WO1996009409A1 - Enrichissement de cellules f×tales, a partir du sang maternel - Google Patents

Enrichissement de cellules f×tales, a partir du sang maternel Download PDF

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Publication number
WO1996009409A1
WO1996009409A1 PCT/US1995/011971 US9511971W WO9609409A1 WO 1996009409 A1 WO1996009409 A1 WO 1996009409A1 US 9511971 W US9511971 W US 9511971W WO 9609409 A1 WO9609409 A1 WO 9609409A1
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Prior art keywords
cells
fetal
suspension
dna
matrix
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PCT/US1995/011971
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English (en)
Inventor
Jurgen Busch
Andreas Radbruch
Stefan Miltenyi
Jürgen HOLTZ
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Miltenyi Biotech, Inc.
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Application filed by Miltenyi Biotech, Inc. filed Critical Miltenyi Biotech, Inc.
Priority to AU35935/95A priority Critical patent/AU691040B2/en
Priority to EP95933174A priority patent/EP0778899A4/fr
Priority to JP8511058A priority patent/JPH10507632A/ja
Publication of WO1996009409A1 publication Critical patent/WO1996009409A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804Nucleic acid analysis using immunogens
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966Animal cells
    • G01N33/56972White blood cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/36Gynecology or obstetrics
    • G01N2800/368Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the field of the invention is prenatal diagnosis.
  • fetal cells found in maternal blood As a source of fetal genetic material.
  • Three types of nucleated fetal cells have been identified so far in maternal blood: placental trophoblasts, fetal erythroblasts and fetal lymphocytes.
  • the observed frequencies of fetal cells in whole maternal blood range from 10" 4 to 10" 8 , which is too low to allow direct detection of fetal alleles by PCR.
  • FACS fluorescence-activated cell sorting
  • Magnetic cell sorting has been used to select for CD71+ cells in maternal blood, however the level of enrichment was insufficient to detect fetal specific DNA by polymerase chain reaction.
  • An improved MACS process whereby fetal cells could be sorted from maternal blood, and which allows multiple, and potentially large, samples to be run on the bench would provide numerous benefits to the field of prenatal diagnosis.
  • Peripheral blood is drawn from the mother, desirably at a site distant from the developing fetus.
  • Fetal nucleated cells are enriched from mononuclear cells of maternal blood, using a two step high-gradient magnetic cell separation (Double MACS). Maternal cells are depleted by specific binding to markers present on adult lymphoid and myeloid cells. In a separate step, fetal cells are enriched by MACS.
  • nucleated erythrocytes are then used as a source of fetal genetic material, which is analyzed for the presence of chromosomal or genetic characteristics by conventional methods such as probes for in situ hybridization, metaphase spreads and polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Figures 1 A and IB show the results of PCR analysis of the HLA-DQ Al locus, using as a source of DNA: maternal peripheral blood mononuclear cells; CD457CD14", CD45+/CD14+, and CD457CD147CD71 + double MACS sorted cells from maternal blood; and peripheral blood mononuclear cells from paternal blood, analyzed with a commercially available HLA-DQ Al typing kit.
  • the DNA in Figure 2B was subjected to a PCR pre-amplification step, as indicated.
  • Figure 2 shows the results of PCR analysis of the D1S80 locus, using as a source of DNA: peripheral blood mononuclear cells; CD457CD14-, CD45+/CD14+ and CD457CD147CD71+ double MACS sorted cells from maternal blood; and peripheral blood mononuclear cells from paternal and child blood.
  • DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods and compositions for the prenatal detection of fetal DNA sequences and chromosomal characteristics are provided.
  • Peripheral blood is drawn from the mother.
  • a preparation is then made of nucleated cells from the maternal blood.
  • Fetal erythrocytes are enriched from the maternal cells by a combination process involving depletion of maternal cells and enrichment of fetal cells, using high-gradient magnetic cell separation (Double MACS), or a combination of high and low gradient magnetic separation.
  • a suspension of blood cells are labeled with superparamagnetic particles specific for cell surface antigens, then sorted by binding to magnetic columns.
  • the double-MACS procedure is most effective when a two-stringency system is used, where the depletion step captures a high percentage of labeled cells and the enrichment step captures a lower percentage of labeled cells.
  • the sorted fetal cells are used as a source of genetic material, which is analyzed to determine fetal sex, or the determination of other genetic characteristics.
  • the use of high-gradient magnetic cell sorting (MACS) to enrich for fetal cells provides several benefits when compared to flow cytometry methods presently used today, particularly for clinical practice.
  • the subject methods require inexpensive reagents and apparatus, which are easily used and maintained. By setting up multiple columns, many samples can be processed at the same time.
  • An automated system can be used to simplify processing of large sample numbers.
  • the very low frequency of fetal cells in maternal circulation favors the use of relatively large maternal blood samples, which cannot easily by sorted with flow cytometry.
  • the mother will be at least about 6 gestational weeks (g.w.), usually at least about 8 g.w. or later in pregnancy. Generally the mother will be from about 8 to 20 g.w., although analysis may be performed later if required.
  • the maternal blood sample is drawn from any site, conveniently by venipuncture. The sample is usually at least about 20 ml, more usually at least about 40 ml and may be as large as about 500 ml, more usually not more than about 250 ml.
  • the blood is treated by conventional methods to prevent clotting, such as the addition of EDTA, heparin or acid-citrate- dextrose solution.
  • a preparation of nucleated cells is made from the sample. Any procedure which can separate nucleated cells from adult erythrocytes is acceptable. The use of Ficoll- Paque density dradients or elutriation is well documented in the literature. Alternatively, the blood cells may be resuspended in a solution which selectively lyses adult erythrocytes, e.g. ammonium chloride potassium, ammonium oxalate, etc.
  • the sample of nucleated peripheral blood cells is selectively depleted of maternal cells.
  • Depletion reagents attached to superparamagnetic particles are bound to cell surface antigens which are present on adult hematopoietic cells, but are low or absent on fetal erythrocytes.
  • Especially useful depletion reagents are antibodies against cell surface antigens.
  • Whole antibodies may be used, or fragments, e.g., Fab, F(ab')2, light or heavy chain fragments, etc.
  • Such antibodies may be polyclonal or monoclonal and are generally commercially available or alternatively, readily produced by techniques known to those skilled in the art.
  • Antibodies selected for use in depletion will have a low level of non-specific staining, and will usually have an affinity of at least about 100 ⁇ M for the antigen.
  • a cocktail of depletion reagents will be used, in order to deplete a wide range of blood cell types. Generally, at least about 95% of adult nucleated peripheral blood cells will be bound by the cocktail of depletion reagents, more usually at least about 99%, and preferably at least about 99.5%.
  • Suitable antigens for depletion include CD45 which is widely expressed on lymphoid cells; CD 14 which is found on monocytes; CD34 which is expressed on progenitor cells, and CD 15 which is primarily found on granulocytes.
  • Other useful cell surface antigens include CDl 1, CD44, CD46, CD48, CD43, CD49d, CD3, CD19, CD56, CD7 and CD5.
  • a cocktail of antibodies specific for CD 14, CD45 and optionally CD34 are used.
  • Other useful combinations of markers for cocktail formulation are CD43 and CD 19; CD3, CD 19, CD56 and CD15; CD7 and CD19; and CD43 and CD5.
  • the depletion reagent antibodies are coupled to superparamagnetic particles, prepared as described in U.S. Patent nos. 4,452,773 and 4,230,685.
  • the microparticles will usually be less than about 100 nm in diameter, and usually will be greater than about 10 nm in diameter.
  • the exact method for coupling is not critical to the practice of the invention, and a number of alternatives are known in the art.
  • Direct coupling attaches the antibodies to the particles, as described in co-pending patent application no. 08/252,112, herein incorporated by reference. Indirect coupling can be accomplished by several methods.
  • the depletion reagent antibodies may be coupled to one member of a high affinity binding system, e.g. biotin, and the particles attached to the other member, e.g.
  • avidin One may also use second stage antibodies which recognize species-specific epitopes of the depletion antibodies, e.g. anti-mouse Ig, anti- rat Ig, etc. Indirect coupling methods allow the use of a single magnetically coupled antibody species with a variety of depletion antibodies.
  • a preferred method uses hapten-specific second stage antibodies coupled to the superparamagnetic particles, as described in co-pending patent application no. 08/252,112.
  • the hapten specific antibodies will usually have an affinity of at least about 100 ⁇ M for the hapten.
  • the depletion antibodies are conjugated to the appropriate hapten.
  • Suitable haptens include digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl, etc. Methods for conjugation of the hapten to antibody are known in the art.
  • the depletion antibodies may be labeled with a fluorochrome, e.g. phycoerythrin, FITC, rhodamine, Texas red, allophycocyanin, etc.
  • the fluorochrome label may be used to monitor microscopically or by flow cytometry the cell composition after the depletion and enrichment steps. Fluorescent labeling may conveniently utilize the same indirect coupling system as the magnetic particles. For example, a cocktail of digoxigenin- coupled depletion antibodies may be used in combination with anti-digoxigenin antibody coupled to magnetic particles, followed by labeling with a fluorochrome conjugated antibody directed to the anti-hapten antibody.
  • the depletion reagent antibodies are added to a suspension of NPBC, and incubated for a period of time sufficient to bind the available cell surface antigens.
  • the incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, so that the efficiency of the magnetic separation is not limited by lack of antibody. The appropriate concentration is determined by titration.
  • the medium in which the cells are separated will be any medium which maintains the viability of the cells.
  • a preferred medium is phosphate buffered saline containing from 0.1 to 0.5% BSA.
  • Various media are commercially available and may be used according to the nature of the cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.
  • dMEM Dulbecco's Modified Eagle Medium
  • HBSS Hank's Basic Salt Solution
  • dPBS Dulbecco's phosphate buffered saline
  • RPMI Dulbecco's phosphate buffered saline
  • Iscove's medium PBS with 5 mM EDTA, etc.
  • the cell suspension may be washed and resuspended in medium as described above prior to incubation with the second stage antibodies.
  • the second stage antibody may be added directly into
  • the suspension of magnetically labeled cells is applied to a column or chamber as described in WO 90/07380, herein incorporated by reference.
  • the matrix may consist of closely packed ferromagnetic spheres, steel wool, wires, magnetically responsive fine particles, etc.
  • the matrix is composed of a ferromagnetic material, e.g. iron, steel, etc. and maybe coated with an impermeable coating to prevent the contact of cells with metal.
  • the matrix should have adequate surface area to create sufficient magnetic field gradients in the separation chamber to permit efficient retention of magnetically labeled cells.
  • the volume necessary for a given separation may be empirically determined, and will vary with the cell size, antigen density on the cell surface, cell number, antibody affinity, etc.
  • a two stringency system is employed, where the depletion step captures a high percentage of labeled cells and the enrichment step captures a lower percentage of labeled cells. This reduces the probability that labeled cells will be carried over from the first separation step into the second.
  • the stringency of the depletion column will be such that at least about 95% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 99% of the labeled cells will be retained, and preferably at least about 99.9% retained.
  • the geometry, matrix composition, magnetic field strength, size and flow rate of the ferromagnetic column will determine the percent of labeled cells that are retained on the column.
  • Factors that will increase the stringency are increased column size and length, decreased flow rate, and a finer matrix composition.
  • a column matrix of fibers with fine particles is preferred for the depletion step.
  • An empirical determination of the stringency may be made by analysis of bound and unbound cells.
  • the labeled cells are bound to the matrix in the presence of a magnetic field, usually at least about 100 mT, more usually at about 500 mT, usually not more than about 2T, more usually not more than about IT.
  • the source of the magnetic field may be a permanent or electromagnet.
  • the unbound cells contained in the eluate are collected as the eluate passes through the column. For greater purity, the unbound cells may be passed a second time over the magnetic column.
  • the unbound cells are used in an enrichment step, to select for fetal nucleated cells.
  • Enrichment reagents attached to superparamagnetic particles are bound to cell surface antigens which are present on fetal cells.
  • Antibody directly coupled to the superparamagnetic particle is preferred.
  • CD71 is present on both adult and fetal activated cells, and is expressed at high levels on fetal erythrocytes. Contamination from CD71 expression on adult hematopoietic progenitor cells may be avoided by the use of CD34 in the depletion reagent cocktail.
  • glycophorin A Another marker of interest is glycophorin A, which is expressed on both adult and fetal erythrocytes.
  • glycophorin A is expressed on both adult and fetal erythrocytes.
  • the possibility of contaminating adult red blood cells does not preclude the use of glycophorin A, as the adult cells are enucleated.
  • the enrichment reagents, superparamagnetic particles, columns and buffers are prepared as described for the depletion reagents, however, the stringency for the enrichment column will be lower than for the depletion column.
  • the stringency of the enrichment column will be such that at least about 50% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 80% of the labeled cells will be retained, usually not more than 95% retained.
  • a column matrix of spheres is preferred for the enrichment step. The cells are bound to the magnetic matrix. After the initial binding, the matrix is washed with any suitable physiological buffer to remove unbound cells. The unbound cells are discarded.
  • the bound cells are released by removing the magnetic field, and eluting in a suitable buffer.
  • the cells may be collected in any appropriate medium which maintains the viability of the cells.
  • Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, PBS- EDTA, PBS. Iscove's medium, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.
  • the separation procedure will perform the depletion step first, followed by the enrichment step. If the enrichment step is to be performed first, then an additional step is necessary after the enrichment, in order to remove the magnetic label from the enriched cells. This may be accomplished by any suitable method.
  • the enriched cell population may be incubated with a solution of dextranase, where the dextranase is present at a concentration sufficient to remove substantially all microparticles from the labeled cells. Usually the reaction will be complete in at least about 1 hour.
  • the depletion step may then be performed as previously described with the dextranase treated cells.
  • the enrichment step may be performed first, and the depletion step modified to use large magnetic spheres in place of the microparticles.
  • the use of such magnetic spheres has been previously described, and the reagents are commercially available.
  • the enriched cell population is incubated with highly magnetic polymer spheres of about 5 to 10 ⁇ m diameter conjugated to the depletion antibody cocktail.
  • the mixture of cells is then placed in close proximity to a magnetic field. Substantially all cells bound to the polymer spheres are bound to the magnet after about 1 minute, and after not more than about 5 minutes. The unbound cells may be decanted and used for analysis.
  • the cells are used as appropriate.
  • the method of harvesting will depend on the type of analysis to be performed. For those procedures which require purified DNA samples the cells will be lysed according to conventional methods. There are a number of suitable procedures described in the literature. If the analysis will be performed on whole chromosomes the cells will be suitably prepared according to conventional methods.
  • the cells are immobilized on a filter, and are then labeled in situ with suitable fluorochrome coupled reagents.
  • the choice of genetic analysis is not critical to the invention. Any conventional method of analysis which requires fetal cells, DNA or chromosomes can be used. Examples include the use of polymerase chain reaction for detection of specific DNA sequences, fluorescence in situ hybridization for chromosome number, metaphase spreads for detection of chromosome complement and Quinicrin mustard staining to detect the presence of the Y chromosome.
  • PCR polymerase chain reaction
  • PCR where DNA from a number of cells is amplified in a single reaction is particularly useful to determine whether paternal alleles are present in the fetus, e.g. Y chromosome alleles, or genes for which the maternal and paternal alleles can be distinguished. It is advantageous in the genetic analysis to discriminate between fetal cells and contaminating maternal cells. This may be accomplished by DNA mosaic detection. Isolated single cells are amplified by PCR, usually in combination with a pre- amplification step. The DNA is amplified and analyzed for the presence of the sequence of interest, and for a paternally derived marker. Any polymorphism which can be assigned to the paternal parent may be used.
  • polymorphic alleles of the major histocompatibility complex e.g. HLA-A, HLA-B, HLA-DP, HLA-DQ and HLA-DR
  • microsatellite repeat polymorphisms e.g. D9S52, APOC2, D19S49, D1S80, D8S320, SE33 etc.
  • polymorphisms in protein sequences etc. Only those samples which differ from the maternal pattern are scored, thereby excluding any contaminating maternal cells.
  • a variation of DNA mosaic detection may also be used in combination with FISH.
  • a probe with a second fluorochrome for a paternally derived marker, or a fetal specific marker, e.g. Hemoglobin F is added. Only those cells which which differ from the maternal pattern are scored.
  • Conditions which can be analysed by the subject methods include fragile X; tris ⁇ mies of chromosome 21, 18, 12, 13, X and Y; and other chromosomal abnormalities; presence of fetal Y chromosome; and detecting the presence of genes correlated with such diseases as Tay-Sachs, muscular dystrophy, cystic fibrosis, hemophilias, and hemoglobinapathies, e.g. sickle cell anemia, thalassemias, etc.
  • kits having the reagents and apparatus necessary to perform the subject invention.
  • a kit may contain for the depletion step: hapten conjugated marker specific antibodies, e.g. anti-CD34, anti-CD45, anti-CD 14, etc.; anti-hapten antibody conjugated to superparamagnetic particles; and column(s) suitable for high stringency selection.
  • Components which may be included for the enrichment step include superparamagnetically coupled marker specific antibody, e.g. anti-CD71, anti- glycophorin A, etc.; and column(s) suitable for low stringency selection. Where required, large magnetic beads may be included.
  • buffers may be included for lysis of adult erythrocytes, cell staining and collection, etc. While single columns may be used, it is anticipated that multiple columns will be run simultaneously, and an apparatus for automated or manual procedures may be provided for such a purpose.
  • Reagents for genetic analysis may also be included, particularly primers for PCR amplification, staining reagents for FISH and filters for cell immobilization.
  • peripheral blood mononuclear cells as prepared above were resuspended in 150 ⁇ l of PBS with 0.01% sodium azide and 1% bovine serum albumin (PBS/BSA/NaN3) and stained with 200 ⁇ l of phycoerythrin conjugated murine anti- human CD45 monoclonal antibodies (KC 56 IgGl Coulter, Krefeld, FRG) diluted 1 to 4 from the stock in PBS/BSA, incubated on ice for 10 min., washed once in PBS/BSA/NaN3, and labelled with 150 ⁇ l of PBS/BS A/NaN3 containing 30 ⁇ l MACS rat anti mouse IgGl -conjugated superparamagnetic microbeads and 30 ⁇ l anti-CD 14- conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, FRG), at 4 * C for 15 min.
  • KC 56 IgGl Coulter, Krefeld, FRG phycoery
  • the negative fraction of the first MACS sort was stained again with 170 ⁇ l of fluorescein isothiocyanate (FITC) conjugated murine anti human CD71 mAb (12 ⁇ g/ml; LOl.l, IgG2a, Becton Dickinson, San Jose, CA, USA) in PBS/BSA/NaN 3 on ice for 10 min.
  • the cells were washed and labelled with 30 ⁇ l MACS rat anti mouse IgG2a+b-conjugated superparamagnetic microbeads (Miltenyi Biotec) in 60 ⁇ l PBS/BSA/NaN ⁇ as described above.
  • the cells were then washed and sorted by MACS.
  • CD71 conjugated to superparamagnetic particles were purchased from Miltenyi Biotec.
  • the labelled cells were applied to an A2 column and sorted essentially as described by Miltenyi et al., supra. For optimal depletion, the flow rate was kept to approx. 0.4 ml/min. by using a 26G needle at the outlet of the MACS column. The cells were then washed off with 1 ml of buffer.
  • a MiniMACS column was used for the second MACS. The cells were labeled with CD71 conjugated microbeads. Negative cells were washed off the column at flow rates of approx. 0.35 ml/min.
  • HLA-DQ Al genotyping was carried out using the Ampli-Type HLA-DQa kit
  • the cycling conditions for the D1S80 PCR were as follows: denaturation at 94 * C for 1 min. after hot start, annealing at 67 * C for 1 min., extension at 72 * C for 1 min, 30 cycles. PCR products were separated on discontinuous native polyacrylamide gels (stacking gel: 3.5% T, 2.7% C, resolving gel: 7%T, 4% C). DNA fragments were detected by silver staining. Alleles differed in size and were classified with the help of the "allelic ladder" of the D1S80 typing kit (Perkin-Elmer).
  • Mononuclear cells were obtained from 20 ml of peripheral blood from 11 pregnant women (weeks 12 to 25 of gestation).
  • CD45+ leukocytes and in some cases CD14+ cells were magnetofluorescently labelled (71 - 92% of cells gated according to scatter) and depleted on a MACS column.
  • the negative fraction contained 0.6 - 2% stained cells. This fraction was magnetically stained again for CD71 and applied to a second MACS.
  • the eluted fraction contained 62 to 87% of CD71+ cells. Both MACS sortings together required approximately 30 minutes of time.
  • Table 1 Table 1
  • Table 1 shows the results of two separations.
  • a simple MACS CD71 + enrichment from ficoll separated PBMC is compared with a double-MACS CD45" /CD147CD71+ enrichment of the same sample.
  • the CD71+ cell population (1.2%) was 9.4 fold enriched after the CD45/CD14 cell depletion (10.2%) and another 32.1 fold by the subsequent CD71 + enrichment of the Double-MACS procedure, leading to a total enrichment of 301fold (78.5%).
  • the direct CD71 + enrichment of the MACS procedure yielded an enrichment rate of only 33 fold (28.9%).
  • CD45 + /CD14 + cells were depleted from 72 - 92% to 0.6% - 2.0% (depletion rates of 242 - 977, average 780).
  • CD457CD14 1" cells were enriched from 0.5 - 2.1% to 1.4 - 10.2% (enrichment rates of 2.8 - 9.4, average 3.9) in the first MACS by depletion of CD45/CD14 cells and in the second MACS, the CD457CD71 + cells were enriched to 62 - 87% (enrichment rates of 31 - 465, average of 130).
  • the overall enrichment rates for CD45" /CD71 + cells varied between 300 and 820 with an average of 500. Recovery rates varied between 38% - 55%.
  • the sorted cells were analyzed by flow cytometry, and DNA was prepared from them for genetic analysis.
  • Paternal alleles were detected in fetal cells from maternal blood in 7 out of 11 analyzed cases.
  • HLA DQ Al genotyping the success rate of detection was 6 out of
  • D1S80 allele (T22), making the detection of a fetal allele impossible.
  • D1S80 polymorphism fetal alleles were detected in 3 out of 11 samples. In these samples fetal
  • DQ Al alleles were also detected. In 3 out of 11 samples D1S80 PCR products from DNA of maternal cells were not observed.
  • the relative frequencies of fetal cells in maternal blood by PCR of HLA DQ Al and D1S80 was estimated by comparing a titration of CD457CD71+ cells to a titration of cells representing the paternal genotype into cells representing the maternal genotype.
  • the D1S80 PCR was able to detect specific allelic length polymorphism with a limit of sensitivity of 1 paternal allele in 20 maternal alleles.
  • the HLA-DQ- A 1 PCR showed a higher sensitivity for detection of a particular allele in die presence of an excess of other different alleles. The sensitivity ranged from 1 in 100 (without PEP) to 1 in 200 (with PEP).
  • CD457CD71 + cells enriched from maternal blood by Double-MACS titration of DNA to the limit of detectability of paternal genes by PCR was consistent with relative frequencies of paternal to maternal alleles of 1 in 20 to 1 in 200.
  • the average amount of DNA obtained from the sorted CD457CD71+ cells of 20 ml of maternal blood was 175 ng, as evaluated photometrically.
  • NAME Sherwood, Pamela J.

Abstract

Procédés pour préparer des cellules f÷tales à partir d'échantillons de sang périphérique maternel. Une suspension de cellules mononucléées est préparée à partir d'un échantillon de sang maternel. Les cellules maternelles sont ensuite appauvries à partir de l'échantillon par l'addition d'anticorps couplés avec des particules magnétiques et spécifiques des marqueurs présents sur les cellules adultes, et la fixation des cellules dans une colonne en présence d'un champ magnétique. A partir de la fraction appauvrie les érythrocytes du f÷tus sont enrichis par triage magnétique des cellules. Ces érythrocytes nucléés sont alors utilisés comme source de matériau génétique du f÷tus, qui est analysé pour mettre en évidence des anomalies chromosomiques ou génétiques, ou pour rechercher la présence d'un chromosome Y, par des procédés conventionnels tels que l'hybridation in situ avec des sondes, l'examen des dispersions en métaphase et la technique d'amplification en chaîne PCR.
PCT/US1995/011971 1994-09-20 1995-09-19 Enrichissement de cellules f×tales, a partir du sang maternel WO1996009409A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU35935/95A AU691040B2 (en) 1994-09-20 1995-09-19 Enrichment of fetal cells from maternal blood
EP95933174A EP0778899A4 (fr) 1994-09-20 1995-09-19 Enrichissement de cellules f tales, a partir du sang maternel
JP8511058A JPH10507632A (ja) 1994-09-20 1995-09-19 母体からの胎児血液の濃縮

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US30922994A 1994-09-20 1994-09-20
US08/309,229 1994-09-20

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WO1998002528A1 (fr) * 1996-07-12 1998-01-22 Domenico Valerio Isolement et culture de cellules foetales de sang maternel peripherique
WO1998010267A1 (fr) * 1996-09-04 1998-03-12 Technical University Of Denmark Systeme a microdebit pour separation et analyse de particules
FR2758884A1 (fr) * 1997-01-30 1998-07-31 Bio Merieux Procede pour isoler, notamment detecter ou quantifier un analyte dans un milieu
WO1998040746A1 (fr) * 1997-03-08 1998-09-17 The University Of Dundee Procedes de diagnostic prenatal
WO2001079851A1 (fr) * 2000-04-13 2001-10-25 Imperial College Innovations Limited Diagnostique prenatal non invasif utilisant des cellules foetales cd45-negatives
WO2008017871A1 (fr) * 2006-08-11 2008-02-14 University Of The West Of England, Bristol Séparation des cellules sanguines
GB2447255A (en) * 2007-03-02 2008-09-10 Oncoprobe Ltd Preparation of enriched target cell samples for use in a chemosensitivity assay
WO2012010666A1 (fr) * 2010-07-21 2012-01-26 Diagast Méthodes d'immunodiagnostic magnétique et nécessaires révélant la présence de complexes anticorps/antigènes dans le cadre du groupage et du phénotypage du sang érythrocytaire
EP2955521A1 (fr) * 2014-06-11 2015-12-16 Centre Hospitalier Universitaire Vaudois (CHUV) Procédés pour séparer des cellules
CN109718880A (zh) * 2019-02-20 2019-05-07 广州睿辰生物科技有限公司 基于双抗法分选胎儿有核红细胞额定微流控芯片及其分选方法

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See also references of EP0778899A4 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998002528A1 (fr) * 1996-07-12 1998-01-22 Domenico Valerio Isolement et culture de cellules foetales de sang maternel peripherique
WO1998010267A1 (fr) * 1996-09-04 1998-03-12 Technical University Of Denmark Systeme a microdebit pour separation et analyse de particules
US7138269B2 (en) 1996-09-04 2006-11-21 Inverness Medical Switzerland Gmbh Microflow system for particle separation and analysis
US6432630B1 (en) 1996-09-04 2002-08-13 Scandinanian Micro Biodevices A/S Micro-flow system for particle separation and analysis
FR2758884A1 (fr) * 1997-01-30 1998-07-31 Bio Merieux Procede pour isoler, notamment detecter ou quantifier un analyte dans un milieu
WO1998034116A1 (fr) * 1997-01-30 1998-08-06 Bio Merieux Procede pour isoler, notamment detecter ou quantifier un analyte dans un milieu
US6342396B1 (en) * 1997-01-30 2002-01-29 Bio Merieux Method for isolating, in particular for detecting or quantifying an analyte in a medium
US6331395B1 (en) 1997-03-08 2001-12-18 The University Of Dundee Prenatal diagnostic methods
GB2326943B (en) * 1997-03-08 1999-06-16 Univ Dundee Detection of fetal red cells in maternal blood
GB2326943A (en) * 1997-03-08 1999-01-06 Univ Dundee Prenatal diagnostic methods
WO1998040746A1 (fr) * 1997-03-08 1998-09-17 The University Of Dundee Procedes de diagnostic prenatal
WO2001079851A1 (fr) * 2000-04-13 2001-10-25 Imperial College Innovations Limited Diagnostique prenatal non invasif utilisant des cellules foetales cd45-negatives
WO2008017871A1 (fr) * 2006-08-11 2008-02-14 University Of The West Of England, Bristol Séparation des cellules sanguines
GB2447255A (en) * 2007-03-02 2008-09-10 Oncoprobe Ltd Preparation of enriched target cell samples for use in a chemosensitivity assay
WO2012010666A1 (fr) * 2010-07-21 2012-01-26 Diagast Méthodes d'immunodiagnostic magnétique et nécessaires révélant la présence de complexes anticorps/antigènes dans le cadre du groupage et du phénotypage du sang érythrocytaire
FR2963108A1 (fr) * 2010-07-21 2012-01-27 Diagast Procede magnetique d'immunodiagnostic et kit pour la mise en evidence de complexe anticorps/antigene de groupe/phenotype sanguin
US9618518B2 (en) 2010-07-21 2017-04-11 Diagast Magnetic immunodiagnostic methods and kit for the demonstration of antibody/antigen complexes in erythrocyte blood grouping and phenotyping
EP2955521A1 (fr) * 2014-06-11 2015-12-16 Centre Hospitalier Universitaire Vaudois (CHUV) Procédés pour séparer des cellules
CN109718880A (zh) * 2019-02-20 2019-05-07 广州睿辰生物科技有限公司 基于双抗法分选胎儿有核红细胞额定微流控芯片及其分选方法

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JPH10507632A (ja) 1998-07-28
CA2200294A1 (fr) 1996-03-28
EP0778899A4 (fr) 2001-08-29
EP0778899A1 (fr) 1997-06-18
AU691040B2 (en) 1998-05-07
AU3593595A (en) 1996-04-09

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