EP0891549A2 - Analyse de cellules foetales - Google Patents

Analyse de cellules foetales

Info

Publication number
EP0891549A2
EP0891549A2 EP97903493A EP97903493A EP0891549A2 EP 0891549 A2 EP0891549 A2 EP 0891549A2 EP 97903493 A EP97903493 A EP 97903493A EP 97903493 A EP97903493 A EP 97903493A EP 0891549 A2 EP0891549 A2 EP 0891549A2
Authority
EP
European Patent Office
Prior art keywords
foetal
agent
cells
binding
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97903493A
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German (de)
English (en)
Inventor
Linda G. University of Nottingham DURRANT
David Tekyung Dept. Obstetrics & Gynaecology LIU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Nottingham
Original Assignee
University of Nottingham
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Nottingham filed Critical University of Nottingham
Publication of EP0891549A2 publication Critical patent/EP0891549A2/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells

Definitions

  • the present invention relates to methods of foetal cell analysis as well as to kits for use in such methods.
  • foetal cells can be carried out by isolating at least two types of foetal cells from a sample of maternal blood. By suitably labelling foetal cells they can easily and simply be isolated and can then be used in conventional analyses.
  • the present invention provides a method of analysing foetal cells, the method comprising isolating at least two types of nucleated foetal cell from a maternal sample.
  • the cell types are nucleated erythrocytes and trophoblast and the maternal sample is a peripheral blood sample.
  • the cells are preferably isolated from the peripheral maternal blood, and desirably the different cell types are isolated from the same maternal sample.
  • maternal blood is sampled from a pregnant human at between eight and sixteen weeks gestation, and desirably at ten weeks.
  • trophoblast cells are isolated prior to isolation of nucleated erythrocytes.
  • the trophoblasts may be isolated by contacting the maternal blood with a trophoblast binding agent prior to subsequent labelling, identification and/or removal.
  • the binding agent may comprise an antibody such as a monoclonal, polyclonal or genetically engineered antibody or active derivatives of any of these.
  • other suitable ligands could be used.
  • the binding agent binds to the 170kD epidermal growth factor receptor (EGFr) antigen or to its 150kD cleavage product.
  • the binding agent is the monoclonal antibody Mab 340 (Durrant et al , Prenatal Diagnosis , 14:131-140 (1994)) or an EGFr binding derivative thereof.
  • the Mab 340 antigen has been deposited with the European Collection of Cell Cultures, CAMR (Centre for Applied Microbiology _ Research) (ECACC) under provisional accession no. 97021428.
  • the present invention also provides antibodies to the EGFr for use in binding cells and or cellular fragments or clusters from samples of peripheral blood or other tissue specimens for application in prenatal diagnosis.
  • Preferred is the antibody Mab 340 described herein and functional equivalents to Mab 340. It will be obvious to those skilled in the art that any antibody, or functional equivalents thereof, with binding specificity to EGFr will be efficacious in this application.
  • the antibodies may be complete immunoglobulin molecules, but they may be Ig fragments for example onovalent or divalent Ig entities such as Fab fragments, single chain Fv's etc.
  • Preferred antibodies are those recognising epitopes to the extracellular domain of the EGFr such as Mab 340. It will be obvious to those skilled in the art that antibodies with binding specificity to other domains of the EGFr normally present either within the cellular membrane or present on the cytoplasmic face of the membrane may be embodied in this invention.
  • peptide or other synthetic mimetic molecules able to bind the EGFr and likewise function to facilitate enrichment and or purification of EGFr bearing cells, cellular fragments or clusters for the purpose of prenatal diagnostic screening.
  • Mab 340 is a particularly advantageous binding agent for use in the methods of the invention.
  • the invention also includes methods wherein the the EGFR binding derivative of Mab 340 comprises at least the CDR regions of one or other of the light or heavy chain variable regions of Mab 340.
  • the EGFR binding derivative of Mab 340 will further comprise one or more of the framework regions of one or other of the light or heavy chain variable regions of Mab 340.
  • sequence information can be used to construct moieties which mimic Mab 340, eg peptides or proteins which although not having the full sequence of Mab 340, nevertheless include the CDRs and optionally the framework regions of the variable portions of Mab 340.
  • an affinity medium eg a solid medium to which the EGFr binding agent will bind
  • the medium may comprise paramagnetic beads or a magnetic colloid such as 51(Cr)-sodium chromate labelled MG63 cells.
  • paramagnetic beads can be coated with Mab 340 which will in turn bind to trophoblast cells in the sample.
  • the cells can then be isolated using a magnet.
  • the beads themselves can be labelled with an agent which binds to an EGFr binding agent, for example where the EGFr binding agent is an antibody the beads may be labelled with rabbit anti-mouse antiseru . In this example, therefore, the anti EGFr antibody will bind to trophoblasts and then the beads bind to the antibody and once again a magnet can then be used to isolate the cells.
  • the magnetic colloid concentration may be in the order of 1/80 and l ⁇ g/ml antibody.
  • the magnetic colloids are pre-labelled with agent prior to introduction to the blood sample.
  • the colloid/sample mixture is incubated at approximately room temperature for about thirty minutes.
  • binding agents may include combinations of monoclonal antibodies preferably mAb 340, or derivatives thereof in use with any other currently existing or future monoclonal antibody with a different binding specifity on the EGFr surface to mAb 340.
  • Combinations of binding agents may include antibody or antibody derivatives in combination with other antibody or antibody derivatives.
  • Combinations of binding agents may include antibody or antibody derivatives in combined use with non-antibody molecules which may be synthetic ligands with functionalised domains, or non-covalently attached functional groups. Combinations of binding agents may consist of entirely of non-antibody molecules capable of binding EGFr.
  • the erythrocytes may be isolated using erythrocyte labelling agents, such as antibodies which selectively bind foetal erythrocytes.
  • erythrocyte labelling agents such as antibodies which selectively bind foetal erythrocytes.
  • an antibody is CD71 antibody.
  • the labelled erythrocytes may then be separated by passing the blood through a medium to which the label is directly or indirectly attracted, for example to reversibly bind therewith.
  • the label comprises anti-transferrin antibody, desirably a monoclonal antibody.
  • the medium may comprise paramagnetic beads coated with an antibody attractant, such as rabbit anti-mouse antiserum.
  • the label may comprise ferromagnetic particles coated with an anti-transferrin antibody, desirably monoclonal, and the medium may comprise a MACS column.
  • Foetal erythrocytes may be subjected to an initial separation by passing the blood sample down a density gradient, such as a triple gradient.
  • the methods of the present invention may further comprise biochemical and/or genetic analysis of isolated foetal cells, principally for the detection of chromosome aneuploides, conditions or disorders.
  • one or more genetic sequences of the foetal cells are amplified for example using the polymerase chain reaction (PCR) or other suitable amplification techniques, to facilitate analysis.
  • a specific sequence on the Y chromosome may be amplified when analysing the sex of a foetus.
  • fluoresence in situ PCR of mRNA for foetal haemoglobin in foetal erythrocytes and/or human chorionic gonadotrophin (HCG) in trophoblasts may be used.
  • a hybridisation techique is used to detect the foetal sequence(s), for example a technique preferably comprising fluoresence in situ hybridisation (FISH).
  • FISH fluoresence in situ hybridisation
  • the present invention further provides an agent for binding and/or labelling for use in the methods of the present invention.
  • the agent may be an antibody, such as a monoclonal, polyclonal or genetically engineered antibody or an active derivative of any of these.
  • the agent can be a ligand or chemical which can associate and preferably bind to the surface of foetal cells.
  • the agent will bind to the 170kD epidermal growth factor receptor (EGFr) antigen or to its 150kD cleavage product.
  • EGFr epidermal growth factor receptor
  • Mab 340 is a particularly advantageous agent and thus an agent comprising Mab 340 or an active derivative thereof (for example a derivative which comprises at least the CDRs of the variable regions of the light or heavy chains of Mab 340) is particularly preferred.
  • the binding/labelling reagent may comprise a foetal erythrocyte binding agent.
  • the present invention provides a primer for use in the amplification of a target nucleotide sequence of a foetal cell.
  • the primer may be hybridisable for amplification of some or all of a sequence coding for foetal haemoglobin.
  • the or a further primer may be hybridisable for amplification of some or all of a sequence coding for human chorionic gonadotrophin.
  • the present invention provides:
  • Mab 340 or an active derivative thereof, in a method of isolating at least two types of foetal cell
  • kits for use in the methods of the invention which comprises at least one trophoblast binding agent optionally in association with a labelling agent.
  • the trophoblast binding agent is Mab 340 or an active derivative thereof and the kit may further comprise an erythrocyte binding agent such as an antibody;
  • a method for analysing foetal cells which comprises: a) obtaining a maternal sample; b) contacting the sample from a) with a trophoblast binding agent; c) contacting the sample with an erythrocyte binding agent; and d) carrying out one or more biochemical and/or genetic analysis steps on the cells isolated in b) and c) .
  • the present invention provides a method for the enrichment of a minority population of cells in the peripheral circulation and their subsequent analysis at the molecular genetic level.
  • this invention is to be used for the capture and analysis of circulating foetal cells for the purpose of prenatal diagnosis of disease and preferably using mAb 340. It is disclosed that mAb 340 most likely recognises and binds an epitope in the extracellular domain of the EGFr.
  • mAb 340 most likely recognises and binds an epitope in the extracellular domain of the EGFr.
  • One further embodiment therefore may be the use of this technology in the enrichment and subsequent analysis of a minority population of malignant cells such as squamous carcinoma cells or any tumour cell expressing the EGFr on its surface.
  • the EGFr is a transmembrane glycoprotein of l70kD with tyrosine kinase activity. The latter is activated following ligand binding leading to autophosphorylation of the receptor and ultimately stimulation of cell proliferation.
  • Over-expression of the EGFr has been reported in a number of human malignancies including cancer of the breast, brain, bladder, head and neck, pancreas and lung. Histological and biological study of several human tumour biopsies and cell lines has shown that overexpression of this receptor is often accompanied by the production of one or more of the known ligands for the receptor namely TGF ⁇ and/or EGF. Such observations have lead to the suggestion that an autocrine loop may be responsible for the growth of tumours of this type.
  • FIGURE 1 shows silver stained SDS-PAGE analysis of 340 antigen purifications from immunoaffinity chromatography
  • FIGURE 2 shows a saturation curve and scatchard plot of radioligand binding assays showing that I 125 EGF binds to 719T cells;
  • FIGURE 3 shows the results of a competitive binding assay of EGF and Mab 340 with I 125 EGF to 719T cells.
  • Trophoblast cells were isolated from the whole blood of women in the first generally one third of the pregnancy using a specific monoclonal antibody, 340 (Durrant et al, supra ) incubated with the blood sample and passed through a separating medium comprising paramagnetic beads coated with rabbit anti-mouse antiserum.
  • Nucleated red blood cells were isolated by separating whole blood on a triple density gradient and staining with either ferromagnetic particles coated with an anti-transferrin monoclonal antibody and separated on a mini MACS column or staining with an anti-transferrin monoclonal antibody and paramagnetic beads coated with rabbit anti-mouse antiserum.
  • the two isolated cell types were sexed using a nested PCR for a specific sequence on the Y chromosome (found in males only) and the sex confirmed by displaying the metaphase chromosomes (karyotyping) of chorionic villus samples.
  • Foetal cells were isolated from nineteen patients between ten to fourteen weeks into pregnancy who were undergoing elective chorionic " villus sampling for the detection of foetal aneuploides.
  • Elective chorionic villus sampling is the conventional method of obtaining trophoblast tissue for analysis of human foetal DNA.
  • the chorion is biopsied either by transcervical or transabdominal routes.
  • the principal disadvantage of this known technique, which the present invention obviates, is that direct biopsys can cause foetal loss.
  • the sensitivity in determining a marie pregnancy in the second study with nucleated erythrocytes alone was 38% and with trophoblasts was 39%, whereas isolating and anaylsing for both correctly predicted a male pregnancy in ten out of eighteen cases or a sensitivity of 56%.
  • the specificity (83-86%) determined by the number of false positives i.e. incorrect diagnosis for male pregnancy was very similar whichever foetal cell was selected.
  • the ten male pregnancies which were correctly diagnosed three were diagnosed on nucleated erythrocytes alone, three were diagnosed only on trophoblasts and four were positive on both cell types.
  • the concentration of foetal cells in maternal blood is particularly low. As few as 20 foetal cells per 20ml of maternal blood may be present, although this number appears to increase due to placenta malformation in Down's pregnancies. Isolated foetal cells are outnumbered by a thousand fold excess of maternal cells which makes identification of the cells with fluorescent in situ hybridisation analysis very difficult to interpret. Identification of the foetal cells by fluorescence in situ polymerase chain reaction (PCR) of mRNA for foetal haemoglobin in erythrocytes and human chorionic gonadotrophin (HCG) in trophoblasts allows more accurate fluorescent in situ hybridisation analysis (FISH).
  • PCR fluorescence in situ polymerase chain reaction
  • HCG human chorionic gonadotrophin
  • magnetic colloids are used to isolate the cells.
  • Initial studies were performed with 51(Cr)-sodium chromate labelled MG63 cells which express high levels of the 340 trophoblast antigen.
  • Optimal isolation and analysis was obtained with a ferrofluid concentration of 1/80 and l ⁇ g/ml 340 monoclonal antibody.
  • Optimal incubation was at room temperature for thirty minutes and pre-labelling the ferrofluid with the antibody proved the most efficient labelling protocol. Using these parameters 74-80% of MG63 cells were isolated and analysed from blood.
  • the isolation and analysis of at least two foetal cell types can be used in the analysis of the cell types for any chromosome aneuploides, disorder, condition or other characteristic.
  • the cells can be analysed for disorders such as Down's syndrome.
  • 5x10* hybrido a 340 cells were pelleted and mRNA isolated using TRIzolTM reagent according to the manufacturers instructions (Life Technologies, Inc. Paisley Scotland). DNA/RNA hybrid was formed using Ready-To-GoTM T-primed First-Stand kit (Pharmacia, Sweden). PCR was carried out using primer sets specific for the mouse heavy and light chain variable regions (Jones, S. T. and Bending, M. , Bio/Technology, 9:88 (1991)). The PCR products were cloned into pCRII (Invitrogen, Netherlands) and six individual clones sequenced from both directions using the Applied Biosystems automated sequencer model 373A. The VH and VL sequences are shown below; single amino acid codes are used and the CDRs are underlined. The sequences have been compared with the Kabat database of proteins of immunological interest to identify the variable region gene families used and to establish their uniqueness.
  • the gel was western blotted onto PVDF (ProBlot Applied Biosystems) according to the protocols supplied with the blotting membrane.
  • the blot was stained with coomassie blue and destained with 10%(v/v)Methanol / Acetic acid.
  • the bands were excised from the blot and protein sequenced obtained using an Applied Biosystems protein sequencer model 473A.
  • N-terminal amino acid sequence was LEEKKVCQG. This peptide shares identity with the mature N-terminal sequence of epidermal growth factor receptor (EGFr).
  • EGF epidermal growth factor receptor
  • a competition binding assay using iodinated EGFr ligand (EGF) and mAb 340 was established to corroborate the finding from the peptide sequence analysis.
  • Cells were harvested with Trypsin/EDTA and washed twice in RPMI 1640 medium before being resuspended at 5 x 10 s /ml. lOO ⁇ l of cell suspension were mixed with 100 ⁇ l of I 125 EGF (Amersham; 100 ⁇ Ci/ ⁇ g, 50 ⁇ Ci/ml) at different concentrations and incubated at 37°C for 1 hour. Cells and bound EGF were separated by centrifugation and washed 2 times in ice-cold Phosphate buffered saline (PBS) containing 0.1% (v/w) bovine serum albumin (BSA). Non- specific binding was measured by adding 100-fold excess of cold EGF.
  • PBS Phosphate buffered saline
  • BSA bovine serum albumin
  • Radioligand binding assays show that I 125 EGF can bind to the 791T cells.
  • a saturation curve was obtained and a non-linear scatchard plot reflects the presence of populations of receptors with varying affinities for EGF (Fig 2).
  • the EGF receptor from A431 cells was first purified in 1980 by the use of affinity chromatography .
  • the purified receptor had an apparent molecular mass of 150 KDa; however, subsequent purification methods that eliminated calcium from the buffers indicated the actual mass to be 170 KDa.
  • Most cell homogenates contain a calcium-activated protease that cleaves the native molecule of 170 KDa to the lower molecular mass of 150 KDa. I "125 EGF binding assays have shown there are two sub-populations of EGFr with different affinities.
  • Ferrofluid was diluted before use (typically 1/40 - 1/80). Cells were fixed in 1% paraformaldehyde for 10 minutes and then permeabilised in 70% Ethanol for 20 minutes. Ferrofluid was labelled with antibody (anti- HbF, anti-G6PDH) for 30 minutes at room temperature prior to Incubating with the fixed and permeabilised cells for 5 mins at 37°C and 55 mins at room temperature. Labelled cells were sorted on an Immunicon magnet for 5 minutes prior to washing.
  • Immunicon ferrofluid was diluted 1/40-1/80 and labelled with biotinylated antibody for 30 mins at room temperature. The cells to be sorted were added for a further 30 mins and then sorted on an Immunicon magnet. Sorted cells were fixed in Acetone (neat) or Paraformaldehyde (1%) for 10 minutes and dropped onto microscope slides and air dried. The slides were incubated with 20% normal rabbit serum in Tris Buffered Saline (TBS) for 30 mins prior to washing x2 in TBS for 5 minutes. Bound antibody was detected using avidin/biotin peroxidase complex and DAB substrate. Specific chromosomes were detected by the fluorescence in situ hybridisation (FISH) technique and commercially available fluorescence hybridisation probes and protocols provided by the manufacturer.
  • FISH fluorescence in situ hybridisation
  • Foetal trophoblasts sorted by the methods above were subjected toa PCR analysis for the presence of DNA encoding HCG.
  • Primers used were as follows; ( 1) 5' hCG 5' CTG GCT GTG GAG AAG ' ;
  • Thermal cycling conditions using primers (l) and (2) comprised a hot start at 80°C for 5 minutes followed by addition of Taq poly erase and 30 cycles of 94°C for 1 minute, 55°C for 1 minute and 72 ⁇ C for 1 minute. This gave a 230bp product which was further amplified using primers (1) and (3) and conditions as above to give a 230bp product indicative of the presence of HCG DNA from foetal trophoblasts.
  • Mononuclear cells were isolated on percol from whole blood of women in the first one third of pregnancy. Trophoblasts were isolated using biotinylated monoclonal antibody 340 which is coated onto ferrofluid and incubated with the mononuclear cells. Labelled cells are sorted on a magnet, washed and dropped onto slides.
  • Unsorted mononuclear cells are fixed and permeablised in 1% paraformaldehyde / 70% ethanol.
  • Nuclear red blood cells are isolated using a biotinylated specific monoclonal antibody (anti-HbF, anti-G6PDH) which is coated onto ferrofluid. Labelled cells are sorted on a magnet, washed and dropped onto slides. Isolated foetal cells are then stained by immunohistochemistry/ FISH.
  • tumour cell line COLO 205 tumour cell line COLO 205
  • sorting using cytokeratin 18 (C18) as the internal antigen have been performed using a foetal cell surrogate, in this case tumour cell line COLO 205 and sorting using cytokeratin 18 (C18) as the internal antigen.
  • COLO 205 cells were harvested and counted with viability assessed by tryphan blue exclusion. 5xl0 6 cells were labelled with 2 MBqs of tritiated thymidine ( 3 H) (Amersham, UK) and incubated at 37°C for at least 6 hours (usually overnight). Cells were harvested and washed twice in RPMI medium (Life Technologies, Paisley Scotland) . Cells were recounted and viability assessed by tryphan blue exclusion. Cells (10 s ) were collected by centrifugation and fixed by resuspension in 1ml 1% (w/v) paraformaldehyde and incubation at 4°C for 20 minutes.
  • Ferrofluid (immunocon corp, Huntingdon Valley, PA, USA) was diluted as required (usually 1/80) and labelled with anti-cytokeratin 18 antibody (C18, Sigma, Poole, Dorset) for 30 minutes at room temperature in a volume of 500 ⁇ l. The antibody concentrations were varied as part of the validation experiments, from 1/50 and 1/200. The ferrofluid/antibody mixture was added to the fixed cells, mixed by gentle shaking, and incubated at 37'C for 5 minutes followed by incubation for a further 55 minutes at room temperature. Cells were sorted using the Immunocon sorting system and pipetted into luraaplates (Canbarra Packard) for 3 H counting using a scintillation counter. Results of cell sorting on an internal antigen using a foetal cell surrogate .Table 2):

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Abstract

L'invention concerne des méthodes d'analyse des cellules foetales, des trousses destinées à être utilisées dans lesdites méthodes et l'utilisation d'agents de liaison appropriés permettant d'isoler les cellules foetales à partir du sang maternel.
EP97903493A 1996-02-16 1997-02-17 Analyse de cellules foetales Withdrawn EP0891549A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9603249.5A GB9603249D0 (en) 1996-02-16 1996-02-16 Foetal cell analysis
GB9603249 1996-02-16
PCT/GB1997/000443 WO1997030354A2 (fr) 1996-02-16 1997-02-17 Analyse de cellules foetales

Publications (1)

Publication Number Publication Date
EP0891549A2 true EP0891549A2 (fr) 1999-01-20

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EP (1) EP0891549A2 (fr)
JP (1) JP2000506608A (fr)
AU (1) AU735692B2 (fr)
CA (1) CA2246372A1 (fr)
GB (1) GB9603249D0 (fr)
WO (1) WO1997030354A2 (fr)

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* Cited by examiner, † Cited by third party
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CN1260046A (zh) * 1997-03-08 2000-07-12 邓迪大学 出生前诊断法
WO1998045413A1 (fr) * 1997-04-08 1998-10-15 Pall Corporation Procede de prelevement de cellules rares dans des produits sanguins
US20090220933A1 (en) 2005-12-08 2009-09-03 Fcmb Aps Detection of fetal cells from maternal blood
WO2017057076A1 (fr) * 2015-09-28 2017-04-06 富士フイルム株式会社 Procédé pour obtenir des érythrocytes nucléés et procédé pour identifier des érythrocytes nucléés
JP2022536121A (ja) 2019-06-07 2022-08-12 アルセディ バイオテック エーピーエス Ｆａｃｓを用いた胎児細胞の単離

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Publication number Priority date Publication date Assignee Title
AU2613988A (en) * 1988-01-04 1989-08-01 E.I. Du Pont De Nemours And Company Multiple stage affinity process for isolation of specific cells from a cell mixture
EP0500727B1 (fr) * 1989-11-13 1998-01-21 Children's Medical Center Corporation Procede non envahissant d'isolation et de detection d'adn f tal
DE4222573C1 (fr) * 1992-05-16 1993-08-12 Dorothee Dr. 4400 Muenster De Ahlert
WO1994025873A1 (fr) * 1993-04-23 1994-11-10 Cellpro, Incorporated Procedes d'enrichissement de cellules souches f×tales provenant du sang maternel
US6322981B1 (en) * 1996-11-27 2001-11-27 The United States Of America As Represented By The Department Of Health And Human Services Rapid method for diagnosing the various forms of alpha-thalassemia

Non-Patent Citations (1)

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Title
See references of WO9730354A3 *

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GB9603249D0 (en) 1996-04-17
AU735692B2 (en) 2001-07-12
JP2000506608A (ja) 2000-05-30
AU1804097A (en) 1997-09-02
CA2246372A1 (fr) 1997-08-21
WO1997030354A2 (fr) 1997-08-21
WO1997030354A3 (fr) 1997-11-20

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