylated anti-avidin antibody. These probes, by way of example, are used to determine the number of chromosomes "X" or "Y", for the determination of sex, or 21, for the determination of trisomy 21.

Claims (19)

 1. Process for obtaining trophoblastic cells from a fetus of an individual mammal, from a sample of peripheral blood taken from the mother carrying said fetus, according to which, successively  (a) the erythrocytes are separated from said sample, to  obtain a leukocyte fraction by negative sorting,  enriched with leukocytes  (b) at least part of the leukocytes are separated from the  leukocyte fraction, labeling leukocytes  to be separated with a specific marker, to  obtain by negative sorting, a pre-enriched fraction  in trophoblastic cells  (c) the trophoblastic cells are separated from the  pre-enriched fraction, by marking said  cells, with a biological marker, for example  an antibody substantially specific for said  cells, to get a positive sort  fraction enriched in trophoblastic cells characterized in that the pre-enrichment step is carried out with a common anti-leukocyte antibody, marking the major part of the leukocytes of the leukocyte fraction, including lymphocytes, polynuclear and monocytes, to obtain a pre- enriched to subsequently distinguish trophoblastic cells from residual maternal leukocytes having ingested trophoblastic cells circulating in maternal blood.  2. Method according to claim 1, characterized in that the common anti-leukocyte antibody is fixed on a solid support, and during the pre-enrichment step the leukocyte fraction is brought into contact with the face of said support having fixed said antibody.  3. Method according to claim 2, characterized in that, during the pre-enrichment step, the solid support in the divided state, in the form of particles, is suspended in the leukocyte fraction.  4. Method according to claim 3, characterized in that the solid support in the divided state consists of magnetized particles, capable of being separated from the suspension medium by the action of a magnetic field.  5. Method according to claim 2, characterized in that the common anti-leukocyte antibody is fixed on the solid support, by means of a secondary antibody, in particular a goat anti-mouse antibody, itself fixed on the solid support, in particular by chemical bonding.  6. Method according to claim 5, characterized in that the anti-mouse goat antibody is fixed on the solid support by its Fc fragment, leaving free a Fab'2 fragment.  7. Method according to claim 1, characterized in that step (b) of pre-enrichment is carried out with an excess of the number of antibody molecules, compared to the number of cell sites estimated in the leukocyte fraction.  8. Process according to claims 4 and 7, characterized in that on each magnetized particle are fixed several common anti-leukocyte antibodies.  9. Method according to claim 3, characterized in that the number of particles, relative to the number of cells in the leukocyte fraction, is between 0.5 and 1.  10. Process according to claim 1, characterized in that the common anti-leukocyte antibody carries a chromophore product, in particular a fluorescent chemical group phycoerytrin.  11. Process according to claim 1, according to which the biological marker marking the trophoblastic cells is an anti-trophoblastic cell antibody, characterized in that said antibody carries a chromophore product, a secondary anti-mouse antibody coupled to fluorescein isothiocyanate. .  12. Process according to claims 10 and 11, characterized in that the common anti-leukocyte antibody and the anti-trophoblastic cell antibody carry respectively different chromophoric products.  13. Process according to claim 1, characterized in that a substantially pure fraction of trophoblastic cells is obtained, by sorting the latter by flow cytometry in the fraction enriched in trophoblastic cells.  14. Method according to claims 12 and 13, characterized in that the flow cytometry is fate under a light flux making it possible to distinguish respectively the leukocytes marked with the anti-leukocyte antibody, and the trophoblastic cells marked with the anti-leukocyte antibody. trophoblastic cells.  15. Process according to claim 1, characterized in that the biological marker marking the trophoblastic cells is a substantially anti-syncythiotrophoblastic cell antibody.  16. Process according to claim 1, characterized in that the sample subjected to successive separations is taken from the venous blood of the mother.  17. Process according to claim 1, characterized in that the leukocyte fraction is obtained from the blood sample, by centrifugation under density gradient.  18. Biological reagent suitable for the method according to any one of claims 1 to 17, comprising a solid support in the divided state, consisting of magnetic particles, and at least one biological marker, in particular an antibody, fixed to said support by via an anti-mouse antibody, characterized in that the anti-mouse antibody is fixed on the solid support by its Fc fragment, leaving a fragment free Fab'2.  19. Biological reagent comprising an anti-trophoblastic cell antibody, characterized in that said antibody carries a chromophore product, in particular a secondary anti-mouse antibody coupled to fluorescein isothiocyanate.