Patent Grant
10591391
Analysis of specific cells can give insight into a variety of diseases. These analyses can provide non-invasive tests for detection, diagnosis and prognosis of diseases, thereby eliminating the risk of invasive diagnosis. For instance, social developments have resulted in an increased number of prenatal tests. However, the available methods today, amniocentesis and chorionic villus sampling (CVS) are potentially harmful to the mother and to the fetus. The rate of miscarriage for pregnant women undergoing amniocentesis is increased by 0.5-1%, and that figure is slightly higher for CVS. Because of the inherent risks posed by amniocentesis and CVS, these procedures are offered primarily to older women, i.e., those over 35 years of age, who have a statistically greater probability of bearing children with congenital defects. As a result, a pregnant woman at the age of 35 has to balance an average risk of 0.5-1% to induce an abortion by amniocentesis against an age related probability for trisomy 21 of less than 0.3%.
To eliminate the risks associated with invasive prenatal screening procedures, non-invasive tests for detection, diagnosis and prognosis of diseases, have been utilized. For example, maternal serum alpha-fetoprotein, and levels of unconjugated estriol and human chorionic gonadotropin are used to identify a proportion of fetuses with Down's syndrome, however, these tests are not one hundred percent accurate. Similarly, ultrasonography is used to determine congenital defects involving neural tube defects and limb abnormalities, but is useful only after fifteen weeks' gestation.
The methods of the present invention allow for the detection of fetal cells and fetal abnormalities when fetal cells are present in a mixed population of cells, even when maternal cells dominate the mixture.
The presence of fetal cells in maternal circulation offers the opportunity to develop a prenatal diagnostic that obviates the risk associated with today's invasive diagnostics procedures. However, fetal cells are rare as compared to the presence of maternal cells in the blood. Therefore, any proposed analysis of fetal cells to diagnose fetal abnormalities requires enrichment of fetal cells. Enriching fetal cells from maternal peripheral blood is challenging, time intensive and any analysis derived therefrom is prone to error. The present invention addresses these challenges.
The present invention relates to methods for determining the presence of fetal cells and fetal abnormalities when fetal cells are present in a mixed sample (e.g. maternal blood sample). In some embodiments, determining the presence of fetal cells or of a fetal abnormality includes comparing the level of genomic DNA from a mixed sample to the level of genomic DNA in a control sample. The control or reference sample can be a mixed sample that has been sufficiently diluted to be free of fetal cells. The mixed sample can contain at least one fetal cell and one non-fetal cell. In other embodiments, the sample comprises up to 50% fetal cells.
In some embodiments, determining the presence of fetal cells and/or abnormalities involves quantifying one or more regions of genomic DNA regions from the mixed sample and determining from the quantification the presence of a fetal abnormality. Preferably, such regions are polymorphic e.g. short tandem repeat (STR) regions.
Examples of fetal abnormalities that can be determined by quantifying regions on one or more chromosomes include trisomy 13, trisomy 18, trisomy 21 (Down Syndrome), Klinefelter Syndrome (XXY) and other irregular number of sex or autosomal chromosomes. Other examples of abnormal fetal genotypes that can be determined by quantifying regions on one or more chromosomes include, but are not limited to, aneuploidy such as, monosomy of one or more chromosomes (X chromosome monosomy, also known as Turner's syndrome), trisomy of one or more chromosomes (such as 13, 18, 21, and X), tetrasomy and pentasomy of one or more chromosomes (which in humans is most commonly observed in the sex chromosomes, e.g. XXXX, XXYY, XXXY, XYYY, XXXXX, XXXXY, XXXYY, XYYYY and XXYYY), triploidy (three of every chromosome, e.g. 69 chromosomes in humans), tetraploidy (four of every chromosome, e.g. 92 chromosomes in humans) and multiploidy. In some embodiments, an abnormal fetal genotype is a segmental aneuploidy. Examples of segmental aneuploidy include, but are not limited to, 1p36 duplication, dup(17)(p11.2p11.2) syndrome, Down syndrome, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, and cat-eye syndrome. In some cases, an abnormal fetal genotype is due to one or more deletions of sex or autosomal chromosomes, which may result in a condition such as Cri-du-chat syndrome, Wolf-Hirschhorn, 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), or 1p36 deletion. In some embodiments, a decrease in chromosomal number results in an XO syndrome.
Furthermore, the methods herein can distinguish maternal trisomy from paternal trisomy, and total aneuploidy from segmental aneuploidy. Segmental aneuploidies can be caused by an intra-chromosomal event such as a deletion, duplication or translocation event. Additionally, the methods herein can be used to identify monoploidy, triploidy, tetraploidy, pentaploidy and other higher multiples of the normal haploid state. In some embodiments, the maternal or paternal origin of the fetal abnormality can be determined.
The genomic DNA region(s) can be quantified by amplifying the regions using, for example, PCR, or preferably quantitative PCR. Alternatively, quantification of the regions can be achieved using capillary gel electrophoresis (CGE). In some embodiments, total genomic DNA is pre-amplified prior to the quantitative amplification step to increase the overall abundance of DNA. Such pre-amplification step can involve the use of multiple displacement amplification.
In some embodiments the genomic DNA regions quantified can be in one chromosome or in 2 or more chromosomes. The polymorphic regions can be quantified on either or both sex chromosomes X and Y, and on autosomal chromosomes including chromosomes 13, 18 and 21.
Prior to analysis a mixed sample suspected of having fetal cells (e.g. a maternal blood sample) can be enriched for fetal cells. Fetal cell enrichment can be accomplished using any method known in the art including size-based separation, affinity (e.g. magnetic) separation, FACS, laser microdisection, and magnetic bead separation. A mixed sample containing as few as 10 fetal cells can be enriched. In some embodiments, the fetal cells in the enriched sample constitute less than 50% of the total number of cells.
In some embodiments, the size-based separation method includes applying a mixed sample into a system that separates a first component of the mixed sample (e.g. fetal cells), which comprises cells that are larger than a critical size, in a first direction, and a second component of the mixed sample (e.g. enucleated maternal red blood cells), which comprises cells that are smaller than a critical size, towards a second exit port. The separation system can be a device that includes one or more arrays of obstacles that form a network of gaps.
In some embodiments, enrichment that is achieved by size-based separation is followed by one or more additional enrichment procedures including magnetic separation, fluorescence activated cell sorting (FACS), laser microdisection, and magnetic bead separation. In some embodiments, a sample enriched by size-based separation is subjected to affinity/magnetic separation and is further enriched for rare cells using fluorescence activated cell sorting (FACS) or selective lysis of a subset of the cells (e.g. fetal cells).
In some embodiments there are provided kits for detecting the fetal abnormalities wherein the kits include separation devices and the reagents needed to perform the genetic analysis. For example, the kit may include arrays for size based enrichment, a device for magnetic enrichment and reagents for performing PCR.
The methods can further comprise inputting the data from the quantification step into data model(s) for the association of DNA quantity with maternal and non-maternal alleles. The invention provides for a computer program product, which includes a computer executable logic recorded on a computer readable medium that can be used for diagnosing a fetal abnormality. The computer program is designed to receive data from one of more quantified DNA genomic regions from a mixed sample containing at least one fetal cell, determine the presence or absence of a fetal abnormality from the data, and generate an output that comprises the evaluation of the fetal abnormality. Methods for using the computer program product are also disclosed.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The present invention provides systems, apparatuses, and methods to detect the presence and condition (e.g. aneuploidy) of fetal cells in a mixed cell population, e.g. a sample wherein fetal cells consist of <50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0.5% of all cells in a mixed sample.
In step 100, a sample containing (or suspected of containing) 1 or more fetal cells is obtained. Samples can be obtained from an animal suspected of being pregnant, pregnant, or that has been pregnant to detect the presence of a fetus or fetal abnormality. Such animal can be a human or a domesticated animal such as a cow, chicken, pig, horse, rabbit, dog, cat, or goat. Samples derived from an animal or human can include, e.g., whole blood, sweat, tears, ear flow, sputum, lymph, bone marrow suspension, lymph, urine, saliva, semen, vaginal flow, cerebrospinal fluid, brain fluid, ascites, milk, secretions of the respiratory, intestinal or genitourinary tracts fluid.
To obtain a blood sample, any technique known in the art may be used, e.g. a syringe or other vacuum suction device. A blood sample can be optionally pre-treated or processed prior to enrichment. 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.
When a blood sample is obtained, a preservative such an anti-coagulation agent and/or a stabilizer can be added to the sample prior to enrichment. This allows for extended time for analysis/detection. Thus, a sample, such as a blood sample, can be enriched and/or analyzed under any of the methods and systems herein within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr from the time the sample is obtained.
In some embodiments, a blood sample can be combined with an agent that selectively lyses one or more cells or components in a blood sample. For example, fetal cells can be selectively lysed releasing their nuclei when a blood sample including fetal 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. In another example, 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 (fnRBC) and maternal nucleated blood cells (mnBC). The fnRBC's can subsequently be separated from the mnBC's using, e.g., affinity to antigen-i or magnetism differences in fetal and adult hemoglobin.
When obtaining a sample from an animal (e.g., blood sample), the amount can vary depending upon animal size, its gestation period, and/or 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.
To detect fetal abnormality, a blood sample can be obtained from a pregnant animal or human within 36, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6 or 4 weeks of gestation.
In step 101, a reference sample is obtained. The reference sample consists of substantially all or all maternal cells. In some embodiments, a reference sample is a maternal blood sample enriched for white blood cells (WBC's) such that it consists of substantially all or all maternal WBC's. In some embodiments, a reference sample is a diluted mixed sample wherein the dilution results in a sample free of fetal cells. For example, a maternal blood sample of 10-50 ML can be diluted by at least 2, 5, 10, 20, 50, or 100 fold to reduce the likelihood that it will include fetal cells.
In step 102, when the sample to be tested or analyzed is a mixed sample (e.g. maternal blood sample), it is enriched for rare cells or rare DNA (e.g. fetal cells, fetal DNA or fetal nuclei) using one or more methods known in the art or disclosed herein. Such enrichment increases the ratio of fetal cells to non-fetal cells; the concentration of fetal DNA to non-fetal DNA; or the concentration of fetal cells in volume per total volume of the mixed sample.
In some embodiments, enrichment occurs by selective lysis as described above. For example, enucleated cells may be selectively lysed prior to subsequent enrichment steps or fetal nucleated cells may be selectively lysed prior to separation of the fetal nuclei from other cells and components in the sample.
In some embodiments, enrichment of fetal cells or fetal nuclei occurs using one or more size-based separation modules. Size-based separation modules include filtration modules, sieves, matrixes, etc., including those disclosed in International Publication Nos. WO 2004/113877, WO 2004/0144651, and US Application Publication No. 2004/011956.
In some embodiments, a size-based separation module includes one or more arrays of obstacles that form a network of gaps. The obstacles are configured to direct particles (e.g. cells or nuclei) as they flow through the array/network of gaps into different directions or outlets based on the particle's hydrodynamic size. For example, as a blood sample flows through an array of obstacles, nucleated cells or cells having a hydrodynamic size larger than a critical size, e.g., 8 microns, 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 critical size, e.g., 8 microns, are directed to a second outlet also located on the opposite side of the array of obstacles from the fluid flow inlet.
An array can be configured to separate cells smaller than a critical size from those larger than the critical size by adjusting the size of the gaps, obstacles, and offset in the period between each successive row of obstacles. For example, in some embodiments, obstacles and/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 or 10 microns in length. In some embodiments, an array for size-based separation includes more than 100, 500, 1,000, 5,000, 10,000, 50,000 or 100,000 obstacles that are arranged into more than 10, 20, 50, 100, 200, 500, or 1000 rows. Preferably, obstacles in a first row of obstacles are offset from a previous (upstream) row of obstacles by up to 50% of the period of the previous row of obstacles. In some embodiments, 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. Furthermore, 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. In some embodiments, a separation module includes multiple discrete arrays of obstacles fluidly coupled such that they are in series with one another. Each array of obstacles has a continuous offset. But each subsequent (downstream) array of obstacles has an offset that is different from the previous (upstream) offset. Preferably, each subsequent array of obstacles has a smaller offset that the previous array of obstacles. This allows for a refinement in the separation process as cells migrate through the array of obstacles. Thus, a plurality of arrays can be fluidly coupled in series or in parallel, (e.g., more than 2, 4, 6, 8, 10, 20, 30, 40, 50). Fluidly 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.
In one embodiment, a size-based separation module comprises an array of obstacles configured to direct rare cells larger than a critical size to migrate along a line-of-sight within the array towards a first outlet or bypass channel leading to a first outlet, while directing cells and analytes smaller than a critical size through the array of obstacles in a different direction towards a second outlet.
A variety of enrichment protocols may be utilized although gentle handling of the cells is preferred to reduce any mechanical damage to the cells or their DNA. This gentle handling also preserves the small number of fetal cells in the sample. Integrity of the nucleic acid being evaluated is an important feature in some embodiments to permit the distinction between the genomic material from the fetal cells and other cells in the sample. In particular, the enrichment and separation of the 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.
In some embodiments, enrichment of fetal cells occurs using one or more capture modules that selectively inhibit the mobility of one or more cells of interest. Preferably a capture module is fluidly coupled downstream to a size-based separation module. Capture modules can include a substrate having multiple obstacles that restrict the movement of cells or analytes greater than a critical size. Examples of capture modules that inhibit the migration of cells based on size are disclosed in U.S. Pat. Nos. 5,837,115 and 6,692,952.
In some embodiments, a capture module includes a two dimensional array of obstacles that selectively filters or captures cells or analytes having a hydrodynamic size greater than a particular gap size, e.g., critical sized. 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. In some embodiments, a two-dimensional array of obstacles is staggered such that each subsequent row of obstacles is offset from the previous row of obstacles to increase the number of interactions between the analytes being sorted (separated) and the obstacles.
Another example of a capture module is an affinity-based separation module. An affinity-based separation module capture analytes or cells of interest based on their affinity to a structure or particle as oppose to their size. One example of an affinity-based separation module is an array of obstacles that are adapted for complete sample flow through, but for the fact that the obstacles are covered with binding moieties that selectively bind one or more analytes (e.g., cell population) of interest (e.g., red blood cells, fetal cells, or nucleated cells) or analytes not-of-interest (e.g., white blood cells). Binding moieties can include e.g., proteins (e.g., ligands/receptors), nucleic acids having complementary counterparts in retained analytes, antibodies, etc. In some embodiments, 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-CD235a, anti-CD36, anti-carbohydrates, anti-selectin, anti-CD45, anti-GPA, and anti-antigen-i.
In some embodiments, a capture module utilizes a magnetic field to separate and/or enrich one or more analytes (cells) that has a magnetic property or magnetic potential. For example, 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. Thus, a sample containing one or more red blood cells and one or more non-red blood cells can be enriched for the red blood cells by first inducing a magnetic property and then separating the above red blood cells from other analytes using a magnetic field (uniform or non-uniform). For example, a maternal blood sample can flow first through a size-based separation module to remove enucleated cells and cellular components (e.g., analytes having a hydrodynamic size less than 6 μms) based on size. Subsequently, the enriched nucleated cells (e.g., analytes having a hydrodynamic size greater than 6 μms) white blood cells and nucleated red blood cells are treated with a reagent, such as CO2, N2 or NaNO2, that changes the magnetic property of the red blood cells' hemoglobin. The treated sample then flows through a magnetic field (e.g., a column coupled to an external magnet), such that the paramagnetic analytes (e.g., red blood cells) will be captured by the magnetic field while the white blood cells and any other non-red blood cells will flow through the device to result in a sample enriched in nucleated red blood cells (including fnRBC's). Additional examples of magnetic separation modules are described in U.S. application Ser. No. 11/323,971, filed Dec. 29, 2005 entitled “Devices and Methods for Magnetic Enrichment of Cells and Other Particles” and U.S. application Ser. No. 11/227,904, filed Sep. 15, 2005, entitled “Devices and Methods for Enrichment and Alteration of Cells and Other Particles”.
Subsequent enrichment steps can be used to separate the rare cells (e.g. fnRBC's) from the non-rare maternal nucleated red blood cells (non-RBC's). In some embodiments, a sample enriched by size-based separation followed by affinity/magnetic separation is further enriched for rare cells using fluorescence activated cell sorting (FACS) or selective lysis of a subset of the cells (e.g. fetal cells). In some embodiments, fetal cells are selectively bound to an anti-antigen i binding moiety (e.g. an antibody) to separate them from the mnRBC's. In some embodiments, the antibody binds to a fetal cell ligand. In some related embodiments the fetal cells are stimulated so as to induce expression of ligands which are targeted by an antibody. In some embodiments the fetal cells are lysed and the nuclei of the fetal cells are separated from other cellular components by binding them with an antobody. In some embodiments, fetal cells are selectively bound to receptors which target fetal cell ligands. In some embodiments, fetal cells are selectively bound to a lectin. In some embodiments, fetal cells or fetal DNA is distinguished from non-fetal cells or non-fetal DNA by forcing the rare cells (fetal cells) to become apoptotic, thus condensing their nuclei and optionally ejecting their nuclei. Rare cells such as fetal cells can be forced into apoptosis using various means including subjecting the cells to hyperbaric pressure (e.g. 4% CO2). The condensed nuclei can be detected and/or isolated for further analysis using any technique known in the art including DNA gel electrophoresis, in situ labeling of DNA nicks (terminal deoxynucleotidyl transferase (TdT))-mediated dUTP in situ nick labeling (also known as 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)).
In some embodiments, when the analyte desired to be separated (e.g., red blood cells or white blood cells) is not ferromagnetic or does not have a magnetic property, a magnetic particle (e.g., a bead) or compound (e.g., Fe3+) can be coupled to the analyte to give it a magnetic property. In some embodiments, a bead coupled to an antibody that selectively binds to an analyte of interest can be decorated with an antibody elected from the group of anti CD71 or CD75. In some embodiments a magnetic compound, such as Fe3+, can be couple to an antibody such as those described above. The magnetic particles or magnetic antibodies herein may be coupled to any one or more of the devices herein prior to contact with a sample or may be mixed with the sample prior to delivery of the sample to the device(s).
Magnetic field used to separate analytes/cells in any of the embodiments herein can uniform or non-uniform 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 may 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. For example, 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.
One or more of the enrichment modules herein (e.g., size-based separation module(s) and capture module(s)) may be fluidly coupled in series or in parallel with one another. For example a first outlet from a separation module can be fluidly coupled to a capture module. In some embodiments, 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.
In any of the embodiments herein, the enrichment steps performed have a specificity and/or sensitivity ≥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. Simultaneously, 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.
Any or all of the enrichment steps can occur with minimal dilution of the sample. For example, in some embodiments the analytes of interest are 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. In some embodiments, any or all of the enrichment steps increase the concentration of the analyte of interest (fetal cell), for example, by transferring them from the fluid sample to an enriched fluid sample (sometimes in a new fluid medium, such as a buffer). The new concentration of the analyte of interest may be at least 2, 4, 6, 8, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 50,000,000, 100,000,000, 200,000,000, 500,000,000, 1,000,000,000, 2,000,000,000, or 5,000,000,000 fold more concentrated than in the original sample. For example, a 10 times concentration increase of a first cell type out of a blood sample means that the ratio of first cell type/all cells in a sample is 10 times greater after the sample was applied to the apparatus herein. Such concentration can take a fluid sample (e.g., a blood sample) of greater than 10, 15, 20, 50, or 100 mL total volume comprising rare components of interest, and it can concentrate such rare component of interest into a concentrated solution of less than 0.5, 1, 2, 3, 5, or 10 mL total volume.
The final concentration of fetal cells in relation to non-fetal cells after enrichment can be about 1/10,000-1/10, or 1/1,000-1/100. In some embodiments, the concentration of fetal cells to maternal cells may be up to 1/1,000, 1/100, or 1/10 or as low as 1/100, 1/1,000 or 1/10,000.
Thus, detection and analysis of the fetal cells can occur even if the non-fetal (e.g. maternal) cells are >50%, 60%, 70%, 80%, 90%, 95%, or 99% of all cells in a sample. In some embodiments, fetal cells are at a concentration of less than 1:2, 1:4, 1:10, 1:50, 1:100, 1:1000, 1:10,000, 1:100,000, 1,000,000, 1:10,000,000 or 1:100,000,000 of all cells in a mixed sample to be analyzed or at a concentration of less than 1×10−3, 1×10−4, 1×10−5, 1×10−6, or 1×10−6 cells/μL of the mixed sample. Over all, the number of fetal cells in a mixed sample, (e.g. enriched sample) has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 total fetal cells.
Enriched target cells (e.g., fnRBC) may be “binned” prior to further analysis of the enriched cells (
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. For example, 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.
For example, the possible cellular content of output from enriched maternal blood which has been passed through a size-based separation module (with or without further enrichment by passing the enriched sample through a MHEM) may consist of: 1) approximately 20 fnRBC; 2) 1,500 nmRBC; 3) 4,000-40,000 WBC; 4) 15×106 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 fnRBC; 2) 150 nmRBC; 3) 400-4,000 WBC; 4) 15×104 RBC. If separated into 10,000 bins, each bin would be expected to contain: 1) 9,980 negative bins and 20 bins positive for one fnRBC; 2) 8,500 negative bins and 1,500 bins positive for one mnRBC; 3) <1-4 WBC; 4) 15×102 RBC. One of skill in the art will recognize that 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.
Analysis may be performed on individual bins to confirm the presence of target cells (e.g. fnRBC) in the individual bin. 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 (
Fetal Biomarkers
In some embodiments fetal biomarkers may be used to detect and/or isolate fetal cells, after enrichment or after detection of fetal abnormality or lack thereof. For example, this may be performed by distinguishing between fetal and maternal nRBCs based on relative expression of a gene (e.g., DYS1, DYZ, CD-71, ε- and ξ-globin) that is differentially expressed during fetal development. In preferred embodiments, biomarker genes are differentially expressed in the first and/or second trimester. “Differentially expressed,” as applied to nucleotide sequences or polypeptide sequences in a cell or cell nuclei, refers to differences in over/under-expression of that sequence when compared to the level of expression of the same sequence in another sample, a control or a reference sample. In some embodiments, expression differences can be temporal and/or cell-specific. For example, for cell-specific expression of biomarkers, differential expression of one or more biomarkers in the cell(s) of interest can be higher or lower relative to background cell populations. Detection of such difference in expression of the biomarker may indicate the presence of a rare cell (e.g., fnRBC) versus other cells in a mixed sample (e.g., background cell populations). In other embodiments, a ratio of two or more such biomarkers that are differentially expressed can be measured and used to detect rare cells.
In one embodiment, fetal biomarkers comprise differentially expressed hemoglobins. Erythroblasts (nRBCs) are very abundant in the early fetal circulation, virtually absent in normal adult blood and by having a short finite lifespan, there is no risk of obtaining fnRBC which may persist from a previous pregnancy. Furthermore, unlike trophoblast cells, fetal erythroblasts are not prone to mosaic characteristics.
Yolk sac erythroblasts synthesize ε-, ξ-, γ- and α-globins, these combine to form the embryonic hemoglobins. Between six and eight weeks, the primary site of erythropoiesis shifts from the yolk sac to the liver, the three embryonic hemoglobins are replaced by fetal hemoglobin (HbF) as the predominant oxygen transport system, and ε- and ξ-globin production gives way to γ-, α- and β-globin production within definitive erythrocytes (Peschle et al., 1985). HbF remains the principal hemoglobin until birth, when the second globin switch occurs and β-globin production accelerates.
Hemoglobin (Hb) is a heterodimer composed of two identical α globin chains and two copies of a second globin. Due to differential gene expression during fetal development, the composition of the second chain changes from ε globin during early embryonic development (1 to 4 weeks of gestation) to γ globin during fetal development (6 to 8 weeks of gestation) to β globin in neonates and adults as illustrated in (Table 1).
In the late-first trimester, the earliest time that fetal cells may be sampled by CVS, fnRBCs contain, in addition to a globin, primarily ε and γ globin. In the early to mid second trimester, when amniocentesis is typically performed, fnRBCs contain primarily γ globin with some adult β globin. Maternal cells contain almost exclusively α and β globin, with traces of γ detectable in some samples. Therefore, by measuring the relative expression of the ε, γ and β genes in RBCs purified from maternal blood samples, the presence of fetal cells in the sample can be determined. Furthermore, positive controls can be utilized to assess failure of the FISH analysis itself.
In various embodiments, fetal cells are distinguished from maternal cells based on the differential expression of hemoglobins β, γ or ε. Expression levels or RNA levels can be determined in the cytoplasm or in the nucleus of cells. Thus in some embodiments, the methods herein involve determining levels of messenger RNA (mRNA), ribosomal RNA (rRNA), or nuclear RNA (nRNA).
In some embodiments, identification of fnRBCs can be achieved by measuring the levels of at least two hemoglobins in the cytoplasm or nucleus of a cell. In various embodiments, identification and assay is from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fetal nuclei. Furthermore, total nuclei arrayed on one or more slides can number from about 100, 200, 300, 400, 500, 700, 800, 5000, 10,000, 100,000, 1,000,000, 2,000,000 to about 3,000,000. In some embodiments, a ratio for γ/β or ε/β is used to determine the presence of fetal cells, where a number less than one indicates that a fnRBC(s) is not present. In some embodiments, the relative expression of γ/β or ε/β provides a fnRBC index (“FNI”), as measured by γ or ε relative to β. In some embodiments, a FNI for γ/β greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 90, 180, 360, 720, 975, 1020, 1024, 1250 to about 1250, indicate that a fnRBC(s) is present. In yet other embodiments, a FNI for γ/β of less than about 1 indicates that a fnRBC(s) is not present. Preferably, the above FNI is determined from a sample obtained during a first trimester. However, similar ratios can be used during second trimester and third trimester.
In some embodiments, the expression levels are determined by measuring nuclear RNA transcripts including, nascent or unprocessed transcripts. In another embodiment, expression levels are determined by measuring mRNA, including ribosomal RNA. There are many methods known in the art for imaging (e.g., measuring) nucleic acids or RNA including, but not limited to, using expression arrays from Affymetrix, Inc. or Illumina, Inc.
RT-PCR primers can be designed by targeting the globin variable regions, selecting the amplicon size, and adjusting the primers annealing temperature to achieve equal PCR amplification efficiency. Thus TaqMan probes can be designed for each of the amplicons with well-separated fluorescent dyes, Alexa fluor®-355 for ε, Alexa Fluor®-488 for γ, and Alexa Fluor-555 for β. The specificity of these primers can be first verified using ε, γ, and β cDNA as templates. The primer sets that give the best specificity can be selected for further assay development. As an alternative, the primers can be selected from two exons spanning an intron sequence to amplify only the mRNA to eliminate the genomic DNA contamination.
The primers selected can be tested first in a duplex format to verify their specificity, limit of detection, and amplification efficiency using target cDNA templates. The best combinations of primers can be further tested in a triplex format for its amplification efficiency, detection dynamic range, and limit of detection.
Various commercially available reagents are available for RT-PCR, such as One-step RT-PCR reagents, including Qiagen One-Step RT-PCR Kit and Applied Biosytems TaqMan One-Step RT-PCR Master Mix Reagents kit. Such reagents can be used to establish the expression ratio of ε, γ, and β using purified RNA from enriched samples. Forward primers can be labeled for each of the targets, using Alexa fluor-355 for ε, Alexa fluor-488 for γ, and Alexa fluor-555 for β. Enriched cells can be deposited by cytospinning onto glass slides. Additionally, cytospinning the enriched cells can be performed after in situ RT-PCR. Thereafter, the presence of the fluorescent-labeled amplicons can be visualized by fluorescence microscopy. The reverse transcription time and PCR cycles can be optimized to maximize the amplicon signal:background ratio to have maximal separation of fetal over maternal signature. Preferably, signal:background ratio is greater than 5, 10, 50 or 100 and the overall cell loss during the process is less than 50, 10 or 5%.
Fetal Cell Analysis
The detection and analysis steps may involve quantifying genomic DNA regions from cells in a sample or enriched sample. In some embodiments, the quantified genomic DNA regions are polymorphic sites such as short tandem repeats (STRs) or variable number of tandem repeats (VNTRs).
In step 103, polymorphic genomic DNA region(s) or whole genome(s) from the mixed sample and optionally reference sample are pre-amplified to increase the overall abundance of DNA used for quantification and analysis. Pre-amplification can be preformed using multiple displacement amplification (MDA) (Gonzalez et al. Envircon Microbiol; 7(7); 1024-8 (2005)) or amplification with outer primers in a nested PCR approach. This permits detection and analysis of fetal DNA even if the total amount of fetal DNA in the mixed (e.g. enriched) sample is only up to 1 μg, 500 ng, 200 ng, 100 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, 1 ng, 500 pg, 200 pg, 100 pg, 50 pg, 40 pg, 30 pg, 20 p, 10 pg, 5 pg, or 1 pg or between 1-5 μg, 5-10 μg, or 10-50 μg. Pre-amplification allows the products to be split into multiple reactions at the next step.
In step 104, polymorphic DNA region(s) such as short tandem repeats (STRs) or variable number of tandem repeats (VNTRs) are selected on suspected trisomic chromosome(s) (e.g., 13, 18, 21, X or Y) or chromosome(s) associated with a condition to be detected and optionally on control (non-trisomic) chromosomes. In some embodiments, 1 or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 DNA polymorphic loci are selected per target chromosome. Multiple polymorphic regions can be analyzed independently or at the same time in the same reaction. The polymorphic DNA regions, e.g. STRs loci, are selected for high heterozygosity (variety of alleles) so that the paternal allele of the fetal cells is more likely to be distinct in length from the maternal alleles. This results in an improved power to detect the presence of fetal cells in the mixed sample and potential fetal abnormalities in such cells. When the polymorphic regions selected are STR loci, di-, tri-, tetra- or penta-nucleotide repeat loci can be used for detection and analysis of fetal cells. Examples of STR loci that may be selected include: D21S1414, D21S1411, D21S1412, D21S11 MBP, D13S634, D13S631, D18S535, AmgXY, XHPRT, as well as those listed in
In step 105, the polymorphic loci selected are amplified. This can be used to detect non-maternal fetal alleles in the mixed sample and to determine the copy number of such alleles. When amplifying more than one polymorphic loci or DNA regions, primers are selected to be multiplexable (fairly uniform melting temperature, absence of cross-priming on the human genome, and absence of primer-primer interaction based on sequence analysis) with other primer pairs. Primers and loci are chosen so that the amplicon lengths from a given locus do not overlap with those from another locus.
In some embodiments, multiple dyes and multi-color fluorescence readout may be used to increase the multiplexing capacity, e.g. of a single CGE. This ensures that the loci are kept distinct in the readout (e.g. CGE readout). In such a case, PCR primer pairs can be grouped and the same end-labeling is applied to the members of a group.
Examples of primers known in the art that correspond to specific STR loci that can be used in the present invention are described in
Examples of PCR techniques that can be used to amplify the DNA regions herein include, but are not limited, to quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real time PCR(RT-PCR), single cell PCR, restriction fragment length polymorphism PCR(PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, in situ polonony PCR, in situ rolling circle amplification (RCA), bridge PCR, picotiter PCR and emulsion PCR. Other suitable amplification methods include the ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) and nucleic acid based sequence amplification (NABSA). Other amplification methods that may be used to amplify specific polymorphic loci include those described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and 6,582,938.
In step 106, the amplified DNA polymorphic regions (e.g. STR loci) from both mixed and reference samples are characterized and quantified using any method known in the art. Examples of such methods include, but are not limited to, gas chromatography, supercritical fluid chromatography, liquid chromatography, including partition chromatography, adsorption chromatography, ion exchange chromatography, size-exclusion chromatography, thin-layer chromatography, and affinity chromatography, electrophoresis, including capillary electrophoresis, capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic capillary chromatography, isotachophoresis, transient isotachophoresis and capillary gel electrophoresis, comparative genomic hybridization (CGH), microarrays, bead arrays, high-throughput genotyping technology, such as molecular inversion probe (MIP), and Genescan.
In one embodiment, capillary gel electrophoresis (CGE) is used to quantify STRs in both the mixed and reference samples. This can be used to detect non-maternal fetal alleles in the mixed sample and to determine the copy number of such alleles. The mixed sample and the reference sample can be analyzed in separate reactions, e.g. separate CGE lanes. Alternatively, the mixed and the reference sample can be run in the same reaction, e.g. same CGE lane, by using two different dye labels, e.g. differently labeled PCR primers. When a reference sample is run through the PCR/CGE process, the alleles show up as peaks in the CGE. It is desirable, but not essential, to associate these peaks with known alleles in the population at each locus. When performing PCR/CGE it may be very useful to reduce the non-linearities in the response of PCR to input DNA copies (i.e. to effect more quantitative PCR) so that the data can be more easily related to models of aneuploidy. This ‘linearization’ can be accomplished by the following procedure:
The above procedure tends to accomplish quantitative PCR while enabling a high degree of multiplexing. Because each CGE run has a slightly different relation between DNA fragment size (and sequence) and mobility, each trace typically will need to undergo a length transformation, such as a low-order (cubic or quartic) polynomial transformation, in order to map to the data from the trace corresponding to the previous amplification point. This mapping can be determined by adjusting the transformation parameters to achieve the best fit of the one data trace to the other, with both normalized to the same total sum of squares or summed peak heights.
The maternal peaks at each locus provide an estimate of the secondary ‘stutter’ structure at each locus due to PCR errors. The locations of these small secondary peaks can be used to blank out length regions that are contaminated by this stutter when looking for and using the non-maternal allele peaks (as described herein for example). Alternatively, more sophisticated ‘deconvolution’ algorithms can be applied to remove the stutter Stoughton, et al., Electrophoresis; 18(1): 1-S (1997).
The sample containing an unknown mixture of fetal and maternal cells is analyzed as in Step (b). This could be done in a separate CGE lane, or in the same CGE lane as the maternal sample by using two different dye labels on the PCR primers. Because each CGE trace has a slightly different relation between DNA fragment size (and sequence) and mobility, these data typically will need to undergo a length transformation, such as a low-order (cubic or quartic) polynomial transformation in order to map one trace onto the other to facilitate peak identification and model fitting. This mapping can be determined by adjusting the transformation parameters to achieve the best fit of the peak locations in one data trace to the other. This mapping will be well determined in the assumed situation where the maternal cells are more numerous than the fetal cells, because the maternal signature will dominate and will be shared in the two data sets.
In step 107, data models are constructed. From the data obtained from the quantifying step different data models can be constructed depending upon different assumptions.
For example, a data model for the CGE patterns in
Let m1 denote the CGE signal obtained from one of the maternal alleles at a given locus and m2 the signal obtained from the other maternal allele, which might be the same allele. Let p denote the CGE signal obtained from the paternal allele at a given locus. Let p1 and p2 denote the CGE signals obtained from the paternal alleles at a given locus when a paternally derived trisomy occurs. Let□ α and β denote the relative number of maternal and fetal cells, respectively. Then in the case of a chromosome with maternal non-dysjunction trisomy, the data will have the form
x=α(□m1+m2)+β(m1+m2+p). (1)
A normal (diploid) chromosome will give
x=α(m1+m2)+β([m1 or m2]+p), (2)
and a paternally derived trisomy will give
x=α(m1+m2)+β([m1 or m2]+p1+p2). (3)
In some embodiments, data and data model is represented as discrete peak masses (or heights) and peak locations or as vectors of values representing the actual peak profiles. In the case of representation by peak characteristics, the ‘addition’ operation in Equations 1-3 denotes summation of peak height or mass at the discrete allele location. In the case where the full peak profiles are represented, summation denotes summation of signals bin by bin over the CGE trace, and in this case it may be helpful to zero the data except in the immediate vicinity of actual peaks. Representation via peak characteristics is preferable when using the PCR linearization technique described above.
To determine aneuploidy, the differences between the structure of the β-term that appears in the first and second equations above is determined. In the first case, there is an additional contribution to both maternal alleles along with the paternal allele, and in the second case there is an additional contribution only to one of the maternal alleles along with the paternal allele. The essence of the presence/absence declaration for fetal cells lies in the evidence for β being greater than zero.
In step 108, the best overall fit of model to data is selected from among all the model sets. This modeling approach optimally uses information contained in the increase of chromosome copy number with aneuploidy and its association with the strength of non-maternal alleles.
In some embodiments, CGE signals representing m1 and m2 at each locus are obtained by profiling the maternal-only sample and mapping the peak locations to the corresponding ones of the mixed sample. The heights of m1 and m2 may be unequal, and this helps correct for PCR amplification biases associated with particular alleles. The values of p, p1, p2, α, and β are determined from the mixed sample data by fitting Equations 1-3 to the data, optionally by using the least squares, or the maximum likelihood methods.
The three models need to be fit to each chromosome with suspected trisomy, e.g. chromosomes 13, 18, 21, X and/or Y. If there are only 3 suspected chromosomes, this results in 27 model variants (3×3×3=27). In Equations 2 and 3, there is also the ambiguity between using m1 or m2 in the β-term, so there are 5 model variants for each chromosome, with 5×5×5=125 total variants over three suspected trisomy chromosomes.
Segmental aneuploidies also could be tested by hypothesizing that different contiguous subsets of loci are contained within the aneuploid region. With each model variant, α and β have to be determined and the parameters describing the paternal alleles have to be determined at each locus for each model variant. The paternal allele peak height and shape can be assumed to be an average of the known maternal ones at that locus, while the paternal allele location needs to be fit to the data. The possible locations for the paternal allele will be the location of m1, the location of m2, and ‘elsewhere in the locus window’ where this latter possibility involves a search over discrete shifts smaller than a typical peak half-width at half maximum. Prior probabilities on the choices of p, taken from population allele frequency data, can be used, if their product lengths can be predicted.
In some cases, because of the number of parameters being fit, suboptimal searches can be used for computational efficiency. For example, one possible approach involves iterative methods, such as the following, which would be applied to each data model variant:
In step 109, the presence or absence of fetal DNA is determined using the models described above. The best overall fit for such models yields the values of β, α that can be called Bmax□, αmax. The likelihood of observing the data given βmax can be compared to the likelihood given β=0. The ratio is a measure of the amount of evidence for fetal DNA. A threshold for declaring fetal DNA is the likelihood ratio of approximately 1000 or more. The likelihood calculation can be approximated by a Chi-squared calculation involving the sum of squared residuals between the data and the model, where each residual is normalized by the expected rms error.
If it is determined that fetal DNA is not present in the mixed sample as calculated above, then the test is declared to be non-informative. On the other hand, if it is decided that fetal DNA is present in the mixed sample, then the likelihoods of the data given the different data model types can be compared to declare trisomy or another condition.
In step 110, the likelihood ratios of trisomy models (Equations 1 and 3) to the normal model (Equation 2) are calculated and these ratios are compared to a predefined threshold. This threshold can be set so that in controlled tests all the trisomic cases are declared aneuploid, and so that it is expected that the vast majority (>99.9%) of all truly trisomic cases are declared aneuploid by the test. In one embodiment, to accomplish a detection rate of >90% or 95% or approximately 99.9%, the likelihood ratio threshold is increased beyond what is necessary to declare all the known trisomic cases in the validation set by a factor of 1000/N, where N is the number of trisomy cases in the validation set.
In step 111, errors that may arise from the experimental procedure used to obtain the data can be taken into account in the model calculation(s). For instance, in the example described above, CGE data contain small additive errors associated with CGE readout, and larger multiplicative errors associated with PCR amplification efficiencies being different from locus to locus and from allele to allele within a locus. By using the maternal-only data to define m1 and m2 peak characteristics at each locus, the effects of PCR amplification biases associated with different primers and different amplicons from the same primers have been mostly controlled. Nevertheless, small variations in the process from day to day and the statistics of small numbers of starting genome copies will cause some random errors to remain. These tend to be multiplicative errors in the resulting CGE peak heights; e.g. two peaks may be 20% different in height although the starting concentrations of the alleles were identical. In one embodiment, it may be assumed that errors are random from peak to peak, and have relatively small additive errors, and larger Poisson and multiplicative error components. The magnitudes of these error components can be estimated from repeated PCR/CGE processing of identical samples. The Chi-square residuals calculation for any data-model fit then can be supported with these modeled squared errors for any peak height or data bin.
In another aspect of the present invention, the presence of fetal cells in a mixed sample and fetal abnormalities in said cells is determined without trying to integrate them in a data-model fitting procedure described above. For example, steps 100-107 can be performed as described above. Then, analysis using Equations 1 and 2 focuses on two indications. First, aneuploidy results in an excess of DNA for the trisomic chromosome, and this is indicated by the difference in mean strengths of the alleles on the trisomic chromosome compared to control chromosomes. A t-test can be applied to the two distributions of m1 and m2 peak heights. These peak heights are normalized to (e.g., divided peak-by-peak by) the corresponding peaks in the maternal-only sample to reduce PCR amplification biases. Second, Equations 1 and 2 show that aneuploidy is associated with less inequality in the heights of m1 and m2 at a given locus, particularly for loci where the paternal allele is distinct from the maternal alleles. Loci are selected where a third (paternal) allele is visibly distinct from two maternal alleles, and the distribution of the inequalities (measured in %) between the m1 and m2 peaks are compared between suspected trisomy chromosomes and control chromosomes. Again, peak heights first are normalized by the maternal-only sample. These two lines of evidence are combined to create an overall likelihood, such as by multiplying the probability values from the two lines of evidence. The presence/absence call is done in a simplified way by looking for loci where a third allele is clearly visible, and comparing the distribution of these peak heights between the maternal and mixed samples. Again, a t-test between these distributions gives the probability of fetal DNA being present.
In another aspect of the invention, the methods herein only determine presence or absence of fetal DNA, and aneuploidy information is known from another sources (e.g. fluorescence in situ hybridization (FISH) assay). For example, it may be desirable only to verify the presence or absence of fetal cells to ensure that a diploid test result is truly due to a normal fetus and not to failure of an assay (e.g. FISH). In this case, the process may be simplified by focusing on detecting the presence of non-maternal alleles without regard to associating them with increases in the maternal allele strengths at the same locus. Thus a process similar to the one outlined above may be used but it is not as necessary to arrange the PCR product lengths so that the products from different loci have distinct length windows in the CGE readout. The alleles from the different loci can be allowed to fall essentially anywhere in the effective measurement length window of the CGE. It also is not necessary to ‘lineate’ the PCR result(s) via multiple CGE readouts at different stages in the PCR cycling as is suggested in step 107.
Therefore, maternal-only and mixed samples are run and mapped to each other to align maternal allele peak locations, as described above. PCR is run to saturation, or nearly to saturation, to be sure to detect the low abundance fetal sequences. The evidence for fetal DNA then arises from extra peaks in the mixed-sample data with respect to the maternal-sample data. Based on typical heterozygosities of approximately 0.7 for highly polymorphic STRs, the chance of not seeing a distinct paternal allele (distinct from both maternal alleles) when fetal DNA is in fact present decreases approximately as (0.72)N, where N is the number of loci included. Thus approximately ten STR loci will provide ˜99.9% probability of detection. In addition, the present invention provides methods to determine when there are insufficient fetal cells for a determination and report a non-informative case. The present invention involves quantifying regions of genomic DNA from a mixed sample. More particularly the invention involves quantifying DNA polymorphisms from the mixed sample. In some embodiments, one or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 DNA polymorphism loci (particularly STRs) per target chromosome are analyzed to verify presence of fetal cells.
Any of the steps above can be performed by a computer program product that comprises a computer executable logic that is recorded on a computer readable medium. For example, the computer program can execute some or all of the following functions: (i) controlling enrichment of fetal cells or DNA from mixed sample and reference sample, (ii) pre-amplifying DNA from both samples, (iii) amplifying specific polymorphic DNA regions from both samples, (iv) identifying and quantifying maternal alleles in the reference sample, (v) identifying maternal and non-maternal alleles in the mixed sample, (vi) fitting data on alleles detected from mixed and/or reference samples into data models, (vii) determining the presence or absence of fetal cells in the mixed sample, (viii) declaring normal or abnormal phenotype for a fetus based on data models or declaring non-informative results, and (ix) declaring a specific fetal abnormality based on the above results. In particular, the computer executable logic can fit data on the quantity of DNA polymorphism(s) (e.g. STR's) into one or more data models. One example of a data model provides a determination of a fetal abnormality from given data signals obtained by molecular analysis e.g. CGE. The computer executable logic provides for a determination of the presence or absence of a trisomy, and distinguish whether the trisomy is paternally derived or if it originates from a maternal non-disjunction event. For example, given the following data signals that can be obtained by molecular analysis (e.g. CGE)
m1, which represents a signal obtained from one of the maternal alleles (m1) at a given locus,
m2, which represents a signal obtained from the other maternal allele, which might be the same allele,
p, which is a signal that is obtained from the paternal allele at a given locus, and
p1 and p2, which are signals obtained from the paternal alleles at one given locus when a paternally derived trisomy occurs, and
letting α and β, which denote the relative number of maternal and fetal cells, respectively, the following determinations can be made. In the case of a chromosome with maternal non-disjunction trisomy, the data will have the form
x=α(□m1+m2)+β(m1+m2+p). (1)
A normal (diploid) chromosome will give
x=α(m1+m2)+β([m1 or m2]+p), (2)
and a paternally derived trisomy will give
x=α(m1+m2)+β([m1 or m2]+p1+p2). (3)
The computer executable logic can work in any computer that may be any of a variety of types of general-purpose computers such as a personal computer, network server, workstation, or other computer platform now or later developed. In some embodiments, a computer program product is described comprising a computer usable medium having the computer executable logic (computer software program, including program code) stored therein. The computer executable logic can be executed by a processor, causing the processor to perform functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.
The program can provide a method of evaluating the presence or absence of trisomy in a mixed cell sample by accessing data that reflects the level of polymorphism(s) at two alleles at two or more given loci in a mixed sample (maternal and fetal cells) and in a sample enriched in fetal cells, relating the levels of polymorphism(s) to the number of maternal and fetal cells (α and β in equations 1-3), and determining the presence or absence of trisomy in the samples.
In one embodiment, the computer executing the computer logic of the invention may also include a digital input device such as a scanner. The digital input device can provide information on the polymorphism levels/quantity. For example, a scanner of this invention can provide an image of the DNA polymorphism (particularly STRs) according to method herein. For instance, a scanner can provide an image by detecting fluorescent, radioactive, or other emission; by detecting transmitted, reflected, or scattered radiation; by detecting electromagnetic properties or other characteristics; or by other techniques. The data detected is typically stored in a memory device in the form of a data file. In one embodiment, a scanner may identify one or more labeled targets. For instance, a first DNA polymorphism may be labeled with a first dye that fluoresces at a particular characteristic frequency, or narrow band of frequencies, in response to an excitation source of a particular frequency. A second DNA polymorphism may be labeled with a second dye that fluoresces at a different characteristic frequency. The excitation sources for the second dye may, but need not, have a different excitation frequency than the source that excites the first dye, e.g., the excitation sources could be the same, or different, lasers.
Another aspect of the invention includes kits containing the devices and reagents for performing the enrichment and genetic analysis. Such kits may include the materials for any individual step disclosed, any combination of devices and reagents or the devices and reagents for performing all of the steps. For example, a kit may include the arrays for size-based enrichment, the device for magnetic separation of the cells and reagents for performing PCR or CGE. Also included may be the reagents for performing multiple displacement amplification. This is an exemplary kit and the kits can be constructed using any combination of disclosed materials and devices. The use of the size-based enrichment provides gentle handling that is particularly advantageous for permitting subsequent genetic analysis.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Dimensions: 100 mm×28 mm×1 mm
Array design: 3 stages, gap size=18, 12 and 8 μm for the first, second and third stage, respectively.
Device fabrication: The arrays and channels were fabricated in silicon using standard photolithography and deep silicon reactive etching techniques. The etch depth is 140 μm. Through holes for fluid access are made using KOH wet etching. The silicon substrate was sealed on the etched face to form enclosed fluidic channels using a blood compatible pressure sensitive adhesive (9795, 3M, St Paul, Minn.).
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 2.0 PSI to the buffer and blood reservoirs to modulate fluidic delivery and extraction from the packaged device.
Experimental conditions: Human fetal cord blood was drawn into phosphate buffered saline containing Acid Citrate Dextrose anticoagulants. 1 mL of blood was processed at 3 mL/hr using the device described above at room temperature and within 48 hrs of draw. Nucleated cells from the blood were separated from enucleated cells (red blood cells and platelets), and plasma delivered into a buffer stream of calcium and magnesium-free Dulbecco's Phosphate Buffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1% Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.) and 2 mM EDTA (15575-020, Invitrogen, Carlsbad, Calif.).
Measurement techniques: Cell smears of the product and waste fractions (
Performance: Fetal nucleated red blood cells were observed in the product fraction (
The device and process described in detail in Example 1 were used in combination with immunomagnetic affinity enrichment techniques to demonstrate the feasibility of isolating fetal cells from maternal blood.
Experimental conditions: blood from consenting maternal donors carrying male fetuses was collected into K2EDTA vacutainers (366643, Becton Dickinson, Franklin Lakes, N.J.) immediately following elective termination of pregnancy. The undiluted blood was processed using the device described in Example 1 at room temperature and within 9 hrs of draw. Nucleated cells from the blood were separated from enucleated cells (red blood cells and platelets), and plasma delivered into a buffer stream of calcium and magnesium-free Dulbecco's Phosphate Buffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1% Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.). Subsequently, the nucleated cell fraction was labeled with anti-CD71 microbeads (130-046-201, Miltenyi Biotech Inc., Auburn, Calif.) and enriched using the MiniMACS™ MS column (130-042-201, Miltenyi Biotech Inc., Auburn, Calif.) 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, Ill.). 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.
Performance: Isolation of fetal cells was confirmed by the reliable presence of male cells in the CD71-positive population prepared from the nucleated cell fractions (
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. After enrichment of fnRBC by any of the methods described herein, the resulting enriched fnRBC are binned by dividing the sample into 100 PCR wells. Prior to binning, enriched samples may be screened by FISH to determine the presence of any fnRBC containing an aneuploidy of interest. Because of the low number of fnRBC in maternal blood, only a portion of the wells will contain a single fnRBC (the other wells are expected to be negative for fnRBC). The cells are fixed in 2% Paraformaldehyde and stored at 4° C. Cells in each bin are pelleted and resuspended in 5 μl PBS plus 1 μl 20 mg/ml Proteinase K (Sigma # P-2308). Cells are lysed by incubation at 65° C. for 60 minutes followed by inactivation of the Proteinase K by incubation for 15 minutes at 95° C. For each reaction, primer sets (DYZ forward primer TCGAGTGCATTCCATTCCG (SEQ ID NO: 159); DYZ reverse primer ATGGAATGGCATCAAACGGAA (SEQ ID NO: 160); and DYZ Taqman Probe 6FAM-TGGCTGTCCATTCCA-MGBNFQ (SEQ ID NO: 161)), 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° C., 10 minutes 95° C. followed by 40 cycles of 95° C. (15 seconds) and 60° C. (1 minute). Following confirmation of the presence of male fetal cells, further analysis of bins containing fnRBC is performed. Positive bins may be pooled prior to further analysis.
Maternal blood is processed through a size-based separation module, with or without subsequent MHEM enhancement of fnRBCs. The enhanced sample is then subjected to FISH analysis using probes specific to the aneuploidy of interest (e.g., triploidy 13, triploidy 18, and XYY). Individual positive cells are isolated by “plucking” individual positive cells from the enhanced sample using standard micromanipulation techniques. Using a nested PCR protocol, STR marker sets are amplified and analyzed to confirm that the FISH-positive aneuploid cell(s) are of fetal origin. For this analysis, comparison to the maternal genotype is typical. An example of a potential resulting data set is shown in Table 2. Non-maternal alleles may be proven to be paternal alleles by paternal genotyping or genotyping of known fetal tissue samples. As can be seen, the presence of paternal alleles in the resulting cells, demonstrates that the cell is of fetal origin (cells #1, 2, 9, and 10). Positive cells may be pooled for further analysis to diagnose aneuploidy of the fetus, or may be further analyzed individually.
Maternal blood is processed through a size-based separation module, with or without subsequent MHEM enhancement of fnRBCs. The enhanced sample is then subjected to FISH analysis using probes specific to the aneuploidy of interest (e.g., triploidy 13, triploidy 18, and XYY). Samples testing positive with FISH analysis are then binned into 96 microtiter wells, each well containing 15 μl of the enhanced sample. Of the 96 wells, 5-10 are expected to contain a single fnRBC and each well should contain approximately 1000 nucleated maternal cells (both WBC and mnRBC). Cells are pelleted and resuspended in 5 μl PBS plus 1 μl 20 mg/ml Proteinase K (Sigma # P-2308). Cells are lysed by incubation at 65° C. for 60 minutes followed by a inactivation of the Proteinase K by 15 minute at 95° C.
In this example, the maternal genotype (BB) and fetal genotype (AB) for a particular set of SNPs is known. The genotypes A and B encompass all three SNPs and differ from each other at all three SNPs. The following sequence from chromosome 7 contains these three SNPs (rs7795605, rs7795611 and rs7795233 indicated in brackets, respectively) (ATGCAGCAAGGCACAGACTAA[G/A]CAAGGAGA[G/C]GCAAAATTTTC [A/G]TAGGG GAGAGAAATGGGTCATT (SEQ ID NO: 162).
In the first round of PCR, genomic DNA from binned enriched cells is amplified using primers specific to the outer portion of the fetal-specific allele A and which flank the interior SNP (forward primer ATGCAGCAAGGCACAGACTACG (SEQ ID NO: 163); reverse primer AGAGGGGAGAGAAATGGGTCATT (SEQ ID NO: 164)). In the second round of PCR, amplification using real time SYBR Green PCR is performed with primers specific to the inner portion of allele A and which encompass the interior SNP (forward primer CAAGGCACAGACTAAGCAAGGAGAG (SEQ ID NO: 165); reverse primer GGCAAAATTTTCATAGGGGAGAGAAATGGGTCATT (SEQ ID NO: 166).
Expected results are shown in
Cell Lysis: Cells are lysed in a proteinase K solution by heating samples for 60 minutes at 65° C., followed by a heating step of 15 minutes at 95° C.
1st round of PCR: A polymerase mix that includes 12 specialized STR primer pairs is added to the crude lysate. A master PCR mix is generated according to the number of samples as per the recipe below. For the reference sample 44 μL of the master mix are added directly to the cell lysate. For the sample recovered from the slide the volume of the reaction is adjusted as necessary. (e.g. 32 μL crude lysate in a 100 μL total reaction volume). A no template or negative control is generated to test for contamination.
Nested PCR: After PCR, optionally, diluted products are added to a second nested primer PCR reaction. Two ul aliquot of each 12-plex PCR reaction is diluted 40 fold (to 80 ul total) with nuclease free water from the PCR kit. The diluted fetal enriched 12-plex reaction could be used as template for a master mix for 8 nested PCR reactions with FAM labeled primers. A second master mix can be generated using the dilution from the maternal reference for 8 nested PCR reactions with VIC labeled primers. The following primer pairs are suggested. A no template or negative control is generated to test for contamination.
The amplification with the nested PCR primers is run with an annealing temperature of 63° C. or 68° C. depending on the primer pair being amplified as indicated in
Detection on ABI 310 Instrument: PCR products are detected on an Agilent Bioanalyzer. The maternal reference VIC labeled PCR reaction products is diluted 10 fold in nano-pure water (17.8 uOhms). Another 10 fold dilution of the fetal enriched FAM-PCR products is generated. The ABI loading buffer is prepared by adding 0.5 μL LIZ 500 size standard to 12 μL Hi Di Formamide (scale as appropriate to the number of samples, include enough buffer for the negative control to test for contamination). 1 μL diluted PCR product is added to 12 ul loading buffer. The sample is heated to 95° C. for 2 minutes and then placed on ice. The samples are loaded onto the ABI 310 as per the manufacturer's instructions.
Analysis: For analysis the ABI fragments output are examined for the expected peak sizes as per the nested STR primer facts table (
The protocol detection limit was determined using fixed cells from cord blood. Cord blood from clinical sample SVH0003C was subjected to erythrocyte lysis and the remaining leukocytes were fixed in a solution of PBS and 2% para-formaldehyde. Cell numbers were estimated from hemocytometer counts and dilutions made into a Tris protienase K (PK) solution. After cell lysis and PK inactivation a PCR cocktail including primers for STR loci TPOX and CSF1P0 was added directly to the crude lysate and amplified as described in Example 6. The products were analyzed on an Agilent bioanalyzer.
An estimated 10 fetal cells were mixed with increasing amounts of maternal cells (approximately 0-4000 cells as measured by hemocytometer counts). After proteinase K lysis, only a 1st round of PCR with primers to STR loci D21S11 and VWA was executed as described in Example 6.
Approximately 100 cells were spotted onto a poly-L-lysine slide and heat dried. Cells were fixed in a MeOH acetic acid solution and rinsed in MeOH. After air drying the slide was treated with 2% para-formaldehyde for 10 minutes then washed in 1×PBS. The slide was dehydrated in passes of EtOH for 1 min each in 70%, 80%, 90%, and finally 100%. A dam was applied around the cells and 30 ul of proteinase K was added on top of the cells and a cover slip adhered over the dam. The slide was incubated on a heat block at 65° C. for 60 minutes and 95° C. for 15 minutes. The lysate solution was then transferred directly to a 100 ul PCR reaction with VWA and LIPOL primer. PCR protocol and analysis were performed as described in Example 6.
Mutliplex PCR reactions from samples with 10 fetal cells in a background of maternal cells generating at 10%, 5% and 2% fetal cells purity concentration were performed as described in Example 6. A dilution of the three multiplex PCR reactions was used as template in nested PCR reactions for STRs TPOX/CYAR04, VWA/D21S11, and D14S1434 as described in Example 6.
Genomic DNA from enriched fetal cells and a maternal control sample will be genotyped for specific STR loci in order to assess the presence of chromosomal abnormalities, such as trisomy. Due to the small number of fetal cells typically isolated from maternal blood it is advantageous to perform a pre-amplification step prior to analysis, using a protocol such as improved primer extension pre-amplification (IPEP) PCR. Cell lysis is carried out in 10 ul High Fidelity buffer (50 mM Tris-HCL, 22 mM (NH.sub.4).sub.2 SO.sub.4 2.5 mM MgCl.sub.2, pH 8.9) which also contained 4 mg/ml proteinase K and 0.5 vol % Tween 20 (Merck) for 12 hours at 48° C. The enzyme is then inactivated for 15 minutes at 94° C. Lysis is performed in parallel batches in 5 ul, 200 mM KOH, 50 mM dithiothreitol for 10 minutes at 65.degree. The batches are then neutralized with 5 ul 900 mM TrisHCl pH 8.3, 300 mM KCl. Preamplication is then carried out for each sample using completely randomized 15-mer primers (16 uM) and dNTP (100 uM) with 5 units of a mixture of Taq polymerase (Boehringer Mannheim) and Pwo polymerase (Boehringer Mannheim) in a ratio of 10:1 under standard PCR buffer conditions (50 mM Tris-HCL, 22 mM (NH.4)2 SO4, 2.5 mM Mg2, pH 8.9, also containing 5% by vol. of DMSO) in a total volume of 60 ul with the following 50 thermal cycles: Step Temperature Time (1) 92° C. 1 Min 30 Sec; (2) 92° C. 40 Min (3) 37° C. 2 Min; (4) ramp: 0.1° C./sec to 55° C. (5) 55° C. 4 Min (6) 68° C. 30 Sec (7) go to step 2, 49 times (8) 8° C. 15′Min.
Dye labeled primers will then be selected from Table 3 based on STR loci on a chromosomes of interest, such as 13, 18, 21 or X. The primers are designed so that one primer of each pair contains a fluorescent dye, such as ROX, HEX, JOE, NED, FAM, TAMARA or LIZ. The primers are placed into multiplex mixes based on expected product size, fluorescent tag compatibility and melting temperature. This allows multiple STR loci to be assayed at once and yet still conserves the amount of initial starting material required. All primers are initially diluted to a working dilution of 10 pM. The primers are then combined in a cocktail that has a final volume of 40 ul. Final primer concentration is determined by reaction optimization. Additional PCR grade water is added if the primer mix is below 40 ul. A reaction mix containing 6 ul of Sigma PCR grade water, 1.25 ul of Perkin Elmer Goldamp PCR buffer, 0.5 ul of dNTPs, 8 ul of the primer cocktail, 0.12 ul of Perkin Elmer Taq Gold Polymerase and 1.25 ul of Mg (25 mM) is mixed for each sample. To this a 1 ul sample containing preamplified DNA from enriched fetal cells or maternal control genomic DNA is added.
The reaction mix is amplified in a DNA thermocyler, (PTC-200; MJ Research) using an amplification cycle optimized for the melting temperature of the primers and the amount of sample DNA.
The amplification product will then analyzed using an automated DNA sequencer system, such as the ABI 310, 377, 3100, 3130, 3700 or 3730, or the Li-Cor 4000, 4100, 4200 or 4300. For example when the amplification products are prepared for analysis on a ABI 377 sequencer, 6 ul of products will be removed and combined with 1.6 ul of loading buffer mix. The master loading buffer mix contains 90 ul deionized formamide combined with 25 ul Perkin Elmer loading dye and 10 ul of a size standard, such as the ROX 350 size standard. Various other standards can be used interchangeably depending on the sizes of the labeled PCR products. The loading buffer and sample are then heat denatured at 95° C. for 3 minutes followed by flash cooling on ice. 2 ul of the product/buffer mix is then electrophoresed on a 12 inch 6% (19:1) polyacrylamide gel on an ABI 377 sequencer.
The results will then be analyzed using ABI Genotyper software. The incorporation of a fluorochrome during amplification allows product quantification for each chromosome specific STR, with 2 fluorescent peaks observed in a normal heterozygous individual with an approximate ratio of 1:1. By comparison in trisomic samples, either 3 fluorescent peaks with a ratio of 1:1:1 (trialleleic) or 2 peaks with a ratio of around 2:1 (diallelic) are observed. Using this method screening may be carried out for common trisomies and sex chromosome aneuploidy in a single reaction.
This application is a Continuation of U.S. application Ser. No. 13/738,268 filed Jan. 10, 2013; which is a Continuation of U.S. application Ser. No. 13/433,232 filed on Mar. 28, 2012; which is a Continuation of U.S. application Ser. No. 12/725,240 filed on Mar. 16, 2010; which is a Continuation of U.S. application Ser. No. 11/763,426 filed on Jun. 14, 2007; which claims the benefit of U.S. Provisional Application Ser. No. 60/804,815 filed on Jun. 14, 2006, each of which applications is incorporated herein by reference. This application also claims the benefit of U.S. Provisional Application No. 60/820,778, filed Jul. 28, 2006. The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 6, 2013, is named 32047-717-306-Seqlisting.txt and is 36 Kilobytes in size.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4508625 | Graham | Sep 1985 | A |
| 4675286 | Calenoff | Jun 1987 | A |
| 4683202 | Mullis | Jul 1987 | A |
| 4789628 | Nayak | Dec 1988 | A |
| 4800159 | Mullis et al. | Jan 1989 | A |
| 4971904 | Luddy | Nov 1990 | A |
| 4977078 | Niimura et al. | Dec 1990 | A |
| 5153117 | Simons | Oct 1992 | A |
| 5215926 | Etchells, III et al. | Jun 1993 | A |
| 5296375 | Kricka et al. | Mar 1994 | A |
| 5300779 | Hillman et al. | Apr 1994 | A |
| 5302509 | Cheeseman | Apr 1994 | A |
| 5304487 | Wilding et al. | Apr 1994 | A |
| 5427663 | Austin et al. | Jun 1995 | A |
| 5427946 | Kricka et al. | Jun 1995 | A |
| 5432054 | Saunders et al. | Jul 1995 | A |
| 5447842 | Simons | Sep 1995 | A |
| 5486335 | Wilding et al. | Jan 1996 | A |
| 5498392 | Wilding et al. | Mar 1996 | A |
| 5508169 | Deugau et al. | Apr 1996 | A |
| 5529903 | Kübler et al. | Jun 1996 | A |
| 5556773 | Youmo | Sep 1996 | A |
| 5629147 | Asgari et al. | May 1997 | A |
| 5639669 | Ledley | Jun 1997 | A |
| 5641628 | Bianchi | Jun 1997 | A |
| 5646001 | Terstappen et al. | Jul 1997 | A |
| 5670325 | Lapidus et al. | Sep 1997 | A |
| 5676849 | Sammons et al. | Oct 1997 | A |
| 5695934 | Brenner | Dec 1997 | A |
| 5707799 | Hansmann et al. | Jan 1998 | A |
| 5709943 | Coleman et al. | Jan 1998 | A |
| 5715946 | Reichenbach | Feb 1998 | A |
| 5726026 | Wilding et al. | Mar 1998 | A |
| 5750339 | Smith | May 1998 | A |
| 5766843 | Asgari et al. | Jun 1998 | A |
| 5770029 | Nelson et al. | Jun 1998 | A |
| 5798042 | Chu et al. | Aug 1998 | A |
| 5837115 | Austin et al. | Nov 1998 | A |
| 5840502 | Van Vlasselaer | Nov 1998 | A |
| 5842787 | Koph-Sill et al. | Dec 1998 | A |
| 5858649 | Asgari et al. | Jan 1999 | A |
| 5866345 | Wilding et al. | Feb 1999 | A |
| 5879883 | Benson et al. | Mar 1999 | A |
| 5891651 | Roche et al. | Apr 1999 | A |
| 5928880 | Wilding et al. | Jul 1999 | A |
| 5952173 | Hansmann et al. | Sep 1999 | A |
| 5962234 | Golbus | Oct 1999 | A |
| 5962237 | Ts'o et al. | Oct 1999 | A |
| 5962332 | Singer et al. | Oct 1999 | A |
| 5972721 | Bruno et al. | Oct 1999 | A |
| 5993665 | Terstappen et al. | Nov 1999 | A |
| 5994057 | Mansfield | Nov 1999 | A |
| 5994517 | Ts'o et al. | Nov 1999 | A |
| 6007690 | Nelson et al. | Dec 1999 | A |
| 6008007 | Fruehauf et al. | Dec 1999 | A |
| 6013188 | Terstappen et al. | Jan 2000 | A |
| 6027923 | Wallace | Feb 2000 | A |
| 6066449 | Ditkoff et al. | May 2000 | A |
| 6074827 | Nelson et al. | Jun 2000 | A |
| 6100029 | Lapidus et al. | Aug 2000 | A |
| 6124120 | Lizardi | Sep 2000 | A |
| 6159685 | Pinkel et al. | Oct 2000 | A |
| 6143496 | Brown et al. | Nov 2000 | A |
| 6143576 | Buechler | Nov 2000 | A |
| 6154707 | Livak et al. | Nov 2000 | A |
| 6156270 | Buechler | Dec 2000 | A |
| 6176962 | Soane et al. | Jan 2001 | B1 |
| 6184029 | Wilding et al. | Feb 2001 | B1 |
| 6184043 | Fodstad et al. | Feb 2001 | B1 |
| 6186660 | Koph-Sill et al. | Feb 2001 | B1 |
| 6190870 | Schmitz et al. | Feb 2001 | B1 |
| 6197523 | Rimm et al. | Mar 2001 | B1 |
| 6200765 | Murphy et al. | Mar 2001 | B1 |
| 6210891 | Nyren et al. | Apr 2001 | B1 |
| 6214558 | Shuber et al. | Apr 2001 | B1 |
| 6235474 | Feinberg | May 2001 | B1 |
| 6258540 | Lo et al. | Jul 2001 | B1 |
| 6265229 | Fodstad et al. | Jul 2001 | B1 |
| 6300077 | Shuber et al. | Oct 2001 | B1 |
| 6329150 | Lizardi et al. | Dec 2001 | B1 |
| 6344326 | Nelson et al. | Feb 2002 | B1 |
| 6361958 | Shieh et al. | Mar 2002 | B1 |
| 6365362 | Terstappen et al. | Apr 2002 | B1 |
| 6368871 | Christel et al. | Apr 2002 | B1 |
| 6376181 | Ramsey et al. | Apr 2002 | B2 |
| 6383759 | Murphy et al. | May 2002 | B1 |
| 6387707 | Seul et al. | May 2002 | B1 |
| 6391559 | Brown et al. | May 2002 | B1 |
| 6394942 | Moon et al. | May 2002 | B2 |
| 6399364 | Reeve et al. | Jun 2002 | B1 |
| 6432630 | Blankenstein | Aug 2002 | B1 |
| 6440706 | Vogelstein et al. | Aug 2002 | B1 |
| 6444461 | Knapp et al. | Sep 2002 | B1 |
| 6454938 | Moon et al. | Sep 2002 | B2 |
| 6479299 | Parce et al. | Nov 2002 | B1 |
| 6511967 | Weissleder et al. | Jan 2003 | B1 |
| 6517234 | Koph-Sill et al. | Feb 2003 | B1 |
| 6540895 | Spence et al. | Apr 2003 | B1 |
| 6566101 | Shuber et al. | May 2003 | B1 |
| 6576478 | Wagner et al. | Jun 2003 | B1 |
| 6582904 | Dahm | Jun 2003 | B2 |
| 6582969 | Wagner et al. | Jun 2003 | B1 |
| 6596144 | Regnier et al. | Jul 2003 | B1 |
| 6596545 | Wagner et al. | Jul 2003 | B1 |
| 6613525 | Nelson et al. | Sep 2003 | B2 |
| 6618679 | Loehriein et al. | Sep 2003 | B2 |
| 6632619 | Harrison et al. | Oct 2003 | B1 |
| 6632652 | Austin et al. | Oct 2003 | B1 |
| 6632655 | Mehta et al. | Oct 2003 | B1 |
| 6637463 | Lei et al. | Oct 2003 | B1 |
| 6645731 | Terstappen et al. | Nov 2003 | B2 |
| 6664056 | Lo et al. | Dec 2003 | B2 |
| 6664104 | Pourahmadi et al. | Dec 2003 | B2 |
| 6673541 | Klein et al. | Jan 2004 | B1 |
| 6674525 | Bardell et al. | Jan 2004 | B2 |
| 6685841 | Lopez et al. | Feb 2004 | B2 |
| 6689615 | Murto et al. | Feb 2004 | B1 |
| 6746503 | Benett et al. | Jun 2004 | B1 |
| 6753147 | Vogelstein et al. | Jun 2004 | B2 |
| 6783928 | Hvichia et al. | Aug 2004 | B2 |
| 6818184 | Fulwyler et al. | Nov 2004 | B2 |
| 6830936 | Anderson et al. | Dec 2004 | B2 |
| 6858439 | Xu et al. | Feb 2005 | B1 |
| 6875619 | Blackburn | Apr 2005 | B2 |
| 6893881 | Fodstad et al. | May 2005 | B1 |
| 6906182 | Ts'o et al. | Jun 2005 | B2 |
| 6911345 | Quake et al. | Jun 2005 | B2 |
| 6913697 | Lopez et al. | Jul 2005 | B2 |
| 6927028 | Lo et al. | Aug 2005 | B2 |
| 6953668 | Israeli et al. | Oct 2005 | B1 |
| 6960449 | Wang et al. | Nov 2005 | B2 |
| 7115709 | Gray et al. | Oct 2006 | B1 |
| 7150812 | Huang et al. | Dec 2006 | B2 |
| 7171975 | Moon et al. | Feb 2007 | B2 |
| 7190818 | Ellis et al. | Mar 2007 | B2 |
| 7192698 | Kinch et al. | Mar 2007 | B1 |
| 7198787 | Fodstad et al. | Apr 2007 | B2 |
| 7208275 | Gocke et al. | Apr 2007 | B2 |
| 7208295 | Faham et al. | Apr 2007 | B2 |
| 7212660 | Wetzel et al. | May 2007 | B2 |
| 7220594 | Foster et al. | May 2007 | B2 |
| 7227002 | Kufer et al. | Jun 2007 | B1 |
| 7229838 | Foster et al. | Jun 2007 | B2 |
| 7250256 | Reinhard et al. | Jul 2007 | B2 |
| 7252976 | Lin et al. | Aug 2007 | B2 |
| 7258987 | Lamorte et al. | Aug 2007 | B2 |
| 7262177 | Ts'o et al. | Aug 2007 | B2 |
| 7262269 | Lam et al. | Aug 2007 | B2 |
| 7264972 | Foster | Sep 2007 | B2 |
| 7272252 | De La Torre-Bueno et al. | Sep 2007 | B2 |
| 7276170 | Oakey et al. | Oct 2007 | B2 |
| 7332277 | Dhallan | Feb 2008 | B2 |
| 7407757 | Brenner | Aug 2008 | B2 |
| 7442506 | Dhallan | Oct 2008 | B2 |
| 7476363 | Unger et al. | Jan 2009 | B2 |
| 7582420 | Oliphant et al. | Sep 2009 | B2 |
| 7645576 | Lo et al. | Jan 2010 | B2 |
| 7655399 | Cantor et al. | Feb 2010 | B2 |
| 7709194 | Lo et al. | May 2010 | B2 |
| 7718367 | Lo et al. | May 2010 | B2 |
| 7727720 | Dhallan et al. | Jun 2010 | B2 |
| 7754428 | Lo et al. | Jul 2010 | B2 |
| 7799531 | Mitchell et al. | Sep 2010 | B2 |
| 7838647 | Hahn et al. | Nov 2010 | B2 |
| 7888017 | Quake et al. | Feb 2011 | B2 |
| RE42315 | Lopez et al. | May 2011 | E |
| 7955794 | Shen et al. | Jun 2011 | B2 |
| 8003354 | Shen et al. | Aug 2011 | B2 |
| 8008018 | Quake et al. | Aug 2011 | B2 |
| 8024128 | Rabinowitz et al. | Sep 2011 | B2 |
| 8137912 | Kapur et al. | Mar 2012 | B2 |
| 8168389 | Shoemaker et al. | May 2012 | B2 |
| 8195415 | Fan et al. | Jun 2012 | B2 |
| 8293470 | Quake et al. | Oct 2012 | B2 |
| 8296076 | Fan et al. | Oct 2012 | B2 |
| 8318430 | Chuu et al. | Nov 2012 | B2 |
| 8372584 | Shoemaker et al. | Feb 2013 | B2 |
| 8515679 | Rabinowitz et al. | Aug 2013 | B2 |
| 8532930 | Rabinowitz et al. | Sep 2013 | B2 |
| 8682592 | Rabinowitz et al. | Mar 2014 | B2 |
| 9017942 | Shoemaker et al. | Apr 2015 | B2 |
| 9347100 | Shoemaker et al. | May 2016 | B2 |
| 9441273 | Quake et al. | Sep 2016 | B2 |
| 20010007749 | Feinberg | Jul 2001 | A1 |
| 20010051341 | Lo et al. | Dec 2001 | A1 |
| 20010053958 | Ried et al. | Dec 2001 | A1 |
| 20020006621 | Bianchi | Jan 2002 | A1 |
| 20020009738 | Houghton et al. | Jan 2002 | A1 |
| 20020012930 | Rothberg et al. | Jan 2002 | A1 |
| 20020012931 | Waldman et al. | Jan 2002 | A1 |
| 20020016450 | Laugharn et al. | Feb 2002 | A1 |
| 20020019001 | Light | Feb 2002 | A1 |
| 20020028431 | Julien | Mar 2002 | A1 |
| 20020058332 | Quake et al. | May 2002 | A1 |
| 20020076825 | Cheng et al. | Jun 2002 | A1 |
| 20020086329 | Shvets et al. | Jul 2002 | A1 |
| 20020110835 | Kumar | Aug 2002 | A1 |
| 20020119469 | Shuber et al. | Aug 2002 | A1 |
| 20020123078 | Seul et al. | Sep 2002 | A1 |
| 20020127575 | Hoke et al. | Sep 2002 | A1 |
| 20020137088 | Bianchi | Sep 2002 | A1 |
| 20020164816 | Quake | Nov 2002 | A1 |
| 20020166760 | Prentiss et al. | Nov 2002 | A1 |
| 20020172987 | Terstappen et al. | Nov 2002 | A1 |
| 20030004402 | Hitt et al. | Jan 2003 | A1 |
| 20030013101 | Balasubramanian | Jan 2003 | A1 |
| 20030017514 | Pachmann et al. | Jan 2003 | A1 |
| 20030022207 | Balasubramanian | Jan 2003 | A1 |
| 20030033091 | Opalsky et al. | Feb 2003 | A1 |
| 20030044388 | Lo et al. | Mar 2003 | A1 |
| 20030044781 | Korlach et al. | Mar 2003 | A1 |
| 20030072682 | Kikinis | Apr 2003 | A1 |
| 20030077292 | Hanash et al. | Apr 2003 | A1 |
| 20030082566 | Sylvan | May 2003 | A1 |
| 20030100102 | Rothberg et al. | May 2003 | A1 |
| 20030119077 | Ts'o et al. | Jun 2003 | A1 |
| 20030119724 | Ts'o et al. | Jun 2003 | A1 |
| 20030129676 | Terstappen et al. | Jul 2003 | A1 |
| 20030152981 | Hulten | Aug 2003 | A1 |
| 20030153085 | Leary et al. | Aug 2003 | A1 |
| 20030159999 | Oakey et al. | Aug 2003 | A1 |
| 20030165852 | Schueler et al. | Sep 2003 | A1 |
| 20030170631 | Houghton et al. | Sep 2003 | A1 |
| 20030170703 | Piper et al. | Sep 2003 | A1 |
| 20030175990 | Heyenga | Sep 2003 | A1 |
| 20030186255 | Williams et al. | Oct 2003 | A1 |
| 20030190602 | Pressman et al. | Oct 2003 | A1 |
| 20030199685 | Pressman et al. | Oct 2003 | A1 |
| 20030204331 | Whitney et al. | Oct 2003 | A1 |
| 20030206901 | Chen | Nov 2003 | A1 |
| 20030219765 | Costa | Nov 2003 | A1 |
| 20030231791 | Torre-Bueno et al. | Dec 2003 | A1 |
| 20030232350 | Afar et al. | Dec 2003 | A1 |
| 20040005582 | Shipwash | Jan 2004 | A1 |
| 20040009471 | Cao | Jan 2004 | A1 |
| 20040018116 | Desmond et al. | Jan 2004 | A1 |
| 20040018509 | Bianchi | Jan 2004 | A1 |
| 20040043506 | Haussecker et al. | Mar 2004 | A1 |
| 20040048360 | Wada et al. | Mar 2004 | A1 |
| 20040053352 | Ouyang et al. | Mar 2004 | A1 |
| 20040072278 | Chou et al. | Apr 2004 | A1 |
| 20040096892 | Wang et al. | May 2004 | A1 |
| 20040137452 | Levett et al. | Jul 2004 | A1 |
| 20040137470 | Dhallan | Jul 2004 | A1 |
| 20040142463 | Walker et al. | Jul 2004 | A1 |
| 20040144651 | Huang et al. | Jul 2004 | A1 |
| 20040157243 | Huang et al. | Aug 2004 | A1 |
| 20040166555 | Braff et al. | Aug 2004 | A1 |
| 20040171091 | Lesko et al. | Sep 2004 | A1 |
| 20040185495 | Schueler et al. | Sep 2004 | A1 |
| 20040203037 | Lo et al. | Oct 2004 | A1 |
| 20040209299 | Pinter et al. | Oct 2004 | A1 |
| 20040214240 | Cao | Oct 2004 | A1 |
| 20040232074 | Peters et al. | Nov 2004 | A1 |
| 20040241707 | Gao et al. | Dec 2004 | A1 |
| 20050014208 | Krehan et al. | Jan 2005 | A1 |
| 20050019792 | McBride et al. | Jan 2005 | A1 |
| 20050037388 | Antonarakis et al. | Feb 2005 | A1 |
| 20050042623 | Ault-Riche et al. | Feb 2005 | A1 |
| 20050042685 | Albert et al. | Feb 2005 | A1 |
| 20050049793 | Paterlini-Brechot | Mar 2005 | A1 |
| 20050061962 | Mueth et al. | Mar 2005 | A1 |
| 20050064476 | Huang et al. | Mar 2005 | A1 |
| 20050095606 | Hoke et al. | May 2005 | A1 |
| 20050100932 | Lapidus et al. | May 2005 | A1 |
| 20050118591 | Tamak et al. | Jun 2005 | A1 |
| 20050129581 | McBride et al. | Jun 2005 | A1 |
| 20050130217 | Huang et al. | Jun 2005 | A1 |
| 20050145496 | Goodsaid et al. | Jul 2005 | A1 |
| 20050147977 | Koo et al. | Jul 2005 | A1 |
| 20050153342 | Chen | Jul 2005 | A1 |
| 20050158754 | Puffenberger et al. | Jul 2005 | A1 |
| 20050164241 | Hahn et al. | Jul 2005 | A1 |
| 20050175996 | Chen | Aug 2005 | A1 |
| 20050181353 | Rao et al. | Aug 2005 | A1 |
| 20050181463 | Rao et al. | Aug 2005 | A1 |
| 20050196785 | Quake et al. | Sep 2005 | A1 |
| 20050207940 | Butler et al. | Sep 2005 | A1 |
| 20050211556 | Childers et al. | Sep 2005 | A1 |
| 20050214855 | Wagner et al. | Sep 2005 | A1 |
| 20050221341 | Shimkets et al. | Oct 2005 | A1 |
| 20050221373 | Enzelberger et al. | Oct 2005 | A1 |
| 20050239101 | Sukumar et al. | Oct 2005 | A1 |
| 20050244843 | Chen et al. | Nov 2005 | A1 |
| 20050250111 | Xie et al. | Nov 2005 | A1 |
| 20050250147 | Macevicz | Nov 2005 | A1 |
| 20050250155 | Lesko et al. | Nov 2005 | A1 |
| 20050250199 | Anderson et al. | Nov 2005 | A1 |
| 20050252773 | McBride et al. | Nov 2005 | A1 |
| 20050255001 | Padmanabhan et al. | Nov 2005 | A1 |
| 20050262577 | Guelly et al. | Nov 2005 | A1 |
| 20050266433 | Kapur et al. | Dec 2005 | A1 |
| 20050272103 | Chen | Dec 2005 | A1 |
| 20050282196 | Costa | Dec 2005 | A1 |
| 20050282293 | Cosmen et al. | Dec 2005 | A1 |
| 20050287611 | Nugent et al. | Dec 2005 | A1 |
| 20060000772 | Sano et al. | Jan 2006 | A1 |
| 20060008807 | O'Hara et al. | Jan 2006 | A1 |
| 20060008824 | Ronaghi et al. | Jan 2006 | A1 |
| 20060024678 | Buzby | Feb 2006 | A1 |
| 20060024711 | Lapidus et al. | Feb 2006 | A1 |
| 20060024756 | Tibbe et al. | Feb 2006 | A1 |
| 20060046258 | Lapidus et al. | Mar 2006 | A1 |
| 20060051265 | Mohamed et al. | Mar 2006 | A1 |
| 20060051775 | Bianchi et al. | Mar 2006 | A1 |
| 20060052945 | Rabinowitz et al. | Mar 2006 | A1 |
| 20060060767 | Wang et al. | Mar 2006 | A1 |
| 20060072805 | Tsipouras et al. | Apr 2006 | A1 |
| 20060073125 | Clarke et al. | Apr 2006 | A1 |
| 20060094109 | Trainer | May 2006 | A1 |
| 20060121452 | Dhallan | Jun 2006 | A1 |
| 20060121624 | Huang et al. | Jun 2006 | A1 |
| 20060128006 | Gerhardt et al. | Jun 2006 | A1 |
| 20060134599 | Toner et al. | Jun 2006 | A1 |
| 20060160105 | Dhallan | Jul 2006 | A1 |
| 20060160150 | Seilhamer et al. | Jul 2006 | A1 |
| 20060160243 | Tang et al. | Jul 2006 | A1 |
| 20060177832 | Brenner | Aug 2006 | A1 |
| 20060183886 | Ts'o et al. | Aug 2006 | A1 |
| 20060205057 | Wayner et al. | Sep 2006 | A1 |
| 20060223178 | Barber et al. | Oct 2006 | A1 |
| 20060228721 | Leamon et al. | Oct 2006 | A1 |
| 20060252054 | Lin et al. | Nov 2006 | A1 |
| 20060252061 | Zabeau et al. | Nov 2006 | A1 |
| 20060252068 | Lo et al. | Nov 2006 | A1 |
| 20060252071 | Lo et al. | Nov 2006 | A1 |
| 20060252087 | Tang et al. | Nov 2006 | A1 |
| 20070015171 | Bianchi et al. | Jan 2007 | A1 |
| 20070017633 | Tonkovich et al. | Jan 2007 | A1 |
| 20070026381 | Huang et al. | Feb 2007 | A1 |
| 20070026413 | Toner et al. | Feb 2007 | A1 |
| 20070026414 | Fuchs et al. | Feb 2007 | A1 |
| 20070026415 | Fuchs et al. | Feb 2007 | A1 |
| 20070026416 | Fuchs | Feb 2007 | A1 |
| 20070026417 | Fuchs et al. | Feb 2007 | A1 |
| 20070026418 | Fuchs et al. | Feb 2007 | A1 |
| 20070026419 | Fuchs et al. | Feb 2007 | A1 |
| 20070026469 | Fuchs et al. | Feb 2007 | A1 |
| 20070027636 | Rabinowitz | Feb 2007 | A1 |
| 20070037172 | Chiu et al. | Feb 2007 | A1 |
| 20070037173 | Allard et al. | Feb 2007 | A1 |
| 20070037273 | Shuler et al. | Feb 2007 | A1 |
| 20070037275 | Shuler et al. | Feb 2007 | A1 |
| 20070042238 | Kim et al. | Feb 2007 | A1 |
| 20070042339 | Toner et al. | Feb 2007 | A1 |
| 20070042360 | Afar et al. | Feb 2007 | A1 |
| 20070042368 | Zehentner-Wilkinson et al. | Feb 2007 | A1 |
| 20070048750 | Peck et al. | Mar 2007 | A1 |
| 20070054268 | Sutherland et al. | Mar 2007 | A1 |
| 20070054287 | Bloch | Mar 2007 | A1 |
| 20070059680 | Kapur et al. | Mar 2007 | A1 |
| 20070059683 | Barber et al. | Mar 2007 | A1 |
| 20070059710 | Luke et al. | Mar 2007 | A1 |
| 20070059716 | Balis et al. | Mar 2007 | A1 |
| 20070059718 | Toner et al. | Mar 2007 | A1 |
| 20070059719 | Grisham et al. | Mar 2007 | A1 |
| 20070059737 | Baker et al. | Mar 2007 | A1 |
| 20070059774 | Grisham et al. | Mar 2007 | A1 |
| 20070059781 | Kapur et al. | Mar 2007 | A1 |
| 20070059785 | Bacus et al. | Mar 2007 | A1 |
| 20070065845 | Baker et al. | Mar 2007 | A1 |
| 20070065858 | Haley | Mar 2007 | A1 |
| 20070071762 | Ts'o et al. | Mar 2007 | A1 |
| 20070072228 | Brauch | Mar 2007 | A1 |
| 20070072290 | Hvichia | Mar 2007 | A1 |
| 20070077578 | Penning et al. | Apr 2007 | A1 |
| 20070092444 | Benos et al. | Apr 2007 | A1 |
| 20070092881 | Ohnishi et al. | Apr 2007 | A1 |
| 20070092917 | Guyon | Apr 2007 | A1 |
| 20070099207 | Fuchs et al. | May 2007 | A1 |
| 20070099219 | Teverovskiy et al. | May 2007 | A1 |
| 20070099289 | Irimia et al. | May 2007 | A1 |
| 20070105105 | Clelland et al. | May 2007 | A1 |
| 20070105133 | Clark et al. | May 2007 | A1 |
| 20070110773 | Asina et al. | May 2007 | A1 |
| 20070117158 | Coumans et al. | May 2007 | A1 |
| 20070122856 | Georges et al. | May 2007 | A1 |
| 20070122896 | Shuler et al. | May 2007 | A1 |
| 20070128655 | Obata | Jun 2007 | A1 |
| 20070131622 | Oakey et al. | Jun 2007 | A1 |
| 20070134658 | Bohmer et al. | Jun 2007 | A1 |
| 20070134713 | Cao | Jun 2007 | A1 |
| 20070135621 | Bourel et al. | Jun 2007 | A1 |
| 20070141587 | Baker et al. | Jun 2007 | A1 |
| 20070141588 | Baker et al. | Jun 2007 | A1 |
| 20070141717 | Carpenter et al. | Jun 2007 | A1 |
| 20070154928 | Mack et al. | Jul 2007 | A1 |
| 20070154960 | Connelly et al. | Jul 2007 | A1 |
| 20070160503 | Sethu et al. | Jul 2007 | A1 |
| 20070160974 | Sidhu et al. | Jul 2007 | A1 |
| 20070160984 | Huang et al. | Jul 2007 | A1 |
| 20070161064 | Kinch et al. | Jul 2007 | A1 |
| 20070166770 | Hsieh et al. | Jul 2007 | A1 |
| 20070170811 | Rubel | Jul 2007 | A1 |
| 20070172903 | Toner et al. | Jul 2007 | A1 |
| 20070178067 | Maier et al. | Aug 2007 | A1 |
| 20070178458 | O'Brien et al. | Aug 2007 | A1 |
| 20070178478 | Dhallan et al. | Aug 2007 | A1 |
| 20070178501 | Rabinowitz et al. | Aug 2007 | A1 |
| 20070187250 | Huang et al. | Aug 2007 | A1 |
| 20070196663 | Schwartz et al. | Aug 2007 | A1 |
| 20070196820 | Kapur et al. | Aug 2007 | A1 |
| 20070196840 | Roca et al. | Aug 2007 | A1 |
| 20070196869 | Perez et al. | Aug 2007 | A1 |
| 20070202106 | Palucka et al. | Aug 2007 | A1 |
| 20070202109 | Nakamura et al. | Aug 2007 | A1 |
| 20070202525 | Quake et al. | Aug 2007 | A1 |
| 20070202536 | Yamanishi et al. | Aug 2007 | A1 |
| 20070207351 | Christensen et al. | Sep 2007 | A1 |
| 20070207466 | Cantor et al. | Sep 2007 | A1 |
| 20070212689 | Bianchi et al. | Sep 2007 | A1 |
| 20070212698 | Bendele et al. | Sep 2007 | A1 |
| 20070212737 | Clarke et al. | Sep 2007 | A1 |
| 20070212738 | Haley et al. | Sep 2007 | A1 |
| 20070231851 | Toner et al. | Oct 2007 | A1 |
| 20070238105 | Barrett et al. | Oct 2007 | A1 |
| 20070259424 | Toner et al. | Nov 2007 | A1 |
| 20070264675 | Toner et al. | Nov 2007 | A1 |
| 20070275402 | Lo et al. | Nov 2007 | A1 |
| 20080020390 | Mitchell et al. | Jan 2008 | A1 |
| 20080023399 | Inglis et al. | Jan 2008 | A1 |
| 20080026390 | Stoughton et al. | Jan 2008 | A1 |
| 20080038733 | Bischoff et al. | Feb 2008 | A1 |
| 20080050739 | Stoughton et al. | Feb 2008 | A1 |
| 20080070792 | Stoughton et al. | Mar 2008 | A1 |
| 20080071076 | Hahn et al. | Mar 2008 | A1 |
| 20080090239 | Shoemaker et al. | Apr 2008 | A1 |
| 20080096216 | Quake | Apr 2008 | A1 |
| 20080096766 | Lee | Apr 2008 | A1 |
| 20080124721 | Fuchs | May 2008 | A1 |
| 20080138809 | Kapur et al. | Jun 2008 | A1 |
| 20080153090 | Lo et al. | Jun 2008 | A1 |
| 20080182261 | Bianchi | Jul 2008 | A1 |
| 20080193927 | Mann et al. | Aug 2008 | A1 |
| 20080213775 | Brody et al. | Sep 2008 | A1 |
| 20080220422 | Shoemaker et al. | Sep 2008 | A1 |
| 20080299562 | Oeth et al. | Dec 2008 | A1 |
| 20080318235 | Handyside | Dec 2008 | A1 |
| 20090029377 | Lo et al. | Jan 2009 | A1 |
| 20090053719 | Lo et al. | Feb 2009 | A1 |
| 20090087847 | Lo et al. | Apr 2009 | A1 |
| 20090170113 | Quake et al. | Jul 2009 | A1 |
| 20090170114 | Quake et al. | Jul 2009 | A1 |
| 20090215633 | Van Eijk et al. | Aug 2009 | A1 |
| 20090280492 | Stoughton et al. | Nov 2009 | A1 |
| 20090291443 | Stoughton et al. | Nov 2009 | A1 |
| 20090317798 | Heid et al. | Dec 2009 | A1 |
| 20100094562 | Shohat et al. | Apr 2010 | A1 |
| 20100112575 | Fan et al. | May 2010 | A1 |
| 20100112590 | Lo et al. | May 2010 | A1 |
| 20100124751 | Quake et al. | May 2010 | A1 |
| 20100124752 | Quake et al. | May 2010 | A1 |
| 20100136529 | Shoemaker et al. | Jun 2010 | A1 |
| 20100216151 | Lapidus et al. | Aug 2010 | A1 |
| 20100216153 | Lapidus et al. | Aug 2010 | A1 |
| 20100255492 | Quake et al. | Oct 2010 | A1 |
| 20100255493 | Quake et al. | Oct 2010 | A1 |
| 20100256013 | Quake et al. | Oct 2010 | A1 |
| 20100291571 | Stoughton et al. | Nov 2010 | A1 |
| 20100291572 | Stoughton et al. | Nov 2010 | A1 |
| 20100311064 | Oliphant et al. | Dec 2010 | A1 |
| 20110003293 | Stoughton et al. | Jan 2011 | A1 |
| 20110015096 | Chiu et al. | Jan 2011 | A1 |
| 20110105353 | Lo et al. | May 2011 | A1 |
| 20110117548 | Mitchell et al. | May 2011 | A1 |
| 20110171638 | Stoughton et al. | Jul 2011 | A1 |
| 20110312503 | Chuu et al. | Dec 2011 | A1 |
| 20120010085 | Rava et al. | Jan 2012 | A1 |
| 20120135872 | Chuu et al. | May 2012 | A1 |
| 20120171666 | Shoemaker et al. | Jul 2012 | A1 |
| 20120171667 | Shoemaker et al. | Jul 2012 | A1 |
| 20120183963 | Stoughton et al. | Jul 2012 | A1 |
| 20120208186 | Kapur et al. | Aug 2012 | A1 |
| 20130189688 | Shoemaker et al. | Jul 2013 | A1 |
| 20130189689 | Shoemaker et al. | Jul 2013 | A1 |
| 20130210644 | Stoughton et al. | Aug 2013 | A1 |
| 20130253369 | Rabinowitz et al. | Sep 2013 | A1 |
| 20130288242 | Stoughton et al. | Oct 2013 | A1 |
| 20130288903 | Kapur et al. | Oct 2013 | A1 |
| 20130295565 | Shoemaker et al. | Nov 2013 | A1 |
| 20130324418 | Fuchs et al. | Dec 2013 | A1 |
| 20140032128 | Rabinowitz et al. | Jan 2014 | A1 |
| 20140087385 | Rabinowitz et al. | Mar 2014 | A1 |
| 20140106975 | Stoughton et al. | Apr 2014 | A1 |
| 20150232936 | Shoemaker et al. | Aug 2015 | A1 |
| 20150344956 | Kapur et al. | Dec 2015 | A1 |
| 20160002737 | Fuchs et al. | Jan 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 2007260676 | Dec 2007 | AU |
| 2655272 | Dec 2007 | CA |
| 0637996 | Jul 1997 | EP |
| 0405972 | May 1999 | EP |
| 1262776 | Dec 2002 | EP |
| 0994963 | May 2003 | EP |
| 0970365 | Oct 2003 | EP |
| 783694 | Nov 2003 | EP |
| 1262776 | Jan 2004 | EP |
| 1388013 | Feb 2004 | EP |
| 0920627 | May 2004 | EP |
| 1418003 | May 2004 | EP |
| 0739240 | Jun 2004 | EP |
| 1462800 | Sep 2004 | EP |
| 0919812 | Oct 2004 | EP |
| 1561507 | Aug 2005 | EP |
| 1409727 | Nov 2005 | EP |
| 1272668 | Feb 2007 | EP |
| 1754788 | Feb 2007 | EP |
| 1757694 | Feb 2007 | EP |
| 1409745 | Apr 2007 | EP |
| 1754788 | Apr 2007 | EP |
| 1770171 | Apr 2007 | EP |
| 1313882 | May 2007 | EP |
| 1803822 | Jul 2007 | EP |
| 951645 | Aug 2007 | EP |
| 1813681 | Aug 2007 | EP |
| 1832661 | Sep 2007 | EP |
| 1757694 | Feb 2008 | EP |
| 2161347 | Mar 2010 | EP |
| 2161347 | Jun 2010 | EP |
| 2366801 | Sep 2011 | EP |
| 2423334 | Feb 2012 | EP |
| 2548972 | Jan 2013 | EP |
| 2589668 | May 2013 | EP |
| 1981995 | Jul 2013 | EP |
| WO 199006509 | Jun 1990 | WO |
| WO 199107660 | May 1991 | WO |
| WO 1991016452 | Oct 1991 | WO |
| WO 199322053 | Nov 1993 | WO |
| WO 199429707 | Dec 1994 | WO |
| WO 199509245 | Apr 1995 | WO |
| WO 199746882 | Dec 1997 | WO |
| WO 199802528 | Jan 1998 | WO |
| WO 199810267 | Mar 1998 | WO |
| WO 9839474 | Sep 1998 | WO |
| WO 9922868 | May 1999 | WO |
| WO 199944064 | Sep 1999 | WO |
| WO 1999061888 | Dec 1999 | WO |
| WO 0006770 | Feb 2000 | WO |
| WO 0040750 | Jul 2000 | WO |
| WO 200062931 | Oct 2000 | WO |
| WO 200135071 | May 2001 | WO |
| WO 200151668 | Jul 2001 | WO |
| WO 1999061888 | Dec 2001 | WO |
| WO 200135071 | Feb 2002 | WO |
| WO 2002012896 | Feb 2002 | WO |
| WO 2002028523 | Apr 2002 | WO |
| WO 2002030562 | Apr 2002 | WO |
| WO 200231506 | Apr 2002 | WO |
| WO 200244318 | Jun 2002 | WO |
| WO 2002073204 | Sep 2002 | WO |
| WO 2003003057 | Jan 2003 | WO |
| WO 03020986 | Mar 2003 | WO |
| WO 2003018757 | Mar 2003 | WO |
| WO 2003019141 | Mar 2003 | WO |
| WO 2003020974 | Mar 2003 | WO |
| WO 2003023057 | Mar 2003 | WO |
| WO 2003031938 | Apr 2003 | WO |
| WO 03044217 | May 2003 | WO |
| WO 2003035894 | May 2003 | WO |
| WO 2003035895 | May 2003 | WO |
| WO 2003044224 | May 2003 | WO |
| WO 2003048295 | Jun 2003 | WO |
| WO 2003069421 | Aug 2003 | WO |
| WO 2003018757 | Sep 2003 | WO |
| WO 2003020974 | Sep 2003 | WO |
| WO 03044217 | Oct 2003 | WO |
| WO 2002073204 | Oct 2003 | WO |
| WO 2003031938 | Nov 2003 | WO |
| WO 2003093795 | Nov 2003 | WO |
| WO 2003023057 | Dec 2003 | WO |
| WO 2003069421 | Dec 2003 | WO |
| WO 2003035895 | Jan 2004 | WO |
| WO 2003035894 | Mar 2004 | WO |
| WO 2004025251 | Mar 2004 | WO |
| WO 2003019141 | Apr 2004 | WO |
| WO 2004029221 | Apr 2004 | WO |
| WO 2004029221 | May 2004 | WO |
| WO 2004037374 | May 2004 | WO |
| WO 2004044236 | May 2004 | WO |
| WO 2004056978 | Jul 2004 | WO |
| WO 2004065629 | Aug 2004 | WO |
| WO 2004076643 | Sep 2004 | WO |
| WO 2003093795 | Oct 2004 | WO |
| WO 2004037374 | Oct 2004 | WO |
| WO 2004088310 | Oct 2004 | WO |
| WO 2004025251 | Nov 2004 | WO |
| WO 2004101762 | Nov 2004 | WO |
| WO 2004113877 | Dec 2004 | WO |
| WO 2004101762 | Feb 2005 | WO |
| WO 2005023091 | Mar 2005 | WO |
| WO 2005028663 | Mar 2005 | WO |
| WO 2005035725 | Apr 2005 | WO |
| WO 2005042713 | May 2005 | WO |
| WO 2005043121 | May 2005 | WO |
| WO 2005047529 | May 2005 | WO |
| WO 2005047532 | May 2005 | WO |
| WO 2005023091 | Jun 2005 | WO |
| WO 2005049168 | Jun 2005 | WO |
| WO 2005058937 | Jun 2005 | WO |
| WO 2005061075 | Jul 2005 | WO |
| WO 2005049168 | Sep 2005 | WO |
| WO 2005084374 | Sep 2005 | WO |
| WO 2005084380 | Sep 2005 | WO |
| WO 2005085476 | Sep 2005 | WO |
| WO 2005085861 | Sep 2005 | WO |
| WO 2005098046 | Oct 2005 | WO |
| WO 2005108621 | Nov 2005 | WO |
| WO 2005109238 | Nov 2005 | WO |
| WO 2005028663 | Dec 2005 | WO |
| WO 2005098046 | Dec 2005 | WO |
| WO 2005116264 | Dec 2005 | WO |
| WO 2005118852 | Dec 2005 | WO |
| WO 2005121362 | Dec 2005 | WO |
| WO 2005085861 | Feb 2006 | WO |
| WO 2006010610 | Feb 2006 | WO |
| WO 2005118852 | Mar 2006 | WO |
| WO 2006023563 | Mar 2006 | WO |
| WO 2005121362 | Apr 2006 | WO |
| WO 2006041453 | Apr 2006 | WO |
| WO 2006043181 | Apr 2006 | WO |
| WO 2005109238 | Jun 2006 | WO |
| WO 2006010610 | Jun 2006 | WO |
| WO 2006043181 | Jun 2006 | WO |
| WO 2006076567 | Jul 2006 | WO |
| WO 2006078470 | Jul 2006 | WO |
| WO 2005043121 | Aug 2006 | WO |
| WO 2006076567 | Sep 2006 | WO |
| WO 2006078470 | Sep 2006 | WO |
| WO 2006097049 | Sep 2006 | WO |
| WO 2006100366 | Sep 2006 | WO |
| WO 2005042713 | Nov 2006 | WO |
| WO 2006023563 | Nov 2006 | WO |
| WO 2006120434 | Nov 2006 | WO |
| WO 2005084380 | Dec 2006 | WO |
| WO 2005116264 | Feb 2007 | WO |
| WO 2007020081 | Feb 2007 | WO |
| WO 2004076643 | Mar 2007 | WO |
| WO 2007024264 | Mar 2007 | WO |
| WO 2007028146 | Mar 2007 | WO |
| WO 2007030949 | Mar 2007 | WO |
| WO 2007033167 | Mar 2007 | WO |
| WO 2007034221 | Mar 2007 | WO |
| WO 2007035414 | Mar 2007 | WO |
| WO 2007024264 | Apr 2007 | WO |
| WO 2007036025 | Apr 2007 | WO |
| WO 2007038264 | Apr 2007 | WO |
| WO 2007041610 | Apr 2007 | WO |
| WO 2007044091 | Apr 2007 | WO |
| WO 2007044690 | Apr 2007 | WO |
| WO 2007048076 | Apr 2007 | WO |
| WO 2007030949 | May 2007 | WO |
| WO 2007034221 | May 2007 | WO |
| WO 2007050495 | May 2007 | WO |
| WO 2007053142 | May 2007 | WO |
| WO 2007053648 | May 2007 | WO |
| WO 2007053785 | May 2007 | WO |
| WO 2007059430 | May 2007 | WO |
| WO 2007062222 | May 2007 | WO |
| WO 2005058937 | Jun 2007 | WO |
| WO 2007067734 | Jun 2007 | WO |
| WO 2007048076 | Jul 2007 | WO |
| WO 2007053648 | Jul 2007 | WO |
| WO 2007075836 | Jul 2007 | WO |
| WO 2007075879 | Jul 2007 | WO |
| WO 2007076989 | Jul 2007 | WO |
| WO 2007079229 | Jul 2007 | WO |
| WO 2007079250 | Jul 2007 | WO |
| WO 2007080583 | Jul 2007 | WO |
| WO 2007082144 | Jul 2007 | WO |
| WO 2007082154 | Jul 2007 | WO |
| WO 2007082379 | Jul 2007 | WO |
| WO 2007050495 | Aug 2007 | WO |
| WO 2007075879 | Aug 2007 | WO |
| WO 2007087612 | Aug 2007 | WO |
| WO 2007089880 | Aug 2007 | WO |
| WO 2007089911 | Aug 2007 | WO |
| WO 2007090670 | Aug 2007 | WO |
| WO 2007092473 | Aug 2007 | WO |
| WO 2007092713 | Aug 2007 | WO |
| WO 2007098484 | Aug 2007 | WO |
| WO 2006100366 | Sep 2007 | WO |
| WO 2007100684 | Sep 2007 | WO |
| WO 2007100911 | Sep 2007 | WO |
| WO 2007101609 | Sep 2007 | WO |
| WO 2007103910 | Sep 2007 | WO |
| WO 2007033167 | Oct 2007 | WO |
| WO 2007038264 | Oct 2007 | WO |
| WO 2007044690 | Oct 2007 | WO |
| WO 2007053785 | Oct 2007 | WO |
| WO 2007059430 | Oct 2007 | WO |
| WO 2005084374 | Nov 2007 | WO |
| WO 2007035414 | Nov 2007 | WO |
| WO 2007044091 | Nov 2007 | WO |
| WO 2007089880 | Nov 2007 | WO |
| WO 2007100911 | Nov 2007 | WO |
| WO 2007126938 | Nov 2007 | WO |
| WO 2007132166 | Nov 2007 | WO |
| WO 2007132167 | Nov 2007 | WO |
| WO 2007082379 | Dec 2007 | WO |
| WO 2007098484 | Dec 2007 | WO |
| WO 2007147018 | Dec 2007 | WO |
| WO 2007147073 | Dec 2007 | WO |
| WO 2007147074 | Dec 2007 | WO |
| WO 2007147076 | Dec 2007 | WO |
| WO 2007147079 | Dec 2007 | WO |
| WO 2007062222 | Jan 2008 | WO |
| WO 2007100684 | Jan 2008 | WO |
| WO 2007075836 | Feb 2008 | WO |
| WO 2007132166 | Feb 2008 | WO |
| WO 2008017871 | Feb 2008 | WO |
| WO 2008045158 | Apr 2008 | WO |
| WO 2007089911 | May 2008 | WO |
| WO 2007132167 | May 2008 | WO |
| WO 2007028146 | Jun 2008 | WO |
| WO 2007067734 | Aug 2008 | WO |
| WO 2008111990 | Sep 2008 | WO |
| WO 2007126938 | Oct 2008 | WO |
| WO 2007082154 | Nov 2008 | WO |
| WO 2007087612 | Nov 2008 | WO |
| WO 2007092473 | Nov 2008 | WO |
| WO 2007082144 | Dec 2008 | WO |
| WO 2007092713 | Dec 2008 | WO |
| WO 2007079229 | Jan 2009 | WO |
| WO 2009013492 | Jan 2009 | WO |
| WO 2009013496 | Jan 2009 | WO |
| WO 2007080583 | Feb 2009 | WO |
| WO 2009019455 | Feb 2009 | WO |
| WO 2007079250 | Mar 2009 | WO |
| WO 2007041610 | Apr 2009 | WO |
| WO 2009019455 | Apr 2009 | WO |
| WO 2010045617 | Apr 2010 | WO |
| WO 2011094646 | Aug 2011 | WO |
| WO 2011102998 | Aug 2011 | WO |
| Entry |
|---|
| Illumina Technical Note: Reproductive Health, pp. 1-5 (2014). |
| Iontorrent Application Note, pp. 1-6 (2016). |
| Hua, R. et al., Prenatal Diagnosis, vol. 34, pp. 1-8 (2014). |
| Breman, A.M. et al., Prenatal Diagnosis, vol. 36, pp. 1009-1019 (2016). |
| Fiddler, M., J. Clin. Med., vol. 3, pp. 972-985 (2014). |
| Hatt, L. et al., Prenatal Diagn., vol. 34, pp. 1-7 (2014). |
| Christensen, B. et al., Fetal Diagn. Ther., vol. 18, pp. 479-484 (2003). |
| Karow, J., “following Improvements in Noninvasive Fetal Cell Isolation, First Prenatal Tests Expected in 2016”, published on genomeweb (www.genomeweb.com/archive/following-improvements-noninvasive-fetal-cell-isolation-first-prenatal-tests-expected-2016; pp. 1-4; (Nov. 2015). |
| Wang, Y. et al., Mol. Cell, vol. 58, pp. 598-609 (2015). |
| Bischoff, F.Z. et al., Clin. Genet., vol. 63, pp. 483-489 (2003). |
| Geifman-Holtzman, O. et al., Am. J. Obstet. Gynecol., vol. 183, pp. 462-468 (2000). |
| Vona, G. et al., Am. J. Pathol., vol. 160, pp. 51-58 (2002). |
| Office action dated Jul. 29, 2014 for U.S. Appl. No. 13/835,926. |
| Office action dated Aug. 29, 2014 for U.S. Appl. No. 13/837,974. |
| Office action dated Sep. 17, 2014 for U.S. Appl. No. 13/863,992. |
| U.S. Appl. No. 60/764,420, filed Feb. 2, 2005, Quake. |
| U.S. Appl. No. 60/949,227, filed Jul. 11, 2007, Kapur. |
| U.S. Appl. No. 11/825,677, filed Jul. 5, 2007, Lopez et al. |
| U.S. Appl. No. 11/909,959, filed Sep. 27, 2007, Duff. |
| U.S. Appl. No. 13/794,503, filed Mar. 11, 2013, Stoughton et al. |
| 717.305 pending claims dated Jan. 10, 2013 for U.S. Appl. No. 13/738,268. |
| Adams, et al. Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project. Science Jun. 21, 1991: vol. 252 No. 5013 pp. 1651-1656 DOI: 10.1126/science.2047873. |
| Adinolfi, et al. Gene Amplification to Detect Fetal Nucleated Cells in Pregnant Women. The Lancet. Aug. 5, 1989:328-329. |
| Adinolfi, et al. Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction. Prenat. Diagn. 1997; 17(13):1299-311. |
| Adinolfi, M. On a Non-Invasive Approach to Prenatel Diagnosis based on the detection of Fetal Nucleated Cells in Maternal Blood Samples. Prenatal Diagnosis. 1991;11:799-804. |
| Advisory action dated Dec. 16, 2013 for U.S. Appl. No. 12/751,940. |
| Ahn, et al. A fully integrated micromachined magnetic particle separator. Journal of Microelectromechanical Systems. 1996; 5(3):151-158. |
| Allard, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. Oct. 15, 2004;10(20):6897-904. |
| Allowed claims dated Dec. 9, 2010 for U.S. Appl. No. 11/701,686. |
| Amendments to the Claims Filed Mar. 29, 2013 with U.S. Patent Office for U.S. Appl. No. 12/751,940. |
| Amendments to the Claims. Filed Aug. 14, 2013 with U.S. Patent Office for U.S. Appl. No. 13/737,730. |
| Amendments to the Claims. Filed Jul. 11, 2013 with U.S. Patent Office for U.S. Appl. No. 13/831,342. |
| Amendments to the Claims. Filed Jul. 22, 2013 with U.S. Patent Office for U.S. Appl. No. 13/794,503. |
| Amendments to the Claims. Filed Jul. 25, 2013 with U.S. Patent Office for U.S. Appl. No. 13/829,971. |
| Amendments to the Claims. Filed Mar. 15, 2013 with U.S. Patent Office for U.S. Appl. No. 13/835,926. |
| Amendments to the Claims. Filed Mar. 15, 2013 with U.S. Patent Office for U.S. Appl. No. 13/837,974. |
| Amendments to the Claims. Filed Nov. 7, 2013 with U.S. Patent Office for U.S. Appl. No. 13/863,992. |
| Andrews, et al. Enrichment of fetal nucleated cells from maternal blood: model test system using cord blood. Prenatal Diagnosis. 1995; 15:913-919. |
| Applicant's Amendment and Response dated Jun. 17, 2009 to Non-Final Office Action dated Jan. 28, 2009 re U.S. Appl. No. 11/701,686. |
| Applicant's Amendment and Response dated Jun. 24, 2010 to Office Action dated Jan. 27, 2010 re U.S. Appl. No. 11/701,686. |
| Applicant's Amendment and Response dated Nov. 13, 2009 to Office Action dated Sep. 11, 2009 re U.S. Appl. No. 11/701,686. |
| Applicant's response dated Jun. 10, 2011 to Office action dated Apr. 25, 2011 for U.S. Appl. No. 12/393,803. |
| Ariga, et al. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion. 2001; 41:1524-1530. |
| Arnould, et al. Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer. Br J Cancer. 2003; 88(10):1587-91. (Abstract only). |
| Babochkina, et al. Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe. Arch Gynecol Obstet. Dec. 2005;273(3):166-9. (Abstract only). |
| Babochkina, T. I. Ph. D. Dissertation—Fetal cells in maternal circulation: Fetal cell separation and FISH analysis. University of Basel, Switzerland. Dec. 8, 2005. (123 pages). |
| Balko, et al. Gene expression patterns that predict sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer cell lines and human lung tumors. BMC Genomics. Nov. 10, 2006;7:289 (14 pages). |
| Barrett, et al. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proc Natl Acad Sci U S A. 2004; 101(51):17765-70. |
| Basch, et al. Cell separation using positive immunoselective techniques. Journal of Immunological Methods. 1983;56:269-280. |
| Bauer, J. Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation. Journal of Chromatography. 1999;722:55-69. |
| Becker, et al. Fabrication of Microstructures With High Aspect Ratios and Great Structural Heights by Synchrotron Radiation Lithography, Galvanoforming, and Plastic Moulding (LIGA Process). Microelectronic Engineering. 1986;4:35-56. |
| Becker, et al. Planar quartz chips with submicron channels for two-dimensional capillary electrophoresis applications. J. Micromech Microeng.1998;9:24-28. |
| Beebe et al. Functional Hydrogel Structures for Autonomous Flow Control Inside Microfluidic Channels. Nature. 2000; 404:588-590. |
| Bennett, et al. Toward the 1,000 dollars human genome. Pharmacogenomics. 2005; 6(4):373-82. |
| Berenson, et al. Cellular Immunoabsorption Using Monoclonal Antibodies. Transplantation.1984 ;38:136-143. |
| Berenson, et al. Positive selection of viable cell populations using avidin-biotin immunoadsorption. Journal of Immunological Methods. 1986;91:11-19. |
| Berg, H. C. Random Walks in Biology, Ch. 4. Princeton University Press. Princeton, NJ. 1993. pp. 48-64. |
| Berger, et al. Design of a microfabricated magnetic cell separator. Electrophoresis. Oct. 2001;22(18):3883-92. |
| Bianchi, et al. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Medical Sciences. 1990;87:3279-3283. |
| Bianchi, et al. Demonstration of fetal gene sequences in nucleated erythrocytes isolated from maternal blood. American Journal of Human Genetics. 1989;45:A252. |
| Bianchi, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. Prenatal Diagnosis. 2002; 22:609-615. |
| Bianchi, et al. Fetal nucleated erythrocytes (FNRBC) in maternal blood: erythroid-specific antibodies improve detection. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 996. |
| Bianchi, et al. Isolation of Male Fetal DNA from Nucleated Erythrocytes (NRBC) in Maternal Blood. The American Pediatric Society and Society for Pediatric Research, (1989) Mar. 1989; 818:139A. |
| Bianchi, et al. Possible Effects of Gestational Age on the Detection of Fetal Nucleated Erythrocytes in Maternal Blood. Prenatal Diagnosis. 1991;11:523-528. |
| Bignell, et al. High-resolution analysis of DNA copy number using oligonucleotide microarrays. Genome Research. 2004; 14(2):287-295. |
| Binladen, et al. The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing. Feb. 2007, PLoS One. 2(2):e197, doi:10.1371/journal.pone.0000197. |
| Blake, et al. Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol. Hum. Reprod. 1999; 5(12):1166-75. |
| Bode, et al. Mutations in the tyrosine kinase domain of the EGFR gene are rare in synovial sarcoma. Mod Pathol. Apr. 2006;19(4):541-7. |
| Boehm, et al. Analysis of Defective Dystrophin Genes with cDNA Probes: Rearrangement Polymorphism, Detection of Deletions in Carrier Females, and Lower Than Expected Frequency of Carrier Mothers in Isolated Cases of Delections. Pediatric Research. Apr. 1989: 139A-820. |
| Bohmer, et al. Differential Development of Fetal and Adult Haemoglobin Profiles in Colony Culture: Isolation of Fetal Nucleated Red Cells by Two-Colour Fluorescence Labelling. Br. J. Haematol. 1998; 103:351-60. |
| Bookout, et al. High-throughput real-time quantifative reverse transcription PCR. Curr. Prot. Mol. Biol. 2005; 15.8.1-15.8.21. |
| Braslavsky, et al. “Sequence information can be obtained from single DNA molecules,” PNAS, Apr. 2003, vol. 100, No. 7, 3960-3964. |
| Brison, et al. General Method for Cloning Amplified DNA by Differential Screening with Genomic Probes. Molecular and Cellular Biology. 1982;2:578-587. |
| Brody, et al. Deformation and Flow of Red Blood Cells in a Synthetic Lattice: Evidence for an Active Cytoskeleton. Biophys. J. 68:2224-2232 (1995). |
| Brown, et al. Applicant-Initiated Interview Summary, with Office Action Summary, and Information Disclosure Statement by Applicant. dated Sep. 16, 2009. |
| Bustamante-Aragones, et al. Detection of a Paternally Inherited Fetal Mutation in Maternal Plasma by the Use of Automated Sequencing. Ann. N.Y. Acad. Sci. 1075: 108-117 (2006), pp. 108-117, XP-002652985. |
| Caggana, M. Microfabricated devices for sparse cell isolation. CNF Project #905-00. Cornell NanoScale Facility. 2003; pp. 38-39. |
| Caggana, M. Microfabricated devices for sparse cell isolation. CNF Project #905-00. Cornell NanoScale Facility. 2004-2005; pp. 32-33. |
| Calin, et al. A microRNA signature Associated with prognosis and progression in chronic lymphocytic leukemia. New England Journal of Medicine. 2005; 353:1793-1801. |
| Cappuzzo, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst. May 4, 2005;97(9):643-55. |
| Cha, The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood. Prenat. Diagn. 2005; 25(7):586-91. |
| Chamberlain, et al. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research. 1988;16:11141-11156. |
| Chan, et al. “DNA Mapping Using Microfluidic Stretching and Single-Molecule Detection of Fluorescent Site-Specific Tags,” Genome Research, 2004, vol. 14, 1137-1146. |
| Chan, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. Jan. 2004;50(1):88-92. |
| Chang, et al. Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel. Lab Chip. 2005; 5:64-73. |
| Cheung, et al. Development and validation of a CGH microarray for clinical cytogenetic diagnosis. Genet Med. 2005; 7(6):422-32. |
| Chiu, et al. “Effects of Blood-Processing Protocols on Fetal and Total DNA Quantification in Maternal Plasma,” Clinical Chemistry, 2001, vol. 47, No. 9, 1607-1613. |
| Chiu, et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ. Jan. 11, 2011;342:c7401. doi: 10.1136/bmj.c7401. |
| Chiu, et al. Non-invasive prenatal diagnosis by single molecule counting technologies. Trends in Genetics, vol. 25, Issue 7, Jul. 2009, pp. 324-331. |
| Chiu, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A. Dec. 23, 2008;105(51):20458-63. |
| Chiu, et al. Patterned Deposition of Cells and Proteins Onto Surfaces by Using Three-Dimensional Microfluidic Systems. Proceedings of the National Academy of Sciences of the United States of America. 2000; pp. 2408-2413. |
| Choesmel, et al. Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance. Breast Cancer Res. 2004;6(5):R556-569. |
| Choolani, et al. Characterization of First Trimester Fetal Erythroblasts for Non-Invasive Prenatal Diagnosis. Mol. Hum. Reprod. 2003; 9:227-35. |
| Chou, et al. A Microfabricated Device for Sizing and Sorting DNA Molecules. Proceedings of the National Academy of Sciences of the United States of America. 1999; pp. 11-13. |
| Chou, et al. Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation. PNAS. 1999; 96(24):13762-13765. |
| Christel, et al. High aspect ratio silicon microstructures for nucleic acid extraction. Solid-state sensor and actuator workshop. Hilton Head, SC. Jun. 8-11, 1998; 363-366. |
| Christensen, et al. Fetal Cells in Maternal Blood: A Comparison of Methods for Cell Isolation and Identification. Fetal Diagn. Ther. 2005; 20:106-12. |
| Chueh, et al. Prenatal Diagnosis Using Fetal Cells from the Maternal Circulation. West J. Med. 159:308-311 (1993). |
| Chueh, et al. Prenatal Diagnosis Using Fetal Cells in the Maternal Circulation. Seminars in Perinatology. 1990;14:471-482. |
| Chueh, et al. The search for fetal cells in the maternal circulation. J Perinat Med. 1991;19:411-420. |
| Cirigliano, et al. “Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies,” Molecular Human Reproduction, 2001, vol. 7, No. 10, 1001-1006. |
| Claims mailed with RCE Response to Final Rejection dated Dec. 31, 2009 for U.S. Appl. No. 11/763,421, filed Jun. 14, 2007 (6 pages). |
| Claims. Filed Nov. 29, 2011 with U.S. Patent Office for U.S. Appl. No. 13/306,520. |
| Claims. Filed Nov. 29, 2011 with U.S. Patent Office for U.S. Appl. No. 13/306,640. |
| Claims. Filed Nov. 29, 2011 with U.S. Patent Office for U.S. Appl. No. 13/306,698. |
| Clayton, et al. Fetal Erythrocytes in the Maternal Circulation of Pregnant Women. Obstetrics and Gynecology. 1964;23:915-919. |
| Collarini, et al. Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood. Cytometry. 2001; 45:267-276. |
| Cotton, et al. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A. Jun. 1988;85(12):4397-401. |
| Craig, et al. Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods. Oct. 2008 ; 5(10): 887-893. DOI:10.1038/nmeth.1251. |
| Cremer, et al. Detection of chromosome aberrations in human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Human Genetics.1986;74:346-352. |
| Cremer, et al. Detection of chromosome aberaations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Human Genetics.1988;80:235-246. |
| Cristofanilli, et al. Circulating tumor cells revisited. JAMA. 2010; 303(11):1092-1093. |
| Cristofanilli, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. Aug. 19, 2004;351(8):781-91. |
| Das, et al. Dielectrophoretic segregation of different human cell types on microscope slides. Anal. Chem. 2005; 77:2708-2719. |
| De Alba, et al. Prenatal diagnosis on fetal cells obtained from maternal peripheral blood: report of 66 cases. Prenat Diagn. Oct. 1999;19(10):934-40. |
| De Kretser, et al. The Separation of Cell Populations using Monoclonal Antibodies attached to Sepharose. Tissue Antigens. 1980;16:317-325. |
| De Luca, et al. Detection of circulating tumor cells in carcinoma patients by a novel epidermal growth factor receptor reverse transcription—PCR assay. Clin Cancer Res. Apr. 2000;6(4):1439-44. |
| Decision re: Institution of Inter Partes Review 37 C.F.R. § 42.108. Entered Oct. 25, 2013. For Case IPR2013-00277. U.S. Pat. No. 8,318,430. |
| Decision: Institution of Inter Partes Review 37 C.F.R. § 42.108. Dated . Entered Oct. 25, 2013. For Case IPR2013-00276. U.S. Pat. No. 8,318,430 B2. |
| Declaration of Cynthia Casson Morton. Dated May 29, 2013. U.S. Pat. No. 8,296,076. |
| Declaration of Cynthia Casson Morton. Dated May 10, 2013. In re: Chuu, et al., U.S. Pat. No. 8,318,430. |
| Declaration of Cynthia Casson Morton. Dated May 10, 2013. In re: Chuu, et al., U.S. Pat. No. 8,318,430, claims 1-18. |
| Declaration of Robert Nussbaum. Dated May 22, 2013. U.S. Pat. No. 8,296,076. |
| Declaration of Robert Nussbaum. Dated May 8, 2013. In re Patent of: Chuu, et al. U.S. Pat. No. 8,318,430. |
| Declaration of Robert Nussbaum. Dated Apr. 16, 2013. In re Patent of: Fan, et al. U.S. Pat. No. 8,318,430, claims 1-18. |
| Delamarche, et al. Microfluidic Networks for Chemical Patterning of Substrates: Design and Application to Bioassays. Journal of the American Chemical Society. 1998; 120:500-508. |
| Delamarche, et al. Patterned Delivery of Immunoglobulins to Surfaces Using Microfluidic Networks. Science. 1997; 276:779-781. |
| Deng, et al. Enumeration and microfluidic chip separation of circulating fetal cells early in pregnancy from maternal blood. American Journal of Obstetrics & Gynecology. Dec. 2008 (vol. 199, Issue 6, p. S134). |
| Deshmukh, et al. Continuous Micromixer With Pulsatile Micropumps. Solid-State Sensor and Actuator Workshop. Hilton Head Island, South Carolina; Jun. 4-8, 2000:73-76. |
| Devotek. “Separation of RNA 8 DNA by Gel Filtration Chromatography,” Edvotek, 1987. 1-9. |
| Dhallan, et al. A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study. The Lancet, vol. 369, Issue 9560, Feb. 10, 2007, pp. 440-442. |
| Di Naro, et al. Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod. 2000; 6(6):571-4. |
| Diehl, et al. Digital quantification of mutant DNA in cancer patients. Curr Opin Oncol. Jan. 2007;19(1):36-42. |
| Dilella, et al. Screening for Phenylketonuria Mutations by DNA Amplification with the Polymerase Chain Reaction. The Lancet. Mar. 5, 1988:497-499. |
| Dohm, et al. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Research. 2008. 36: e105 doi: 10.1093\nark\gkn425. |
| Doyle, et al. Self-Assembled Magnetic Matrices for DNA Separation Chips. Science 295:2237 (2002). |
| Dragovich, et al. Anti-EGFR-targeted therapy for exophageal and gastric cancers: an evolving concept. Jornal of Oncology. 2009; vol. 2009, Article ID 804108. |
| Dressman, et al. “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations.” PNAS, Jul. 2003, vol. 100. No. 15, 8817-8822. |
| Drmanac, et al. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing. Science Jun. 11, 1993: vol. 260 No. 5114 pp. 1649-1652 DOI: 10.1126/science.8503011. Jun. 11, 1993. |
| Eigen, et al. Sorting Single Molecules: Application to Diagnostics and Evolutionary Biotechnology. Proceedings of the National Academy of Sciences of the United States of America. 1994; 91:5740-5747. |
| Emanuel, et al. Amplification of Specific Gene Products from Human Serum. GATA, 1993, vol. 10, No. 6, 144-146. |
| European office action dated Jun. 26, 2012 for EP Application No. 11159371.1. |
| European office action dated Jul. 17, 2013 for EP Application No. 11159371.1, 8 pages. |
| European office action dated Oct. 8, 2012 for EP Application No. 11175845. |
| European office action dated Dec. 13, 2013 for EP Application No. 11159371.1. |
| European office action dated Dec. 16, 2013 for EP Application No. 12175907. |
| European office action dated Dec. 18, 2012 for EP Application No. 11159371.1. |
| European Search Opinion dated Jul. 31, 2009 for EP07763674.4. |
| European Search Report Office action dated Dec. 21, 2010 for EP07763674.4. |
| European search report and search opinion dated Jan. 2, 2013 for EP Application No. 12175907.0. |
| European search report and search opinion dated Mar. 16, 2012 for EP Application No. 11182181. |
| European search report and search opinion dated Apr. 9, 2013 for EP Application No. 12180149.2. |
| European search report and search opinion dated Nov. 17, 2011 for EP Application No. 11175845. |
| European Search Report dated Jul. 31, 2009 for EP07763674.4. |
| European search report dated Nov. 9, 2009 for Application No. 7784442.1. |
| European search report dated Dec. 21, 2009 for Application No. 07798579.4. |
| European search report dated Dec. 22, 2009 for Application No. 07798580.2. |
| European search report dated Dec. 22, 2009 for Application No. 07784444.7. |
| Applicant's Response with Allowed Claims dated Dec. 2, 2010 issued in U.S. Appl. No. 11/701,686. |
| Pending Claims filed with the USPTO on Jun. 24, 2010 for U.S. Appl. No. 11/701,686. |
| Extended European Search Report for Application No. 11159371 dated Aug. 10, 2011, 10 pages. |
| Falcidia, et al. Fetal Cells in maternal blood: a six-fold increase in women who have undergone mniocentesis and carry a fetus with Down syndrome: a multicenter study. Neuropediatrics. 2004; 35(6):321-324. (Abstract only). |
| Fan, et al. Detection of aneuploidy with digital polymerase chain reaction. Anal Chem. Oct. 1, 2007; 79(19):7576-9. |
| Fan, et al. Highly parallel SNP genotyping. Cold Spring Harb. Symp. Quant. Biol. 2003; 68:69-78. |
| Fan, et al. Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy. Am J Obstet Gynecol. May 2009;200(5):543.e1-7. |
| Fan, et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. Oct. 21, 2008;105(42):16266-71. |
| Fan, et al. Single cell degenerate oligonucleotide primer-PCR and comparative genomic hybridization with modified control reference. Journal of Ahejian University—Science A. 2001; 2(3):318-321. |
| Farber, et al. Demonstration of spontaneous XX/XY chimerism by DNA fingerprinting. Human Genetics. 1989;82:197-198. |
| Farooqui, et al. Microfabrication of Submicron Nozzles in Silicon Nitride. Journal of Microelectromechanical Systems. 1992; 1(2):86-88. |
| Feinberg, et al. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. Jul. 1, 1983;132(1):6-13. |
| Fiedler, et al. Dielectrophoretic Sorting of Particles and Cells in a Microsystem. Analytical Chemistry. 1998; pp. 1909-1915. |
| Findlay, et al. Using MF-PCR to diagnose multiple defects from single cells: implications for PGD. Mol Cell Endocrinol. 2001; 183 Suppl 1:S5-12. |
| Fleischmann, et al. Whole-Genome Random Sequencing and Assembly of Haemophilus influenzae Rd. Science Jul. 28, 1995: vol. 269 No. 5223 pp. 496-512 DOI: 10.1126/science.7542800. |
| Freemantle, M. Downsizing Chemistry. Chemical analysis and synthesis on microchips promise a variety of potential benefits. Chemical & Engineering News. 1999; pp. 27-36. |
| Fu, et al. An integrated miscrofabricated cell sorter. Anal Chem. 2002;74:2451-2457. |
| Fu, et al. A Microfabricated Fluorescence-Activated Cell Sorter. Nature Biotechnology.1999; 17:1109-1111. |
| Fuhr, et al. Biological Application of Microstructures. Topics in Current Chemistry. 1997; 194:83-116. |
| Fullwood, et al. Next Generation DNA sequencin of paired-end tags (PET) for transcriptome and genome analyses. Genome Research. 2009. 19:521-532. |
| Furdui, et al. Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems. Lab Chip. Dec. 2004;4(6):614-8. |
| Ganshirt-Ahlert, et al. Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol. 1992;166:1350-1355. |
| Ganshirt-Ahlert, et al. Noninvasive prenatal diagnosis: Triple density gradient, magnetic activated cell sorting and FISH prove to be an efficient and reproducible method for detection of fetal aneuploidies from maternal blood. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 182. |
| Gardella, et al. Second Petition for Inter Partes Review Under 35 U.S.C. §§ 311-319 and 37 C.F.R. § 42.100 ET SEQ.(Claims 19-30). Dated May 10, 2013, for U.S. Pat. No. 8,318,430. |
| Gardella, G. First Petition for Inter Partes Review Under 35 U.S.C. §§ 311-319 and 37 C.F.R. § 42.100 ET SEQ. (Claims 1-18). Dated May 10, 2013. U.S. Pat. No. 8,318,430. |
| Gardella, G. Petition for Inter Partes Review Under 35 U.S.C. §§ 311-319 and 37 C.F.R. § 42.100 ET SEQ. Dated May 24, 2013. U.S. Pat. No. 8,296,076. |
| GenomeWeb. Immunicon inks biomarker assay, lab services deal with merck serona. Available at C:\Documents and Settings\fc3\Local Settings\Temporary Internet Files\OLK35E\141896-1.htm. Accessed on Sep. 11, 2007. |
| Ghia, et al. Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci. J Exp Med. Dec. 1, 1996;184(6):2217-29. |
| Giddings, J. C. Chemistry ‘Eddy’ Diffusion in Chromatography. Nature. 1959;184:357-358. |
| Giddings, J. C. Field-Flow Fractionation: Analysis of Macromolecular, Colloidal, and Particulate Materials. Science. 1993;260:1456-1465. |
| Gonzalez, et al. Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environ Microbiol. 2005; 7(7):1024-8. |
| Graham. Efficiency comparison of two preparative mechanisms for magnetic separation of erthrocytes from whole blood. J. Appl. Phys. 1981; 52:2578-2580. |
| Greaves, et al. Expression of the OKT Monoclonal Antibody Defined Antigenic Determinants in Malignancy. Int. J. Immunopharmac. 1981;3:283-299. |
| Guetta, et al. Analysis of fetal blood cells in the maternal circulation: challenges, ongoing efforts, and potential solutions. Stem Cells Dev. 2004;13(1):93-9. |
| Gunderson, et al. A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet. 2005; 37(5):549-54. |
| Hahn, et al. “Prenatal Diagnosis Using Fetal Cells and Cell-Free Fetal DNA in Maternal Blood: What is Currently Feasible?” Clinical Obstetrics and Gynecology, Sep. 2002, vol. 45, No. 3, 649-656. |
| Hahn, et al. Current applications of single-cell PCR. Cell. Mol. Life Sci. 2000; 57(1):96-105. Review. |
| Hahn, et al. Micro system for isolation of fetal DNA from maternal plasma by preparative size separation. Clin Chem. Dec. 2009;55(12):2144-52. doi: 10.1373/clinchem.2009.127480. Epub Oct. 1, 2009. |
| Hamabe, et al. Molecular study of the Prader-Willi syndrome: deletion, RFLP, and phenotype analyses of 50 patients. Am J Med Genet. Oct. 1, 1991;41(1):54-63. |
| Han, et al. Separation of Long DNA Molecules in a Microfabricated Entropic Trap Array. Science. 2000;288:1026-1029. |
| Hardenbol, et al. Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 2005;15(2):269-75. |
| Hardenbol, et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 2003; 21(6):673-8. |
| Harris, et al. Single-molecule DNA sequencing of a viral genome. Science. Apr. 4, 2008;320(5872):106-9. |
| Hartmann, et al. Gene expression profiling of single cells on large-scale oligonucleotide arrays. Nucleic Acids Research. 2006; 34(21): e143. (11 pages). |
| Herzenberg, et al. Fetal cells in the blood of pregnant women: Detection and enrichment by flourescence-activated cell sorting. Proc. Natl. Acad. Sci. 1979;76:1453-1455. |
| Holt, et al. The new paradigm of flow cell sequencing. DOI: 10.1101/gr.073262.107 Genome Res. 2008. 18: 839-846. |
| Holzgreve, et al. Fetal Cells in the Maternal Circulation. Journal of Reproductive Medicine. 1992;37:410-418. |
| Hong, et al. A nanoliter-scale nucleic acid processor with parallel architecture. Nat. Biotechnol. 2004; 22(4):435-9. |
| Hong, et al. Molecular biology on a microfluidic chip. Journal of Physics: Condensed Matter, 2006, vol. 18, S691-S701. |
| Hosono, et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. May 2003;13(5):954-64. |
| Hromadnikova, et al. “Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies.” Bio Med Central, May 2002, 1-5. |
| Huang, et al. A DNA prism for high-speed continuous fractionation of large DNA molecules. Nature Biotechnology. 2002;20:1048-1051. |
| Huang, et al. Continuous Particle Separation Through Deterministic Lateral Displacement. Science 304:987-90 (2004). |
| Huang, et al. Electric Manipulation of Bioparticles and Macromoledules on Microfabricated Electrodes. Analytical Chemistry. 2001; pp. 1549-1559. |
| Huang, et al. Role of Molecular Size in Ratchet Fractionation. 2002; 89(17):178301-1-178301-4. |
| Huh, et al. Gravity-driven microhydrodynamics-based cell sorter (microHYCS) for rapid, inexpensive, and efficient cell separation and size-profiling. 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnology in Medicine and Biology. Madison, Wisconsin USA; May 2-4, 2002:466-469. |
| Hviid T. In-Cell PCT method for specific genotyping of genomic DNA from one individual in a micture of cells from two individuals: a model study with specific relevance to prenatal diagnosis based on fetal cells in maternal blood. Molecular Diagnostics and Genetics. 2002; 48:2115-2123. |
| Hviid, T. In-cell polymerase chain reaction: strategy and diagnostic applications. Methods Mol Biol. 2006;336:45-58. |
| International preliminary report on patentability dated Oct. 29, 2008 for PCT/US2007/003209. |
| International search report and written opinion dated Mar. 16, 2010 for PCT Application No. US2009/57136. |
| International Search Report and Written Opinion dated Sep. 18, 2008 for PCT/US2007/003209. |
| International search report dated Jan. 16, 2008 for PCT Application No. US2007/71247. |
| International search report dated Jan. 25, 2008 for PCT Application No. US2007/71250. |
| International search report dated Nov. 15, 2007 for PCT Application No. US2007/71149. |
| International search report dated Nov. 26, 2007 for PCT Application No. US2007/71256. |
| International search report dated Feb. 25, 2008 for PCT Application No. US07/71148. |
| International search report dated Feb. 25, 2008 for PCT Application No. US2007/71248. |
| Ishikawa, et al. Allelic dosage analysis with genotyping microarrays. Biochem Biophys Res Commun. Aug. 12, 2005;333(4):1309-14. |
| Iverson, et al. Detection and Isolation of Fetal Cells From Maternal Blood Using the Flourescence-Activated Cell Sorter (FACS). Prenatal Diagnosis 1981;1:61-73. |
| Jan, et al. Fetal Erythrocytes Detected and Separated from Maternal Blood by Electronic Fluorescent Cell Sorter. Texas Rep Biol Med.1973;31:575. |
| Jeon, et al. Generation of Solution and surface Gradients Using Microfluidic Systems. Langmuir. 2000, pp. 8311-8316. |
| Jiang, et al. Genome amplification of single sperm using multiple displacement amplification. Nucleic Acids Res. 2005; 33(10):e91. (9 pages). |
| Jiang, et al. Old can be new again: HAPPY whole genome sequencing, mapping and assembly. Int J Biol Sci. 2009;5(4):298-303. Epub Apr. 15, 2009. |
| Joint Claim Construction and Prehearing Statement. Case: 12-cv-05501-SI; Dated May 3, 2013. |
| Kamholz, et al. Quantitative Analysis of Molecular Interaction in a Microfluidic Channel: the T-Sensor. Analytical Chemistry. 1999; pp. 5340-5347. |
| Kan, et al. Concentration of Fetal Red Blood Cells From a Mixture of Maternal and Fetal Blood by Anti-i Serum—An Aid to Prenatal Diagnosis of Hemoglobinopathies. Blood. 1974; 43:411-415. |
| Kartalov et al.: “Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis.”, Nucleic Acids Research, 2004, vol. 32, No. 9, 2004, pp. 2873-2879, XP-002652987. |
| Kasakov, et al. Extracellular DNA in the blood of pregnant women. Tsitologiia. 1995;37(3):232-6. (English translation only). |
| Kazakov, et al. Extracellular DNA in the blood of pregnant women Tsitologiia. 1995; 37(3): 232-6. |
| Kenis, et al. Microfabrication Inside Capillaries Using Multiphase Laminar Flow Patterning. Science. 1999; 285:83-85. |
| Kim, et al. Polymer microstructures formed by moulding in capillaries. Nature. 1995;376:581-584. |
| Kimura, et al. Deletional mutant EGFR detected in circulating tumor-derived DNA from lung cancer patients treated with gefitinib. American Association for Cancer Research 96th Annual Meeting. Apr. 16-20, 2005. Abstract 479. |
| Kimura, et al. The DYRK1A gene, encoded in chromosome 21 Down syndrome critical region, bridges between (β-amyloid production and tau phosphorylation in Alzheimer disease. Human Molecular Genetics, Nov. 29, 2008, vol. 16, No. 1, 15-23. |
| Klein, C. A. Single cell amplification methods for the study of cancer and cellular ageing. Mech. Ageing Dev. 2005; 126(1):147-51. |
| Klein, et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci U S A. 1999; 96(8):4494-9. |
| Kobayashi, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. Feb. 24, 2005;352(8):786-92. |
| Kogan, et al. An Improved Method for Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences, Application to Hemophilia A. The New England Journal of Medicine.1987;317:985-990. |
| Korenberg, et al. Down syndrome phenotypes: the consequences of chromosomal imbalance. PNAS 1994; 91:4997-5001. |
| Krabchi, et al. Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenic techniques. Clin. Genet. 2001; 60:145-150. |
| Krivacic, et al. A rare-cell detector for cancer. PNAS. 2004;101:10501-10504. |
| Kulozik, et al. Fetal Cell in the Maternal Circulation: Detection by Direct AFP-Immunoflourescence. Human Genetics. 1982;62:221-224. |
| Kurg, et al. Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology. Genet Test. 2000;4(1):1-7. |
| Lander, et al. Initial sequencing and analysis of the human genome. Nature 409, 860-921 (Feb. 15, 2001) | DOI:10.1038/35057062; Accepted Jan. 9, 2001. |
| Leutwyler, K. Mapping Chromosome 21. Available at http://www.scientificamerican.com/article.cfm?id=mapping-chromosome-21. Accessed Feb. 3, 2010. |
| Levett, et al. A large-scale evaluation of amnio-PCR for the rapid prenatal diagnosis of fetal trisomy. Ultrasound Obstet Gynecol. 2001; 17(2):115-8. |
| Li , et al. Transport, Manipulation, and Reaction of Biological Cells On-Chip Using Electrokinetic Effects. Analytical Chemistry., 1997; pp. 1564-1568. |
| Li, et al. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988;335:414-417. |
| Li, et al. Size separation of circulatory Dna in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin Chem. Jun. 2004;50(6):1002-11. |
| Li, et. al. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. Nov. 2008;18(11):1851-8. doi: 10.1101/gr.078212.108. Epub Aug. 19, 2008. |
| Lichter , et al. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hyridization using recombinant DNA libraries. Hum Genet. 1988;80:224-234. |
| Lieberfarb, et al. Genome-wide loss of heterozygosity analysis from laser capture microdissected prostate cancer using single nucleotide polymorphic allele (SNP) arrays and a novel bioinformatics platform dChipSNP. Cancer Res. Aug. 15, 2003;63(16):4781-5. |
| Liu, et al. Development and validation of a T7 based linear amplification for genomic DNA. BMC Genomics. 2003; 4(1):19. (11 pages). |
| Lo, et al. Detection of fetal RhD sequence from peripheral blood of sensitized RhD-negative pregnant women. British Journalof Haematology, 1994, vol. 87, 658-660. |
| Lo, et al. Detection of single-copy fetal DNA sequence from maternal blood. The Lancet, Jun. 16, 1990, vol. 335, 1463-1464. |
| Lo, et al. Digital PCR for the molecular detection of fetal chromosomal aneuploidy. PNAS. Aug. 7, 2007; 104(32):13116-13121. |
| Lo, et al. Fetal DNA in Maternal Plasma. Ann. N. Y. Acad. Sci, Apr. 2000, vol. 906, 141-147. |
| Lo, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med. Feb, 2007;13(2):218-23. |
| Lo, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998; 339:1734-1738 Dec. 10, 1998 DOI: 10.1056/NEJM199812103392402. |
| Lo, et al. Prenatal diagnosis: progress through plasma nucleic acids. Nature. Jan. 2007, vol. 8, 71-76. |
| Lo, et al. Prenatal sex determination by DNA amplification from material peripheral blood. The Lancet.Dec. 9, 1989:1363-1365. |
| Lo, et al. Presence of fetal DNA in maternal plasma and serum. The Lancet, Aug. 16, 1997, vol. 350, 485-487. |
| Lo, et al. Quantitative Analysis of Fetal NA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis. Am J. Hum. Genet., 1998, vol. 62, 768-775. |
| Lo, Y. M. Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art. BJOG, 2009, vol. 116, 152-157. |
| Loken , et al. Flow Cytometric Analysis of Human Bone Marrow: I. Normal Erythroid Development. Blood. 1987;69:255-263. |
| Lun, et al. Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma. Clinical Chemistry, 2008, vol. 54, No. 10, 1664-1672. |
| Mahr, et al. Fluorescence in situ hybridization of fetal nucleated red blood cells. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1621. |
| Maloney et al. “Microchimerism of maternal origin persists into adult life,” J. Clin. Invest. 104:41-47 (1999). |
| Marcus, et al. Microfluidic Single-Cell mRNA Isolation and Analysis. American Chemical Society, Mar. 2006; 76:3084-3089. |
| Marcus, et al. Parallel Picoliter RT-PCR Assays Using Microfluidics. Analytical Chemistry, Feb. 1, 2006, vol. 78, No. 3, 956-958. |
| Margulies, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005; 437:376-80. |
| Marks, et al. Epidermal growth factor receptor (EGFR) expression in prostatic adenocarcinoma after hormonal therapy: a fluorescence in situ hybridization and immunohistochemical analysis. The Prostate. 2008; 68:919-923. |
| Martin, et al. “A method for using serum or plasma as a source of NDA for HLA typing,” Human Immunology. 1992; 33:108-113. |
| Martin, New technologies for large-genome sequencing. Genome, 1989, vol. 31, No. 2 : pp. 1073-1080 DOI: 10.1139/g89-184. |
| Mavrou, et al. Identification of nucleated red blood cells in maternal circulation: A second step in screening for fetal aneuploidies and pregnancy complications. Prenat Diagn. 2007; 247:150-153. |
| McCabe, et al. DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening. Hum Genet.1987;75:213-216. |
| McCarley, et al. Patterning of surface-capture architectures in polymer-based microanalytical devices. In Kutter, et al. Eds. Royal Society of Chemistry Special Publication. 2005;130-132. (Abstract only). |
| Mehrishi , et al. Electrophoresis of cells and the biological relevance of surface charge. Electrophoresis.2002;23:1984-1994. |
| Melville, et al. Direct magnetic separation of red cells from whole blood. Nature. 1975; 255:706. |
| Meng, et al.: “Design and Synthesis of a Photocleavable Fluorescent Nucleotide 3′-O-Allyl-dGTPPC-Bodipy-FL-510 as a Reversible Terminator for DNA Sequencing by Synthesis”, J. Org. Chem. 2006, 71, pp. 3248-3252, XP-002652986. |
| Metzker, et al. Sequencing technologies—the next generation. Nature Reviews Genetics 11, 31-46 (Jan. 2010) | DOI:10.1038/nrg2626. |
| Mirzabekov, et al. DNA sequencing by hybridization—a megasequencing method and a diagnostic tool? Trends in Biotechnology, vol. 12, Issue 1, Jan. 1994, pp. 27-32. |
| Mohamed, et al. A Micromachined Sparse Cell Isolation Device: Application in Prenatal Diagnostics. Nanotech 2006 vol. 2; 641-644. (Abstract only). |
| Mohamed, et al. Biochip for separating fetal cells from maternal circulation. J Chromatogr A. Aug. 31, 2007;1162(2):187-92. |
| Mohamed, et al. Development of a rare cell fractionation device: application for cancer detection. IEEE Trans Nanobioscience. 2004; 3(4): 251-6. |
| Moore, et al. Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter. J Biochem Biophys Methods. 1998;37:11-33. |
| Moorhead, et al. Optimal genotype determination in highly multiplexed SNP data. Eur. J. Hum. Genet. 2006;14(2):207-15. (published online Nov. 23, 2005). |
| Mueller , et al. Isolation of fetal trophoblast cells from peripheral blood of pregnant women. The Lancet. 1990;336:197-200. |
| Muller, et al. Moderately repeated DNA sequences specific for the short arm of the human Y chromosome are present in XX makes and reduced in copy number in an XY female. 1986;14:1325-1340. |
| Mullis, et al. Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biolgy 1986;51:263-273. |
| Murakami, et al. A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia. Leukemia. Oct. 1998;12(10):1645-50. |
| Murthy, et al. Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R. Hum. Mutat. 2005; 26(2):145-52. |
| Myers, et al. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. Dec. 13, 1985;230(4731):1242-6. |
| Nagrath, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007; 450: 1235-1241 (with Supplemental pp. 1-10). |
| Nannya, et al. A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res. Jul. 15, 2005;65(14):6071-9. |
| Nelson, et al. Genotyping Fetal DNA by Non-Invasive Means: Extraction From Maternal Plasma. Vox Sang. 2001;80:112-116. |
| Newcombe, R. G. Two-sided confidence intervals for the single proportion: comparison of seven methods. Statistics in Medicine. 1998; 17:857-872. |
| Ng, et al. “The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia,” Clinical Chemistry, 2003, vol. 49, No. 5, 727-731. |
| Notice of Allowance and Issue Fee dated Dec. 9, 2010 issued in U.S. Appl. No. 11/701,686. |
| Notice of allowance dated Jun. 21, 2012 for U.S. Appl. No. 12/815,647. |
| Notice of allowance dated Oct. 5, 2012 for U.S. Appl. No. 11/763,421. |
| Notice of allowance dated Dec. 23, 2011 for U.S. Appl. No. 12/230,628. |
| Notice of allowance dated Dec. 29, 2011 for U.S. Appl. No. 11/763,245. |
| Notice of allowance dated Jul. 12, 2011 with allowed claims for U.S. Appl. No. 12/393,803. |
| Oakey et al. Laminar Flow-Based Separations at the Microscale. Biotechnology Progress. 2002; pp. 1439-1442. |
| Office action (Ex parte Quayle) dated May 13, 2011 for U.S. Appl. No. 11/763,421. |
| Office Action dated Jan. 12, 2009 for U.S. Appl. No. 11/763,133. |
| Office Action dated Jan. 27, 2010 for U.S. Appl. No. 11/701,686. |
| Office action dated Jan. 28, 2009 for U.S. Appl. No. 11/701,686. |
| Office action dated Jan. 30, 2012 for Application No. CN 200780030309.3. |
| Office action dated Feb. 4, 2010 for U.S. Appl. No. 11/067,102. |
| Office action dated Feb. 15, 2011 for U.S. Appl. No. 11/763,426. |
| Office action dated Mar. 4, 2009 for U.S. Appl. No. 11/228,454. |
| Office action dated Mar. 7, 2012 for U.S. Appl. No. 12/816,043. |
| Office action dated Mar. 11, 2010 for U.S. Appl. No. 11/763,245. |
| Office action dated Mar. 20, 2013 for U.S. Appl. No. 12/815,674. |
| Office action dated Mar. 29, 2011 for U.S. Appl. No. 11/763,245. |
| Office action dated Apr. 4, 2008 for U.S. Appl. No. 11/067,102. |
| Office action dated Apr. 4, 2012 for EP Application No. 07784444.7. |
| Office action dated Apr. 4, 2013 for U.S. Appl. No. 12/689,517. |
| Office action dated Apr. 5, 2012 for U.S. Appl. No. 12/751,931. |
| Office action dated Apr. 9, 2013 for U.S. Appl. No. 13/306,520. |
| Office action dated Apr. 12, 2012 for U.S. Appl. No. 12/815,647. |
| Office action dated Apr. 13, 2009 for U.S. Appl. No. 11/067,102. |
| Office action dated Apr. 24, 2012 for EP Application No. 07784442.1. |
| Office action dated Apr. 25, 2011 for U.S. Appl. No. 12/393,803 with pending claims. |
| Office action dated May 4, 2009 for U.S. Appl. No. 11/763,431. |
| Office action dated May 6, 2011 for U.S. Appl. No. 11/763,133. |
| Office action dated May 12, 2011 for U.S. Appl. No. 12/230,628. |
| Office action dated May 18, 2011 for U.S. Appl. No. 12/413,467. |
| Office action dated May 26, 2011 for U.S. Appl. No. 11/762,750. |
| Office action dated May 31, 2013 for U.S. Appl. No. 13/835,926. |
| Office action dated Jun. 3, 2013 for U.S. Appl. No. 13/306,640. |
| Office action dated Jun. 3, 2013 for U.S. Appl. No. 13/837,974. |
| Office action dated Jun. 4, 2012 for U.S. Appl. No. 11/762,747. |
| Office action dated Jun. 5, 2012 for U.S. Appl. No. 12/393,833. |
| Office action dated Jun. 5, 2013 for U.S. Appl. No. 12/751,940. |
| Office action dated Jun. 14, 2010 for U.S. Appl. No. 11/763,426. |
| Office action dated Jun. 15, 2007 for U.S. Appl. No. 11/067,102. |
| Office action dated Jun. 18, 2012 for U.S. Appl. No. 12/751,908. |
| Office action dated Jun. 18, 2012 for U.S. Appl. No. 12/751,940. |
| Office action dated Jun. 22, 2012 for Applicatioin No. AU 2007260676. |
| Office action dated Jul. 2, 2010 for EP Application No. 07784442.1. |
| Office action dated Jul. 9, 2012 for U.S. Appl. No. 11/762,750. |
| Office action dated Jul. 10, 2009 for U.S. Appl. No. 11/763,421. |
| Office action dated Jul. 10, 2012 for U.S. Appl. No. 13/433,232. |
| Office action dated Jul. 26, 2011 for U.S. Appl. No. 11/763,245. |
| Office action dated Aug. 1, 2008 for U.S. Appl. No. 11/067,102. |
| Office action dated Aug. 1, 2012 for U.S. Appl. No. 12/815,674. |
| Office action dated Aug. 2, 2010 for EP Application No. 07784444.7. |
| Office action dated Aug. 7, 2013 for U.S. Appl. No. 13/863,992. |
| Office action dated Aug. 27, 2010 for U.S. Appl. No. 11/762,747. |
| Office Action dated Sep. 8, 2010 for U.S. Appl. No. 11/701,686. |
| Office action dated Sep. 10, 2010 for U.S. Appl. No. 11/762,750. |
| Office Action dated Sep. 11, 2009 for U.S. Appl. No. 11/701,686. |
| Office action dated Sep. 14, 2012 for U.S. Appl. No. 12/393,833. |
| Office action dated Sep. 17, 2010 for U.S. Appl. No. 11/067,102. |
| Office action dated Sep. 22, 2011 for U.S. Appl. No. 12/815,647. |
| Office action dated Sep. 23, 2009 for EP Application No. EP07763674.4 with pending claims. |
| Office action dated Sep. 23, 2009 for EP Application No. EP07763674.4. |
| Office action dated Sep. 27, 2010 for U.S. Appl. No. 12/413,485. |
| Office action dated Sep. 27, 2013 for U.S. Appl. No. 12/816,043. |
| Office action dated Oct. 24, 2011 for U.S. Appl. No. 11/762,747. |
| Office action dated Oct. 25, 2012 for U.S. Appl. No. 12/816,043. |
| Office action dated Oct. 29, 2010 for U.S. Appl. No. 12/230,628. |
| Office action dated Nov. 3, 2009 for U.S. Appl. No. 11/763,133. |
| Office action dated Dec. 1, 2009 for U.S. Appl. No. 11/763,426. |
| Office action dated Dec. 2, 2008 for U.S. Appl. No. 11/762,747. |
| Office action dated Dec. 3, 2008 for U.S. Appl. No. 11/763,426. |
| Office action dated Dec. 10, 2013 for CA Application No. 2655272. |
| Office action dated Dec. 13, 2013 for U.S. Appl. No. 13/306,698. |
| Office action dated Dec. 16, 2013 for U.S. Appl. No. 13/863,992. |
| Office action dated Dec. 31, 2009 for U.S. Appl. No. 11/763,421. |
| Office action dated Dec. 31, 2009 for U.S. Appl. No. 11/762,750. |
| Office action dated Dec. 31, 2011 for U.S. Appl. No. 11/763,421. |
| Office action dated Mar. 7, 2013 for European Application No. 11182181. |
| Olson, et al. A Common Language for Physical Mapping of the Human Genome. “A common language for physical mapping of the human genome.” Science 245.4925 (1989): 1434-1435. Sep. 29, 1989. |
| Olson, et al. An In Situ Flow Cytometer for the Optical Analysis of Individual Particles in Seawater. Available at http://www.whoi.edu/science/B/Olsonlab/insitu2001.htm. Accessed Apr. 24, 2006. |
| Oosterwijk, et al. Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment. Prenat Diagn. 1998;18(10):1082-5. |
| Ottesen,et al. Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria. Science. Dec. 1, 2006; 314(5804):1464-1467. (Abstract only). |
| Owen, et al. High gradient magnetic separation of erythrocytes. Biophys. J. 1978; 22:171-178. |
| Paez, et al. Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res. May 18, 2004;32(9):e71. |
| Pallavicini, et al. Analysis of fetal cells sorted from maternal blood using fluorescence in situ hybridization. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1031. |
| Parano, et al. Fetal Nucleated red blood cell counts in peripheral blood of mothers bearing Down Syndrome fetus. Neuropediatrics. 2001; 32(3):147-149. (Abstract only). |
| Parano, et al. Noninvasive Prenatal Diagnosis of Chromosomal Aneuploidies by Isolation and Analysis of Fetal Cells from Maternal Blood. Am. J. Med. Genet. 101:262-7 (2001). |
| Paterlini-Brechot, et al. Circulating tumor cells (CTC) detection: Clinical impact and future directions. Cancer Letter. 2007. (In press, 25 pages.) Available at www.sciencedirect.com. |
| Paul, et al. Single-molecule dilution and multiple displacement amplification for molecular haplotyping. Biotechniques. 2005; 38(4):553-4, 556, 558-9. |
| Pawlik, et al. Prodrug Bioactivation and Oncolysis of Diffuse Liver Metastases by a Herpes Simplex Virus 1 Mutant that Expresses the CYP2B1 Transgene. Cancer. 2002;95:1171-81. |
| Peixoto, et al. Quantification of multiple gene expression in individual cells. Genome Res. Oct. 2004;14(10A):1938-47. |
| Pending Claims and Preliminary Amendment filed Nov. 19, 2010 for U.S. Appl. No. 11/763,133. |
| Pending claims and preliminary amendment filed Dec. 10, 2010 for U.S. Appl. No. 11/763,245. |
| Pending claims filed with the USPTO on Apr. 26, 2010 for U.S. Appl. No. 11/067,102. |
| Peng, et al. Real-time detection of gene expression in cancer cells using molecular beacon imaging: new strategies for cancer research. Cancer Res. 2005; 65(5):1909-17. |
| Pertl, et al. “Fetal DNA in Maternal Plasma: Emerging Clinical Applications,” Obstetrics and Gynecology, Sep. 2001, vol. 98, No. 3, 483-490. |
| Petersen, et al. The Promise of Miniaturized Clinical Diagnostic Systems. IVD Technol. 4:43-49 (1998). |
| Pfaffl, et al. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. May 1, 2002;30(9):e36. |
| Pinkel, et al. Cytogenetic Analysis Using Quantitative, High-sensitivity, Fluorescence Hybridization. Genetics. 1986;83:2934-2938. |
| Pinkel, et al. Fluorescence in situ Hybridization with Human Chromosome-specific Libraries: Detection of Trisomy 21 and Translocations of Chromosome 4. Genetics.1988;85:9138-9142. |
| Pinkel, et al. Detection of structural chromosome abberations in metaphase in metaphase spreads and interphase nuclei by in situ hybridization high complexity probes which stain entire human chromosomes. The American Journal of Human Genetics. Sep. 1988. Supplemental to vol. 43, No. 3: 0471. |
| Pinzani, et al. Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum Pathol. 2006; 37(6):711-8. |
| Pohl et al. Principle and applications of digital PCR. Expert Rev Mol Diagn. Jan. 2004;4(1):41-7. |
| Poon, et al. “Circulating fetal DNA in maternal plasma,” ClinicalChimica Acta, 2001, vol. 313, 151-155. |
| Potti, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006; 12(11):1294-1300. |
| Preliminary Amendments to the Claims. Filed Jul. 11, 2013 with U.S. Patent Office for U.S. Appl. No. 13/738,268. |
| Price, et al. Prenatal Diagnosis with Fetal Cells Isolated from Maternal Blood by Multiparameter Flow Cytometry. Am. J. Obstet. Gynecol. 1991; 165:1731-7. |
| Prieto, et al. Isolation of fetal nucleated red blood cells from maternal blood in normal and aneuploid pregnancies. Clin Chem Lab Med. Jul. 2002;40(7):667-72. |
| Product literature for GEM, a system for blood testing: GEM Premier 3000. Avaiable at http://www.ilus.com/premier_gem3000_iqm.asp. Accessed Apr. 24, 2006. |
| Pruitt, et al. RefSeq and LocusLink: NCBI gene-centered resources. Nucl. Acids Res. (2001) vol. 29 No. 1: 137-140. DOI: 10.1093/nar/29.1.137. |
| Purwosunu, et al. Clinical potential for noninvasive prenatal diagnosis through detection of fetal cells in maternal blood. Taiwan J Obstet Gynecol. Mar. 2006;45(1):10-20. |
| Raeburn, P. Fetal Cells Isolated in Women's Blood. Associated Press (Jul. 28, 1989) [electronic version]. |
| Rahil, et al. Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes, European Journal of Human Genetics, 2002, vol. 10, 462-466. |
| Request for Continued Examination by applicant with claim set dated Mar. 26, 2010 in Response to Final Office Action dated Nov. 3, 2009 for U.S. Appl. No. 11/763,133. |
| Request for Continued Examination by applicant with claim set dated Mar. 26, 2010 in Response to Final Office Action dated Dec. 1, 2009 for U.S. Appl. No. 11/763,426. |
| Response dated Nov. 24, 2010 to Office action dated Jun. 14, 2010 with Pending Claims for U.S. Appl. No. 11/763,426. |
| Response filed Dec. 26, 2012 with claims for U.S. Appl. No. 12/393,833. |
| Rickman, et al. Prenatal diagnosis by array-CGH. European Journal of Medical Genetics. 2005; 48:232-240. |
| Rolle, et al. Increase in number of circulating disseminated epithelia cells after surgery for non-small cell lung cancer monitored by MAINTRAC is a predictor for relapse: a preliminary report. World Journal of Surgical Oncology. 2005; 9 pages. |
| Rosato, M. Verinata Health, Inc.'s Preliminary Patent Owner Response Pursuant to 37 C.F.R. §42.107(a). Dated Jul. 29, 2013. For Case IPR2013-00276. U.S. Pat. No. 8,318,430. |
| Rosato, Verinata Health Inc.'s Preliminary Patent Owner Response Pursuant to 37 C.F.R. §42.107(a). Dated Jul. 29, 2013. For Case IPR2013-00277. U.S. Pat. No. 8,318,430. |
| Ruan, et al. Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Molecular & Cellular Proteomics. 2006; 5(12): 2364-73. |
| Sakhnini, et al. Magnetic behavior of human erythrocytes at different hemoglobin states. Eur Biophys J. Oct. 2001;30(6):467-70. |
| Saleeba, et al. Chemical cleavage of mismatch to detect mutations. Methods Enzymol. 1993;217:286-95. |
| Samura, et al. Diagnosis of trisomy 21 in fetal nucleated erythrocytes from maternal blood by use of short tandem repeat sequences. Clin. Chem. 2001; 47(9):1622-6. |
| Samura, et al. Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin. Hum. Genet. 2000;107(1):28-32. |
| Sato, et al. Individual and Mass Operation of Biological Cells Using Micromechanical Silicon Devices. Sensors and Actuators. 1990;A21-A23:948-953. |
| Schaefer, et al. The Clinical Relevance of Nucleated Red Blood Cells counts. Sysmex Journal International. 2000; 10(2):59-63. |
| Schröder, et al. Fetal Lymphocytes in the Maternal Blood. The Journal of Hematolog:Blood. 1972;39:153-162. |
| Sehnert, et al. Optimal Detection of Fetal Chromosomal Abnormalities by Massively Parallel DNA Sequencing of Cell-Free Fetal DNA from Maternal Blood. Clin Chem. Apr. 25, 2011. [Epub ahead of print]. |
| Sethu, et al. Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis. Anal. Chem. 76:6247-6253 (2004). |
| Shen, et al. High-throughput SNP genotyping on universal bead arrays. Mutat. Res. 2005; 573:70-82. |
| Shendure, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science. 2005; 309:1728-32. |
| Shendure, et al. Next-generation DNA sequencing. Nature. 2008; 26(10):1135-1145. |
| Sherlock, et al. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann. Hum. Genet. 1998; 62:9-23. |
| Sitar, et al. The Use of Non-Physiological Conditions to Isolate Fetal Cells from Maternal Blood. Exp. Cell. Res. 2005; 302:153-61. |
| Sohda, et al. The Proportion of Fetal Nucleated Red Blood Cells in Maternal Blood: Estimation by FACS Analysis. Prenat. Diagn. 1997; 17:743-52. |
| Solexa Genome Analysis System. 2006; 1-2. |
| Sparkes, et al. “New Molecular Techniques for the Prenatal Detection of Chromosomal Aneuploidy,” JOGC, Jul. 2008, No. 210, 617-621. |
| Stipp, D. IG Labs Licenses New Technology for Fetal Testing. The Wall Street Journal. Aug. 10, 1990:B5. |
| Stoecklein, et al. SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am J Pathol. 2002; 161(1):43-51. |
| Stoughton, et al. Data-adaptive algorithms for calling alleles in repeat polymorphisms. Electrophoresis. 1997;18(1):1-5. |
| Sun, et al. Whole-genome amplification: relative efficiencies of the current methods. Leg Med. 2005; 7(5):279-86. |
| Supplemental Amendment and Transmittal of Terminal Disclaimer dated Oct. 18, 2011 for U.S. Appl. No. 12/230,628. |
| Sykes, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques. Sep. 1992;13(3):444-9. |
| Tanaka, et al. “Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray,” PNAS, Aug. 2000, vol. 97, No. 16, 9127-9132. |
| Tettelin, et al. The nucleotide sequence of Saccharomyces cerevisiae chromosome VII. Nature. May 29, 1997;387(6632 Suppl):81-4. |
| Thomas, et al. Specific Binding and Release of Cells from Beads Using Cleavable Tettrametric Antibody Complexes. Journal of Immunological Methods 1989;120:221-231. |
| Tibbe, et al. Statistical considerations for enumeration of circulating tumor cells. Cytometry A. Mar. 2007;71(3):154-62. |
| Toner, et al. Blood-on-a-Chip. Annu. Rev. Biomed. Eng. 7:77-103, C1-C3 (2005). |
| Trask, et al. Detection of DNA Sequences and Nuclei in Suspension by In Situ Hybridization and Dual Beam Flow Cytometry. Science.1985;230:1401-1403. |
| Troeger, et al. Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod. 1999; 5(12):1162-5. |
| Tufan, et al., Analysis of Cell-Free Fetal DNA from Maternal Plasma and Serum Using a Conventional Multiplex PCR: Factors Influencing Success. 2005. Turk. J. Med. Sci. 35:85-92. |
| Uitto, et al. Probing the fetal genome: progress in non-invasive prenatal diagnosis. Trends Mol Med. Aug. 2003;9(8):339-43. |
| Van Raamsdonk, et al. Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization. Nucl. Acids. Res. 2001; 29(8):e42. |
| Venter, et al. The Sequence of the Human Genome. Science Feb. 16, 2001; vol. 291 No. 5507 pp. 1304-1351 DOI: 10.1126/science.1058040. |
| Verinata Health. Complaint for Patent Infringement. Case: 12-cv-05501-SI; Dated Oct. 25, 2012. |
| Verinata Health. Plaintiffs' Opposition to Defendants' Motion to Dismiss Plaintiffs' Claims for Patent Infringement Against Laboratory Corporation of America and Claims for Enhanced Damages Against All Defendants. Case: 12-cv-05501-SI; Dated Jan. 25, 2013. |
| Verinata Health: Joint Claim Construction and Prehearing Statement. Case: 12-cv-05501-SI; Dated May 3, 2013. |
| Voelkerding, et al. Digital fetal aneuploidy diagnosis by next-generation sequencing. Clin Chem. Mar. 2010;56(3):336-8. |
| Vogelstein, et al. “Digital PCR.” Proc Natl. Acad Sci. USA, Aug. 1999, vol. 96., 9236-9241. |
| Voldberg, et al. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol. Dec. 1997;8(12):1197-206. |
| Volkmuth, et al. DNA electrophoresis in microlithographic arrays. Nature. 1992; 358:600-602. |
| Volkmuth, et al. Observation of Electrophoresis of Single DNA Molecules in Nanofabricated Arrays. Presentation at joint annual meeting of Biophysical Society and the American Society for Biochemistry and Molecular Biology. Feb. 9-13, 1992. |
| Von Eggeling, et al. Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms. Hum Genet. Feb. 1997;99(2):266-70. |
| Vona, et al. Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood. Am J Pathol. Jan. 2002;160(1):51-8. |
| Voss, et al. Efficient low redundancy large-scale DNA sequencing at EMBL. Journal of Biotechnology, vol. 41, Issues 2-3, Jul. 31, 1995, pp. 121-129. |
| Voullaire, et al. Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn. 1999; 19(9):846-51. |
| Vrettou, et al. Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Human Mutation. 2004; 23(5):513-21. |
| Wachtel, et al. Fetal Cells in the Maternal Circulation: Isolation by Multiparameter Flow Cytometry and Confirmation by Polymerase Chain Reaction. Human Reproduction. 1991;6:1466-1469. |
| Wang, et al. Allele quantification using molecular inversion probes (MIP). Nucleic Acids Research. 2005; 33(21); e183 (14 pages). |
| Wapner, et al. First-trimester screening for trisomies 21 and 18. N. Engl. J. Med. 2003; 349:1405-1413. |
| Warren, et al. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. PNAS. Nov. 21, 2006; 103(47):17807-17812. |
| Washizu, et al. Handling Biological Cells Utilizing a Fluid Integrated Circuit. IEEE Industry Applications Society Annual Meeting Presentations. Oct. 2-7, 1988;: 1735-40. |
| Washizu, et al. Handling Biological Cells Utilizing a Fluid Integrated Circuit. IEEE Transactions of Industry Applications. 1990; 26: 352-8. |
| Weigl, et al. Microfluidic Diffusion-Based Separation and Detection. Science. 1999; pp. 346-347. |
| Wheeler, et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872-876 (Apr. 17, 2008) | DOI:10.1038/nature06884; Accepted Mar. 4, 2008. |
| White, et al. Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics. Mar. 19, 2009;10:116. |
| Williams, et al. Comparison of cell separation methods to entrich the proportion of fetal cells in material blood samples. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1049. |
| Xiong, et al. “A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences,” Nucleic Acids Research, Apr. 19, 2004, vol. 32, No. 12, e98. |
| Yang, et al. Prenatal diagnosis of trisomy 21 with fetal cells i maternal blood using comparative genomic hybridization. Fetal Diagn Ther. 2006; 21:125-133. |
| Yang, et al. Rapid Prenatal Diagnosis of Trisomy 21 by Real-time Quantitative Polymerase Chain Reaction with Amplification of Small Tandem Repeats and S1OOB in Chromosome 21. Yonsei Medical Journal, 2005, vol. 46, No. 2, 193-197. |
| Yu, et al. Objective Aneuploidy Detection for Fetal and Neonatal Screening Using Comparative Genomic Hybridization (CGH). Cytometry. 1997; 28(3): 191-197. (Absbract). |
| Zavala, et al. Genomic GC content prediction in prokaryotes from a sample of genes. Gene. Sep. 12, 2005;357(2):137-43. |
| Zhao, et al. An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays. Cancer Res. May 1, 2004;64(9):3060-71. |
| Zhen, et al. Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood. Prenat Diagn. 1998; 18(11):1181-5. |
| Zheng, et al. Fetal cell identifiers: results of microscope slide-based immunocytochemical studies as a function of gestational age and abnormality. Am J Obstet Gynecol. May 1999;180(5):1234-9. |
| Zhu, et al. Single molecule profiling of alternative pre-mRNA splicing. Science. Aug. 8, 2003;301(5634):836-8. |
| Zimmerman et al. QIAGEN News. 2003. e12. Available via uri: <b2b.qiagen.com/literature/qiagennews/weeklyarticle/apr03/e12/default.aspx>. |
| Zimmerman, et al. Novel real-time quantitative PCR test for trisomy 21. Jan. 1, 2002. Clinical Chemistry, American Association for Clinical Chemistry. 48:(2) 362-363. |
| Zimmermann, Bernhard. “Molecular Diagnosis in Prenatal Medicine,” Ph.D. Thesis, 2004. |
| Zuska, P. Microtechnology Opens Doors to the Universe of Small Space, MD&DI Jan. 1997, p. 131. |
| Fan, et al. Highly Parallel Genomic Assays. Aug. 2006. Nat. Rev. Genet. 7(8):632-44. |
| Office action dated Feb. 5, 2014 for U.S. Appl. No. 12/689,517. |
| Office action dated Mar. 20, 2014 for U.S. Appl. No. 12/816,043. |
| Office action dated Mar. 21, 2014 for U.S. Appl. No. 12/815,674. |
| Office action dated Apr. 28, 2014 for U.S. Appl. No. 12/689,548. |
| Office action dated Nov. 7, 2014 for U.S. Appl. No. 13/831,342. |
| Office action dated Nov. 24, 2014 for U.S. Appl. No. 12/689,548. |
| U.S. Appl. No. 14/705,239, filed May 6, 2015, Kapur, et al. |
| Advisory action dated Mar. 4, 2015 for U.S. Appl. No. 12/689,548. |
| European office action dated May 22, 2015 for EP Application No. 07798579.4. |
| International Preliminary Report on Patentability dated Dec. 16, 2008 for PCT Application No. US2007/071248. |
| Notice of allowance dated Jan. 26, 2015 for U.S. Appl. No. 13/835,926. |
| Office action dated Jan. 26, 2015 for CA Application No. 2655272. |
| Office action dated Jan. 30, 2014 for U.S. Appl. No. 13/837,974. |
| Office action dated Feb. 23, 2015 for U.S. Appl. No. 12/689,517. |
| Office action dated Mar. 11, 2015 for U.S. Appl. No. 13/737,730. |
| Office action dated Apr. 7, 2015 for U.S. Appl. No. 13/829,971. |
| Office action dated Apr. 29, 2015 for U.S. Appl. No. 13/794,503. |
| Office action dated May 8, 2015 for U.S. Appl. No. 12/816,043. |
| Office action dated May 11, 2015 for U.S. Appl. No. 13/863,992. |
| Office action dated Jul. 16, 2015 for U.S. Appl. No. 13/837,974. |
| Office action dated Jul. 21, 2015 for U.S. Appl. No. 12/689,548. |
| Office action dated Aug. 1, 2014 for U.S. Appl. No. 12/816,043. |
| Office action dated Aug. 19, 2015 for U.S. Appl. No. 12/689,517. |
| Office action dated Sep. 18, 2015 for U.S. Appl. No. 12/816,043. |
| Office action dated Dec. 10, 2014 for U.S. Appl. No. 12/751,940. |
| Office action dated Dec. 12, 2014 for U.S. Appl. No. 13/738,268. |
| Bianchi, et al. Large amounts of cell-free fetal DNA are present in amniotic fluid. Clinical chemistry 47.10 (2001): 1867-1869. |
| Bischoff, et al. Cell-free fetal DNA and intact fetal cells in maternal blood circulation: implications for first and second trimester non-invasive prenatal diagnosis. Human reproduction update 8.6 (2002): 493-500. |
| Brown, et al. Aneuploidy detection in mixed DNA samples by methylation-sensitive amplification and microarray analysis. Clinical chemistry 56.5 (2010): 805-813. |
| Brown, et al. Validation of QF-PCR for prenatal aneuploidy screening in the United States. Prenatal diagnosis 26.11 (2006): 1068-1074. |
| Chim, et al. Detection of the placental epigenetic signature of the Maspin gene in maternal plasma. Proceedings of the National Academy of Sciences of the United States of America 102.41 (2005): 14753-14758. |
| Chiu, et al. Noninvasive prenatal diagnosis by analysis of fetal DNA in maternal plasma. Clinical Applications of PCR (2006): 101-109. |
| Evans, et al. Digital PCR for noninvasive detection of aneuploidy: power analysis equations for feasibility. Fetal diagnosis and therapy 31.4 (2012): 244-247. |
| Grundevik, et al. Molecular Diagnostics of Aneuploidies. Chalmers University of Technology. May 17, 2005. |
| Kaiser, J. An earlier look at baby's genes. Science 309.5740 (2005): 1476. |
| Khattabi, et al. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study. PloS one 11.5 (2016): e0155009. |
| Mann, et al. Strategies for the rapid prenatal diagnosis of chromosome aneuploidy. European Journal of Human Genetics 12.11 (2004): 907-915. |
| Notice of allowance dated Jul. 27, 2016 for U.S. Appl. No. 12/816,043. |
| Office action dated Mar. 14, 2016 for U.S. Appl. No. 12/816,043. |
| Office action dated Sep. 16, 2016 for U.S. Appl. No. 12/689,517. |
| Office action dated Dec. 1, 2016 for U.S. Appl. No. 13/794,503. |
| Office action dated Dec. 27, 2016 for U.S. Appl. No. 13/863,992. |
| Opposition dated Apr. 10, 2014 by Olswang against EP Application No. EP07763674.4. |
| Opposition dated Jun. 12, 2015 by Premaitha Health PLC against EP Application No. EP07763674.4. |
| Poon, et al. Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clinical chemistry 48.1 (2002): 35-41. |
| Sekizawa, et al. Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood. Journal of Obstetrics and Gynaecology Research 33.6 (2007): 747-764. |
| Tong, et al. Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: theoretical and empirical considerations. Clinical Chemistry 52.12 (2006): 2194-2202. |
| Vogelstein, et al. Allelotype of colorectal carcinomas. Science 244 (4): 207-211. 1989. |
| Wong, et al. Circulating placental RNA in maternal plasma is associated with a preponderance of 5′ mRNA fragments: implications for noninvasive prenatal diagnosis and monitoring. Clinical chemistry 51.10 (2005): 1786-1795. |
| Yu, et al. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proceedings of the National Academy of Sciences 111.23 (2014): 8583-8588. |
| Zhou, et al. Counting alleles reveals a connection between chromosome 18q loss and vascular invasion. Nature biotechnology 19.1 (2001): 78-81. |
| Zhou, et al. Counting alleles to predict recurrence of early-stage colorectal cancers. The Lancet 359.9302 (2002): 219-225. |
| Zimmerman, et al. Digital PCR: a powerful new tool for noninvasive prenatal diagnosis? 2008 Prenat Diagn 28, 1087-1093. |
| U.S. Appl. No. 60/951,438, filed Jul. 23, 2007, Lo et al. |
| Amendment to the Claims dated Jun. 16, 2014 for U.S. Appl. No. 13/863,992. |
| Amendment to the Claims dated Dec. 5, 2013 for U.S. Appl. No. 12/751,940. |
| Amendments to the Claims. Filed Oct. 14, 2014 with U.S. Patent Office for U.S. Appl. No. 13/831,342. |
| Chang, et al. Assessment of Plasma DNA Levels, Allelic Imbalance, and CA 125 as Diagnostic Tests for Cancer. Nov. 20, 2002. J. Nat'l Cancer Inst. 94(22):1697-1703. |
| Coble, et al. Characterization of New MiniSTR Loci to Aid Analysis of Degraded DNA. Jan. 2005. J. Forensic Sci. 50(1):43-53. |
| Dahl, et al. Multigene Amplification and Massively Parallel Sequencing for Cancer Mutation Discovery. May 29, 2007. PNAS USA 104(22):9387-9392. |
| Declaration of Atul J. Butte, M.D. PhD. in Support of Patent Owner's Response to Inter Partes Review of U.S. Pat. No. 8,316,430. US Patent Office. Dated Jan. 16, 2014. |
| Deposition of Dr. Cynthia Casson Morton. US Patent Office. Dated Dec. 10, 2013. |
| Deposition of Dr. Robert Nussbaum. US Patent Office. Dated Dec. 11, 2013. |
| Deutsch, et al. Detection of aneuploidies by paralogous sequence quantification. J.Med Genet. Dec. 2004;41(12):908-15. |
| European office action dated Aug. 22, 2013 for EP Application No. 07798579.4. |
| Hall. Advanced Sequencing Technologies and their Wider Impact in Microbiology. 2007. J. Exp. Biol. 209:1518-1525. |
| Jama, et al. Quantification of cell-free DNA levels in maternal plasma by STR analysis. Mar. 24-28, 2010. ACMG Annual Clinical Genetics Meeting Poster 398. Availabble at http://acmg.omnibooksonline.com/2010/data/papers/398.pdf. Accessed Apr. 5, 2013. |
| Koide, et al. Fragmentation of Cell-Free Fetal DNA in Plasma and Urine of Pregnant Women. Jul. 2005. Prenat. Diagn. 25(7):604-7. |
| Leon, et al. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. Mar. 1977;37(3):646-50. |
| Liu, et al. Feasibility Study of Using Fetal DNA in Maternal Plasma for Non-invasive Prenatal Diagnosis. Acta Obstet. Gynecol. Scand. May 2007; 86(5):535-41. |
| Lo, et al. Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21. Oct. 1999. Clin. Chem. 45(10):1747-51. |
| Lo, et al. Rapid Clearance of Fetal DNA from Maternal Plasma. Jan. 1999. Am. J. Hum. Genet. 64(1):218-24. |
| Office action dated Jan. 28, 2014 for U.S. Appl. No. 13/835,926. |
| Office action dated Mar. 15, 2016 for U.S. Appl. No. 12/751,940. |
| Office action dated Mar. 31, 2016for U.S. Appl. No. 13/863,992. |
| Pathak, et al. Circulating Cell-Free DNA in Plasma/Serum of Lung Cancer Patients as a Potential Screening and Prognostic Tool. Oct. 2006. Clin. Chem.52(10):1833-42. |
| Pertl, et al. Detection of Male and Female DNA in Maternal Plasma by Multiplex Fluorescent Polymerase Chain Reaction Amplification of Short Tandem Repeats. Jan. 2000. Hum. Genet. 106(1):45-9. |
| REPLI-g® Mini and Midi Kits pamphlet from Qiagen (Oct. 2005). |
| Su, et al. Human Urine Contains Small, 150 to 250 Nucleotide-Sized, Soluble DNA Derived from the Circulation and may be Useful in the Detection of Colorectal Cancer. May 2005. J. Mol. Diagn. 6(2):101-7. |
| Swarup, et al. Circulating (cell free) nucleic acids—A promising, non-invasive tool for early detection of several human diseases. 2007 FEBS Letters 581:795-799. |
| Thomas, et al. Sensitive Mutation Detection in Heterogeneous Cancer Specimens by Massively Parallel Picoliter Reactor Sequencing. Jul. 2006. Nature Medicine. 12(7):852-855. |
| Wright, et al. The use of cell-free fetal nucleic acids in maternal blood for noninvasive prenatal diagnosis. Hum Reprod Update. Jan.-Feb. 2009;15(1):139-51. |
| Zhang, et al. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A. Jul. 1, 1992;89(13):5847-51. |
| Notice of allowance dated Jan. 22, 2016 for U.S. Appl. No. 13/837,974. |
| Notice of allowance dated Oct. 27, 2015 for U.S. Appl. No. 13/829,971. |
| Office action dated Dec. 22, 2015 for U.S. Appl. No. 13/794,503. |
| Office action dated Aug. 16, 2016 for U.S. Appl. No. 13/863,992. |
| Bailey et al., “Recent Segmental Duplications in the Human Genome,” Science, Aug. 2002, 297(5583):1003-1007. |
| Extended European Search Report in Application No. 18170287.9, dated Sep. 19, 2018, 10 pages. |
| Jauniaux et al., “Very early prenatal diagnosis on coelomic cells using quantitative fluorescent polymerase chain reaction,” Reproductive BioMedicine Online, Jan. 2003, 6:494-498. |
| Leary et al., “Digital karyotyping,” Nature Protocols, 2007, 2(8):1973-1986. |
| Wang et al., “Digital karyotyping,” Proc. Natl. Acad. Sci., U.S.A, Dec. 2002, 99(25):16156-16161. |
| Number | Date | Country | |
|---|---|---|---|
| 20130280709 A1 | Oct 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60804815 | Jun 2006 | US | |
| 60820778 | Jul 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13738268 | Jan 2013 | US |
| Child | 13830871 | US | |
| Parent | 13433232 | Mar 2012 | US |
| Child | 13738268 | US | |
| Parent | 12725240 | Mar 2010 | US |
| Child | 13433232 | US | |
| Parent | 11763426 | Jun 2007 | US |
| Child | 12725240 | US |
