[go: up one dir, main page]

WO2009009769A2 - Diagnostic d'anomalies fœtales au moyen d'hématies nucléées - Google Patents

Diagnostic d'anomalies fœtales au moyen d'hématies nucléées Download PDF

Info

Publication number
WO2009009769A2
WO2009009769A2 PCT/US2008/069879 US2008069879W WO2009009769A2 WO 2009009769 A2 WO2009009769 A2 WO 2009009769A2 US 2008069879 W US2008069879 W US 2008069879W WO 2009009769 A2 WO2009009769 A2 WO 2009009769A2
Authority
WO
WIPO (PCT)
Prior art keywords
sample
syndrome
cells
red blood
blood cells
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.)
Ceased
Application number
PCT/US2008/069879
Other languages
English (en)
Other versions
WO2009009769A3 (fr
Inventor
Ravi Kapur
Diana Bianchi
Tom Barber
Mehmet Toner
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.)
General Hospital Corp
Tufts Medical Center Inc
Artemis Health Inc
Curate Biosciences LLC
Original Assignee
General Hospital Corp
Tufts Medical Center Inc
GPB Scientific Inc
Artemis Health Inc
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 General Hospital Corp, Tufts Medical Center Inc, GPB Scientific Inc, Artemis Health Inc filed Critical General Hospital Corp
Publication of WO2009009769A2 publication Critical patent/WO2009009769A2/fr
Publication of WO2009009769A3 publication Critical patent/WO2009009769A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1029—Particle size
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/385—Congenital anomalies

Definitions

  • the present invention relates to systems, apparatus, and methods for determining the presence of a fetal or maternal abnormal condition by enumerating fetal nucleated red blood cells isolated from a sample from a pregnant woman.
  • Implementation of the invention can include one or more of the following features.
  • a method for determining the presence of a fetal abnormal condition including enumerating nucleated red blood cells in a blood sample from a pregnant woman and determining the presence of a fetal abnormal condition based on the number of nucleated red blood cells in the blood sample.
  • a method for determining the presence of aneuploidy in a fetus including a) enumerating nucleated red blood cells in a sample from a pregnant woman and b) assigning a likelihood of said pregnant woman's fetus being aneuploid based on statistical averages of nucleated red blood cells from blood samples from pregnant women carrying euploid fetuses compared with statistical averages of nucleated red blood cells from blood samples from pregnant women carrying aneuploid fetuses.
  • a method for determining the presence of a fetal abnormal condition including a) enumerating nRBCs in a first blood sample from a pregnant woman (b) and either: (i) detecting the presence or level of one or more serum markers in the first or a second blood sample from the pregnant woman, (ii) measuring space in nuchal fold of her fetus; or (iii) or both (i) and (ii), and determining the presence of the fetal abnormal condition in the fetus from results from steps (a) and (b).
  • the method can include the step of enriching nucleated red blood cells from enucleated red blood cells or white blood cells.
  • the enriching can be based on cell size and/or magnetic property.
  • the enriching can include using arrays of obstacles.
  • the enriching can include rendering nucleated red blood cells magnetic.
  • the enriching can include using arrays of obstacles and rendering nucleated red blood cells magnetic.
  • the sample can be taken in the first trimester of pregnancy.
  • the said pregnant woman can be under the age of 35.
  • the nRBCs can be enriched in a flow-through microfluidic device.
  • the enumerating of nRBCs can be performed by flow cytometry, fluorescence imaging, or radioactive imaging.
  • the method can further include performing fluorescence in situ hybridization on said nucleated red blood cells with chromosome-specific probes.
  • the method can include determining the genetic characteristics of said pregnant woman's fetus.
  • the serum markers can be comprised of papA, free ⁇ HCG, unconjugated estriol (UE3), AFP, HCG, or inhibin.
  • the aneuploidy can be trisomy 21.
  • the aneuploidy can be trisomy 8, trisomy 9, trisomy 12, trisomy 13, trisomy 18, trisomy 21, XXX, XXY, XYY, XXXY, XXYY, XYYY, XXXXX, XXXYY, XXYYY, or triploidy.
  • the fetal abnormal condition can be Klinefelter Syndrome, dup(17)(pl 1.2pl 1.2) syndrome, Down syndrome, Pre-eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup(22)(qll.2qll.2) syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie- Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Prader- Willi syndrome, Kallmann syndrome, microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, Azospermia (factor a),
  • the method can include determining the origin of the cells enumerated in step (b).
  • the sample can be a peripheral blood sample. In another embodiment, the sample can be an amniotic sample.
  • a method for determining a condition in a fetus of a subject including enriching one or more nucleated red blood cells from a first sample from said subject, performing a maternal serum marker screen on said first sample or a second sample from said subject, optionally, performing a Nuchal Translucency (NT) sonographic test on said first sample, said second sample, or a third sample from said subject, determining a condition of said fetus based on: (1) the number of nucleated red blood cells isolated from said first sample, (2) the results from said maternal serum marker screen; and (3) optionally, the results from said Nuchal Translucency test.
  • NT Nuchal Translucency
  • the condition can be selected from the group consisting of trisomy 8, trisomy 9, trisomy 12, trisomy 13, trisomy 18, trisomy 21, XXX, XXY, XYY, XXXY, XXYY, XYYY, XXXXX, XXXY, XXYYY, XYYYY, Klinefelter Syndrome, dup(17)(pl l.2pl l.2) syndrome, Down syndrome, Pre-eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merzbacher disease, du ⁇ (22)(ql 1.2ql 1.2) syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome,
  • the first sample, second sample, or third sample can be a peripheral blood sample.
  • the maternal serum marker screen can be AFP, MSAFP, Double Marker Screen, Double Screen, Triple Marker Screen, Triple Screen, Quad Screen, 1st Trimester Screen, 2nd Trimester Screen, Integrated Screen, Combined Screen, Contingency Screen, Repeated Measures Screen or Sequential Screen.
  • the subject can be under the age of 35.
  • the sample can be taken in the first trimester of pregnancy.
  • the enriching can be based on cell size and/or magnetic property.
  • the enriching can include using arrays of obstacles.
  • the enriching can include rendering nucleated red blood cells magnetic.
  • the enriching can include using arrays of obstacles and rendering nucleated red blood cells magnetic.
  • a method for determining the presence of a maternal abnormal condition including enumerating nucleated red blood cells in a blood sample from a pregnant woman and determining the presence of a maternal abnormal condition based on the number of nucleated red blood cells in the blood sample.
  • the condition can be Pre-eclampsia.
  • Figures 1A-1D illustrate some of the operational principles of a size-based separation module.
  • Figures 2A-2C illustrate one embodiment of an affinity separation module.
  • Figure 3 illustrates one embodiment of a magnetic separation module.
  • Figures 4A-4D illustrate schematics of a size-based separation module.
  • Figure 5A illustrates a schematic representation of a high-gradient magnet, designed to generate 1.2 Tesla to about 3 Tesla/mm.
  • Figure 5B illustrates a schematic representation of a capillary disposed adjacent to the magnet shown in
  • Figure 5C is a graph of the field strength of the magnet as a function of the position of the capillary.
  • Figure 6 is a summary of the results of the Phase I study performed at Site B.
  • Figure 7 is a summary of the results of the Phase I study performed at Site C.
  • Figure 8 lists the descriptive statistics and effect sizes for the combined nRBC enumeration and number of certitude trisomic events data from both sites.
  • Figure 9 is a summary of the results of a larger clinical study.
  • Figure 1OA is a summary of the results of a simulated clinical study.
  • Figure 1OB is a summary of the sensitivity rates calculated from the simulated clinical study.
  • the present invention provides systems, apparatus, and methods to diagnose conditions in a fetus based on the number of nucleated red blood cells collected from a sample from the mother. Furthermore, the present invention also provides methods to diagnose or prognosticate a condition in a fetus based on the number of nucleated red blood cells (nRBCs) collected from a sample from a mother using the aforementioned systems, apparatus, and methods. Further, serum marker screen data and/or nuchal translucency data taken from the same mother can be combined with nucleated red blood cell enumeration to diagnose or prognosticate a condition in a fetus.
  • nRBCs nucleated red blood cells
  • the invention also relates to a method for identifying a characteristic associated with a condition in a subject comprising obtaining a plurality of control samples, obtaining a plurality of case samples, applying each of said samples to a device comprising a plurality of obstacles that deflect a first analyte (such as a nucleated red blood cell or a trophoblast) from said sample in a direction away from a second analyte (such as an enucleated red blood cell) of said blood sample wherein said first analyte and said second analyte have a different hydrodynamic diameter, analyzing said first analyte from said samples to determine a characteristic of said first analyte, and performing an association study based on said characteristic.
  • a first analyte such as a nucleated red blood cell or a trophoblast
  • Samples containing rare cells can be obtained from a mammal pregnant with a fetus in need of a diagnosis or prognosis.
  • a sample can be obtained from mammal suspected of being pregnant, pregnant, or that has been pregnant to detect the presence of a fetus or detect a fetal condition (such as an abnormal fetal condition).
  • the mammal of the present invention can be a human or a domesticated mammal such as a cow, pig, horse, rabbit, dogs, cat, or goat.
  • Samples derived from a mammal or human can include, e.g., whole blood, amniotic fluid, or cervical swabs.
  • a blood sample can be obtained using any technique known in the art (such as withdrawal with a syringe a hypodermic needle connected to a Vacutainer tube, or other vacuum device.
  • a blood sample can be optionally pre-treated or processed prior to enrichment (such as by the addition of sodium heparin).
  • Examples of pre-treatment steps include the addition of a reagent such as a stabilizer, a preservative, a fixant, a lysing reagent, a diluent, an anti-apoptotic reagent, an anti-coagulation reagent, an anti-thrombotic reagent, magnetic property regulating reagent, a buffering reagent, an osmolality regulating reagent, a pH regulating reagent, and/or a cross-linking reagent.
  • a reagent such as a stabilizer, a preservative, a fixant, a lysing reagent, a diluent, an anti-apoptotic reagent, an anti-coagulation reagent, an anti-thrombotic reagent, magnetic property regulating reagent, a buffering reagent, an osmolality regulating reagent, a pH regulating reagent, and/or a cross-linking reagent.
  • a preservative such an anti-coagulation reagent and/or a stabilizer can be added to the sample prior to enrichment. This allows for extended time for analysis/detection.
  • a sample such as a blood sample
  • a sample can be enriched and/or analyzed under any of the methods and systems herein within 30 days, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18 hrs, 17 hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9 hrs, 8 hrs, 7 hrs, 6 hrs, 5 hrs, 4 hrs, 3 hrs, 2 hrs, 1 hrs, 45 min, 30 min, 20 min, or 15 min from the time the sample is obtained.
  • a blood sample can be combined with a reagent that selectively lyses one or more cells or components in the blood sample.
  • fetal nucleated cells can be selectively lysed releasing their nuclei when a blood sample comprising fetal nucleated cells is combined with deionized water.
  • Such selective lysis allows for the subsequent enrichment of fetal nuclei using, e.g., size or affinity based separation.
  • platelets and/or enucleated red blood cells are selectively lysed to generate a sample enriched in nucleated cells, such as fetal nucleated red blood cells (friRBC's) or maternal nucleated blood cells (mnBC).
  • fhRBC's can be subsequently separated from mnRBC's or maternal nucleated red blood cells (mnRBC) using, e.g., antigen-i affinity or differences in hemoglobin.
  • the amount of sample collected can vary depending upon mammal size, its gestation period, and the condition being screened. In some embodiments, up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mL of a sample is obtained. In some embodiments, 1-50, 2-40, 3-30, or 4-20 mL of sample is obtained. In some embodiments, more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mL of a sample is obtained.
  • a blood sample can be obtained from a pregnant mammal or human within 36, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6 or 4 weeks of gestation or even after a pregnancy has terminated.
  • a sample e.g. blood sample
  • enrichment occurs by selective lysis as described above.
  • nRBCs are enriched using one or more size-based separation modules.
  • Nucleated RBCs can be infrequent in number compared to other nucleated cells found in maternal peripheral blood.
  • enrichment of fetal trophoblasts occurs using one or more size-based separation modules.
  • size-based separation modules include filtration modules, sieves, matrixes, etc.
  • size-based separation modules contemplated by the present invention include those disclosed in International Publication No. WO 2004/113877, which is herein incorporated by reference in its entirety.
  • Other size based separation modules are disclosed in International Publication No. WO 2004/0144651, which is herein incorporated by reference in its entirety.
  • Yet other size based separation modules are disclosed in United States Publication No. US 2006-0223178 Al, which is herein incorporated by reference in its entirety.
  • a size-based separation module comprises one or more arrays of obstacles forming a network of gaps.
  • the obstacles are configured to direct particles (e.g. cells) as they flow through the array/network of gaps into different directions or outlets based on the particle's hydrodynamic size.
  • nucleated cells or cells having a hydrodynamic size larger than a predetermined size are directed to a first outlet located on the opposite side of the array of obstacles from the fluid flow inlet, while the enucleated cells or cells having a hydrodynamic size smaller than a predetermined size (e.g., 4, 5, 6, 7, 8, 9, or 10 microns) are directed to a second outlet also located on the opposite side of the array of obstacles from the fluid flow inlet.
  • a predetermined size e.g., 4, 5, 6, 7, 8, 9, or 10 microns
  • An array can be configured to separate cells smaller or larger than a predetermined size by adjusting the size of the gaps, obstacles, and offset in the period between each successive row of obstacles.
  • obstacles or gaps between obstacles can be up to 10, 20, 50, 70, 100, 120, 150, 170, or 200 microns in length or about 2, 4, 6, 8, 10, 20, 30 or 40 microns in length.
  • an array of obstacles for size-based separation includes more than 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, or 200,000 obstacles that are arranged into more than 10, 20, 50, 100, 200, 500, 1000 or 2000 rows.
  • Obstacles in a first row of obstacles can be offset from a previous (upstream) row of obstacles by up to 50% the period of the previous row of obstacles.
  • obstacles in a first row of obstacles are offset from a previous row of obstacles by up to 45, 40, 35, 30, 25, 20, 15 or 10% the period of the previous row of obstacles.
  • the distance between a first row of obstacles and a second row of obstacles can be up to 10, 20, 50, 70, 100, 120, 150, 170 or 200 microns.
  • a particular offset can be continuous (repeating for multiple rows) or non-continuous.
  • a separation module includes multiple discrete arrays of obstacles fluidly coupled such that they are in series with one another. Each array of obstacles can have a continuous offset.
  • Each subsequent (downstream) array of obstacles can have an offset that is different from the previous (upstream) offset.
  • each subsequent array of obstacles can have a smaller offset that the previous array of obstacles.
  • a plurality of arrays can be fiuidly coupled in series or in parallel, (e.g., more than 2, 4, 6, 8, 10, 20, 30, 40, 50).
  • Fiuidly coupling separation modules (e.g., arrays) in parallel allows for high-throughput analysis of the sample, such that at least 1, 2, 5, 10, 20, 50, 100, 200, or 500 mL per hour flows through the enrichment modules or at least 1, 5, 10, or 50 million cells per hour are sorted or flow through the device.
  • Figure IA illustrates an example of a size-based separation module.
  • Obstacles (which can be of any shape) are coupled to a flat substrate to form an array of gaps.
  • a transparent cover or lid can be used to cover the array.
  • the obstacles form a two-dimensional array with each successive row shifted horizontally with respect to the previous row of obstacles, where the array of obstacles directs component having a hydrodynamic size smaller than a predetermined size in a first direction and component having a hydrodynamic size larger that a predetermined size in a second direction.
  • the hydrodynamic size can be between 4-10 ⁇ m or between 6-8 ⁇ m.
  • the flow of sample into the array of obstacles can be aligned at a small angle (flow angle) with respect to a line-of-sight of the array.
  • the array is coupled to an infusion pump to perfuse the sample through the obstacles.
  • the flow conditions of the size-based separation module described herein are such that cells are sorted by the array with minimal damage. This allows for downstream analysis of intact cells and intact nuclei to be more efficient and reliable.
  • a size-based separation module comprising an array of obstacles can be configured to direct cells larger than a predetermined size to migrate along a line-of-sight within the array (e.g. towards a first outlet or bypass channel leading to a first outlet), while directing cells and analytes smaller than a predetermined size to migrate through the array of obstacles in a different direction than the larger cells (e.g. towards a second outlet).
  • nRBC's are directed to a first output while enucleated RBCs are directed to a second output.
  • gentle handling of the cells can reduce any mechanical damage to the cells or their DNA. This gentle handling can serve to preserve the small number of fetal cells or nucleated red blood cells in the sample.
  • integrity of the nucleic acid being evaluated is an important feature to permit the distinction between the genomic material from the fetal cells and other cells in the sample.
  • enrichment and separation of fetal cells using the arrays of obstacles produces gentle treatment which minimizes cellular damage and maximizes nucleic acid integrity permitting exceptional levels of separation and the ability to subsequently utilize various formats to very accurately analyze the genome of the cells which are present in the sample in extremely low numbers.
  • An enrichment device of the invention can comprise one or more size -based separation modules fluidically coupled upstream to one or more capture modules.
  • the capture modules can be configured to selectively enrich the nRBC's from other larger cells not comprising hemoglobin.
  • a capture module can selectively bind cells of interest such as nRBC's.
  • Capture modules can include a substrate having multiple obstacles that restrict the movement of cells or analytes greater than a predetermined size. Examples of capture modules that inhibit the migration of cells based on size are disclosed in U.S. Patent No. 5,837,115 and 6,692,952, which are herein incorporated by reference in their entirety.
  • a capture module captures analytes (e.g., analytes of interest or not of interest) based on their affinity.
  • an affinity-based separation module can include an array of obstacles with binding moieties attached, which selectively bind one or more analytes of interest (e.g., red blood cells, fetal cells, or nRBCs) or analytes not-of-interest (e.g., enucleated RBCS or white blood cells).
  • analytes of interest e.g., red blood cells, fetal cells, or nRBCs
  • analytes not-of-interest e.g., enucleated RBCS or white blood cells.
  • Arrays of obstacles adapted for separation by capture can include obstacles having one or more shapes and can be arranged in a uniform or non-uniform order.
  • a two-dimensional array of obstacles is staggered such that each subsequent row of obstacles is offset from the previous row of obstacles to mcrease the number of interactions between
  • Binding moieties coupled to the obstacles can include e.g., proteins (e.g., hgands/receptors), nucleic acids having complementary counterparts in retained analytes, antibodies, etc.
  • an affinity-based separation module comprises a two-dimensional array of obstacles covered with one or more antibodies selected from the group consisting of: anti-CD71, anti-CD45, anti CD-36, anti-GPA and anti- CD34
  • Figure 2A illustrates a path of a first analyte through an array of posts wherein an analyte that does not specifically bind to a post continues to migrate through the array, while an analyte that does bind a post is captured by the array.
  • Figure 2B is a picture of antibody coated posts.
  • Figure 2C illustrates coupling of antibodies to a substrate (e.g., obstacles, side walls, etc.) as contemplated by the present invention. Examples of such affinity-based separation modules are desc ⁇ bed in WO 2004/029221, which is herein incorporated by reference in its entirety.
  • a capture module utilizes a magnetic field to separate and/or enrich one or more analytes (cells) based on a magnetic property or magnetic potential in an analyte.
  • analytes cells
  • red blood cells which are slightly diamagnetic (repelled by magnetic field) in physiological conditions can be made paramagnetic (attributed by magnetic field) by deoxygenation of the hemoglobin into methemoglobin.
  • This magnetic property can be achieved through physical or chemical treatment of the red blood cells.
  • Cells containing hemoglobin can be enriched by treating them with a reagent to render the cells magnetically responsive.
  • These cells can then be enriched from a mixed population of cells (e.g., a raw blood sample or a size enriched sample) by flowing the sample through a magnetic field (e.g., uniform or non-uniform).
  • a mixed population of cells e.g., a raw blood sample or a size enriched sample
  • a magnetic field e.g., uniform or non-uniform
  • the reagent is sodium nitrite.
  • an enrichment device can have both one or more size based separation module(s) and one or more capture module(s) in series. This allows for a maternal blood sample to flow first through a size-based separation module to remove enucleated cells and cellular components (e.g., analytes having a hydrodynamic size less than 4, 5, or 6 ⁇ ms) based on size.
  • a size-based separation module to remove enucleated cells and cellular components (e.g., analytes having a hydrodynamic size less than 4, 5, or 6 ⁇ ms) based on size.
  • the size enriched larger cells e.g., analytes having a hydrodynamic size greater than 4, 5, or 6 ⁇ ms
  • white blood cells and nucleated red blood cells can be treated with a reagent, such as CO 2, N 2, Na 2 S 2 O 4 , or NaNO 2 , that alters a magnetic property of the red blood cells' hemoglobin.
  • a reagent such as CO 2, N 2, Na 2 S 2 O 4 , or NaNO 2 , that alters a magnetic property of the red blood cells' hemoglobin.
  • the treated sample can then flow through a micro channel, channel or a column coupled to an external magnet, or a column containing large magnetic obstacles.
  • Paramagnetic analytes e.g., nucleated red blood cells
  • This device enriches a sample for nRBCs (including mnRBC's and/or friRBC's). Additional examples of magnetic separation modules are described ni US 2006-0223178 and US 2007-0196820, which are herein incorporated by reference in their entirety.
  • Other means of rendering cells magnetic include by adsorption of magnetic cations.
  • Paramagnetic cations include, for example, Cr + 3, Co +2 , Mn +2 , Ni +2 , Fe +3 , Fe +2 , La +3 , Cu +2 , GD +3 , Ce +3 , Tb +3 , Pr +3 , Dy +3 , Nd +3 , Ho +3 , Pm +3 , Er +3 , Sm +3 , Tm +3 , Eu +3 , Yb +3 , and Lu +3 (U.S. Patent Application Publication No. 20060078502).
  • red blood cells can be rendered paramagnetic with chromium by contacting cells with an aqueous solution of chromate ions (Eisenberg et al. U.S. Patent No. 4,669,481).
  • Cells may be rendered magnetic by conjugating a magnetic agent to a targeting compound that binds to the cell surface.
  • Suitable targeting compounds include, for example, proteins, antibodies, hormones, and ligands.
  • cells may be rendered magnetic by coating magnetic nanoparticles with strepavidin or avidin; biotinylating the cells, and contacting the cells with the coated nanoparticles (WO/2000/071169).
  • Magnetic agents can also be treated to form magnetodendrimers by any means known in the art, for example the method of Bulte et al., (Magneto Dendrimers as a New Class of Cellular Contrast Agents. Pro. Internat. Soc).
  • Supermagnetic iron oxides which may be used in the current invention include (magneto) ferritins, (magneto) liposomes, (magneto) dendrimers, dysprosium, and gadolinium-or-iron- containing macromolecular chelates.
  • the superparamagnetic iron oxide can be magnetic iron oxide nanoparticles (MION), for example, MION-46L.
  • MION-46L is a dextran-coated magnetic nanoparticle with a superparamagnetic maghemite-or magnetite-like inverse spinel core structure.
  • a sample enriched by size-based separation followed by affinity/magnetic separation is further enriched using fluorescence activated cell sorting (FACS) or selective lysis of a subset of the enriched cells.
  • FACS fluorescence activated cell sorting
  • subsequent enrichment involves isolation of rare cells or rare DNA (e.g. fetal cells or fetal DNA) by selectively initiating apoptosis in the cells of interest.
  • This can be accomplished, for example, by subjecting a sample that includes rare cells (e.g. a mixed sample) to hyperbaric pressure (increased levels of CO 2 ; e.g. 4% CO 2 ).
  • This selectively initiates condensation and/or apoptosis in the rare or fragile cells in the sample (e.g. fetal cells).
  • the rare cells e.g. fetal cells
  • their nuclei will condense and optionally be ejected from the rare cells.
  • the rare cells or nuclei can be detected using any technique known in the art to detect condensed nuclei, including DNA gel electrophoresis, in situ labeling fluorescence labeling, and in situ labeling of DNA nicks using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick labeling (TUNEL) (Gavrieli, Y., et al. J. Cell Biol. 119:493- 501 (1992)), and ligation of DNA strand breaks having one or two-base 3' overhangs (Taq polymerase - based in situ ligation; Didenko V., et al. J. Cell Biol. 135:1369-76 (1996)).
  • TdT terminal deoxynucleotidyl transferase
  • TUNEL terminal deoxynucleotidyl transferase
  • TUNEL terminal deoxynucleotidyl transferase
  • ejected nuclei can be detected using a size based separation module adapted to selectively enrich nuclei and other analytes smaller than a predetermined size (e.g. 4, 5, or 6 microns) and isolate them from cells and analytes having a hydrodynamic diameter larger than a predetermined size (e.g. 4, 5, or 6 microns).
  • a predetermined size e.g. 4, 5, or 6 microns
  • the present invention contemplates detecting fetal cells/fetal DNA and optionally using such fetal DNA to diagnose or prognosticate a condition in a fetus.
  • detection and diagnosis can occur by obtaining a blood sample from a pregnant female, enriching the sample for cells and analytes larger than 8 microns using, for example, an array of obstacles adapted for size-base separation where the predetermined size of the separation is 8 microns (e.g. the gap between obstacles is up to 8 microns). Then, the enriched product can be further enriched for nRBCs by oxidizing the sample to make the hemoglobin paramagnetic and flowing the sample through one or more magnetic regions. This selectively captures the nRBCs and removes other cells (e.g. white blood cells) from the sample.
  • the enriched product can be further enriched for nRBCs by oxidizing the sample to make the hemoglobin paramagnetic and flowing the sample through one or more magnetic regions. This selectively captures the nRBCs and removes other cells (e.g. white blood cells) from the sample.
  • the fhRBC's can be enriched from mnRBC's in the second enriched product by subjecting the second enriched product to hyperbaric or hypobaric pressure or other stimulus that selectively causes the fetal cells to begin apoptosis and condense / eject their nuclei.
  • Condensed nuclei can then be identified/isolated using e.g. laser capture microdissection or a size based separation module that separates components smaller than 3, 4, 5 or 6 microns from a sample. Such fetal nuclei can then by analyzed using any method known in the art or described herein.
  • a magnetic particle e.g., a bead
  • compound e.g., Fe 3+
  • a bead coupled to an antibody that selectively binds to an analyte of interest can be decorated with one or more antibodies selected from the group of anti CD-71, anti CD-34, anti CD GPA, anti-CD45, anti-CD36.
  • a magnetic compound such as Fe 3+
  • the magnetic particles or magnetic antibodies herein can be coupled to any one or more of the devices herein prior to contact with a sample or can be mixed with the sample prior to delivery of the sample to the device(s). Magnetic particles can also be used to decorate one or more analytes (cells of interest or not of interest) to increase the size prior to performing size-based separation.
  • Magnetic field used to separate analytes/cells in any of the embodiments herein can uniform or nonuniform as well as external or internal to the device(s) herein.
  • An external magnetic field is one whose source is outside a device herein (e.g., container, channel, obstacles).
  • An internal magnetic field is one whose source is within a device contemplated herein.
  • An example of an internal magnetic field is one where magnetic particles can be attached to obstacles present in the device (or manipulated to create obstacles) to increase surface area for analytes to interact with to increase the likelihood of binding.
  • Analytes captured by a magnetic field can be released by demagnetizing the magnetic regions retaining the magnetic particles. For selective release of analytes from regions, the demagnetization can be limited to selected obstacles or regions.
  • the magnetic field can be designed to be electromagnetic, enabling turn-on and turn-off off the magnetic fields for each individual region or obstacle at will.
  • Figure 3 illustrates an embodiment of a device configured for capture and isolation of cells expressing the transferrin receptor from a complex mixture.
  • Monoclonal antibodies to CD71 receptor are readily available off-the-shelf and can be covalently coupled to magnetic materials, such as, but not limited to any ferroparticles including but not limited to ferrous doped polystyrene and ferroparticles or ferro-colloids (e.g., from Miltenyi and Dynal).
  • the anti CD71 bound to magnetic particles can then be flowed into the device.
  • the antibody coated particles are drawn to the obstacles (e.g., posts), floor, and walls and are retained by the strength of the magnetic field interaction between the particles and the magnetic field.
  • the particles between the obstacles and those loosely retained with the sphere of influence of the local magnetic fields away from the obstacles can be removed by a rinse with a buffer or wash fluid.
  • a fluid sample such as a blood sample is first flowed through one or more size-base separation module.
  • size-base separation modules can be fluidly connected in series and/or in parallel.
  • waste e.g., cells having hydrodynamic size less than 4 microns
  • the product e.g., cells having hydrodynamic size greater than 4 microns
  • Cells in the product can be subsequently enriched by rendering them magnetically responsive.
  • the product is modified (e.g., by addition of one or more reagents) such that the hemoglobin in the red blood cells becomes paramagnetic.
  • the product is exposed to magnetically responsive beads (e.g., ferrous beads) with cell specific binding moieties (e.g. antibodies). Subsequently, the product is flowed through one or more magnetic fields. The cells that are trapped by the magnetic field can then be analyzed using the one or more methods herein.
  • One or more of the enrichment modules herein can be fluidly coupled in series or in parallel with one another.
  • a first outlet from a separation module can be fluidly coupled to a capture module.
  • the separation module and capture module are integrated such that a plurality of obstacles acts both to deflect certain analytes according to size and direct them in a path different than the direction of analyte(s) of interest, and also as a capture module to capture, retain, or bind certain analytes based on size, affinity, magnetism or other physical property.
  • the enrichment steps performed can have a specificity and/or sensitivity greater than 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 99.95%
  • the retention rate of the enrichment module(s) herein is such that >50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9 % of the analytes or cells of interest (e.g., nucleated cells or nucleated red blood cells or nucleated from red blood cells) are retained.
  • the enrichment modules are configured to remove >50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9 % of all unwanted analytes (e.g., red blood-platelet enriched cells) from a sample.
  • unwanted analytes e.g., red blood-platelet enriched cells
  • the analytes of interest can be retained in an enriched solution that is less than 50, 40, 30, 20, 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5 fold diluted from the original sample.
  • any or all of the enrichment steps increase the concentration of the analyte of interest (e.g., nrBCs, mnRBCs, friRBCs, fetal cells or trophoblasts), for example, by transferring them from the fluid sample to an enriched fluid sample (sometimes in a new fluid medium, such as a buffer).
  • a method for determining the likelihood of the presence of a condition in a fetus, such as an abnormal condition.
  • the number of nRBCs in a sample from a pregnant female can be determined (such as by counting) and a likelihood of a fetal abnormal condition is determined based on the comparison between statistical averages of nRBCs from samples from pregnant females with normal fetuses with statistical averages of nRBCs from samples from pregnant females with fetuses with an abnormal condition.
  • the likelihood of the presence of an abnormality in a fetus can be calculated by determining the number of nRBCs in a sample from a mother of the fetus and comparing them to a pre-determined threshold number for nRBCs obtained from samples from mothers with known normal fetuses and/or with mothers with known abnormal fetuses.
  • the biologic fluids that can be sampled and compared include, but are not limited to, blood, amniotic, cervical, or vaginal fluids.
  • the fetal abnormal condition is a genetic abnormality such as an aberration in chromosome number, an error in DNA sequence, an error in methylation status or an error in chromosome imprinting.
  • a fetal abnormal condition can include aneuploidy, segmental aneuploidy, Alpha- 1-antitry ⁇ sin (AlA) deficiency, Achondroplasia, ⁇ -thalassemia, Bloom syndrome, Cystic Fibrosis (CF), Familial Dysautonomia (Riley Day syndrome), Familial Mediterranean Fever (FMF), Fibrodysplasia Ossificans Progressiva (FOP), Hutchinson-Gilford Progeria syndrome, Lesch-Nyhan Syndrome (LNS) & Variant (LNV), Multiple Sclerosis (MS), Polycystic kidney disease (PKD), Tay Sachs, Tuberous sclerosis, Wilson Disease, or Wolman disease.
  • AlA Alpha- 1-antitry ⁇ sin
  • Achondroplasia ⁇ -thalassemia
  • Bloom syndrome Cystic Fibrosis
  • FMF Familial Dysautonomia
  • FMF Familial Mediterranean Fever
  • FOP Fibrodysplasia Oss
  • a method for determining the likelihood of the presence of an aneuploidy or segmental aneuploidy in a fetus or assessing an increased risk of aneuploidy or segmental aneuploidy in a fetus.
  • the number of nRBCs in a sample from a mother of is determined (such as by counting) and a likelihood of a fetal aneuploidy is determined based on the comparison between statistical averages of nRBCs from samples from mothers with diploid fetuses compared with statistical averages of nRBCs from samples from mothers with aneuploid fetuses.
  • the likelihood of the presence of an aneuploidy in a fetus can be calculated by determining the number of nRBCs in a sample from a mother of the fetus and comparing them to predetermined threshold numbers for nRBCs obtained from samples from mothers with known diploid fetuses and with mothers with known aneuploid fetuses.
  • the biologic fluids that can be sampled and compared include blood, amniotic, cervical, or vaginal fluids.
  • Aneuploidy means the condition of having less than or more than the normal diploid number of chromosomes. In other words, it is any deviation from euploidy.
  • Aneuploidy includes conditions such as monosomy (the presence of only one chromosome of a pair in a cell's nucleus), trisomy (having three chromosomes of a particular type in a cell's nucleus), tetrasomy (having four chromosomes of a particular type in a cell's nucleus), pentasomy (having five chromosomes of a particular type in a cell's nucleus), triploidy (having three of every chromosome in a cell's nucleus), and tetraploidy (having four of every chromosome in a cell's nucleus).
  • segmental aneuploidy means having less than or more than the normal diploid number of chromosomal segments. Examples of segmental aneuploidy include, but are not limited to, Ip36 duplication, dup( 17)( ⁇ l 1.2p 11.2) syndrome, Pelizaeus-Merzbacher disease, du ⁇ (22)(ql 1.2ql 1.2) syndrome, and cat-eye syndrome.
  • An abnormal or aneuploid condition of a fetus or an increased risk for such a condition can be determined when the total number of nRBCs in the sample is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 70, 90, 100, 150, 200, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 nRBC / mL.
  • Sample volume useful in the disclosed methods can range from 10ml to 100 mL, such as 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 45 ml, 50 ml, 55 ml, 60 ml, 65 ml, 70 ml, 75 ml, 80 ml, 85 ml, 90 ml, 95 ml, or 100 ml. Samples can be obtained from a pregnant woman at first trimest
  • the presence of a maternal condition can be determined based on enumerating nucleated red blood cells from a sample.
  • Maternal conditions that can be determined include severe infection, hypoxia, preeclampsia, diabetes, solid tumors, acute and chronic hematological malignancies, leukemia, myeloproliferative syndromes (e.g. myelosclerosis and caricinomatosis), benign hematological conditions (e.g. hemolysis, hemorrhage, nutritional anaemia, infectious mononucleosis, myelodysplasia, Hb-SS, thalassaemia), septicemia, inflammatory bowel disease, chronic lung disease, fractures, myocardial infarction, and liver disease.
  • the biologic sample can be processed in order to enrich for nRBCs relative to enucleated cells prior to enumerating the number of nRBCs present in the sample.
  • nRBCs can be enriched by a variety of methods including, but not limited to, one or more of the following, performed at the same time or in sequence: enrichment based on cell size, affinity selection based on anti-CD45, anti-CD36, anti- GPA, anti-CD71 and anti-CD34 antibodies, or affinity selection based of nRBCs rendered magnetically responsive.
  • Size based separation can involve, for example, flowing a maternal sample mother through a microfluidic device that selectively directs cells and particles larger than a certain size to a first outlet and cells or particles smaller than a certain size to a second outlet.
  • This can enrich nucleated cells (e.g., nRBCs) relative to non-nucleated cells (such as enucleated red blood cells).
  • the nRBCs can also be enriched relative to nucleated cells (such as white blood cells).
  • this can be accomplished using, affinity selection, whereby white blood cells are selected using an antibody that selectively binds white blood cells relative to red blood cells.
  • this can also be accomplished using magnetic separation.
  • nRBCs can be rendered magnetically responsive by treating them with a reagent that alters a magnetic property of the cells, such as by altering the oxidation or reduction state of the hemoglobin in said cells.
  • the magnetic beads can be bound to nRBCs for affinity selection.
  • magnetic separation involves adding a reagent that alters the magnetic property of hemoglobin, e.g., sodium nitrite oxidation of hemoglobin to methamoglobin. This renders the hemoglobin containing red blood cells magnetically responsive.
  • a reagent that alters the magnetic property of hemoglobin, e.g., sodium nitrite oxidation of hemoglobin to methamoglobin.
  • the red blood cells can be separated from the white blood cells.
  • an affinity separation following a size-based separation can be used to enrich nucleated red blood cells from a sample.
  • the cells can be lysed in a way such that the nuclei of the cells remain intact. In some embodiments, lysing occurs prior to enumerating the number of nRBCs present.
  • the systems and methods herein can further be utilized for performing association studies.
  • the systems and methods herein are used to perform association studies based on data collected from a plurality of control samples and a plurality of case samples.
  • fluid samples e.g., blood samples
  • case individuals individuals with a phenotypic condition
  • control individuals such as a phenotypic condition
  • Samples from each individual can then be enriched for a first or a plurality of analytes (e.g., nRBCs, mnRBCs, fnRBCs or trophoblasts).
  • Such analytes can then be enumerated and/or characterized and data collected. This data can be subsequently be used to perform an association study.
  • Data can be stored in an electronic database.
  • the association study can be performed using a computer executable logic for identifying one or more characteristics associated with case or control samples. For example an association study between the number of nRBCs in a sample and a specific fetal abnormal condition can be used to develop a diagnostic or prognostic test.
  • the system is an analyzer system.
  • fluid samples obtained from individuals for an association study are blood samples.
  • the analytes (such as nucleated red blood cells) enriched from such samples can be ones that have a hydrodynamic size greater than 4 microns, or greater than 6, 8, 10, 12, 14, or 16 microns.
  • samples obtained from individuals are enriched for one or more cells selected from the group consisting of: a RBC, a fetal RBC, a trophoblast, a fetal fibroblast, a white blood cell (WBCs), an infected WBC, a stem cell, an epithelial cell, an endothelial cell, an endometrial cell, a progenitor cell.
  • the cells that are enriched are those that are found in vivo at a concentration of less than 1 x 10 '', 1 x 10 "2 , or 1 x 10 "3 cells/ ⁇ L.
  • the cells can be at least 99% of the cells of interest (those enriched) from the sample are retained. Enrichment for purposes of conducting an association study can increase the concentration of a first cell type of interest by at least 10,000 fold.
  • the enriched analytes can then be analyzed to determine one or more characteristics.
  • characteristics can include, e.g., the presence or absence of an analyte in a sample, quantity of an analyte, ratio of two analytes (e.g., endothelial cells and epithelial cells), morphology of one or more analytes, genotype of analyte, proteome of analyte, RNA composition of analyte, gene expression within an analyte, microRNA levels, or other characteristic traits of the analytes enriched are subsequently used to perform an association study.
  • an analyzer system can be configured to perform an analysis step such as detecting, enumerating, or analyzing analytes of interest, e.g., nucleated red blood cells (mnRBCs or fiiRBCs), trophoblasts or cell fragments (such as a nucleus or a chromosome).
  • An analyzer system comprises an analyzer and, optionally, at least one of a computer, a monitor and a command interface (e.g., a keyboard, mouse, trackball or joystick).
  • exemplary analyzers include, but are not limited to, a cell counter, a fluorescent activated cell sorting (FACS) machine, or a microscope.
  • the number of analytes of interest (such as mnRBCs or fiiRBCs) detected in a sample can be used by the analyzer or a user to determine a diagnosis or prognosis of a fetal condition such as an abnormal condition.
  • an analyzer system compares (and optionally stores) data collected with known data points.
  • an analyzer system compares (and optionally stores) data collected from case samples and control samples and performs an association study. For example an analyzer system can compare the statistical averages of nRBCs from samples from mothers with normal fetuses with statistical averages of nRBCs from samples from mothers with abnormal fetuses. This comparison can be used to determine a threshold value which can be used to determine a diagnosis or prognosis based on the results obtained for a subject of interest (e.g. a pregnant female)
  • an analyzer system comprises a computer executable logic that detects a probe signal from one or more probes that selectively bind an enriched analyte of interest, or components thereof.
  • the computer executable logic also analyzes such signals for their intensity, size, shape, aspect ratio, and/or distribution. The computer executable logic can then general a call based on results of analyzing the probe signals.
  • probes whose signals can be detected/analyzed by an analyzer include, but are not limited to, a fluorescent probe (e.g., for staining chromosomes such as X, Y, 13, 18 and 21 in fetal cells), a chromogenic probe, a direct immunoagent (e.g.
  • an analyzer can detect a chromogenic probe, which can provide a faster read time than a fluorescent probe.
  • an analyzer comprises a computer executable logic that performs karyotyping, in situ hybridization (ISH) (e.g., florescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), nanogold in situ hybridization (NISH)), restriction fragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR) techniques, flow cytometry, electron microscopy, quantum dot analysis, or detects single nucleotide polymorphisms (SNPs) or levels of RNA.
  • ISH in situ hybridization
  • FISH florescence in situ hybridization
  • CISH chromogenic in situ hybridization
  • NISH nanogold in situ hybridization
  • RFLP restriction fragment length polymorphism
  • PCR polymerase chain reaction
  • flow cytometry electron microscopy
  • quantum dot analysis or detects single nucleotide polymorphisms (SNPs) or levels of RNA.
  • two or more probes are used, which can emit different wavelengths.
  • Methods for using FISH to detect rare cells are disclosed in Zhen, D.K., et al. (1999) Prenatal Diagnosis, 18(11), 1181 - 1185, Cheung, MC, (1996) Nature Genetics 14, 264 - 268, which are incorporated herein by reference for all purposes.
  • Methods for using CISH are disclosed in Arnould, L. et al British Journal of Cancer (2003) 88, 1587-1591; and US 2002/0019001, which are incorporated herein by reference in their entirety.
  • an analyzer when analyzing nucleated red blood cells enriched from maternal blood, can be configured to detect nucleated red blood cells or components thereof.
  • analysis of fetal cells (such as fhPJBCs) or components thereof is used to determine the sex of a fetus; the presence/absence of a genetic abnormality (e.g., chromosomal, DNA or RNA abnormality); or one or more SNPs.
  • an analyzer uses flow cytometry to enumerate the number of cells (nucleated red blood cells, mnRBCs, fnRBCs or trophoblasts) enriched from a maternal blood sample.
  • Flow cytometry generally uses an apparatus that comprises a beam of light (usually laser light) of a single wavelength that is directed onto a hydro-dynamically focused stream of fluid.
  • a number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).
  • FSC Forward Scatter
  • SSC Segment Scatter
  • fluorescent detectors SSC
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle can be excited into emitting light at a lower frequency than the light source.
  • FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
  • nRBCs from the test sample can be imaged prior to, during or after enumeration.
  • FISH can be performed on the nucleated red blood cells prior to, during or after enumeration.
  • the results of the enumeration step of nRBCs or aneuploid nRBCs can be compared to threshold or predetermined values to determine if there is an increased likelihood of certain genetic characteristics of the fetus. For example, if the number of nRBCs in a peripheral blood sample from a pregnant female exceeds a threshold value then an increased likelihood of an abnormal fetal condition can be determined.
  • Fetal conditions in which the likelihood of occurrence can be calculated include abnormal conditions such as aneuploidy and segmental aneuploidy, for example: trisomy 8, trisomy 9, trisomy 12, trisomy 13, trisomy 18, trisomy 21, XXX, XXY, XYY, XXXY, XXYY, XYYY, XXXXX, XXXYY, XXYYY or XYYYYY.
  • Klinefelter Syndrome dup(17)(pl l.2pl 1.2) syndrome, Down syndrome, Pre-eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup(22)(ql 1.2ql 1.2) syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome, microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, Azospermia (factor a), Azosper
  • the fetal abnormal condition to be detected is due to one or more deletions in a sex or autosomal chromosome, for example: Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, Hereditary neuropathy with liability to pressure palsies, Smith- Magenis syndrome, Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome, Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, Azospermia (factor a), Azospermia (factor b), Azospermia (factor c) and Ip36 deletion.
  • the fetal abnormal condition is an abnormal decrease in chromosomal number, such
  • a condition in a fetus in a subject can be determined by enriching one or more nucleated red blood cells from a biologic sample obtained from said subject and determining a condition of the fetus based on the number of nucleated red blood cells isolated from the biologic sample.
  • Sources of biologic samples include blood, amniotic fluid, and cervical swabs.
  • a method for determining the likelihood or increased likelihood of the presence of an abnormal condition in a fetus by determining the number of nucleated red blood cells (nRBCs) as well as performing one or more maternal serum marker screens, and using the combined results to assign a likelihood of the fetus being abnormal. This can be based on statistical averages of nRBCs and corresponding maternal serum marker screening results from samples from mothers with abnormal fetuses.
  • nRBCs nucleated red blood cells
  • the likelihood of the presence of an abnormal condition in a fetus can be determined by enumerating the number of nRBCs and results from maternal serum marker screens in a sample from the mother of the fetus and comparing them to threshold number of nRBCs and maternal serum marker levels.
  • the biologic fluids that can be sampled and compared include blood, amniotic, cervical, or vaginal fluids.
  • a condition of a fetus in a subject can be determined by enriching one or more nucleated red blood cells from a biologic sample obtained from said subject and combining this with the detection of serum markers and/or using diagnostic ultrasound to measure space in nuchal fold of the fetus, and determining a condition of the fetus based on the number of nucleated red blood cells isolated and the presence and/or concentration of the serum markers.
  • Examples of maternal serum marker screening include but are not limited to alpha-fetoprotein (AFP), maternal serum alpha-fetoprotein (MSAFP), Double Marker Screen, Double Screen, Triple Marker Screen, Triple Screen, Quad Screen, Quad Marker Screen, Penta Screen, Penta Marker Screen, 1 st Trimester Screen, 2 nd Trimester Screen, Integrated Screen, Combined Screen, Contingency Screen, Repeated Measures Screen or Sequential Screen.
  • AFP alpha-fetoprotein
  • MSAFP maternal serum alpha-fetoprotein
  • Double Marker Screen Double Screen
  • Triple Screen Triple Marker Screen
  • Triple Screen Triple Screen
  • Quad Screen Quad Marker Screen
  • Penta Screen Penta Marker Screen
  • 1 st Trimester Screen 1 st Trimester Screen
  • 2 nd Trimester Screen Integrated Screen
  • Contingency Screen Contingency Screen
  • the specific serum markers include but are not limited to Pregnancy Associated Plasma Protein-A (papA), free ⁇ HCG, unconjugated estriol (UE3), alpha- fetoprotein (AFP), human Chorionic Gonadotropin (HCG), inhibin, D-inhibin A (DIA), and Invasive Trophoblast Antigen (ITA, or hhCG).
  • papA Pregnancy Associated Plasma Protein-A
  • UE3 unconjugated estriol
  • AFP alpha- fetoprotein
  • HCG human Chorionic Gonadotropin
  • HCG human Chorionic Gonadotropin
  • DIA D-inhibin A
  • ITA Invasive Trophoblast Antigen
  • the Penta Screen uses AFP, hCG, uE3, inhibin, and ITA as markers.
  • a Nuchal Translucency (NT) test is used in combination with enumeration of nRBCs and optionally detection of serum markers to determine a more accurate diagnosis of fetal abnormal condition (such as fetal aneuploidy).
  • the results are correlated with the Mother's age, for example with whether or not a human mother is under the age of 35.
  • the 1 st Trimester Screen includes the use of papA, free ⁇ HCG, ITA, and an NT test individually or in combination (the combination of papA, free ⁇ HCG, and NT is sometimes refered to as the Combined Screen).
  • the 2 nd Trimester Screen includes the use of AFP, hCG, uE3, DIA, and ITA individually or in combination.
  • the Integrated Screen includes the use of papA and NT in the 1 st trimester, and combines the results with a Quad Screen (AFP, hCG, uE3, inhibin) in the 2 nd trimester.
  • the Sequential Screen comprises a 1 st Trimester Screen followed by a Quad Screen plus papA and NT in the 2 nd trimester.
  • the Contingency Screen is a staged screen, including a 1 st Trimester Screen, followed if necessary by a Quad Screen.
  • the Sequential Screen includes the Integrated Screen followed by a 2 nd Trimester Screen.
  • the Repeated Measures Screen includes measuring a serum marker such as papA in the 1 st and 2 nd trimesters.
  • the conditions that can be diagnosed include aneuploidy and segmental aneuploidy, such as trisomy 8, trisomy 9, trisomy 12, trisomy 13, trisomy 18, trisomy 21, XXX, XXY, XYY, XXXY, XXYY, XYYY, XXXXX, XXXY, XXYYY, XYYYY, Klinefelter Syndrome, dup(17)(pl l.2pll.2) syndrome, Down syndrome, Pre-eclampsia, Pre-term labor, Edometriosis, Pelizaeus-Merzbacher disease, dup(22)(ql 1.2ql 1.2) syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis,
  • a business performs an association study to link the number of nucleated red blood cells in a biological sample with various conditions.
  • the condition is fetal abnormality or fetal aneuploidy.
  • the business can perform the assays necessary to enumerate nRBC's in the sample.
  • the business can provide a screen based on the enumerated nRBC's.
  • the business can provide a screen based on the combination of enumerated nRBC's and the results of a diagnostic ultrasound and/or serum marker tests.
  • Such serum marker tests can include alpha- fetoprotein (AFP), maternal serum alpha-fetoprotein (MSAFP), Double Marker Screen, Double Screen, Triple Marker Screen, Triple Screen, Quad Screen, Quad Marker Screen, Penta Screen, Penta Marker Screen, 1 st Trimester Screen, 2 nd Trimester Screen, Integrated Screen, Combined Screen, Contingency Screen, Repeated Measures Screen or Sequential Screen.
  • AFP alpha- fetoprotein
  • MSAFP maternal serum alpha-fetoprotein
  • Double Marker Screen Double Screen
  • Triple Marker Screen Triple Screen
  • Quad Screen Quad Marker Screen
  • Penta Screen Penta Marker Screen
  • Penta Marker Screen 1 st Trimester Screen
  • 2 nd Trimester Screen Integrated Screen
  • Contingency Screen Contingency Screen
  • Such serum markers could include Pregnancy Associated Plasma Protein-A (papA), free ⁇ HCG, and Invasive Trophoblast Antigen (ITA) for the 1 st trimester, and/or unconjugated estriol (UE3), alpha-fetoprotein (AFP), human Chorionic Gonadotropin (HCG), inhibin, and/or D-inhibin A (DIA) for the 2 nd trimester.
  • papA Pregnancy Associated Plasma Protein-A
  • ITA Invasive Trophoblast Antigen
  • UE3 unconjugated estriol
  • AFP alpha-fetoprotein
  • HCG human Chorionic Gonadotropin
  • DIA D-inhibin A
  • the clinical service provider conducts fetal testing in regional or localized free-standing facilities or alternately, on-site at hospitals or at physician offices.
  • the clinical service provider can be mobile and can be scheduled to perform the testing services on-site at pre- established times or on call.
  • the clinical service providers include CLIA certified
  • a confirmation step can also be included.
  • the confirmation step can confirm (i) the presence of fetal cells in the sample, and/or (ii) a fetal abnormal condition y.
  • a confirmation step can comprise performing one or more assay on the enriched nRBCs, for example: fluorescent in-situ hybridization (FISH), polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), nucleic acid analysis such as high-throughput sequencing, SNP detection, RNA expression analysis, or comparative genomic hybridization (CGH) array analysis.
  • FISH fluorescent in-situ hybridization
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • nucleic acid analysis such as high-throughput sequencing
  • SNP detection SNP detection
  • RNA expression analysis or comparative genomic hybridization (CGH) array analysis.
  • CGH comparative genomic hybridization
  • enriched product is binned into a microliter plate such that statistically each well has only 1 or 0 fetal cells.
  • qPCR can then be performed on individual wells to detect the presence of a Y chromosome (e.g., using a DYZ probe, SRY probe or any other probe specific for the Y chromosome).
  • FISH probes are applied to the enriched product to detect sex chromosomes X and Y.
  • Cells that are potential male fetus cells (express Y chromosome) are then microdissected and can be further analyzed using qPCR for the Y chromosome.
  • the enriched cells can flow through a FACS sorter and fetal cells can be identified using probes that are specific to fetal cells or fetal hemoglobin.
  • fetal specific probes include CD34, and antibodies to fetal globins such as epsilon and gamma. Confirmation can also be accomplished by binning the enriched cells and then determining the levels of expression (mRNA) of various globins such as epsilon, gamma, and beta globins in each well.
  • mRNA levels of expression
  • Binning may comprise distribution of enriched cells across wells in a plate (such as a 96 or 384 well plate), microencapsulation of cells in droplets that are separated in an emulsion, or by introduction of cells into microarrays of nanofluidic bins.
  • Fetal cells are then identified using methods that may comprise the use of biomarkers (such as fetal (gamma) hemoglobin), allele-specif ⁇ c SNP panels that could detect fetal genome DNA, detection of differentially expressed maternal and fetal transcripts (such as Affymetrix chips), or primers and probes directed to fetal specific loci (such as the multi-repeat DYZ locus on the Y- chromosome).
  • biomarkers such as fetal (gamma) hemoglobin
  • allele-specif ⁇ c SNP panels that could detect fetal genome DNA, detection of differentially expressed maternal and fetal transcripts (such as Affymetrix chips), or primers and probes directed to fetal specific loci (such
  • Binning sites that contain fetal cells are then be analyzed for aneuploidy and/or other genetic defects using a technique such as CGH array detection, ultra deep sequencing (such as Solexa, 454, or mass spectrometry), STR analysis, or SNP detection.
  • Enriched target cells may be "binned" prior to further analysis of the enriched cells.
  • Binning is any process which results in the reduction of complexity and/or total cell number of the enriched cell output.
  • Binning may be performed by any method known in the art or described herein.
  • One method of binning is by serial dilution. Such dilution may be carried out using any appropriate platform (e.g., PCR wells, microtiter plates) and appropriate buffers.
  • Other methods include nanofluidic systems which can separate samples into droplets (e.g., BioTrove, Raindance, Fluidigm). Such nanofluidic systems may result in the presence of a single cell present in a nanodroplet.
  • Binning may be preceded by positive selection for target cells including, but not limited to, affinity binding (e.g. using anti-CD71 antibodies). Alternately, negative selection of non-target cells may precede binning.
  • output from a size-based separation module may be passed through a magnetic hemoglobin enrichment module (MHEM) which selectively removes WBCs from the enriched sample by attracting magnetized hemoglobin-containing cells.
  • MHEM magnetic hemoglobin enrichment module
  • the possible cellular content of output from enriched maternal blood which has been passed through a size-based separation module may consist of: 1) approximately 20 fhRBC; 2) 1,500 mnRBC; 3) 4,000-40,000 WBC; 4) 15x10 6 RBC. If this sample is separated into 100 bins (PCR wells or other acceptable binning platform), each bin would be expected to contain: 1) 80 negative bins and 20 bins positive for one fhRBC; 2) 150 mnRBC; 3) 400-4,000 WBC; 4) 15xlO 4 RBC.
  • each bin would be expected to contain: 1) 9,980 negative bins and 20 bins positive for one fhRBC; 2) 8,500 negative bins and 1,500 bins positive for one mnRBC; 3) ⁇ l-4 WBC; 4) 15xlO 2 RBC.
  • the number of bins may be increased or decreased depending on experimental design and/or the platform used for binning. Reduced complexity of the binned cell populations may facilitate further genetic and/or cellular analysis of the target cells by reducing the number of non-target cells in an individual bin.
  • [00105J Analysis may be performed on individual bins to confirm the presence of target cells (e.g. nRBC, mnRBC or fnRBC) in the individual bin.
  • target cells e.g. nRBC, mnRBC or fnRBC
  • Such analysis may consist of any method known in the art including, but not limited to, FISH, PCR, STR detection, SNP analysis, biomarker detection, and sequence analysis.
  • a peripheral maternal venous blood sample enriched by the methods herein can be analyzed to determine pregnancy or a condition of a fetus (e.g., sex of fetus or aneuploidy).
  • the analysis step for fetal cells may further involve comparing the ratio of maternal to paternal genomic DNA on the identified fetal cells.
  • Any of the techniques herein can be used for prenatal as well as postnatal diagnosis as the fetal cells remain in circulation for a period of time after delivery of the fetus.
  • Figures 4A-4D shows a schematic of the device used to separate nucleated cells from maternal blood.
  • Dimensions 64 mm * 32 mm * lmm
  • Device fabrication The arrays and channels were fabricated in silicon using standard photolithography and deep silicon reactive etching techniques. The etch depth is 150 ⁇ m. Through holes for fluid access are made using KOH wet etching. The silicon substrate was anodically bonded on the etched face to form enclosed fluidic channels with a glass piece (9795, 3M, St Paul, MN).
  • Device packaging The device was mechanically mated to a plastic manifold with external fluidic reservoirs to deliver blood and buffer to the device and extract the generated fractions.
  • Device operation An external pressure source was used to apply a pressure of 1.0 PSI to the buffer and blood reservoirs to modulate fluidic delivery and extraction from the packaged device.
  • the buffer used consists of 1% BSA with 2mM EDTA in Dulbecco's Phosphate Buffer (iDPBS).
  • Figures 5A and 5B show a schematic of the magnetic separation module used to separate hemoglobin- containing cells from non-hemoglobin-containing cells. This process helps to further separate nucleated cells from maternal blood after enrichment using the process described in Example 1.
  • Figure 5C is a graph of the field strength of the magnet as a function of the position of the capillary.
  • Device operation Prior to device operation, the sample is centrifuged for 10 minutes at 300g. The sample is then treated with sodium nitrite at 50M for 10 min. The nucleated cells are then passed through the magnetic column (the magnetic separation module) where nucleated red blood cells are retained. In the column, the magnetic field strength is about 1 Tesla, the magnetic field gradient is about 3000 Tesla/m, and the flow velocity is about 0.4 mm/sec. White blood cells are rinsed out of the column using Dulbecco PBS buffer with 1% BSA and 2 mM EDTA, and collected as the negative fraction. The nucleated red blood cells are eluted from the column using the same buffer at a flow velocity of 4 mm/s and collected as the positive fraction.
  • An external pressure source was used to apply a pressure of 1.4 PSI to the buffer and sample reservoirs to modulate fluidic delivery and extraction from the packaged device.
  • Example 1 The device and process described in detail in Example 1 was used in combination with magnetic affinity enrichment techniques to isolate fetal cells from maternal blood.
  • the nucleated cell fraction was labeled with anti-CD71 microbeads (130-046-201, Miltenyi Biotech Inc., Auburn, CA) and enriched using the MiniMACSTM MS column (130-042-201, Miltenyi Biotech Inc., Auburn, CA) according to the manufacturer's specifications. Finally, the CD71-positive fraction was spotted onto glass slides.
  • Measurement techniques Spotted slides were stained using fluorescence in situ hybridization (FISH) techniques according to the manufacturer's specifications using Vysis probes (Abbott Laboratories, Downer's Grove, IL). Samples were stained from the presence of X and Y chromosomes. In one case, a sample prepared from a known Trisomy 21 pregnancy was also stained for chromosome 21.
  • FISH fluorescence in situ hybridization
  • Example 4 Confirmation of the presence of male fetal cells in enriched samples.
  • Confirmation of the presence of a male fetal cell in an enriched sample is performed using qPCR with primers specific for DYZ, a marker repeated in high copy number on the Y chromosome.
  • the resulting enriched fnRBC can be binned by dividing the sample into multiple, i.e. 100 PCR wells.
  • enriched samples Prior to binning, enriched samples can be screened by FISH to determine the presence of any fnRBC containing an aneuploidy of interest.
  • primer sets (DYZ forward primer TCGAGTGCATTCCATTCCG; DYZ reverse primer ATGGAATGGCATCAAACGGAA; and DYZ Taqman Probe 6FAM-TGGCTGTCCATTCCA-MGBNFQ), TaqMan Universal PCR master mix, No AmpErase and water are added.
  • the samples are run and analysis is performed on an ABI 7300: 2 minutes at 50 0 C, 10 minutes 95°C followed by 40 cycles of 95°C (15 seconds) and 60 0 C (1 minute). Following confirmation of the presence of male fetal cells, further analysis of bins containing fnRBC is performed. Positive bins can be pooled prior to further analysis.
  • Example 5 Clinical study of device and methodology in subjects with confirmed normal or aneuploidv fetuses
  • Figures 6 and 7 provide a summary of the results of a study performed at Sites B and C respectively, on the blood obtained from four women with normal fetuses and from five women with aneuploidy fetuses.
  • Column 1 lists the subject identification numbers.
  • Column 2 the total volume of blood obtain for the studies is listed.
  • Column 3 lists the total number of nRBCs obtained from the blood along with the number of nRBCs per ml, disclosed in the parenthesizes.
  • Column 4 lists the total Certitude number and Certitude number per ml of blood drawn.
  • the Certitude number for XX is disclosed along with the Certitude number for XX per ml of blood.
  • Column 6 lists the confirmed karyotype of the fetus.
  • column 7 the Certitude number corrected for the presence of XX cells is provided along with the corrected number per ml of blood.
  • Column 8 lists the mother's age, while in column 9 the gestational age is provided.
  • the date of the blood draw post the performance of the subject diagnosis is given in column 10.
  • Column 11 lists the interval in hours between the blood draw and the time the cells were plated.
  • Column 12 lists the temperature of the blood sample on arrival at the analysis facility.
  • Figure 8 lists the means and standard deviations for the results of the clinical studies for Sites B and C from Figures 6 and 7 for nRBCs enumration and FISH analysis.
  • nRBC enumeration blood from a total of 17 women was analyzed with 10 carrying normal fetuses and 7 with fetuses with an abnormal condition (ie, abnormal fetuses).
  • the mean number of nRBCs per ml for women with normal fetuses was 12.6, while women with abnormal fetuses the mean number was 22.9.
  • the standard deviations on these values were respectively 9.9 and 14.5.
  • the present invention contemplates measuring total nRBCs in a sample to provide information on a fetal abnormal condition. Due to high standard deviations and low sample size, further testing is required.
  • Example 6 Further clinical study of device and methodology in subjects with confirmed normal or aneuploidv fetuses.
  • Figure 9 lists the means and standard deviations for results of clinical studies for nRBC enumeration.
  • the mean and standard deviation (SD) of number of nRBCs for women with normal fetuses, a gestational age of less than 15 weeks, and a maternal age of less than 35 was 7.7 and 8.5 respectively.
  • the mean and SD for women with normal fetuses, a gestational age of 15 or more weeks, and a maternal age of less than 35 was 18.8 and 11.3 respectively.
  • the mean and SD for women with abnormal fetuses, a gestational age of 15 or more weeks, and a maternal age of less than 35 was 29.5 and 26.9 respectively.
  • the mean and SD for women with normal fetuses, a gestational age of less than 15 weeks, and a maternal age of 35 or more was 13.3 and 21.3 respectively.
  • the mean and SD for women with abnormal fetuses, a gestational age of less than 15 weeks, and a maternal age of 35 or more was 27.9 and 30.0 respectively.
  • the mean and SD for women with normal fetuses, a gestational age of 15 or more weeks, and a maternal age of 35 or more was 23.8 and 22.0 respectively.
  • the mean and SD for women with abnormal fetuses, a gestational age of 15 or more weeks, and a maternal age of 35 or more was 28.3 and 22.9 respectively. There were no samples in the abnormal fetus, a gestational age less than 15, and maternal age of less than 35 category.
  • Example 7 Simulation study using the FaSTER trial data set to demonstrate possible higher sensitivity and specificity generated from combining nRBC enumeration with maternal serum marker screen results.
  • the FaSTER (First and Second Trimester Evaluation of Risk) trial data set is a large, national, multicenter study in which numerous woman around the United States were tested using first and second-trimester screening methods for the prenatal detection of Down syndrome (Am J Obstet Gynecol. 2004 Oct;191(4): 1446-51).
  • a simulation is designed to compare maternal age, serum markers and nRBC alone and in combination as risk predictors for Down Syndrome.
  • Cases are selected from the FaSTER data set, and nRBCs values are assigned to normals and trisomy 21 cases assuming normal distributions and using means and standard deviations estimated from data shown in Figure 1OA.
  • the data that is used is limited to the subset of subjects with adequate serum screen data.
  • Figure 1OB shows the sensitivities at a 5% false positive fraction in women ⁇ 35 years of age and the sensitivities at a 5% and 15% false positive fraction in women 35 years and older. Note that among older women, the false positive fractions are shown to be higher than those observed among younger women.
  • nRBC can substantially improve the sensitivity of tests for women under age 35, for a fixed false positive rate of 5%.
  • the addition of nRBC improves sensitivity from 23% to 54% for a test based on maternal age alone.
  • the addition of nRBC improves sensitivity from 71% to 79% for a test based on maternal age and first trimester (IT) serum markers. Based on the results of the initial Artemis study, there appears to be an opportunity to improve on the performance of currently used screening tests.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de diagnostic d'une affection chez un foeus par enrichissement et numération des hématies en circulation, éventuellement en association avec les résultats des criblages des marqueurs sériques maternels.
PCT/US2008/069879 2007-07-11 2008-07-11 Diagnostic d'anomalies fœtales au moyen d'hématies nucléées Ceased WO2009009769A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94922707P 2007-07-11 2007-07-11
US60/949,227 2007-07-11

Publications (2)

Publication Number Publication Date
WO2009009769A2 true WO2009009769A2 (fr) 2009-01-15
WO2009009769A3 WO2009009769A3 (fr) 2009-04-09

Family

ID=40229506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/069879 Ceased WO2009009769A2 (fr) 2007-07-11 2008-07-11 Diagnostic d'anomalies fœtales au moyen d'hématies nucléées

Country Status (1)

Country Link
WO (1) WO2009009769A2 (fr)

Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010121294A1 (fr) * 2009-04-21 2010-10-28 Genetic Technologies Limited Procédés d'obtention de matériel génétique foetal
CN103235133A (zh) * 2013-04-28 2013-08-07 成都中医药大学 一种胎儿9号染色体异常疾病的筛查试剂盒
CN103278633A (zh) * 2013-04-28 2013-09-04 成都中医药大学 一种孕妇流产史筛查试剂盒
US8949036B2 (en) 2010-05-18 2015-02-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9228234B2 (en) 2009-09-30 2016-01-05 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9424392B2 (en) 2005-11-26 2016-08-23 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9499870B2 (en) 2013-09-27 2016-11-22 Natera, Inc. Cell free DNA diagnostic testing standards
US9639657B2 (en) 2008-08-04 2017-05-02 Natera, Inc. Methods for allele calling and ploidy calling
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10017812B2 (en) 2010-05-18 2018-07-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US10316362B2 (en) 2010-05-18 2019-06-11 Natera, Inc. Methods for simultaneous amplification of target loci
CN110093247A (zh) * 2019-05-07 2019-08-06 西安交通大学 一种富集捕获不同规格尺寸靶标细胞的微流控芯片
CN110471426A (zh) * 2019-09-02 2019-11-19 哈尔滨工程大学 基于量子狼群算法的无人驾驶智能车自动避碰方法
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
US10894976B2 (en) 2017-02-21 2021-01-19 Natera, Inc. Compositions, methods, and kits for isolating nucleic acids
US20210198733A1 (en) 2018-07-03 2021-07-01 Natera, Inc. Methods for detection of donor-derived cell-free dna
US11052392B2 (en) 2002-09-27 2021-07-06 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11378498B2 (en) 2006-06-14 2022-07-05 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US11408031B2 (en) 2010-05-18 2022-08-09 Natera, Inc. Methods for non-invasive prenatal paternity testing
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11781187B2 (en) 2006-06-14 2023-10-10 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US12024738B2 (en) 2018-04-14 2024-07-02 Natera, Inc. Methods for cancer detection and monitoring
US12084720B2 (en) 2017-12-14 2024-09-10 Natera, Inc. Assessing graft suitability for transplantation
US12100478B2 (en) 2012-08-17 2024-09-24 Natera, Inc. Method for non-invasive prenatal testing using parental mosaicism data
US12146195B2 (en) 2016-04-15 2024-11-19 Natera, Inc. Methods for lung cancer detection
US12152275B2 (en) 2010-05-18 2024-11-26 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US12203946B2 (en) 2014-05-15 2025-01-21 Kellbenx Incorporated Preparation of fetal nucleated red blood cells (NRBCs) for diagnostic testing
US12202890B1 (en) 2014-11-07 2025-01-21 Kellbenx Incorporated Antibodies for detection and enrichment of fetal cells and their use
US12221653B2 (en) 2010-05-18 2025-02-11 Natera, Inc. Methods for simultaneous amplification of target loci
US12260934B2 (en) 2014-06-05 2025-03-25 Natera, Inc. Systems and methods for detection of aneuploidy
US12305235B2 (en) 2019-06-06 2025-05-20 Natera, Inc. Methods for detecting immune cell DNA and monitoring immune system
US12398389B2 (en) 2018-02-15 2025-08-26 Natera, Inc. Methods for isolating nucleic acids with size selection
US12460264B2 (en) 2016-11-02 2025-11-04 Natera, Inc. Method of detecting tumour recurrence
US12486542B2 (en) 2024-06-04 2025-12-02 Natera, Inc. Detecting mutations and ploidy in chromosomal segments

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070059716A1 (en) * 2005-09-15 2007-03-15 Ulysses Balis Methods for detecting fetal abnormality
US20070059719A1 (en) * 2005-09-15 2007-03-15 Michael Grisham Business methods for prenatal Diagnosis

Cited By (109)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11052392B2 (en) 2002-09-27 2021-07-06 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US12065703B2 (en) 2005-07-29 2024-08-20 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10260096B2 (en) 2005-07-29 2019-04-16 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US10392664B2 (en) 2005-07-29 2019-08-27 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10266893B2 (en) 2005-07-29 2019-04-23 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10227652B2 (en) 2005-07-29 2019-03-12 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10711309B2 (en) 2005-11-26 2020-07-14 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11306359B2 (en) 2005-11-26 2022-04-19 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9695477B2 (en) 2005-11-26 2017-07-04 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9430611B2 (en) 2005-11-26 2016-08-30 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10240202B2 (en) 2005-11-26 2019-03-26 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9424392B2 (en) 2005-11-26 2016-08-23 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10597724B2 (en) 2005-11-26 2020-03-24 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11378498B2 (en) 2006-06-14 2022-07-05 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US11781187B2 (en) 2006-06-14 2023-10-10 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US9639657B2 (en) 2008-08-04 2017-05-02 Natera, Inc. Methods for allele calling and ploidy calling
US9447467B2 (en) 2009-04-21 2016-09-20 Genetic Technologies Limited Methods for obtaining fetal genetic material
WO2010121294A1 (fr) * 2009-04-21 2010-10-28 Genetic Technologies Limited Procédés d'obtention de matériel génétique foetal
US10216896B2 (en) 2009-09-30 2019-02-26 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10522242B2 (en) 2009-09-30 2019-12-31 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10061890B2 (en) 2009-09-30 2018-08-28 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10061889B2 (en) 2009-09-30 2018-08-28 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9228234B2 (en) 2009-09-30 2016-01-05 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11525162B2 (en) 2010-05-18 2022-12-13 Natera, Inc. Methods for simultaneous amplification of target loci
US8949036B2 (en) 2010-05-18 2015-02-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US12410476B2 (en) 2010-05-18 2025-09-09 Natera, Inc. Methods for simultaneous amplification of target loci
US12270073B2 (en) 2010-05-18 2025-04-08 Natera, Inc. Methods for preparing a biological sample obtained from an individual for use in a genetic testing assay
US12221653B2 (en) 2010-05-18 2025-02-11 Natera, Inc. Methods for simultaneous amplification of target loci
US11111545B2 (en) 2010-05-18 2021-09-07 Natera, Inc. Methods for simultaneous amplification of target loci
US10174369B2 (en) 2010-05-18 2019-01-08 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US12152275B2 (en) 2010-05-18 2024-11-26 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10538814B2 (en) 2010-05-18 2020-01-21 Natera, Inc. Methods for simultaneous amplification of target loci
US10557172B2 (en) 2010-05-18 2020-02-11 Natera, Inc. Methods for simultaneous amplification of target loci
US12110552B2 (en) 2010-05-18 2024-10-08 Natera, Inc. Methods for simultaneous amplification of target loci
US10316362B2 (en) 2010-05-18 2019-06-11 Natera, Inc. Methods for simultaneous amplification of target loci
US10590482B2 (en) 2010-05-18 2020-03-17 Natera, Inc. Amplification of cell-free DNA using nested PCR
US12020778B2 (en) 2010-05-18 2024-06-25 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US10597723B2 (en) 2010-05-18 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
US9334541B2 (en) 2010-05-18 2016-05-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10017812B2 (en) 2010-05-18 2018-07-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10731220B2 (en) 2010-05-18 2020-08-04 Natera, Inc. Methods for simultaneous amplification of target loci
US10774380B2 (en) 2010-05-18 2020-09-15 Natera, Inc. Methods for multiplex PCR amplification of target loci in a nucleic acid sample
US10793912B2 (en) 2010-05-18 2020-10-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11746376B2 (en) 2010-05-18 2023-09-05 Natera, Inc. Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
US11519035B2 (en) 2010-05-18 2022-12-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11482300B2 (en) 2010-05-18 2022-10-25 Natera, Inc. Methods for preparing a DNA fraction from a biological sample for analyzing genotypes of cell-free DNA
US10655180B2 (en) 2010-05-18 2020-05-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11408031B2 (en) 2010-05-18 2022-08-09 Natera, Inc. Methods for non-invasive prenatal paternity testing
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11286530B2 (en) 2010-05-18 2022-03-29 Natera, Inc. Methods for simultaneous amplification of target loci
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11312996B2 (en) 2010-05-18 2022-04-26 Natera, Inc. Methods for simultaneous amplification of target loci
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US12100478B2 (en) 2012-08-17 2024-09-24 Natera, Inc. Method for non-invasive prenatal testing using parental mosaicism data
CN103235133A (zh) * 2013-04-28 2013-08-07 成都中医药大学 一种胎儿9号染色体异常疾病的筛查试剂盒
CN103278633A (zh) * 2013-04-28 2013-09-04 成都中医药大学 一种孕妇流产史筛查试剂盒
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
US9499870B2 (en) 2013-09-27 2016-11-22 Natera, Inc. Cell free DNA diagnostic testing standards
US11371100B2 (en) 2014-04-21 2022-06-28 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11319596B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11390916B2 (en) 2014-04-21 2022-07-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11408037B2 (en) 2014-04-21 2022-08-09 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US12305229B2 (en) 2014-04-21 2025-05-20 Natera, Inc. Methods for simultaneous amplification of target loci
US11414709B2 (en) 2014-04-21 2022-08-16 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US12203142B2 (en) 2014-04-21 2025-01-21 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11486008B2 (en) 2014-04-21 2022-11-01 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10351906B2 (en) 2014-04-21 2019-07-16 Natera, Inc. Methods for simultaneous amplification of target loci
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11530454B2 (en) 2014-04-21 2022-12-20 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10597709B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US10597708B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplifications of target loci
US12203946B2 (en) 2014-05-15 2025-01-21 Kellbenx Incorporated Preparation of fetal nucleated red blood cells (NRBCs) for diagnostic testing
US12260934B2 (en) 2014-06-05 2025-03-25 Natera, Inc. Systems and methods for detection of aneuploidy
US12202890B1 (en) 2014-11-07 2025-01-21 Kellbenx Incorporated Antibodies for detection and enrichment of fetal cells and their use
US11946101B2 (en) 2015-05-11 2024-04-02 Natera, Inc. Methods and compositions for determining ploidy
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US12146195B2 (en) 2016-04-15 2024-11-19 Natera, Inc. Methods for lung cancer detection
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US12460264B2 (en) 2016-11-02 2025-11-04 Natera, Inc. Method of detecting tumour recurrence
US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10577650B2 (en) 2016-12-07 2020-03-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10533219B2 (en) 2016-12-07 2020-01-14 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11530442B2 (en) 2016-12-07 2022-12-20 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10894976B2 (en) 2017-02-21 2021-01-19 Natera, Inc. Compositions, methods, and kits for isolating nucleic acids
US12084720B2 (en) 2017-12-14 2024-09-10 Natera, Inc. Assessing graft suitability for transplantation
US12398389B2 (en) 2018-02-15 2025-08-26 Natera, Inc. Methods for isolating nucleic acids with size selection
US12024738B2 (en) 2018-04-14 2024-07-02 Natera, Inc. Methods for cancer detection and monitoring
US12385096B2 (en) 2018-04-14 2025-08-12 Natera, Inc. Methods for cancer detection and monitoring
US12234509B2 (en) 2018-07-03 2025-02-25 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US20210198733A1 (en) 2018-07-03 2021-07-01 Natera, Inc. Methods for detection of donor-derived cell-free dna
CN110093247A (zh) * 2019-05-07 2019-08-06 西安交通大学 一种富集捕获不同规格尺寸靶标细胞的微流控芯片
CN110093247B (zh) * 2019-05-07 2020-11-17 西安交通大学 一种富集捕获不同规格尺寸靶标细胞的微流控芯片
US12305235B2 (en) 2019-06-06 2025-05-20 Natera, Inc. Methods for detecting immune cell DNA and monitoring immune system
CN110471426A (zh) * 2019-09-02 2019-11-19 哈尔滨工程大学 基于量子狼群算法的无人驾驶智能车自动避碰方法
US12486542B2 (en) 2024-06-04 2025-12-02 Natera, Inc. Detecting mutations and ploidy in chromosomal segments

Also Published As

Publication number Publication date
WO2009009769A3 (fr) 2009-04-09

Similar Documents

Publication Publication Date Title
WO2009009769A2 (fr) Diagnostic d'anomalies fœtales au moyen d'hématies nucléées
US20230168160A1 (en) Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US20080026390A1 (en) Diagnosis of Fetal Abnormalities by Comparative Genomic Hybridization Analysis
Huang et al. A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women
US20090181421A1 (en) Diagnosis of fetal abnormalities using nucleated red blood cells
EP2029778B1 (fr) Diagnostic d'anomalies foetales
US20060223178A1 (en) Devices and methods for magnetic enrichment of cells and other particles
CN101305087A (zh) 用于磁富集细胞和其他颗粒的装置和方法
US20100273675A1 (en) Methods for detecting fetal abnormality
US20080090239A1 (en) Rare cell analysis using sample splitting and dna tags
US20070059774A1 (en) Kits for Prenatal Testing
US20080113358A1 (en) Selection of cells using biomarkers
US20070059683A1 (en) Veterinary diagnostic system
US20070059718A1 (en) Systems and methods for enrichment of analytes
US20070059719A1 (en) Business methods for prenatal Diagnosis
US20070059781A1 (en) System for size based separation and analysis
US20090081689A1 (en) Reagents and methods to enrich rare cells from body fluids
JP2009509143A (ja) 分析改善システムおよび方法
CN110114474A (zh) 获得胎儿细胞核酸的方法
KR101794218B1 (ko) 비침습적 산전 진단을 위한 산모 혈액 내 유핵 적혈구의 농축 분리 방법
Zhang et al. Quantitative abnormalities of fetal trophoblast cells in maternal circulation in preeclampsia
KR20220044517A (ko) 태아 세포의 단리, 검출 및 분석을 위한 조성물 및 방법
HK40002659A (en) Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08781738

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08781738

Country of ref document: EP

Kind code of ref document: A2