JP2009509143A - Analysis improvement system and method - Google Patents

Abstract

The present invention relates to one or more sizing modules adapted to increase the first analyte in a sample to a concentration of at least 10,000 times, wherein the first analyte has an initial concentration in the sample of 1 ×. 10 ⁻³ specimens / μL or less, and relates to an analyzer for analyzing the first contribution in a concentrated medium. A method is also provided for using the screening module to identify characteristics associated with a patient condition, ie, a fetal abnormality.
[Selection figure] None

Description

  Analysis of specific cells provides insight into various diseases. Such analysis has enabled invasion testing for disease detection, diagnosis, and prediction, eliminating the risk of invasion diagnosis. For example, the number of fetal tests has increased due to social evolution. However, the methods available today, amniocentesis and chorionic virus sampling (CVS) are potentially harmful to the mother and fetus. The miscarriage rate of pregnant women who have undergone amniocentesis increases by 0.5-1%, which is slightly higher in CVS. Because of the inherent risk of amniocentesis and CVS, these treatments are primarily applied to women over 35 years of age who are likely to have older children, ie, children who are statistically congenital. As a result, a 35 year old pregnant woman had to weigh an average 0.5-1% risk of amniocentesis that caused miscarriage for a probability of 21 trisomy less than 0.3% at this age.

  Several non-invasive methods have been developed for the diagnosis of certain congenital diseases. For example, maternal plasma alpha-fetoprotein, unbound estriol levels and human chorionic gonadotropin can be used to identify fetal Down syndrome potential, but these tests are not 100% accurate. Similarly, ultrasonography is used to examine congenital diseases such as neural tube disease and limb abnormalities, but this is only useful after the 15th week of pregnancy.

  The emergence of fetal cells in the blood of pregnant women has provided an opportunity for fetal diagnosis instead of amniocentesis and has now eliminated the risk of invasive diagnosis. However, fetal cells were sampled with a small number of maternal cells in the large number of blood in the background, which was time consuming and error prone.

  Several devices have been made to select the number of cells. Such cell sorting techniques can be divided into two categories: (1) Sorting of stained cells using various cell specific markers such as fluorescence activated cell sorting (FACS) and magnetically active cell sorting (MACS). And (2) isolating living cells using biophysical parameters specific to the individual of interest. These methods have various limitations such as high cost, low production, skill required, lack of specificity. As a result, there has been no improved clinically acceptable improved method for individual selection of few cells, especially fetal cells, from surrounding blood samples, sufficient for clinical diagnosis. Therefore, an improved method for selecting specific types of cells from a mixture, which eliminates the limitations of the prior art, has been desired.

  The present invention relates to a system comprising one or more sizing modules adapted to increase a first analyte in a sample to a concentration of at least 10,000 times, the first analyte being an initial in the sample The concentration is 1 × 10 −3 analyte / μL or less, and the analyzer comprises computer-executable logic for analyzing the first analyte in the medium that selectively enriches the first analyte.

  In some embodiments, the analyzer further comprises a microscope, microarray, or cell counter.

  In some embodiments, the computer-executable logic detects discoloration due to the presence of fetal hemoglobin, globin γ, globin ε, GPA, i antigen, CD46, selectin, CD45, or combinations thereof.

  In some embodiments, the computer-executable logic detects the strength of the probe that selectively binds the first analyte. In some embodiments, the computer-executable logic performs a three-dimensional analysis of the first specimen.

  In some embodiments, the analyzer has a dual scan function.

  In some embodiments, the computer-executable logic depicts the first analyte.

  In some embodiments, particularly when the first specimen is a fetal cell obtained from maternal blood, the computer-executable logic may determine, for the fetal cell, fetal sex, trisomy, or one or more fetal cells. Detect chromosomal abnormalities in

  In some embodiments, one or more size-based sorting modules deterministically guide the first analyte in a first direction, and a first hydrodynamically smaller size than the first analyte. A two-dimensional obstacle array for guiding two specimens in a second direction;

  In some embodiments, two or more sizing modules are fluidly connected in parallel with each other.

  In some embodiments, the system is adapted for high throughput analysis of at least 10 mL / hr of fluid.

  In some embodiments, the system further comprises one or more extraction regions connected to a sorting region that selectively extracts the first or second analyte from a fluid sample.

  In some embodiments, the system comprises one or more capture modules, one of the extraction modules comprising a two-dimensional obstacle array.

  In some embodiments, the capture module obstacle is bound to an antibody. Such antibodies selectively bind red blood cells, white blood cells, fetal blood cells, fetal nuclear blood cells, cancer cells, epithelial cells, stem cells, progenitor cells, foam cells, platelets, across a second analyte in the sample. . In some embodiments, the antibody is selected from the group consisting of anti-CD71, anti-CD46, anti-carbohydrate, anti-selectin, anti-CD451, anti-GPA, anti-antigen I, anti-EpCam.

  In some embodiments, the liquid sample is a pregnant blood sample and the first analyte is nucleated fetal red blood cells.

  In some embodiments, the liquid sample is a blood sample and the first analyte is selected from the group consisting of epithelial cells, endothelial cells, progenitor cells, stem cells, foam cells, or cancer cells.

  In some embodiments, the sample is a blood sample and is 5 mL or less.

  In some embodiments, the liquid sample is a blood sample of a woman under 12 weeks of gestation.

  In some embodiments, the spacing between obstacles in the sizing or extraction module is 1000 microns or less.

  In some embodiments, the system further comprises a reservoir containing magnetic particles. This reservoir is fluidly connected to the sizing or extracting module.

  In another embodiment, the present invention comprises a sorting module suitable for removing more than 99.5% of enucleated cells from a blood sample and retaining more than 99% of nucleated cells from the blood sample. In some embodiments, the system further comprises an analyzer that is fluidly connected to the sorting module and configured to analyze one or more nucleated cells and a database that stores data from the analysis.

  The invention further provides a system useful for enriching, for example, selected types of cellular specimens in a sample, and a method for analyzing patient status based on specimens from case and control samples. In particular, the system according to the present invention comprises one or more first enrichment regions comprising a first fluid flow path for a first specimen and a second fluid flow path for a second specimen. A plurality of obstacles to be defined, wherein the first specimen and the second specimen have different hydrodynamic diameters; and the first specimen or the second specimen is liquid-connected to the one or more concentrated areas. An analyzer for acquiring the data of the specimen and a database for storing the data.

  In the present invention, the first specimen is an erythrocyte (RBC), fetal RBC, trophoblast, fetal fibroblast, leukocyte (WBC), infected WBC, stem cell, epithelial cell, endothelial cell, stem cell, cancer cell, Examples include those selected from the group consisting of viral cells, bacterial cells, and protozoa, and examples in which the cell type is alive at a concentration of 1 × 10 −3 cells / μL or less.

  In a particular embodiment, the gap between obstacles in the first enrichment region of the system is about 1000 microns. In a further embodiment, the system comprises one or more second enrichment regions, the second enrichment region captures the first analyte or the second analyte, and the second enrichment region comprises A liquid connection is made to the first concentration region.

  In an embodiment, the one or more first enrichment regions are configured to retain 99% or more of the first analyte, and the one or more first enrichment regions are the first concentration region. The analyte concentration is configured to increase by a factor of at least 100,000.

  The present invention also relates to a method for identifying a characteristic associated with a patient condition, the method comprising: obtaining a plurality of control samples; obtaining a plurality of case samples; and each of the samples from the sample as a first analyte. Applying to a device having a plurality of obstacles for deflecting the blood sample in a direction different from the second specimen, wherein the first specimen and the second specimen have different diameters; Analyzing the first specimen from a sample to identify characteristics of the first specimen, and performing a related study based on the characteristics.

  In an embodiment, the characteristic is the appearance or disappearance of the first specimen, and in another embodiment, the characteristic is the number of the first specimen. In various contemplated embodiments, the characteristics include the morphology of the first specimen, the genotype of the first specimen, the protein metabolism of the first specimen, the RNA structure of the first specimen, and / Or gene expression of the first specimen.

  In certain embodiments, the plurality of control samples comprises at least 100 control samples. In certain embodiments, the plurality of case samples includes at least 100 case samples. In another embodiment, the control sample and case sample are blood samples. Moreover, the blood of the nuclear blood sample of an Example is 100 mL or less.

  In an embodiment of the present invention, the specimen is a cell type, and in a particular embodiment, the specimen is an epithelial cell, a cancer cell, or a fetal cell.

  In another embodiment, the present invention relates to a method for detecting biohazard analytes including but not limited to bacteria, protozoa, viral viral pathogens, toxins in the environment and other samples.

  In particular, the present invention relates to a method for detecting a biohazard analyte in a sample, wherein the biohazard analyte is found below a concentration of 1 × 10 −3 analyte / μL of the sample and the sample is applied to a concentrator / module And analyzing the concentrated biohazard specimen.

  The concentrator, in some embodiments, is a two-dimensional array of obstacles that guides the biohazard specimen deterministically in a first direction and one or more non-biohazard specimens in a second direction. With In addition or in the alternative to the obstacle array, in some embodiments, the concentrator comprises a two-dimensional array of obstacles that selectively capture the biohazard analyte. The gap between obstacles in the array described above is preferably 1000 microns, 500 microns, 100 microns, 50 microns, or 10 microns.

  In some embodiments, the concentrator is configured to retain 99% or more of the biohazard specimen.

  In some embodiments, the concentrator is configured to concentrate the biohazard analyte by a factor of at least 10,000.

  The present invention realizes an embodiment in which the concentrator has about 98% of the specificity obtained with the concentrator and more than 98% of the sensitivity.

  The invention includes embodiments where the biohazard specimen is a pathogen selected from the group comprising bacteria, protozoa, and viruses. More specifically, in an example of the present invention, the biohazard specimen is a plague, anthrax, botulinum, wild gonorrhoeae, coxiella, Brucella sp., Burkholderia bone, burkholderia pseudobone, Streptococcus, Ebola virus, Lassa virus, SARS, ulcer, alphavirus, rash typhoid rickettsia, parrot disease Chlamydia, Salmonella species, E. coli O157: H7, Vibrio cholerae, Cryptosporidium parvum, Nipah virus, Hantavirus, and A pathogen selected from the group comprising these chimeras.

  In some examples, the biohazard specimen is a cell type or a toxin.

  In particular embodiments of the invention, the sample is a water sample, an air sample, a plant or animal sample, or a soil sample. In certain embodiments, the sample is a fluid sample. In a related embodiment, the present invention further includes the step of melting the sample into a fluid sample.

  In a further embodiment of the invention, the invention provides a method wherein the enrichment removes 99% or more of the second analyte from the sample.

  In one aspect, the invention provides feeding a maternal blood sample from a pregnant woman to an analyzer configured to take an image of one or more fetal cells concentrated from the blood sample; Identifying a fetal abnormality from a maternal blood sample by analyzing a signal from one or more nucleic acid probes connected to each other, analyzing the signal, and generating a diagnostic output based on the analyzing step On how to do. Such probes may be dedicated to chromosomes such as X chromosome, Y chromosome, chromosome 21, chromosome 13, and chromosome 18, for example.

  In some embodiments, the analysis includes identifying the number of probe signals, identifying the size of the probe signal, identifying the shape of the probe signal, identifying the aspect ratio of the probe signal, Or a step of specifying a distribution of the probe signal. In some embodiments, the analysis includes capturing an image of fetal red blood cells obtained from the maternal blood sample, and inputting probe intensities for a plurality of nucleic acid probes connecting fetal nucleic acids of interest. Analyzing the probe intensity and generating a diagnostic output in response to the result of the analysis. In one embodiment, the probe is dedicated to chromosomes. In one embodiment, the probe is selected from the group consisting of X chromosome, Y chromosome, chromosome 21, chromosome 13, and chromosome 18. In one embodiment, the analyzing step includes identifying the number of probes.

  In one aspect, the invention relates to a computer program product for detecting a fetal condition, wherein the computer code for detecting fetal nucleated red blood cells (fnRBC) in a sample and one or more fetal nucleic acids of interest are coupled. Computer code for receiving probe intensity from a nucleic acid probe, computer code for analyzing the received intensity, computer code for generating a call according to the analysis result of the probe intensity, and computer reading for storing the computer code With possible media. In one embodiment, the computer readable medium is a memory, hard drive, floppy disk, CD = ROM, flash memory, or tape. In one embodiment, the probe is dedicated to chromosomes. In one embodiment, the chromosome is selected from the group consisting of X chromosome, Y chromosome, chromosome 21, chromosome 13, and chromosome 18. In one embodiment, the probe is a colorimetric probe. In one embodiment, the probe is a fluorescent probe.

  The present invention provides a prenatal test kit, comprising a size sorting module configured to separate one or more first type cells from a maternal blood sample, wherein the first type cells are It is found at a concentration of less than 1% in all blood cells in a pregnant woman's body and comprises a series of instructions for analyzing the one or more enriched cells to make a prenatal diagnosis.

  In some embodiments, the kit also includes one or more reagents (in bottles or containers). Such reagents can be selected from the group consisting of PCR reagents, lysis reagents, nucleic acid probes, and labeling reagents. An example of a labeling reagent is a FISH reagent or a FISH probe. Preferably, the FISH probe selectively connects chromosomes selected from the group consisting of X chromosome, Y chromosome, chromosome 13, chromosome 18, and chromosome 21. In some embodiments, the kit may comprise a microarray.

  In one aspect, the sizing sorting module deterministically guides the one or more first type of cells in a first direction and directs the one or more second type of cells to the second. A two-dimensional obstacle array guiding in the direction of This first type of cell may be a fetal cell (in the kit used to diagnose cancer, this first surrounding cell is an epithelial cell or a circulating cancer cell). In some embodiments, the second type of cells is enucleated red blood cells or platelets.

  In any of the examples herein, the size sorting module can retain 99% or more of the first type of cells and remove 99% of the enucleated red blood cells.

  The present invention may further include a sizing module that is fluidly connected to the obstacle array, wherein the obstacle is selected from the group consisting of anti-CD71, anti-CD36, anti-selectin, anti-GPA, anti-CD45, and anti-antibody i. Connected to the antibody.

  Diagnosis of the fetus includes fetal sex, presence of chromosome 13, chromosome 18 and chromosome 21 (Down syndrome), Turner syndrome (X chromosome damage), Kleinfelter syndrome (XXY), or the number of other sex chromosomes or autosomes Or Wolf-Hirschhorn syndrome (4p-), cat cry syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13), Angelman syndrome (15q11.2) -Q13), Miller-Dieker syndrome (17q13.3), Smith-Magenis syndrome (17p11.2), DiGeorge syndrome and membrane-cardio-facial syndrome (22q11.2), Kalman syndrome (Xp22.3), steroid monkey Fatase deficiency (STS) (Xp 2.3), X linkage ichthyosis (Xp22.3), and retinoblastoma (can be the appearance of conditions selected from the group consisting of 13q14).

  The present invention relates to a method for diagnosing a health condition of an animal, comprising: obtaining a fluid sample from the animal; and at least 10, Concentrating by a factor of 000 and analyzing one or more concentrated first specimens to determine the health status of the animal. Concentration is preferably performed using one or more sizing modules. A sizing screening module comprises a two-dimensional obstacle array that generates a deterministic flow path for a first analyte from the fluid sample and a deterministic flow path for a second analyte from the fluid sample; This specimen is different in hydrodynamic size from the second specimen. In some embodiments, the first flow path extends to the first outlet and the second flow path extends to the second outlet. The method includes analyzing one or more concentrated first analytes to determine the health status of the animal. In some embodiments, the first specimen is a cancer cell, fetal cell, or pathogen.

  In some embodiments, the animal is a domestic animal. In some embodiments, the livestock is selected from the group consisting of cattle, chickens, pigs, horses, fish, carp, dogs, cats, and goats. In one embodiment, the first specimen is a cancer cell. In some embodiments, the first analyte is a fetal cell. In some embodiments, the first specimen is a pathogen. In one embodiment, the pathogen is a bacterium, virus, or protozoan. In some embodiments, the analyzing step comprises performing a DNA analysis. In some embodiments, the analyzing step comprises performing an RNA analysis. In some embodiments, the analyzing step comprises performing a protein analysis. In one embodiment, the fluid sample is a blood sample.

  In some embodiments, the method further comprises applying a reagent to the sample, the reagent increasing the size of the first analyte by at least 10%. In one embodiment, the reagent application step occurs before applying the sample to the obstacle array. In some embodiments, the reagent application step comprises the reagent comprising a quantum dot, antibody, phage, aptamer, fluorophore, enzyme, or bead.

  In some embodiments, the analyzing step includes counting the number of enriched first analytes. In some embodiments, the condition is the sex of the animal's fetus. In some embodiments, the condition comprises microbial infection of an animal. In some embodiments, the condition includes cancer. In some embodiments, the first outlet is fluidly connected to one or more capture regions that include a plurality of obstacles that selectively capture the first analyte. In some embodiments, the plurality of obstacles to selectively capture are bound to one or more binding moieties that selectively bind red blood cells, fetal cells, cancer cells, or epithelial cells. ing. In some embodiments, the capture moiety comprises an antibody or fragment thereof.

  The invention also relates to a business method in which a screening service and diagnosis for analyzing fetal status is provided. In particular, the present invention comprises the steps of obtaining a blood sample from a pregnant or / or pregnant mammal, screening the blood sample to identify fetal cells from the mammalian fetus, The present invention relates to a business method for providing a fetal screening service, comprising analyzing and determining the status of the fetus and providing a report of the status in exchange for a service fee. In a related embodiment, the business implements the service.

  In another embodiment, the business authorizes a CLIA laboratory to perform the analysis step. The present invention also includes embodiments in which the report is performed for a health care provider or health insurance company.

  In a particular embodiment of the invention, the screening is performed in a fluid system configured to separate the fetal cells from maternal cells by guiding the cells in different directions depending on size.

  In another embodiment, the fetal status determined is trisomy 13, trisomy 18, trisomy 21 (Down syndrome), Turner syndrome (X chromosome damage), Klinefelter syndrome (XXY), and sex or autosomal number abnormalities Is selected from the group consisting of other states in which. In certain embodiments, the condition is Wolf-Hirschhorn syndrome (4p-), cat cry syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13), Angelman syndrome (15q11.2-q13), Miller-Dieker syndrome (17q13.3), Smith-Magenis syndrome (17p11.2), DiGeorge syndrome and membrane-cardio-facial syndrome (22q11.2), Kalman syndrome (Xp22.3) ), Steroid sulfatase deficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22.3), and retinoblastoma (13q14).

  The present invention also relates to a business method of business that provides diagnostics, including commercial diagnostics that perform prenatal testing for fetal genetic defects, wherein the diagnostics analyze maternal blood. In a related embodiment, the business manufactures the diagnostic. In another related embodiment, the diagnostic is made from a polymer material. In certain embodiments, the diagnosis is disposable.

  In certain embodiments, the diagnostic product analyzes maternal blood by separating fetal nucleated red blood cells (fnRBC) from enucleated red blood cells. In an embodiment, the diagnosis detects a genetic abnormality of the fnRBC. A related example includes a business model in which the genetic abnormality is detected using a label that binds a nucleic acid. In a further related embodiment, the label is a fluorescent label or a colorimetric label.

  The present invention also relates to a business method for separating fetal cells from maternal blood, performed by exchanging fees or cross licenses. Such a business method includes obtaining a blood sample from a pregnant or living mammal (eg, a human) and concentrating one or more fetal cells from the blood sample. This service is performed by exchanging fees or cross licenses.

  While preferred embodiments of the invention are illustrated and disclosed herein, those skilled in the art will appreciate that such embodiments are merely illustrative. Those skilled in the art will envision various modifications, variations, and substitutions without departing from the invention. Various alternatives to the embodiments of the invention disclosed herein may occur in practicing the invention. The accompanying claims are intended to define the scope of the invention, and the methods, structures, and equivalents within the scope of these claims are encompassed herein.

  The present invention provides systems, devices, and methods for separating, sorting, and concentrating rare analytes (eg, tissues, cells, and cellular components) from a sample, fluid sample, or more preferably a whole blood sample.

Table 1 Blood cell types, concentrations, and cells

  In some embodiments, the device is used to sort or concentrate analytes or cells from a mixed fluid, wherein the analytes or cells are 1x10-3, 1x10-4, 1x10-5, 1x10- of a fluid sample. The concentration is 6 or 1 × 10 −6 cells / μL or less. In some embodiments, the apparatus is used to sort or concentrate an analyte or cell from a mixed fluid, wherein the analyte or cell is 1: 100, 1: 1000, 1 of all cells in the sample. : 10,000, 1: 100,000, 1: 1,000,000, 1: 10,000,000, 1: 100,000,000 or less.

  In a preferred embodiment, the present invention provides a system and apparatus for sorting and enriching one or more cells from a blood sample. For example, fetal cells are enriched or separated from a maternal blood sample by the present system and method. Also, epithelial, endothelial, progenitor, foam, stem, and cancer cells can be concentrated from a blood sample. After sorting and / or enriching these and / or other analytes or rare cells from the fluid sample, the system is used to detect and analyze such analytes. Sample analysis can be used for various applications disclosed herein.

I. Sample Collection / Preparation The system and method includes obtaining one or more samples from the source to be analyzed. Samples can be obtained from water sources, food, soil, air, animals, etc. If a solid sample (eg, a tissue sample or a soil sample) is obtained, such a solid sample may be thawed or melted prior to subsequent concentration and / or analysis. If a gas sample is obtained, it may be similarly melted or melted.

  In some embodiments, where a sample is supplied from an animal, it is desirable that it be supplied from an animal, more preferably from a human. Examples of fluid samples obtained from animals include, but are not limited to, whole blood, sweat, tears, saliva, lymph fluid, bone marrow suspension, lymph fluid, urine, saliva, semen, vaginal fluid, cerebrospinal fluid, brain fluid Ascites, breast milk, respiratory secretions, intestinal and urogenital systems, and amniotic fluid. Preferably, the liquid sample obtained from the animal is a blood sample. When analyzing a liquid sample from an animal, the animal is, for example, livestock such as cows, chickens, pigs, horses, rabbits, dogs, cats, goats and the like. In a preferred embodiment, the animal is a human and the blood sample is a whole blood sample. A blood sample obtained from an animal can be used, for example, for testing / diagnosis of the condition of the animal or for prenatal testing from a pregnant animal. In the preferred embodiment, the system obtains a blood sample from a pregnant person to test the fetal condition and abnormalities.

  Liquid samples can be obtained using various known techniques in the field. For example, a syringe or other suction device is used to collect blood. A liquid sample such as blood is preferably collected in a suction tube or bag.

  In some embodiments, a liquid sample obtained from an animal is supplied directly to the device, and in other embodiments, it is pre-treated or processed before being supplied to the device of the present invention. For example, blood collected from an animal may be treated with one or more reagents before being supplied to the device of the present invention, or collected in a container containing such reagents in advance. Reagents used herein include, but are not limited to, stabilizers, preservatives, fixatives, melting agents, diluents, anti-apoptotic agents, anti-thrombotic agents, magnetic modifiers, and / or cross-linking agents.

  Examples of methods for processing liquid samples and supplying them to analyzers include US Application No. 11 / 071,270 “Sytem For Delivering a Diluted Solution” filed Mar. 3, 2004 and US application filed Sep. 15, 2005. It is described in “Methods and Systems for Fluid Delivery”, which is not assigned an application number, both of which are incorporated by reference for all purposes.

  When a blood sample is obtained from an animal, the blood volume varies depending on the size of the animal, the pregnancy period, the examination state, and the like. In some examples, 50 mL, 40 mL, 30 mL, 20 mL, 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, or 1 mL or less of a liquid sample (eg, blood) is taken from the animal. In some examples, 1-50 mL, 2-40 mL, 3-30 mL, or 4-20 mL of blood is collected from an individual. In other embodiments, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mL or more of liquid sample Are collected from animals.

  The entire collected sample is applied to the device for enrichment and / or sorting of rare analytes such as fetal cells and epithelial cells. In some embodiments, samples are taken at successive time intervals and applied to the device for further analysis.

  In some embodiments, the systems and methods provide for enrichment, sorting, and analysis of rare cells (eg, fetal cells, epithelial cells, or cancer cells) from 10 mL, 5 mL, or 3 mL blood samples. . In some examples, the systems and methods can be used to concentrate rare cells from large blood samples greater than 20 mL, 50 mL, or 100 mL. Any of the above capabilities is, for example, less than a day, or within 12, 10, 11, 9, 8, 7, 6, 5, 4, 3, 2 hours, or 60, 50, 40, 30, 20, 10 Can occur within minutes.

  In some embodiments, the blood sample is combined with a melt that selectively lyses one or more cells or components in the blood sample, such as fetal cells or components of blood cells. For example, maternal blood samples containing fetal cells are combined with water or other osmolality modifiers to selectively lyse fetal cells prior to selection and concentration of fetal cell components by the system. You may let them.

  Preferably, the blood sample is one week, six days, five days, four days, three days, two days, one day, twelve hours, six hours, three hours, two hours, or one hour after blood has been collected. To be supplied to the system within. In some embodiments, a blood sample is provided to the system as soon as it is collected from the animal. Preferably, the sample is supplied to the system at a temperature of 4-37 ° C.

II Concentration The present invention incorporates the enrichment of a rare analyte from a sample. In some embodiments, the rare analyte is a cell or cell component. Examples of rare cells include, but are not limited to, platelets, white blood cells, fetal nucleated red blood cells from maternal blood, epithelial cells, endothelial cells, progenitor cells, cancer cells, bacteria, viruses, progenitor cells, and chimeras thereof including. Examples of cellular components include, but are not limited to, mitochondria, ribozymes, endoplasmic reticulum, Golgi apparatus, proteins, protein complexes, and nucleic acids. These sorting is preferably achieved by size. The sample of the present invention is a solid, gas, or liquid sample. The solid sample is preferably melted or melted prior to the concentration step.

  Concentration can be performed by one or more methods and apparatus known in the art, particularly International Publication Nos. 2004/029221 and 2004/113877, U.S. Publication No. 2004/0144651, U.S. Pat. No. 5,641,628, 5,837,115, and 6,692,952, U.S. Application Nos. 60 / 703,833, 60 / 704,067, 60 / 668,415, 10 / 778,831, 11 / 071,679, and 11/146. 581, all of which are incorporated herein by reference for all purposes. In preferred embodiments, analyte enrichment or sorting is performed by one or more size-based sorting modules (eg, mesh, matrix, electrophoresis modules) and optionally one or more capture modules (eg, affinity-type). This is realized using a sorting module, an antibody, and a magnetic bead.

1. Size Sorting The size sorting module sorts analytes from a liquid sample based on the hydrodynamic size of the analyte in the sample. In a preferred embodiment, the sizing module comprises a two-dimensional array of one or more obstacles that make up the gap array. The obstacle array is preferably two-dimensional and preferably constitutes a zigzag array. These arrays are configured so that liquid passing through the gaps in the array is unevenly distributed in the continuous gaps. The deflection angle is, for example, at least 10, 20, 30, 40, 50, 60, 70% of the pitch. Preferably, the sorting module may be configured to deflect analytes larger than the reference size away from the obstacle array and into the bypass channel. In some embodiments, the sizing module includes 10, 100, 1,000, 10,000, 100,000 or more obstacles. If the obstacles are aligned in a two-dimensional array, this array can be, for example, 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 400. , 600, 800, 1000 or more obstacle rows.

  In the preferred embodiment, the gaps, obstacles, or both are medium scale (1 mm or less in one direction). FIG. 1 shows an example of a size type selection module. Obstacles (which may be of various shapes) are attached to a flat substrate to form an array of gaps. A transparent cover or lid is used to cover the array. Obstacles constitute a two-dimensional array, and each successive row is arranged in a zigzag manner up and down. The average flow rate is designed by this field array. In some embodiments, the obstacle array is designed to pass and process 1 mL, 2 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 200 mL, or 500 mL of liquid sample at least every hour. The sample stream of the sizing screening module may be arranged at a small angle (flow angle) with respect to the line of sight of the array. Optionally, a sizing sorting module may be coupled to the infusion pump to pass the sample through the obstacle.

  This sizing module allows analytes (eg, cells) with a hydrodynamic size larger than a reference size to move along the line of sight of the array, while those with a hydrodynamic size smaller than the reference size move the flow in different directions. You may comprise so that it may flow. The hydrodynamic size of the analyte depends on the physical dimensions of the analyte, the permeability of the liquid medium, and the shape and deformability of the analyte.

  FIG. 2 shows this example; the first flow path A for the first specimen is of a first hydrodynamic size. The more tortuous second flow path within the obstacle is a deterministic path for the second specimen that has a smaller hydrodynamic size than the first specimen. The second specimen is seen to flow more in the average flow in the array than the first specimen. This flows through the deterministic channel B. In addition, a third specimen having a hydrodynamic size smaller than that of the first and second specimens flows exclusively through the obstacles in the array and the channel C in the average channel.

Multiplexing In any of the embodiments herein, one or more obstacle arrays are liquid connected in series or parallel. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 , 100 or more sorting modules are liquid-connected in parallel. Preferably about 10-20 such modules are liquid connected in parallel. When more than one sorting module is fluidly connected in parallel, high throughput analysis of the sample being tested is achieved (eg, 1,1.5,2,2.5,3,3.5,4,4.4 per hour). 5, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mL liquid sample, or more preferably 5 mL fluid sample per hour).

  FIG. 3 shows one embodiment of multiplexing. In FIG. 3, two obstacle arrays are arranged next to each other, that is, in a mirror image. In this configuration, the critical size of the two arrays may be the same or different. In addition, these arrays are configured such that the main flow is at the boundary of the two arrays, the edge of each array, or a combination thereof. Such a double array also includes a central region between the two arrays for collecting particles larger than the reference size or modifying the sample (eg, by buffer exchange, reaction, or labeling). Yeah. In FIG. 3, the central region or bypass channel is provided in an obstruction in the shape of a piece of cheese and the backflow is incorrect.

  Providing a plurality of arrays in parallel with one device increases the throughput of sample processing, and enables parallel processing of multiple types of samples and sample portions with different fragments or operations. Moreover, the flow rate of the liquid processed by the sorting module increases. When processing the same sample in parallel, the outlet may or may not be liquid connected. For example, when multiple arrays are the same reference size, the outlets are connected to achieve high throughput sample processing. In another example, if the reference size of the array is not all the same or if all the particles in the array are not processed in the same way, the outlet will not be liquid connected. In some embodiments, multiplexing is achieved by mounting multiple duplex arrays on a single device. Double or single arrays can be arranged in various possible three-dimensional relationships with each other. In some embodiments, the multiplex device comprises two or more obstacle arrays that are liquid-connected in series. For example, the outlet from the main flow of one device may be connected to the inlet of the second device. Alternatively, a small flow outlet of one device may be connected to a second device.

  In another embodiment, multiple arrays may be used to sort a wide size range of analytes. For example, a device may include three arrays that are liquid-connected in series, although various other numbers of arrays may be used. Usually, the cut-off size in the first array (the most upstream array) is larger than the cut-off size of the second array (the next array downstream of the first array), and the cut-off size of the first array Is configured to be smaller than the passage size of the second array. The same applies to subsequent arrays. The first array deflects (removes) analytes that stagnate in the second array. Similarly, the second array deflects (removes) analytes that stagnate in the third array.

  As described above, in a multi-stage array, for example, large particles such as cells that cause downstream clogging are deflected first, but these deflected particles are blocked by bypassing the downstream stage. Need to prevent. Thus, the device of the present invention comprises a bypass channel that removes the output from the array. Although the case of removing particles larger than the reference size is described here, the bypass channel can also be used to remove output from various portions of the array.

  In either embodiment, the sorting module is preferably 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.99. Liquid samples with specificities of 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or more Select the specimen of interest (especially fetal cells and epithelial cells). In either embodiment, the sorting module is preferably 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%, 99.95% or more of interest from a liquid sample Select a specimen (especially fetal cells or epithelial cells).

  Furthermore, in any of the examples, the specimens of interest are 5, 2, 1, 5 × 10−1, 2 × 10−1, 1 × 10−1, 5 × 10−2, 2 × 10−2, 1 × 10−2, 5 × 10−3, and 2 × 10−3. 1x10-3, 5x10-4, 2x10-4, 1x10-4, 5x10-5, 2x10-5, 1x10-5, 5x10-6, 2x10-6, 1x10-6, 5x10-7, 2x10-7, or It can be concentrated from an initial concentration of 1 × 10 −7 analyte / μL liquid sample or less. Also, in any embodiment, the sorting module includes less than 1% of all analytes (eg, cells) in the sample, or all analytes (eg, cells in a sample (eg, a blood sample obtained from an animal such as a human)). 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, 0.001%, 0.0005%, 0.0002%, 0.0001%, 0.00005%, 0.00002%, 0.00001%, 0.000005%, 0.00002%, 0.000001% or less can be selected. The sorting module can increase the concentration of such an analyte of interest by changing from a liquid sample to a concentrated sample (sometimes in a new liquid medium such as a buffer). The new concentration of the analyte in the concentrated sample is 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50 from at least one sample. , 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 times more concentrated.

Inlet / Outlets Furthermore, the number of inlets and / or outlets may vary depending on the intended use of the device. In the preferred embodiment, a single obstacle array has two or more outlets. An example of such an array is shown in FIG. 4, where 14 pairs of arrays are linked for high throughput of sample processing. Each array has a first inlet for supplying a sample and a second inlet for supplying a reagent such as a buffer to the array. Each array also has a first outlet for waste (unwanted product) and a second outlet for product (analyte of interest).

  In some embodiments, the size sorting module includes a first outlet that is directed away from the normal flow direction to remove large analytes, and a small sample that flows in the normal flow direction through the obstacle array. And a second outlet to be taken out. Additional outlets may be provided to collect fragments at various points in the sorting process. Further, in some embodiments, one or more inlets are provided in a single two-dimensional array. This inlet may be, for example, a stabilizer, preservative, fixative, melting agent, diluent, anti-apoptotic agent, labeling agent, anticoagulant, antithrombotic agent, buffer solution, osmolality adjusting agent, pH adjusting agent, Additional samples and / or reagents such as stabilizers, PCR reagents, detergents, and / or crosslinkers may be supplied.

  In some embodiments, the analyte of interest (eg, fetal cells) is selectively melted, and then a liquid sample containing the cellular components of the cells of interest is applied to the sorting module. Cell components of interest in the cells are separated from other cells in the blood sample based on size using the methods disclosed herein or known techniques. When supplying the sorter with the sample at the same time as the sample, or when the sample is first mixed with the melt and then fed to the sorter, the sorter is, for example, the nucleus, mitochondria, ribozyme, lysosome, endoplasmic reticulum, or Golgi apparatus Configured to deflect / separate one or more organelles. For example, in some embodiments, the maternal blood sample is mixed with a melting agent that selectively melts fetal nucleated red blood cells. Such a melting agent is, for example, a drug known in this field for selectively melting water and fetal cells. The blood sample is then supplied to the complaint and selectively deflects all or nearly all other analytes from the blood sample, thereby enriching the concentration of fetal erythrocyte organelles (eg, nuclei). . In such an embodiment, the nuclei are discharged from the “waste” outlet. In another embodiment, the melting agent is supplied with the blood sample from the second inlet. In this embodiment, melting occurs simultaneously with sorting.

  In some embodiments, one or more analytes may be contacted with a binder portion (eg, a magnetic bead) that selectively binds the analyte and increases its size (hydrodynamic size). The unbound portion is removed because of its reduced size (from the “waste” outlet), and the bound analyte is altered by size and removed from another outlet.

  The configuration and / or shape of the device may be designed in various ways. For example, an annular well outlet and outlet may be used (see the annular inlet in FIG. 4). An entrance area free of obstructions is incorporated into the design to ensure that blood cells that reach the obstruction area are evenly distributed. Similarly, the exit area is provided with an exit area free of obstacles so that existing cells can be collected uniformly without damage.

Bypass channel As the fluid sample analyte and / or cells flow through the obstacle array, the hydrodynamic size larger than the reference size is deflected to the bypass channel. Bypass channels have channels wider than the average gap between obstacles. Furthermore, the width of the bypass channel is equal to or greater than the largest element (largest cell) that is separated from the sample. For example, in some embodiments, the width of the bypass channel in the sorting module is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 microns or more. In some embodiments, the main channel width is 100, 90, 80, 70, 60, 50, 40, 30, 20 microns or less.

  The bypass channel may form the periphery of the obstacle or its outer edge. Such obstacles preferably prevent backflow or turbulence of large cells that have reached the bypass channel. In some embodiments, the obstruction in the bypass channel is a cross-section cheese piece with a wedge-shaped pointed end pointing downstream (see FIG. 3).

  In some embodiments, a single bypass channel is used to share this bypass channel in one or more stages (arrays). In some embodiments, multiple bypass channels are used. For example, each of the multiple stages may have its own bypass channel. In one embodiment, large specimens (eg fetal cells, epithelial cells, tumor cells) are deflected into the mainstream and then into the bypass channel to prevent clogging. Small cells that do not clog go to the second stage where they are further sorted by size. This design is repeated as many times as desired. At each stage, the bypass channel is fluidly connected to the outlet so that multiple deflections can be recovered from the sample. The bypass channel may also be designed to keep the flow through the device constant, to remove a certain amount of flow so that the flow of the array is not disturbed, or to increase the flow rate in certain areas. On Saturdays, the boundaries of the array may be designed to form a unique flow pattern (eg, supply flow or extract flow).

  In either embodiment, each array has a maximum pass size several times larger than the cutoff size. This result can be obtained by combining a large gap and a small branching ratio ε. In some embodiments, this ε is approximately 1/2, 1/3, 1/10, 1/30, 1/100, 1/300, 1/1000. Also, in such an embodiment, the shape of the obstacle affects the shape of the flow through the gap, but the obstacle may narrow in the flow direction to shorten the array. A single stage array may comprise a bypass channel as described herein.

Obstacle shape The size and shape of the obstacles in the sizing module may be uniform or may vary to form a uniform or non-uniform pattern. For example, the obstacle may have a cylindrical, moon-shaped, or square cross section. In a preferred embodiment, the obstacle is cylindrical so that it has a round cross-sectional shape. Preferably, the obstacle diameter (longest cross-sectional length) is 4-40 microns, 5-30 microns, 6-20 microns, or 7-10 microns. In some embodiments, the diameter of the screening interface is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 30, 40, 50 microns or more. In some embodiments, the obstacle diameter is 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 microns or less. The distance between the screening obstacles may vary. In some embodiments, the distance between obstacles is at least 10, 25, 50, 75, 100, 250, 500, 750 μm. In some embodiments, the distance between obstacles is a maximum of 1000, 750, 500, 250, 100, 75, 50, 25 μm. Furthermore, the diameter, width, and length of the obstacle are at least 5, 10, 25, 50, 75, 100, and 250 μm, and the maximum is 500, 250, 100, 75, 50, 25, and 10 μm. The height of the obstacle may vary, but is preferably equal to or greater than the largest specimen to be selected. In some embodiments, the height of the sorting obstacle is 10-500 microns, 20-200 microns, 30-100 microns, 40-50 microns. In some embodiments, the screening obstacle is 1500, 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 microns or less.

Specimen Size In some embodiments, the sorting module comprises a first sorting area adapted to separate analytes (rare cells) from a liquid sample, the hydrodynamic size of the specimen being 20, 19, 18 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 micron or more. More preferably, the sorting module comprises a first sorting area adapted to sort an analyte from a liquid sample, the hydrodynamic size of the analyte being 15, 14, 13, 12, 11, 10, 9, 8 , Greater than 7, 6, 5, 4 microns. More preferably, it comprises a first sorting area adapted to sort the analyte from the liquid sample, the hydrodynamic size of the analyte being greater than 10, 9, 8, 7, 6, microns.

  In one embodiment, the sorting module comprises a first sorting area and a second sorting area, the first sorting area having a hydrodynamic size of at least 15, 20, 25, 30, 35, 40 microns or more. Wherein the second sorting region is configured to sort a sample having a hydrodynamic size of at least 10, 15, 20, 25, 30, 35 microns or more. The reference size is larger than the reference size of the second area. The first and second sorting areas are in liquid connection (liquid passage) with each other, and the second sorting area is continuously provided downstream of the second sorting area. In some embodiments, the sorting area may also comprise a third sorting area configured to sort components having a hydrodynamic size of at least 5, 10, 15, 20, 25, 30 microns or more. Well, here, the reference size of the second region is larger than the reference size of the first region. The third sorting area is liquid-connected downstream of the second sorting area. This sorting module may optionally comprise additional areas as described above, each of which sorts out very small components from the sample.

  In one embodiment, the sorting module is configured to guide an analyte having a hydrodynamic size (eg, diameter) of 15 microns or greater to the main channel away from the direction of flow of the smaller components; the second sorting region is Configured to guide components having a hydrodynamic size (e.g., size) of 7.5 microns or more in the sample to the main channel away from the flow direction of the smaller components; the third sorting region is the fluid in the sample A component having a mechanical size (eg, size) of 5 microns or more is configured to be guided to the main channel away from the flow direction of the smaller component. The above example is particularly useful for sorting red blood cells from a blood sample.

  Of course, the sorting module described above may be configured to sort smaller or larger components from the liquid sample. For example, in some embodiments, the sorting module is configured to sort all components greater than 4 microns in diameter (eg, fetal nucleated red blood cells, nucleated red blood cells, white blood cells). In some embodiments, the sorting module is configured to separate nucleated cells in the blood sample from non-nucleated cells.

  In some embodiments, the sorting device is used to concentrate a cell type or component of interest in a liquid sample (eg, blood sample, urine sample, other body sample), where the cell type or component of interest is Seen in vivo at a concentration of 50, 40, 30, 20, 10% or less of total blood cells, or more preferably 0.9, 0.8, 0.7, 0.6, 0. 5,0.4,0.3,0.2,0.1% or less, more preferably 0.09,0.08,0.07,0.06,0.05,0 of whole blood cells. 0.04, 0.03, 0.02, 0.01 or less, more preferably 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003 of whole blood cells. , 0.002, 0.001 or less, more preferably 0. 009, 0.0008, 0.0007, 0.0006, 0.0005, 0.0004, 0.0003, 0.0002, 0.0001 or less, more preferably 0.00009, 0.00008, 0.00007, 0.00006, 0.00005, 0.00004, 0.00003, 0.00002, 0.00001 or less.

Specificity / sensitivity In either embodiment, the size sorter can be used to sort one or more cell types from a mixed cell population (eg, whole blood) with improved effectiveness. . For example, a size sorter can perform ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80%, ≧ 90%, ≧ 91%, ≧ 92%, ≧ 92% of all nucleated cells from a whole blood sample after sorting. Ensure 93%, ≧ 94%, ≧ 95%, ≧ 96%, ≧ 97%, ≧ 98%, ≧ 99%, ≧ 99.9%, more preferably all nucleated fetal erythrocytes from the maternal blood sample ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80%, ≧ 90%, ≧ 91%, ≧ 92%, ≧ 93%, ≧ 94%, ≧ 95%, ≧ 96%, ≧ 97%, ≧ Ensure 98%, ≧ 99%, ≧ 99.9%. Similarly, the device is ≧ 50%, ≧ 60%, ≧ 70%, ≧ 80%, ≧ 90%, ≧ 91%, ≧ 92%, ≧ 93% of all epithelial cells from a whole blood sample after sorting, ≧ 94%, ≧ 95%, ≧ 96%, ≧ 97%, ≧ 98%, ≧ 99%, ≧ 99.9%, or ≧ 50%, ≧ 60% of all cancer cells from blood samples ≧ 70%, ≧ 80%, ≧ 90%, ≧ 91%, ≧ 92%, ≧ 93%, ≧ 94%, ≧ 95%, ≧ 96%, ≧ 97%, ≧ 98%, ≧ 99%, ≧ Secure 99.9%. At the same time, the sorting module also allows ≧ 95%, ≧ 96%, ≧ 97%, ≧ 98% of all nucleated cells from the whole blood sample after sorting unwanted analytes (eg red blood cells and platelets) from the liquid sample, Remove ≧ 99%, ≧ 99.9%. FIG. 8 shows some examples of specificity and sensitivity obtained with one embodiment of this sizing screening module.

  Any or all of the above steps can occur with minimal dilution of the product. In some embodiments, the desired analyte of interest is 50, 40, 30, 20, 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.5 of the original sample. , 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5 times or less in a solvent and separated. In some embodiments, any or all of the above steps may occur while concentrating the desired product. For example, the concentrated analyte of interest is at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5 than the original sample in the final concentrated solution. 0.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 100,000, 500,000 times concentrated Has been. For example, if the first cell type is concentrated 10 times from a blood sample, the ratio of the first cell type in all the cells in the sample after application to this apparatus will be 10 times larger. Such concentration is used for a total volume of 10 mL or 20 mL or more of a liquid sample (eg, blood sample) containing a rare component of interest, and such a rare component of interest can be concentrated to a total volume of 5 mL or less.

  In one example, a reagent is added to the sample that selectively or non-selectively increases the hydrodynamic size of the analyte in the sample. This conditioned sample is passed through the obstacle array of the present invention. If the specimen is enlarged and the hydrodynamic size is increased, the gap size can be more easily manufactured with a large obstacle array. In a preferred embodiment, the enlargement step and the concentration by size are achieved with an integrated method of the device. Suitable reagents include various hypotonic solvents such as deionized water, 2% sugar solution, or non-aqueous solvents. Other reagents include beads such as magnetic or polymers that bind selectively (eg, through antibodies and avidin biotin) or non-selectively.

  In another example, a reagent is added to the sample that selectively or non-selectively reduces the hydrodynamic size of the analyte in the sample. If the particle size in the sample is reduced non-uniformly, the difference in particle mechanical size between specimens increases. For example, nucleated cells are separated from enucleated cells by contracting the cells with high osmotic pressure. These enucleated cells shrink into very small particles, but nucleated cells do not shrink below the nucleus size. An example of a shrinking agent is a hyperosmotic solvent.

  In an alternative embodiment, an affinity functional bead is used to increase the volume of the particle of interest relative to the other particles in the sample, thereby making the gap size obstacle larger and easier to manufacture. Can be used.

  In either embodiment, the liquid may be passed dynamically or statically through the device. The liquid may be delivered using an electric field, centrifugal force, pressure driven flow, electroosmotic flow, or capillary action. In the preferred embodiment, the average direction of the field is parallel to the walls of the channel containing the array.

Separation by capture The system may optionally comprise one or more capture modules. The capture module concentrates the analyte of interest (eg, cells) from the liquid sample by limiting or prohibiting its movement or movement, or by multiplexing with the capture moiety. In some embodiments, the capture module uses affinity-based sorting, but affinity-based sorting is just an option.

  This capture module is highly unique and selective. In either embodiment, the capture module is preferably 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97 in separating the analyte of interest (eg, fetal cells or epithelial cells) from the liquid sample. %, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.%. Specificity of 9%, 99.95% or more. In any embodiment, the capture module is preferably 50%, 60%, 70%, 80%, 90%, 95%, 96% in separation of the analyte of interest (eg, fetal cells or epithelial cells) from the liquid sample. 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99 .9%, 99.95% or higher sensitivity.

  Furthermore, in any of the embodiments, the analyte of interest is captured by the capture module in the form of 5, 2, 1, 5x10-1, 2x10-1, 1x10-1, 5x10-2, 2x10-2, 1x10-2, 5x10-3, 2x10-3, 1x10-3, 5x10-4, 2x10-4, 1x10-4, 5x10-5, 2x10-5, 1x10-5, 5x10-6, 2x10-6, 1x10-6, 5x10-7, 2x10- It can be concentrated (ie separated) from an initial concentration of 7 or 1 × 10 −7 analyte / μL liquid sample. Also, in any embodiment, the capture module may contain less than 1% of all analytes (eg, cells) in a sample, or all analytes (eg, cells) in a sample (eg, a blood sample obtained from an animal such as a human). 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, 0.001%, 0.0005%, 0.0002%, 0.0001%, 0.00005%, 0.00002%, 0.00001%, 0.000005%, 0.00002%, 0.000001% or less can be selected. The capture module can bring such analyte concentrations of interest at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000 from the original sample. 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 , 100,000,000,000, 200,000,000, 500,000,000, 1,000,000,000, 2,000,000,000, or 5,000,000,000 times more.

  In some embodiments, the capture module includes a channel having an obstacle array. These negotiations may be one or more shapes. The array is preferably two dimensional and the obstacles may be uniform or non-uniform in order. In the preferred embodiment, this is a two-dimensional, uniform array with obstacles in a zigzag arrangement.

  Examples of capture modules are disclosed in International Publication No. 2004/029221, and U.S. Patent Nos. 5,641,628, 5,837,115, 6,692,952, which are incorporated herein for all purposes. It is incorporated by.

Shape and size To increase the amount of binding, it is desirable to increase the surface area of the obstacle or to increase the number of free contact between the sample and the obstacle. Accordingly, the capture obstacle of the present invention may increase its surface area and / or the number of contact with the sample in various shapes and forms. In addition, the shape and size of the obstacle may vary depending on the specimen to be captured and the sample concentration. The larger the specimen captured by the capture module, the more obstacles will be captured. In some embodiments, the height of the obstacle is 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 microns or less. In some embodiments, the height of the obstacle is 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 microns or more.

  Similarly, the size of the gap between the obstacles varies depending on the size of the obstacle to be captured. In some embodiments, the gap between obstacles is 50, 40, 30, 20, 10 microns or less. In some embodiments, the gap between the obstacles is no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 times the granular mechanical size of the specimen of interest. In some embodiments, the gap between obstacles is less than or equal to the granulodynamic size of the analyte of interest. In such an embodiment, the analyte of interest is captured between obstacles. The present invention contemplates both arrays that are wider than the analyte of interest and that are narrower than the analyte of interest. In some embodiments, limited gaps (same or less in width as the analyte of interest) are uniformly or non-uniformly distributed across the obstacle array. Preferably, the limited gaps are non-uniformly distributed across the obstacle array.

  In some embodiments, the diameter of each obstacle is 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60. , 50, 40, 30, 20 microns or less. In another embodiment, the diameter of each obstacle is greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 microns.

  In some embodiments, the obstacle in the capture array selectively (and selectively reversibly) binds one or more components of the liquid sample reversibly or irreversibly. One obstruction includes, for example, one or more capture moieties that selectively have an affinity for cells or components in a liquid sample. Such capture moieties specifically bind cells or components of interest such as fetal cells, red blood cells, white blood cells, platelets, epithelial cells, cancer cells, endothelial cells, or other rare cells. For example, in some embodiments, the capture moiety comprises an antibody (or fragment thereof) that specifically binds red blood cells or epithelial cells. Such antibodies include, for example, anti-CD71 and EpCAM antibodies, respectively. In a preferred embodiment, such antibodies are single cells. Other antibodies that can be capture moieties include, but are not limited to, anti-CD235a, anti-CD36, anti-selectin, anti-carbohydrate, anti-CD45, anti-GPA, anti-antigen i. FIG. 6 shows an embodiment of the present invention in which fetal cells are bound to an obstacle bound to a binding moiety (anti-CD71). FIG. 7A shows the path of the first analyte through the post array, where the analyte not specifically bound to the post continues to move through the array and the array not bound to the post is captured in the array. FIG. 7B is a photograph of a post covered with antibodies. FIG. 7C shows antibody binding to a substrate (eg, an obstacle, sidewall, etc.) implemented in accordance with the present invention.

  Along with the sorting module, the capture module may have multiple regions, each of which selectively binds different cells and / or components of interest. A system comprising a module having a plurality of capture regions comprises two or more capture regions that are liquid connected in series with each other. Furthermore, one system may include a plurality of sorting modules that are liquid-connected in parallel to increase the amount of sample to be analyzed simultaneously.

  When enriching the first type of cells from a mixed population of cells (eg blood), preferably at least 60%, 70%, 80%, 90%, 95%, 98 that can bind to the surface of the capture module. %, 99% of the cells are removed from the mixture. The surface coating of the capture module is preferably designed to minimize nonspecific binding of cells. For example, 99%, 98%, 95%, 90%, 80%, 70% of cells or analytes that cannot bind to the binding moiety are not bound to the surface of the capture module. By selective binding of the capture module, a specific analyte (a collection of active cells) can be separated from the mixture of cells. The device is provided with obstacles to increase the surface area of the device so that analytes (eg cells) interact, increasing the likelihood of binding within the chamber containing the obstacles. The flow condition is handled very gently in the device and does not need to be mechanically deformed to pass between obstacles. Positive or negative pressure delivery or flow from a fluid stream is used to move cells into or out of the microfluidic device (eg, capture module) of the present invention.

  Preferably, the method comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% of the desired analyte (eg, cells) relative to the initial mixture, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99. 95% is secured, and there is a possibility of concentrating a desired set of specimens by a factor of 100, 1000, 10,000, 100,000, 1,000,000 compared to the quantity of specimens in the sample. .

  In some embodiments, the capture module has 10, 100, 1,000, 10,000, 100,000 or more obstacles. When aligning such obstacles in a two-dimensional array, the array may be, for example, 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, It has 200, 400, 600, 800, 1000 rows of obstacles.

Magnetic In some embodiments, the capture module uses magnetic particles, magnetic fields, and / or magnetic devices / device components for the purpose of separating and / or concentrating one or more analytes.

  The magnetic particles of the present invention may be of various sizes and / or shapes. In some embodiments, the diameter of the magnetic particles is 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 500 nm or less. In some embodiments, the diameter of the magnetic particles is between 10-1000 nm, 20-800 nm, 30-600 nm, 40-400 nm, 50-200 nm. In some embodiments, the magnetic particle diameter is 10 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 5000 nm or more. The magnetic particles may be dried or immersed in a liquid. The mixture of the liquid sample containing magnetic particles and the second liquid medium is accomplished by a variety of known means including the unassigned name “Methods and Systems for Fluid Delivery” filed on September 15, 2005. be able to.

  In some embodiments, the analyte in the sample (the analyte of interest or not) is ferromagnetic or not magnetic, and such an analyte utilizes one or more other magnetic fields. Can be separated or removed from any specimen (specimens of interest or not). FIG. 8 shows an example of this capture mechanism, where a first analyte is bound to an antibody that specifically binds the first analyte, and this antibody is also bound to a nanobead. When the mixture of the specimen including the first specimen-nanobead complex and the second specimen are supplied within the market price, the first specimen-nanobead complex is captured, and other cells move in the magnetic field. Continue. The first specimen is released when the magnetic field is removed.

  The magnetic field may be internal or external to the device. The external magnetic field is sourced outside the device (eg, container, channel, obstacle). The internal magnetic field is where the source is in the device here.

  In some embodiments, if the analyte to be sorted (eg, the analyte of interest or not) is not ferromagnetic or non-magnetic, the magnetic particles are combined with a binding moiety that selectively binds such analyte. May be. Examples of binding moieties include, but are not limited to, polypeptides, antibodies, nucleic acids and the like. In preferred embodiments, the binding moiety is an antibody that selectively binds to the analyte of interest (eg, red blood cells, cancer cells, epithelial cells). Thus, in some embodiments, the magnetic particles are covered with an antibody (preferably a monoclonal antibody) selected from the group consisting of anti-CD71, anti-CD45, anti-EpiCAM, or various antibodies disclosed herein.

  The magnetic particles may be combined with one or more devices herein prior to contact with the sample, or may be mixed into the sample prior to supplying the sample to the device.

  In some embodiments, the system may include a reservoir that contains reagents (eg, magnetic particles) that can change the magnetism of an analyte that is captured or not captured. This reservoir is preferably in fluid communication with one or more of the devices / modules. For example, in some embodiments, a magnetic reservoir is connected to the sizing module, and in another embodiment, the magnetic reservoir is connected to the capture module.

  The exact nature of the reagent depends on the nature of the analyte. Examples of reagents include transition metal oxidizing or reducing agents, reagents that oxidize or reduce hemoglobin, magnetic beads that bind to analytes, or chelate, oxidize, or bind iron or other magnetic materials or particles. Contains reagents. This reagent acts to change the magnetism of the specimen, and applies or enhances the adsorption force to the magnetic field, imparts or strengthens the repulsive force to the magnetic field, or removes the magnetism from which the specimen is affected by the magnetic field.

  Magnetic particles that react to a magnetic field can be applied to the apparatus and method of the present invention. Desirable particles are those whose surface chemistry can be altered chemically or physically by, for example, chemical reaction, physical adsorption, entanglement, or electrostatic interaction.

  The capture moiety may be coupled to the magnetic particles by various means known in the art. Examples are chemical reactions, physical adsorption, entanglement, or electrostatic interactions. The capture moiety bound to the magnetic particle depends on the nature of the analyte of interest. Examples of capture moieties include, but are not limited to, proteins (eg, antibodies, avidin, cell surface receptors), charged or uncharged polymers (eg, polypeptides, nucleic acids, synthetic polymers), hydrophobic or hydrophilic polymers, small cells ( For example, biotin, receptor ligands, chelating agents), carbohydrates, ions and the like. Such capture moieties are in particular cells (eg bacterial, pathogenic, fetal cells, fetal blood cells, cancer cells, blood cells) organelles (eg nuclei), viruses, peptides, proteins, carbohydrates, polymers, nucleic acids, Supramolecular complexes, other biological molecules (eg organic or inorganic molecules), small molecules, ions, or combinations (chimeras) or fragments thereof. Examples of capture moieties specifically used for fetal cells include anti-CD71, anti-CD36, anti-selectin, anti-GPA, anti-carbohydrate, and complete transferrin. Thus, in another embodiment, the capture moiety is specialized for fetal cells.

  If the magnetic properties of the analyte change, it can be used to affect the separation or concentration of the analyte relative to other components of the sample. This separation or concentration includes attracting a desired analyte to the magnetic field by positive selection using a magnetic field, or attracting an uninteresting analyte by negative selection. In either case, a collection of specimens containing the desired specimen is collected for analysis or further processing.

  The device that performs this magnetic sorting may be a variety of devices that generate a magnetic field (eg, any of the devices or reservoirs described herein). In one embodiment, a MACS sequence is used to affect the selection of magnetically modified analytes. When an analyte becomes magnetically reacted with a reagent (eg, using any of the reagents described herein), it is bound to the MACS sequence, thereby concentrating the desired sample relative to other components of the sample Is realized.

  In another embodiment, the sorting is accomplished using an apparatus such as a microfluidic device that preferably comprises a plurality of magnetic obstacles. If the sample in the sample is modified to react magnetically (for example, by strengthening the inherent magnetism of the sample or by binding a magnetically reacting particle to the sample), the sample will bind to the obstacle and thereby bind Concentration of the collected specimen is achieved. Alternatively, negative selection may be used. In this example, magnetic reaction particles are bound to a desired specimen so that the desired specimen does not magnetically react or to an undesired specimen. In this case, an undesired sample is held on the obstacle, while a desired sample is not held, thereby concentrating the desired sample.

  The magnetic region of the device can be made of hard or soft magnetic materials such as, but not limited to, rare earth materials, neodymium-iron-boron, iron-chromium-cobalt, nickel-iron, cobalt-platinum, and strontium ferrite. it can. Part of the device can be manufactured directly from a magnetic material, or the magnetic material may be applied to another material. The use of hard magnetic material can simplify the design of the device because a magnetic field is generated without the need for other actuations. However, soft magnetic materials can easily release the bound analyte by demagnetizing the material and send it downstream. Depending on the magnetic material, the application process includes cathodic sputtering, sintering, electrodeposition, or a thin film coating of a composite of polymer-bonded magnetic powder. A preferred embodiment is a thin film coating of obstacles (eg, silicon posts) micromachined by rotational molding using a polymer composite such as polyimide-strontium ferrite (polyimide acts as a binder and strontium ferrite acts as a magnetic filler). It is. After coating, the polymer magnetic coating is cured and stable mechanical properties are obtained. After curing, the device is briefly exposed to an external induction field, which preferably determines the orientation of the permanent magnets within the device. The magnetic flux density of the magnetic field from the post and the intrinsic coercive force can be controlled by the amount of magnetic filler.

  In another embodiment, a conductive material is micropatterned on the outer surface of the encapsulated microfluidic device. This pattern consists of a single electrical circuit with a spatial period of about 100 microns. By controlling the design of this electrical circuit and the magnitude of the current flowing through the circuit, it is possible to improve the periodic range of high and low magnetic strength in the satirized microfluidic device.

  The magnetic particles may be distributed uniformly throughout the device, or may be distributed in spatially dispersed areas. Furthermore, the magnetic particles can be used to create structures in the device. For example, two magnetic regions on the opposite side of the channel may be used to attract magnetic particles to form a “bridge” that connects these two regions.

  As described above, the present invention constitutes an analyzer for concentrating specimens such as bacteria, viruses, fungi, cells, cell components, viruses, nucleic acids, proteins, protein complexes, carbohydrates, fragments or combinations (chimeras) thereof, and the like. To do. Furthermore, instead of magnetic properties, the device can be used to perform various operations on the specimen in the sample. Such operations include enlargement or concentration of the particles, subdivision of the size formula, or modification of the particles themselves or the liquid carrying the particles. Preferably, the device concentrates rare specimens (rare cells) from a mixture of different components, for example by exchanging liquid in suspension or bringing a reagent into contact with the specimen. It is used to modify. Such an apparatus, for example, has little mechanical lysis and intracellular activity of cells, and can highly concentrate while limiting the stress applied to the cells.

  Although the example of a cell was demonstrated initially, the apparatus of this invention can be used for the various test substance of the size which can be sorted with the apparatus of this invention.

  The apparatus of the present invention can be used, for example, on a concentrated sample that is in contact with an analyte and interacts hydrodynamically with each other or affects flow distribution around another analyte. For example, the method can sort white blood cells from red blood cells of whole blood of a human donor. Human blood usually contains 45% or less of cells by volume. As the cells pass through the array, they are physically contacted and / or bound to each other mechanically.

  The method of the present invention includes the step of selecting one or more analytes from a sample based on the magnetic properties of the one or more analytes. In one embodiment, a reagent is added to the sample that modifies the magnetic properties of the analyte. This modification is achieved by magnetic particles. In one embodiment, particles (eg, magnetic particles) are bound to the surface of the device and the desired analyte (eg, fetal cells, pathogen cells, cancer cells, or bacterial cells) in the sample within the device. ) Is secured. Thus, the analyte or analyte of interest will then bind to the surface of the device. In another embodiment, the desired specimen is secured in the device through a mechanism based on size, shape, and deformability. In another embodiment, negative selection is employed, where the desired analyte is not bound to magnetic particles. In any embodiment, a specimen (for example, a specimen bound to magnetic particles) may be held using a MACS sequence. In the case of positive selection, it is desirable that at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the specimen is secured in the apparatus. The surface of the device is desirably designed to minimize non-specific binding of non-target analytes. For example, at least 99%, 98%, 95%, 90%, 80%, 70% of non-target specimens remain in the device. By selective retention within the device, it is possible to achieve selection of a particular analyte collection from a mixture such as blood, saliva, urine, soil, air, or water samples.

  The selective securing of the specimen can be achieved by introducing magnetic particles into the apparatus of the present invention. The capture moiety may be bound to magnetic particles and act on the specific binding of the analyte of interest. In another embodiment, magnetic particles are deployed so that only specimens of a selected size, shape, or deformability can pass through the device. Combinations of these examples can also be recalled. For example, one device may be configured to hold a particular specimen based on size, and the other device may be based on binding. In addition, one device is designed to constrain more than one analyte of interest, for example in series, parallel, or interspersed within the device, or two or more capture moieties are the same magnetic particle or For example, distributed to adjacent particles bound to the same obstacle or region. Furthermore, multiple capture moieties specialized for the same analyte (eg, anti-CD71 and anti-Cd36) may be applied to the same or different magnetic particles, eg, to the same or different obstacles or regions.

  Magnetic particles may be attached to obstacles present in the device (or operated to mold the obstacles) to increase the number of surfaces that interact with the specimen to facilitate binding. The flow condition is usually such that the specimen is handled very gently in the device so that no damage occurs. The application of positive or negative pressure or flow from the column may be applied to send the analyte to or from the microfluidic device of the present invention. This device allows gentle processing and maximizes the frequency with which each analyte collides with one or more magnetic particles. The analyte of interest collides with the magnetic particles and interacts with various capture moieties. This capture portion can coexist with an obstacle designed to be magnetically adsorbed in the device. Due to this interaction, the target specimen in the defined location is captured and held. Alternatively, the specimen is held by the wedge as per the device based on size, shape, or deformability, for example. The captured specimen is released by demagnetizing the magnetic region holding the magnetic particles. To selectively release the specimen from the area, demagnetization may be limited to the selected obstacle or area. For example, the magnetic field may be designed electromagnetically, allowing the magnetic field to be turned on and off freely in individual areas or obstacles. In another example, the analyte may be resolved by releasing the binding between the analyte and the capture moiety, eg, by chemical separation or by releasing non-covalent interactions. For example, several iron particles can be linked to a single cell antibody via a DNA circle and DNAse can be used to attach and detach analytes from the iron particles. Alternatively, the technician may selectively release using a protease (eg, papain) that breaks down the antibody. In particularly strong magnetic regions, the repulsive force of the magnetic particles may be increased to release the magnetic particles from the magnetic region. In another embodiment, the captured analyte is not released and is analyzed and processed further as it is secured.

  In one embodiment, the device is configured to capture and separate cells expressing the transferrin receptor from the complex mixture. Single cell antibodies to the CD71 receptor are off-the-shelf products covalently bonded to magnetic materials such as, but not limited to, polystyrene-doped iron and iron particles or iron colloids (eg, from Milterny and Dynal). . The CD71 mAB coupled to the magnetic particles is poured into the apparatus. Antibody coated particles are fed to obstacles (eg, posts), floors, walls, and retained by the strength of the magnetic field action between the particles and the magnetic field. Particles between obstacles and those that are loosely held in the area of local magnetic field away from the obstacles are removed by rinsing (this flow rate is the hydrodynamic shear stress on the specimen away from the obstacles) Is adjusted to be greater than the magnetic field strength).

  In addition to the above examples, the device is soluble in blood-borne pathogens, bacterial and viral loads, aqueous media in pathogen testing in the food industry, environmental sampling for chemical and biological hazards It can be used for the isolation and detection of pathogens in Further, the magnetic particles are allowed to coexist with the capture portion or the hybrid candidate drug. The capture of the cells of interest is further analyzed to allow the captured cells to interact with the immobilized hybrid drug. Thus, the device separates a subpopulation of cells from a complex mixture for use in high-throughput drug development processes and for secondary cell-based candidate drug testing, and with these hybrid candidate drugs. It can be used for both checking the reaction. In another embodiment, the device can be used to study receptor ligand action, for example by localizing capture moieties such as receptors that flow on magnetic particles or within a complex mixture of candidate ligands (or agonists or antagonists). It is. The ligand of interest is captured and binding can be detected, for example, by secondary staining with a fluorescent probe. This example can quickly identify the presence or absence of a known ligand from a complex mixture extracted from tissue or cell digest, or can identify candidate drug compounds.

Capture combined with sizing screening In the embodiment here, the sizing screening module and the capture module are preferably in liquid connection. For example, the first outlet of the sorting module is in liquid connection with the capture module. The average flow rate of the sample through the capture module can be the same as or different from the sorting module. In some embodiments, the average flow rate of the sample through the capture module is 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mL / hour. Greater than.

  In some embodiments, the sorting module and the capture module are integrally configured, and a plurality of obstacles deflects a specific sample according to size to guide the specific sample to a flow path different from the direction of the related sample. As both capturing based on size, affinity, magnetism, or other physical properties.

III Detection / Analysis In any embodiment, the detection and / or analysis of concentrated specimens (eg, rare cells) and their components (other and eb, each and chromosome) can be performed in whole or in part on humans or analyzers. Can be done. If the concentrated analyte is a cell, it is permeated or lysed prior to detection / analysis. The analyzer of the present invention is automated for the detection / analysis of high-throughput concentrated specimens (eg, rare cells from blood or dangerous specimens). The detection and analysis by the analyzer may be performed in continuous steps or may be combined in one step. Preferably, detection and analysis are performed in a single step.

  The analyzer may include various known sample analyzers, such as a microscope, a microarray, a cell counter, and the like. The analyzer may further comprise one or more of a computer, database, memory system, system output (eg, computer screen or printer). In a preferred embodiment, the analyzer comprises program code including a series of instructions for performing detection or analysis on a computer readable medium, such as a floppy disk, CD-ROM, hard drive, flash memory, tape, or concentrated sample. Other digital storage media may be included. In some embodiments, computer-executable logic or analyzer program code is stored on a storage medium, which is loaded and / or executed on a computer, or transmission of electrical wiring, cables, optical fibers, electromagnetic radiation, etc. Transmitted over the medium. When implemented on a general-purpose microprocessor, the computer-executable logic configures the microprocessor to generate specific logic circuits. Preferably, the computer-executable logic performs some or all of the tasks described herein including sample provisioning, enrichment, detection and / or analysis.

  In some embodiments, the analyzer is sized and fluidly connected to a baseline-based module or capture module. In some embodiments, concentrated analytes (eg, cells of interest) are removed from the capture module / size sorting module and fed to a glass slide or cell sorter for analysis. In the preferred embodiment, the cell sorter holds a plurality of specimens (eg, cells) in an addressable location. An example of such an embodiment is disclosed in US Pat. No. 6,692,952, incorporated herein by reference for all purposes. Such a module may comprise an actuator configured to selectively release cells from an addressable location.

  In some embodiments, the analyzer is configured to perform a detection step, such as visualizing one or more analytes of interest. Visualization of the analyte of interest is accomplished through a transparent cover or lid that covers the obstacles of the sizing and / or capture module. In some embodiments, the analyzer comprises a microscope, preferably an optical microscope, a bright field optical microscope, a fluorescence microscope, an electron microscope (preferably liquid connected to the capture module). In some embodiments, the analyzer has a dual scan function (eg, using an optical microscope and a fluorescence microscope). Preferably, the analyzer provides a three-dimensional image of the concentrated analyte (including the analyte of interest). For example, a computer code can detect all nucleated red blood cells, including fetal nucleated red blood cells in a concentrated sample. In some embodiments, the analyzer comprises an imaging device such as a camera or video camera. Such an imaging device is used to acquire an image of a specimen (including a specimen of interest). For example, the imaging device can acquire one or more fnRBC images acquired from a maternal blood sample. Any of the above can be controlled by computer-executable logic that renders and stores the concentrated analyte.

  In some embodiments, the analyzer is configured to perform an analysis step that counts analytes of interest, such as cancer cells, endothelial cells, epithelial cells, and the like. Such an analyzer comprises, for example, a cell counter. The number of analytes of interest detected in the sample can be used to make a diagnosis (e.g., cancer) or predict a condition. In some embodiments, the analyzer compares (and optionally stores) data collected from known data points. In some examples, the analyzer compares (and optionally stores) data collected from case samples and control samples and performs accompanying studies.

  In some embodiments, the analyzer comprises computer-executable logic that detects the probe signal from one or more probes that selectively bind the concentrated analyte, the analyte of interest, or a component thereof. In some embodiments, the computer-executable logic also analyzes the strength, size, shape, aspect ratio, and / or distribution of such signals. This computer-executable logic may then generate instructions based on the analysis result of the probe signal.

  Examples of the probe of the signal that can be detected / analyzed by the detector include, but are not limited to, a fluorescent probe (for example, staining a chromosome such as X, Y, 13, 18, and 21 in fetal cells), a chromogenic probe, and an indirect immunizing agent. (Eg, unclassified primary antibody conjugated to a secondary enzyme), quantum dots, or other probes that emit photons. In some embodiments, the analyzer detects chromogenic probes that have significantly faster lead times than fluorescent probes. In some embodiments, the analyzer comprises a karyotype, in situ hybridization (: ISH) (eg, fluorescence in situ hybridization (FISH), chromogen in situ hybridization (CISH), nanogold Field hybridization (NISH)), restriction fragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR) technology, flow cytometry, electron microscopy, quantum dots, and single nucleotide polymorphism (SNP) or RNA Comprising computer-executable logic that implements a nucleic acid array that detects levels. In some embodiments, two or more probes are used. For example, a single cell or component of interest may be analyzed using multiple FISH probes or other DNA probes. A method using FISH to detect rare cells is described in Zhen, DK, et. Al. (1999) Prenatal Diagnosis, 18 (11), 1181-1185, Cheung, MC., (1996) Nature Genetics 14, 264. -268, incorporated herein by reference for all purposes. Methods using CISH are disclosed in Arnould, L. et al British Journal of Cancer (2003), 88, 1587-1591, and US Patent Publication 2002/0019001, which are hereby incorporated by reference for all purposes.

  For example, when analyzing concentrated fetal cells from maternal blood, the analyzer is configured to detect fetal cells or components thereof. In some embodiments, analysis of fetal cells or components thereof detects fetal sex identification, presence / absence of genetic abnormalities (eg, chromosomal / DNA / RNA abnormalities), one or more SNPs. Used for. Examples of chromosomal abnormalities that can be detected with this analyzer include, but are not limited to, Angelman syndrome (15q11.2-q13), cat cry syndrome (5p-), DiGeorge syndrome and membrane-heart-facial syndrome (22q11.2). ), Miller-Dieker syndrome (17q13.3), Prader-Willi syndrome (15q11.2-q13), retinoblastoma (13q14), Smith-Magenis syndrome (17p11.2), trisomy 13, trisomy 16, trisomy 18, There are trisomy 21 (Down syndrome), triploidy, Williams syndrome (7q11.23), Wolf-Hirschhorn syndrome (4p-). Examples of sex chromosomal abnormalities detectable with this analyzer include, but are not limited to, Kalman syndrome (Xp22.3), steroid sulfatase deficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22.3), It includes Kleinfelder syndrome (XXY), fragile X chromosome syndrome, Turner syndrome (X chromosome damage), super female or trichromosome X, single chromosome X and the like. Other less well-known chromosomal abnormalities that can be detected / analyzed by this analyzer include, but are not limited to, deletions (small missing parts), microdeletions (minor absence of substances containing only a single gene), translocations. Includes loci (part of the chromosome is joined to other chromosomes), inversion (part of the chromosome is excised and inserted in the opposite direction).

  In some embodiments, the detector detects a specimen (eg, a cell) stained for an antibody selected from the group consisting of γ and ε globin, glycophorin A (GPA), I antibody, CD35. In particular, the analyzer can detect cells stained with anti-ε or anti-γ globin antibodies or combinations thereof. A combination of γ and ε globin is found in 95-100% of fNRBC at 10-24 weeks of gestation. A1 Mufti et al., (2001) Haematologica 85, 357-362; Choolani et al., (2003) Mol. Hum. Reprod., 9, 227-235. This ε-γ combination, or γ globin alone, has been shown to stain fNRBC. See Bohmer, (1998); Choolani et al. (2003); Christensen et al., (2005) Fetal Diagn. Ther. 20, 106-112; Hennerbichler et al., (2002) Cytometry, 48, 87-92. Antibodies for both globins are known to those skilled in the art and can be obtained from various vendors. Staining can be achieved with a positive or negative binary score, or to varying degrees indicating the amount of antibody in the specimen.

  In some examples, the analyzer detects analytes (eg, cells) stained for GPA and / or CD71. GPA exists throughout the erythroid lineage. Thus, it can be used to identify each red blood cell regardless of maturity. GPA is thought to be found only in erythroid lineage cells and is usually rarely found in circulating cells and increases during pregnancy. FACS testing shows that during pregnancy, the combination of CD71 and GPA appears at least 0.15% of mononuclear cells. Price et al., (1991) Am. J. Obstet Gynecol., 165, 1713-1717; Sohda et al., (1997) Prenat. Diagn., 17, 743-752. In some embodiments, the analyzer is configured to detect CD71 and GPA specific probes.

  In some embodiments, the detector detects an analyte (eg, a cell) stained for I antibody. This I antibody was first disclosed in the 1950s using patient polyclonal serum. The continuous data indicates that the two forms of antigen “I” or “i” represent adult and fetal cells, respectively. More recent structural evidence has defined these antigens as linear or branched repeats of N-acetylactosamine. The “i” structure appears due to the action of two enzymes, β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyl transferase. The rearrangement of the “i” antigen to “I” occurs via an enzyme (β-1,6-N-acetylglucosamine rearrangement enzyme). The genes and chromosomal loci for these enzymes have recently been identified. Yu et al., (2001) Blood, 98, 3840-3845. More recently, antibodies for i antigens have long been used in the field of fetal cells. Kan et al., (1974) Blood 43, 411-415. In recent years, these are used for examination of fetal cells obtained by fractional density centrifugation. Sitar et al. (2005) Exp. Cell. Res., 203, 153, 161. Thus, antibodies and antibody fragments that specifically bind i antigens can be used to enrich, separate, and detect fetal cells by the present methods and compositions. In addition, the i antigen identifies many fetal cells in maternal blood samples (Sitar et al) and improves the speed of reading results.

  In some embodiments, the analyzer comprises computer-executable logic or computer program code that provides a series of instructions that identify / characterize a rare analyte, such as a rare cell in a concentrated sample. This code further provides instructions to describe these rare specimens and save these images. In one embodiment, computer-executable logic directs the microscope to identify rare cells (eg, fetal cells or epithelial cells), the code further includes, for example, ε globin, γ globin, fetal hemoglobin, GPA, A series of instructions are provided for virginity of probes that bind selectively rare cells or components thereof, such as antibodies that specifically bind i antigen, CD71, EpCAM, or combinations thereof.

  For example, in some embodiments, computer-executable logic identifies fetal nucleated red blood cells in a sample, identifies and counts rare cell components such as chromosomes, and in particular chromosomes 13, 18, 21, X and Detect probes that bind to Y, detect one or more single nucleotide polymorphisms (SNPs), detect mutations in gene sequences, detect mRNA levels, detect microRNA levels Provide instructions such as. This computer-executable logic also detects and / or compares the intensity of probes from one or more nucleic acid probes bound to, for example, fetal nucleic acids of interest (eg, chromosomes X, Y, 13, 18, 21). And a code for generating a call related to the probe strength analysis result.

  9A-D are diagrams showing an embodiment of the present invention. FIG. 9A shows a computer connected to a microscope connected to a slide or cell array device. FIG. 9B shows cells visualized with a bright field microscope. FIG. 9C shows XXY cells. FIG. 9D shows an image of cells in the field of view. This also illustrates various features of the code that detect various probe intensity levels.

  In either embodiment, the analyzer comprises computer-executable logic that controls the flow rate of the sample through one or more of the various modules disclosed herein.

IV Applications This apparatus / module and method can be used for the various applications described above, without limitation.

Prenatal Screening In some embodiments, the system and method can be used to perform a prenatal screening. For example, a peripheral blood sample from a pregnant animal (preferably a human) is obtained and concentrated using one or more of the methods and devices disclosed herein. Preferably, the maternal blood sample is first concentrated using one or more sizing modules, and other samples of granulodynamic size (ie fetal nucleated red blood cells and maternal white blood cells) greater than 4 microns are collected from the blood sample. Separate from other specimens. The enriched sample containing fetal nucleated red blood cells and maternal white blood cells is then further screened using one or more capture modules. Preferably, the capture module positively sorts fetal blood cells from white blood cells (selectively and releasably binds). The capture module preferably does not use magnetic particles. In some embodiments, the capture module comprises one or more obstacle arrays covered with anti-CD71 single cell antibodies. Cells captured in such devices are then genetically analyzed using one or more of FISH testing, PCR amplification, RNA analysis, DNA analysis, and the like. In some examples, a FISH test is used to detect the presence or absence of aneuploidy. In some examples, DNA or RNA analysis is used to analyze one or more of SNP or mRNA levels in enriched fetal cells. An analyzer with computer-executable logic that detects fetal cell lifetime analysis can be used to automate the system. The analyzer may further comprise a microscope or a microarray.

b. Cancer Diagnosis In some examples, the system and method can be utilized to perform a cancer diagnosis. For example, a peripheral blood sample or other fluid is obtained from a suspected or cancerous animal. The sample is flowed through one or more sizing modules, and analytes with a granulodynamic size greater than 8, 10, 12, 14, 16, 18, 20 microns are separated from the sample. In some embodiments, the cells to be enriched are one or more selected from the group consisting of infected WBC, stem cells, progenitor cells, epithelial cells, endothelial cells, endometrial cells, tumor cells, cancer cells. It is a cell. In some embodiments, the concentrated analyte may optionally be passed through one or more of the capture modules described above.

  The enriched cells are then analyzed, for example, number of epithelial cells in the sample, number of endothelial cells in the sample, ratio of epithelium / endothelium in the sample, profile of all cells larger than the reference size, migration of all cells larger than the reference size Patterns, changes in characteristics based on one or more second samples taken from the same animal at different points at the same time, etc. are determined.

  In some embodiments, the analysis selectively captures enriched cells, cells of a specific size range, or involves cells (eg, cancer cells or epithelial cells that display one or more cancer markers on the surface). Applying to one or more capture modules for selectively capturing. In some histories, enriched cells are further analyzed to determine the presence or absence of cancer markers between cells. In any of the embodiments, cells may be detected, counted and analyzed using an analyzer.

  Neoplastic conditions for which diagnosis or prediction according to the present invention is carried out are breast cancer, bone cancer, prostate cancer, liver cancer, tongue cancer, brain cancer, laryngeal cancer, gallbladder cancer, pancreatic cancer, rectal cancer, epithelium Body cancer, thyroid cancer, kidney cancer, nerve cell cancer, head cancer, neck cancer, colon cancer, stomach cancer, bronchial cancer, kidney cancer, basal cell cancer, squamous cell carcinoma, neoplastic skin cancer, osteosarcoma, Ewing sarcoma, veticulum cell carcinoma, myeloma, giant cell tumor, small cell lung cancer, gallstone tumor, islet cell tumor, primary brain tumor, acute and chronic lymph gland and granulocytic tumor, hairy cell tumor, adenoma, hyperplasia, Medullary carcinoma, pheochromocytoma, mucosal neuroma, interstellar ganglion hyperplastic comeal neuroma, Marfan syndrome body tumor, Wilms tumor, seminoma, ovarian tumor, leiomyosarcoma, cervical dysplasia Vivid tumor, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, local skin disorder, mycosis fungoides, rhabdomyosarcoma, Kaposi sarcoma, malignant hypercalcemia, renal cell tumor, true polycythemia , Adenocarcinoma, glioblastoma multiforme, acute granulocytic leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, lymphoma, malignant melanoma, and epidermoid carcinoma Including those selected from the group consisting of

  In some embodiments, the system and method are utilized to perform a veterinary diagnosis. Veterinary diagnosis preferably involves obtaining a fluid sample (eg, a blood sample) from a domesticated animal. Examples of domesticated animals include, but are not limited to, cows, chickens, pigs, horses, rabbits, dogs, cats, fish, goats. The sample is then concentrated using one or more sizing modules, for example greater than 4, 6, 8, 10, 12, 14, 16, 18, 20 microns, or a granular mechanical size range (eg 6- Only a granulodynamic size specimen (such as 12 microns or 8-10 microns) is isolated. The concentrated analyte may optionally be subjected to one or more additional concentration steps prior to analysis. For example, in some embodiments, the concentrated analyte is optionally flowed through one or more of the above-described capture modules.

  In some embodiments, the analyte enriched from the sample is fetal cells. Such cells are then examined to determine fetal sex and fetal status.

  In some embodiments, the analyte concentrated from the sample is a pathogen. Examples of pathogens concentrated from animals include, but are not limited to, bacteria, viruses, protozoa (of course, such applications are applicable to humans as well as domestic animals). Once concentrated, the cells are analyzed using a detector / analyzer implemented here. Such analyzers can perform Gram positive tests, viral load tests, FISH tests, PCR tests, etc. to identify pathogen infection types, their sources, therapeutic treatments, and the like.

  In some examples, the analyte enriched from the sample is an endothelial cell or circulating cancer cell. Such cells are further analyzed to determine the origin of the cancer in which the animal suffered, the severity of the condition, the effectiveness of the therapeutic treatment, and the like.

d. Biodefense In some embodiments, the system and method can be utilized to detect the presence of a biodefense or biological hazard (eg, biohazard analyte). Biohazard specimens include, but are not limited to, organisms that infect humans, animals, and plants (eg, parasites, viruses, bacteria, fungi, prions, rickettsia), cellular components (eg, recombinant DNA), and causes of other biological diseases There are biologically active agents (toxins, allergens, toxicants) that can have a significant impact on the environment or society. Examples of pathogens that can be biohazard specimens are: plague, anthrax, botulinum, wild gonorrhoeae, coxiella, brucella, burkholderia bone, burkholderia pseudobone, streptococcus, ebola virus, lassa virus, Selected from the group comprising SARS, acne, alphavirus, rash typhoid rickettsia, parrot disease Chlamydia, Salmonella species, E. coli O157: H7, Vibrio cholerae, Cryptosporidium parvum, Nipah virus, Hantavirus, and chimeras thereof Including things. Biohazard materials can be detected using the present methods and systems, for example, food samples, water samples, air samples, soil samples, or biochemical samples from animals and plants.

  In some embodiments, samples analyzed with the present methods and systems are 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001 of all analytes in the sample. %, 0.000001% or less of biohazard specimens. Further, in any of the examples, the biohazard specimen has an initial concentration of 5, 2, 1, 5 × 10−1, 2 × 10−1, 1 × 10−1, 5 × 10−2, 2 × 10−2, 1 × 10−2, 5 × 10−3, 2x10-3, 1x10-3, 5x10-4, 2x10-4, 1x10-4, 5x10-5, 2x10-5, 1x10-5, 5x10-6, 2x10-6, 1x10-6, 5x10-7, 2x10- 7 or 1 × 10 −7 biohazard analyte / μL liquid sample or less. When analyzing a non-liquid sample, it is desirable to melt or liquefy the sample by various known means.

  Samples to be analyzed for biohazardous material are passed to one or more size-based sorting modules. Preferably, such sizing module increases the concentration of biohazard analyte by at least 1,000 or 10,000 times. The concentrated analyte may also optionally be passed through one or more of the capture modules described above.

e. Research The system and method can also be used to conduct research. For example, in some embodiments, the systems and methods are utilized to conduct related studies based on data collected from multiple control samples and multiple case samples. For example, a liquid sample (for example, a blood sample) has 10, 20, 50, 100 or more individuals (individuals with phenotypic conditions) and 10, 20, 50, 100 or more control samples (no disease expression). Individual). The sample from each individual is then enriched for the first or plurality of analytes. Such analytes are then counted and / or characterized. The data from the above steps is then used to conduct related studies. The data is preferably stored in an electronic database. Related studies are performed using computer-executable logic that identifies one or more features associated with a case sample or a control sample.

  In a preferred embodiment, the liquid sample taken from the individual for the relevant study is a blood sample. In a preferred embodiment, the analyte concentrated from such a sample has a hydrodynamic size greater than 4 microns, or greater than 6, 8, 10, 12, 14, 16 microns. In some embodiments, a sample taken from an individual is RBC, fetal RBC, trophoblast, fetal trophoblast, red blood cell (WBC), infected WBC, stem cell, epithelial cell, endothelial cell, endometrial cell, progenitor One or more cells selected from the group consisting of cells, cancer cells, viral cells, bacterial cells, and strict animals are enriched. Preferably, the cells to be concentrated are those having a concentration in vivo of 1 × 10-1, 1 × 10-2, or 1 × 10-3 cells / μL or less. Preferably, 99% or more of the (enriched) cells of interest are secured from the sample. Concentration to perform related studies can enrich the first cell tumor of interest by at least 10,000-fold.

  The concentrated analyte is then analyzed to determine one or more characteristics. Such characteristics include, for example, the presence or absence of the analyte in the sample, the amount of the analyte, the ratio of the two analytes (eg, endothelial and epithelial cells), the form of one or more analytes, the genotype of the analyte , Specimen proteome, specimen RNA structure, gene expression in specimen, microRNA level, or other properties of the enriched specimen, which are then used to conduct related studies.

  If the characteristic is associated with a control sample, this characteristic is subsequently used to diagnose the absence of disease development in the patient being tested. If the characteristic is associated with a case sample, this characteristic is subsequently used to diagnose the presence of the disease manifestation in the patient being tested.

  Examples of disease manifestations contemplated by the present invention include, but are not limited to, cancer, endometriosis, infection (eg, HIV, bacterial), inflammation, ischemia, trauma, fetal abnormalities, and the like.

V. Kits The present invention contemplates kits that concentrate an analyte from a liquid sample.

  In some embodiments, such a kit includes one or more sorting modules coupled to a capture module configured to selectively enrich fetal cells from a maternal blood sample.

  The sorting module is highly sensitive, and it is desirable to concentrate fetal cells with a sensitivity of 98% or 99% or more. In some embodiments, one or more capture modules are fluidly connected to the sorting module. The kit may further comprise a series of instructions for analyzing the concentrated fetal cells for prenatal screening. Prenatal checkups that can be performed with this kit include, but are not limited to: fetal sex, presence of chromosome 13, chromosome 18, chromosome 21 (Down's syndrome), Turner syndrome (X chromosome damage), Kleinfelter syndrome (XXY), or other Expression of abnormalities in the number of sex chromosomes or autosomes, or Wolf-Hirschhorn syndrome (4p-), cat cry syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13), Angelman syndrome (15q11.2-q13), Miller-Dieker syndrome (17q13.3), Smith-Magenis syndrome (17p11.2), DiGeorge syndrome and membrane-cardio-facial syndrome (22q11.2), Kalman syndrome ( Xp22.3), steroid sulfur Synthetase deficiency (STS) (Xp22.3), X linkage ichthyosis (Xp22.3), and there is a retinoblastoma.

  In some embodiments, the kit includes one or more sorting modules coupled to a capture module that selectively enriches epithelial or cancer cells from a blood sample. Such modules are highly sensitive and it is desirable to concentrate fetal cells with a sensitivity of 98% or 99% or more. Preferably both the sorting module and the capture module are on the same substrate. The kit may further comprise one or more labeling reagents that detect cancer origin, cancer metastasis, treatment efficacy, prognosis, and the like. Such reagents comprise antibodies that specifically bind cell surface cancer markers. The kit may further comprise a series of instructions for analyzing the enriched cells to make a cancer diagnosis.

  Examples of cancers that can be diagnosed using this method include, but are not limited to, breast cancer, bone cancer, prostate cancer, liver cancer, tongue cancer, brain cancer, laryngeal cancer, gallbladder cancer, pancreatic cancer, rectal cancer, parathyroid cancer, Thyroid cancer, kidney cancer, nerve cell cancer, head cancer, neck cancer, colon cancer, stomach cancer, bronchial cancer, kidney cancer, basal cell cancer, squamous cell carcinoma, neoplastic skin cancer, osteosarcoma, Ewing sarcoma, veticulum Cell carcinoma, myeloma, giant cell tumor, small cell lung cancer, gallstone tumor, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumor, hairy cell tumor, adenoma, hyperplasia, medullary cancer, Pheochromocytoma, mucosal neuroma, interstellar ganglion hyperplastic comeal neuroma, Marfan syndrome somatic tumor, Wilms tumor, seminoma, ovarian tumor, leiomyosarcoma, cervical dysplasia and its tumor, Nerve bud Retinoblastoma, soft tissue sarcoma, malignant carcinoid, local skin disorder, mycosis fungoides, rhabdomyosarcoma, Kaposi's sarcoma, malignant hypercalcemia, renal cell tumor, true polycythemia, adenocarcinoma, polymorphic nerve Includes glioblastoma, acute granulocytic leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, lymphoma, malignant melanoma, and epidermoid carcinoma.

VI. Business Method The system and method can be used in diagnostic services and / or cytodiagnostic products. The diagnostic product may comprise, for example, one or more sorting modules, one or more capture modules, detectors, analyzers, or combinations thereof.

Prenatal Diagnosis Service In some embodiments, the business method provides a prenatal inspection service. Such a business obtains a blood sample from a mammal seeking to diagnose a fetus. In some embodiments, the business can either draw blood from a pregnant patient (animal) or receive blood directly from a pregnant patient. This business concentrates fetal cells in a blood sample and performs one or more screening tests on the fetal cells to determine fetal status. Examples of conditions to be determined include, but are not limited to, fetal gender, genetic abnormalities such as chromosomes 13, 18, 21, X, Y, conditions associated with known SNPs, Wolf-Hirschhorn syndrome (4p-), cat crying Syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13), Angelman syndrome (15q11.2-q13), Miller-Dieker syndrome (17q13.3), Smith-Magenis Syndrome (17p11.2), DiGeorge syndrome and membrane-heart-facial syndrome (22q11.2), Kalman syndrome (Xp22.3), steroid sulfatase deficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22) .3), and retinoblastoma (13q14). The present invention may address all other genetic diseases.

  This business method then provides a status report in exchange for a service fee. This report may be provided directly to the tested patient or may be provided to a health care facility, a patient insurance company, or the government.

  In some examples, the business may outsource enrichment and analysis to a CLIA laboratory. In another example, the business may perform an enrichment step and entrust the analysis step to a third party (eg, a CLIA laboratory).

  FIG. 10A is a diagram illustrating an example of the business method disclosed herein. A blood sample (eg, 16-20 mL of blood) is taken from a pregnant woman by this business or CLIA Institute, a patient health care provider. The business or CLIA laboratory performs one or more of the following steps: (a) flowing the sample through a sizing screening module configured to remove red blood cells and platelets from the sample; (b) anti-CD71. Passing through a capture module that binds to the antibody and selectively binds the red blood cell beyond the white blood cell; (c) concentrating the sample using a magnetic bead (eg, repeating the previous concentration step by coating with CD71) (D) Arrange the enriched cells (eg on a cytospin 2D slide); (e) Add to the enriched cells one or two FISH probes that specifically bind the X and / or Y chromosomes; (f) Detect FISH probe using analysis / detection module; (g) from enriched cells to nucleated red blood cells or Providing a report on the presence or absence of detection of (h) fetal abnormalities, patients for example tested, a medical facility, the insurance company; more preferably identify fetal nucleated red blood cells, selectively characterize these.

  FIG. 10 is a diagram illustrating another embodiment of the business method disclosed herein. 32-40 mL of blood is collected from a pregnant woman. The sample is run through an automated sizing module to remove red blood cells and platelets from the sample. This automatic sorting module is connected to a supply device. This sample is then flowed through a capture module connected to an anti-GPA antibody. This sample is then concentrated using a magnetic bead (eg, coating with CD71 and repeating the previous concentration step). The remaining enriched cells are arranged on cytospin 2D slides with FISH probes for chromosomes X, Y, 13, 18, 21. The FISH probe is then read using the analysis / detection module described above, or preferably a multispectral sensitive imaging system, to identify and classify nucleated RBCs. Finally, a report is created for the tested patient, health care provider, or insurance company that diagnoses the fetus due to the presence or absence of fetal abnormalities.

Diagnostic Service-Oncology In some embodiments, the business method provides an oncology testing service. By this method, a liquid sample (eg blood) is taken from the patient to be diagnosed. The business then performs one or more enrichment steps to enrich one or more of the cancer cells, tumor cells, epithelial cells, endothelial cells, or many cells that indicate the presence of cancer from the sample. The cells are concentrated from the liquid sample using any of the systems and methods disclosed herein. After enrichment, the cells are further analyzed (eg, counting, specific biomarker tests, etc.) to determine if this patient has cancer, the origin of the cancer, effective treatment for this patient, cancer metastasis, etc. The Reports generated by this business are provided directly to the patient or to the patient's health care provider or insurance company.

Diagnostic Service-Infection In some embodiments, the business method provides an infection test service. This service collects a liquid sample (eg, urine or blood) from a mammal whose infection is diagnosed. In some embodiments, the business may collect blood and obtain a sample directly from the patient (animal). In some embodiments, samples are delivered to this business. The business then conducts a screening test of the sample to concentrate one or more infected cells (eg, infected leukocytes) or infectious tissues such as bacterial cells, viruses, protozoa. Infectious tissue can be enriched by this business with the systems and methods disclosed herein. Examples of circulating pathogens that can be selected / concentrated by this method include viruses (eg, HIV, influenza, SARS), bacteria (such as E. coli, H influenza, S pneumonia, M meningitis), protozoa (protozoa malaria, Trypanosoma, brusei, etc.). In some embodiments, the method is used for the separation and detection of HIB infected cells in a blood sample. Reports generated by this business are provided directly to the patient or to the patient's health care provider or insurance company.

Diagnostic Products In some embodiments, the business methods of the present invention commercialize diagnostic products that are adapted to concentrate one or more analytes from a blood sample. For example, one business method involves one or more devices / modules for sorting and selectively analyzing fetal cells herein, individually or optionally as a kit with one or more reagents (eg, labeling reagents). Is intended to sell. Such a kit may include instructions for prenatal testing. Another business method is to sell one or more devices / modules that select and selectively analyze circulating cancer cells individually or optionally as a kit with one or more reagents (eg, a labeling reagent). I intend to do that. Such a kit may have instructions for conducting a cancer screening. Another business method is to sell one or more devices / modules that sort and selectively analyze circulating epithelial cells individually or optionally as a kit with one or more reagents (eg, a labeling reagent). I intend to do that. Such a kit may have instructions for conducting a cancer screening.

  In a preferred embodiment, the diagnostic product comprises one or more sorting modules and optionally one or more capture modules. The diagnostic product may optionally comprise a detection / analysis tool (eg, computer code or software) for condition detection.

  In some embodiments, the business method produces a diagnostic tool. In some embodiments, the business method outsources the manufacture of diagnostic tools to a third party. In either embodiment, the diagnostic tool is preferably made of a polymeric material and is selectively disposable.

Specimen Isolation In some embodiments, the business method segregates one or more specimens using the systems and methods herein for a cost or exchange or cross license. The sample is, for example, a blood sample or other body sample. In some embodiments, CLIA laboratories or other third-party organizations provide blood samples to this business, and rare cells such as fetal cells, epithelial cells, cancer cells, etc. are extracted from the blood samples. Use to isolate. In some embodiments, the business obtains a blood sample from one or more abnormal individuals and separates one or more therapeutic blood components such as platelets, leukocytes, circulating stem cells, etc. from such blood samples. To do. Such blood components are then sold for a fee by this business. Such blood products have research and / or therapeutic purposes.

VII. Manufacture In this example, an obstacle photoresist pattern is formed on a silicon-on-insulator (SOI) wafer using standard photolithographic techniques. The SOI wafer is composed of a 100 μm thick Si (100) wafer on top of a 1 μm thick SiO 2 layer on top of a 500 μm thick Si (100) wafer. To optimize photoresist adhesion, the SOI wafer may be exposed to hexamethyldisilazane vapor prior to the photoresist coating. The UV reactive photoresist is spin coated on the wafer, baked at 90 ° C for 30 minutes, exposed to UV light through a chrome contact mask for 300 seconds, developed with developer for 5 minutes, and post-baked at 90 ° C for 30 minutes. Is done. This process parameter may vary depending on the nature and thickness of the photoresist. The pattern of the chrome contact mask is transferred to the photoresist and determines the contour of the obstacle.

  When the same photoresist pattern as the obstacle is formed, etching is started. SiO2 acts as a stopper for the etching process. This etch may also be controlled to stop at a given depth without the use of a stopper layer. The photoresist pattern is transferred to a 100 μm thick Si layer by plasma etching. A uniform obstacle may be obtained by using complex deep etching. For example, the substrate is subjected to fluorine-rich plasma with SF6 flowing for 15 seconds, and then the system is switched to fluorocarbon high-purity plasma with only C4F8 flowing for 10 seconds, where the protective film is entirely covered. Covered with. In subsequent etching cycles, when subjected to ion bombardment, the polymer is preferentially removed from the horizontal plane, and this cycle is repeated a plurality of times, for example until reaching the SiO2 layer.

  To bond the binding portion to the obstruction surface, the substrate is subjected to an oxygen plasma before the surface is changed to produce a silicon dioxide layer that is adsorbed by the binding portion. The substrate is then rinsed twice with distilled deionized water and air dried. The sample was immersed in a 2% v / v aqueous solution of 3-[(2-aminoethyl) amino] propyltrimethoxysilane prepared immediately before for 30 seconds to immobilize the silane on the exposed glass, and then distilled further. Washed with ionized water. The substrate is then dried and baked in nitrogen gas. Next, the substrate is immersed in a 2.5% glutaraldehyde solution in sarin buffered with phosphate phosphate at room temperature for 1 hour. The substrate is then rinsed again and soaked in a 0.5 mg / mL solution of distilled deionized water, such as anti-CD71, for 15 minutes at room temperature, waiting for the binder to bind to the obstacle. The substrate is then rinsed again with distilled deionized water and sterilized by immersion in 70% ethanol overnight.

  There are a number of techniques other than the methods described above for fixing the fixed part on the obstacle and on the surface of the device. Simply physisorbing on the surface is a simplification and cost choice. Another approach is to use self-assembled monolayers (eg, thiol on gold) that have government functions at various binding moieties. Additional methods may be used depending on the bonding parts to be bonded and the materials used to construct the device. Surface modification methods are well known in the art. In addition, certain cells preferentially bind to the modified surface of the material. For example, some cells preferentially bind to chemical groups that appear on positively charged, negatively charged, or hydrophobic surfaces, or on certain polymers.

  The next step involves fabricating the flow device by bonding the top layer to microfabricated silicon containing obstacles. The top substrate is glass so that cells can be seen during and after capture. Thermal bonding or UV curable epoxy is used to form the flow chamber. The top and bottom are compression fitted using, for example, a silicone gasket. Such compression coupling can be released. Other bonding methods (eg, wafer bonding) are known in the art. Which method is used depends on the nature of the material used.

  The cell depletion device can be made of different materials. Different manufacturing techniques may be used depending on the choice of material. The device can be made using a hot embossing technique with a plastic such as polystyrene. Obstacles and other necessary structures are stamped into the plastic to form the bottom surface. The top layer is then bonded to the bottom layer. Injection molding is another approach that can create such a device. The entire chamber made of poly (demethylsiloxane) (PDMS) may be used with soft lithographic printing, or only obstructions may be molded with PDMS and then bonded to the glass structure to create a closed chamber. Good. Yet another approach is obstruction formation using epoxy casting technology, where UV or temperature curable epoxies are used for masters with opposite replicas of the desired structure. A laser or other type of microfabrication approach may be used to form the flow chamber. Other suitable polymers that can be used to make the device are polycarbonate, polyethylene, poly (methyl methacrylate). Furthermore, the apparatus of the present invention may be manufactured using a metal such as iron or nickel, for example, by conventional metal processing. The device may be manufactured in one piece using three-dimensional manufacturing techniques (eg, stereolithography). Other manufacturing methods are also known.

Example 1. Silicon Device Multiplex 14 Triple Stage Duplex Array FIGS. 11A-11F are examples of sizing screening modules of the present invention and are characterized as follows:
Size: 90mmx34mmx1mm
Array design: 3 stages, gap size = 18, 12, and 8 μm for the first, second, and third stages, respectively. Branching ratio = 1/10. Duplex: Single bypass channel Device design: Multiple 14-array duplex; with flow register for flow stabilization Device manufacture: Arrays and channels using standard photolithography and silicon reactive planographic technology Made of silicon. The etching depth is 150 μm. A through hole for liquid access is provided using KOH wet etching. The etched surface of the silicon substrate is sealed, and a sealed fluid channel is formed using a pressure sensitive adhesive (9795, 3M, St Paul, MN) that does not react with blood.
Device package: The device is mechanically connected to a plastic manifold having an external fluid reservoir for supplying blood and buffer to the device and removing the generated fraction.
Device Operation: Using an external pressure source, apply a pressure of 2.4 PSI to the buffer and blood reservoir to regulate and remove the fluid supply from the packaging device.
Test Status: Accepted adult human blood is collected with a K2EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ). Raw blood is processed within 9 hours at room temperature using the apparatus of the above-described embodiment. Nucleated cells from blood were separated from enucleated cells (red blood cells and platelets) and contained 1% bovine serum albumin (BSA) (A8414-100ML, Sigma-Aldrich, St Louis, MO) to remove calcium and magnesium. Plasma is supplied to the buffer flow of Dulbecco phosphate buffered saline (14190-144, Invitrogen, Carlsbad, CA).
Instrumentation technique: A complete blood count is determined using a Coulter impedance hematology analyzer (COULTER® Ac · T diff ™, Beckman Coulter, Fullerton, Calif.).
Performance: FIGS. 12A-12F are typical of blood analyzers made from blood samples and waste (buffer, plasma, red blood cells, platelets) and product (buffer and nucleated cell) fractions produced by the device. A histogram is shown.

Example 2 Silicon Device Multiplex 14 Single-Stage Duplex Array FIGS. 13A-13D are examples of apparatus of the present invention and are characterized as follows:
Size: 90mmx34mmx1mm
Array design: 1 stage, gap size = 24 μm. Branching ratio = 1/60. Duplex: Double bypass channel Device design: Multiple 14-array duplex; with flow register for flow stabilization Device manufacture: Arrays and channels using standard photolithography and silicon reactive planographic technology Made of silicon. The etching depth is 150 μm. A through hole for liquid access is provided using KOH wet etching. The etched surface of the silicon substrate is sealed, and a sealed fluid channel is formed using a pressure sensitive adhesive (9795, 3M, St Paul, MN) that does not react with blood.
Device package: The device is mechanically connected to a plastic manifold having an external fluid reservoir for supplying blood and buffer to the device and removing the generated fraction.
Device Operation: Using an external pressure source, apply a pressure of 2.4 PSI to the buffer and blood reservoir to regulate and remove the fluid supply from the packaging device.
Test status: Human blood from an approved adult donor is collected with a K2EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ). Raw blood is processed within 9 hours at room temperature using the apparatus of the above-described embodiment. Nucleated cells from blood were separated from enucleated cells (red blood cells and platelets) and contained 1% bovine serum albumin (BSA) (A8414-100ML, Sigma-Aldrich, St Louis, MO) to remove calcium and magnesium. Plasma is supplied to the buffer flow of Dulbecco phosphate buffered saline (14190-144, Invitrogen, Carlsbad, CA).
Instrumentation technique: A complete blood count is determined using a Coulter impedance hematology analyzer (COULTER® Ac · T diff ™, Beckman Coulter, Fullerton, Calif.).
Performance: The device operates at 17 mL / hour, achieves over 99% red blood cell removal, secures over 95% nucleated cells, and removes over 98% platelets.

Example 3 FIG. Separation of Fetal Cord Blood FIGS. 14A-14D are schematic views of an apparatus used to separate nucleated cells from fetal cord blood.
Size: 100mmx28mmx1mm
Array design: 3 stages. Gap size = 18, 12, and 8 μm for the first, second, and third stages, respectively. Branching ratio = 1/10. Duplex: single bypass channel Device design: Multiple 10 array duplex; with flow register for flow stabilization Device manufacture: Arrays and channels using standard photolithography and silicon reactive planographic technology Made of silicon. The etching depth is 140 μm. A through hole for liquid access is provided using KOH wet etching. The etched surface of the silicon substrate is sealed, and a sealed fluid channel is formed using a pressure sensitive adhesive (9795, 3M, St Paul, MN) that does not react with blood.
Device package: The device is mechanically connected to a plastic manifold having an external fluid reservoir for supplying blood and buffer to the device and removing the generated fraction.
Device Operation: An external pressure source is used to apply a pressure of 2.0 PSI to the buffer and blood reservoirs to regulate and remove the fluid supply from the packaging device.
Test situation: Human umbilical cord blood is supplied in phosphate buffered saline containing citrate glucose anticoagulant. 1 mL of blood is processed within 3 hours at room temperature at 3 mL / hour using the apparatus described above. Nucleated cells from blood are separated from enucleated cells (erythrocytes and platelets), and 1% bovine serum albumin (BSA) (A8414-100ML, Sigma-Aldrich, St Louis, MO) and 2 mM EDTA (15575-020, Plasma is supplied to the buffer flow of Dulbecco phosphate buffered saline (14190-144, Invitrogen, Carlsbad, Calif.) Containing Invitrogen, Carlsbad, Calif.) And calcium and magnesium removed.
Measurement technique: Cell smears and waste fractions (FIGS. 15A-15B) are prepared and modified Wright-Giemsa staining is performed (WG16, SigmaAldrich, St. Louis, MO).
Performance: Fetal nucleated red blood cells are observed in the product fraction (FIG. 15A) and absent in the waste fraction (FIG. 15B).

Example 4 Separation of fetal cells from maternal blood The device and process described in detail in Example 1 was used in conjunction with immunomagnetic affinity enrichment techniques to demonstrate the possibility of separating fetal cells from maternal blood.

Test situation: Blood from an approved pregnant donor with a male fetus is collected with a K2EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ) immediately after the abortion. Raw blood is processed within 9 hours at room temperature using the apparatus of Example 1 described above. Nucleated cells from blood were separated from enucleated cells (red blood cells and platelets) and contained 1% bovine serum albumin (BSA) (A8414-100ML, Sigma-Aldrich, St Louis, MO) to remove calcium and magnesium. Plasma is supplied to the buffer flow of Dulbecco phosphate buffered saline (14190-144, Invitrogen, Carlsbad, CA). The fraction of nucleated cells was then labeled with anti-CD71 microbeads (130-046-201, Miletenyi Biotech Inc., Auburn, Calif.) And the MiniMACS (TM) column (130-046-201, Miletenyi Biotech Inc., Concentrate according to the manufacturer's instructions using Auburn, CA). Finally, the CD71 positive fraction is placed on a glass slide.
Metrology technology: Spotted slides are stained with Vysis probe (Abbott Laboratories, Downer's Grove, IL) using fluorescent in situ hybridization (FISH) technology according to the manufacturer's instructions. Samples are stained by expression of X and Y chromosomes. In some cases, samples from known trisomy 21 pregnant women are also stained with chromosome 21.
Performance: Separation of fetal cells was confirmed by the reliable appearance of male cells in the CD71 positive population prepared from the nucleated cell fraction (FIG. 16). In a single abnormal case study, trisomy 21 pathology was also identified (FIG. 17).

  The following examples illustrate specific embodiments of the device of the present invention. Each device description provides the number of consecutive stages, the size of the gap between each stage, ε (flow angle), and the number of channels per device (array / chip). Each device is made of silicon using DRIER, and each device comprises a thermal oxide layer.

This apparatus comprises five stages in a single array.

This apparatus comprises a plurality of stages, each stage having a bypass channel and being duplicated. The height of the device is 125 μm.

  FIG. 18A shows a mask used for manufacturing a size type sorting apparatus. 18B-18D are enlarged views of this mask, where the inlet, array, and outlet are defined. Figures 19A-19G show SEMs of the size sorting module.

This apparatus comprises a plurality of stages, each stage having a bypass channel and being duplicated. “Fins” are provided on the sides of the bypass channel to prevent liquid from the bypass channel from reentering the array. The chip also includes an on-chip chip flow register, i.e., an inlet and outlet that are more fluid resistant than the array. The height of the device is 117 μm.

  FIG. 20A shows a mask used for manufacturing a size type sorting apparatus. FIGS. 20B-20D show the configuration of this mask portion, where the inlet, array, and outlet are defined. FIGS. 21A-21F show the SEM of the size sorting module used in this example.

This apparatus comprises a plurality of stages, each stage having a bypass channel and being duplicated. “Fins” are provided on the sides of the bypass channel to prevent liquid from the bypass channel from reentering the array. The chip also includes an on-chip chip flow register, i.e., an inlet and outlet that are more fluid resistant than the array. The height of the device is 117 μm.

  FIG. 14A shows a mask used in the manufacture of the device. FIGS. 14B-14D show the configuration of this mask portion, where the inlet, array, and outlet are defined. Figures 22A-22F show SEMs of the apparatus.

This apparatus comprises a plurality of stages, each stage having a bypass channel and being duplicated. The “fins” are optimized using Femlab to prevent liquid from the bypass channel from re-entering the array. The edge closest to the array of fins follows the shape of the array. The chip also includes an on-chip chip flow register, i.e., an inlet and outlet that are more fluid resistant than the array. The height of the device is 139-142 μm.

  FIG. 23A shows a mask used in the manufacture of the device. FIGS. 23B-23D are partial enlarged views of this mask, in which the inlet, array, and outlet are defined. Figures 24A-24S show SEMs of the apparatus.

The apparatus comprises a single stage, dual apparatus having a bypass channel provided to receive the output from the ends of both arrays. The obstacle for this device is oval. The boundary of the array is the Femlab model. The chip also includes an on-chip chip flow register, i.e., an inlet and outlet that are more fluid resistant than the array. The height of the device is 152 μm.

  FIG. 13A shows a mask used in the manufacture of the device. 13B-13D are partial enlarged views of this mask, in which the inlet, array, and outlet are defined. Figures 25A-25C show SEMs of actual devices.

  All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Those skilled in the art will recognize a variety of various modifications and variations of the disclosed methods and systems of the present invention without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific embodiments, the invention as claimed should not be limited to such specific embodiments. In particular, those skilled in the art will appreciate that various variations of the modes disclosed to practice the present invention are within the scope of the present invention.

  Other embodiments are in the claims.

FIG. 1 is a diagram showing an embodiment of a size type selection module. FIG. 2 is a diagram showing an embodiment of a size type selection module through which individual specimens having different hydrodynamic sizes pass. FIG. 3 is a diagram showing an embodiment of a size type selection module including a cheese piece-shaped bypass obstacle. FIG. 4 is a diagram showing an embodiment of a plurality of size type selection modules parallel to each other. FIG. 5 is a table showing the sorting capability of one embodiment of the size type sorting module. FIG. 6 is a photograph showing cells captured by the capture module. 7A-7C are diagrams illustrating various embodiments of the capture module. FIG. 8 is a diagram illustrating an embodiment of the capture module. 9A-9D are diagrams illustrating various aspects of the detection module. 10A-B are diagrams illustrating an example of the business method disclosed herein. 11A-11F are diagrams showing an example of the size type selection module of the present invention. 12A-12F are typical histograms generated by hematology specimens from blood samples generated by the device. FIGS. 13A-13D are diagrams illustrating various embodiments of sizing screening modules. 14A-14D are diagrams illustrating various embodiments of a sizing screening module. 15A-15B are diagrams showing cell smears and waste fractions of the product cells. 16A-16F are diagrams showing cell smears and waste fractions of the product cells. FIG. 17 is a diagram showing the trisomy 21 pathology of isolated fetal nucleated red blood cells. 18A to 18D are views showing examples of masks used for manufacturing a size type sorting module. 19A to 19G are diagrams illustrating examples of SEMs of the size type selection module. 20A-20D are diagrams showing an example of a mask used to manufacture a size type sorting module. 21A to 21F are diagrams illustrating examples of SEMs of the size type selection module. 22A to 22F are diagrams showing examples of SEM of the size type selection module. FIGS. 23A-23D are diagrams showing masks and portions of a size selection module. 24A to 24S are diagrams illustrating examples of SEMs of the size type selection module. 25A to 25C are diagrams illustrating examples of the size type selection module.

Claims (117)

One or more sizing modules adapted to increase the first analyte in the sample to a concentration of at least 10,000 times, wherein the first analyte has an initial concentration in the sample of 1 × 10 ⁻³ Specimen / μL or less,
A system comprising an analyzer comprising computer-executable logic for analyzing a first analyte in a medium enriched in the first analyte.   The system of claim 1, wherein the analyzer further comprises a microscope, a microarray, or a cell counter.   2. The system of claim 1, wherein the computer-executable logic detects discoloration due to the presence of fetal hemoglobin, globin γ, globin ε, globin β, GPA, i antigen, CD36, selectin, CD71, or combinations thereof. A system characterized by that.   The system of claim 1, wherein the computer-executable logic detects the strength of a probe that selectively binds a first analyte.   The system of claim 1, wherein the computer-executable logic performs a three-dimensional analysis of the first specimen.   2. The system according to claim 1, wherein the analyzer has a dual scan function.   The system of claim 1, wherein the computer-executable logic depicts the first analyte.   The system of claim 1, wherein the computer-executable logic detects trisomy, fetal gender, cellular gene or chromosomal abnormality of interest, or a combination thereof.   2. The system of claim 1, wherein each of the size-based sorting modules deterministically guides the first sample in a first direction, and selects a second sample having a hydrodynamic size smaller than the first sample. A system comprising a two-dimensional obstacle array for guiding in a second direction.   10. The system according to claim 9, wherein the first specimen is a nucleated red blood cell.   10. The system according to claim 9, wherein the first specimen is fetal nucleated red blood cells.   The system of claim 1, wherein the system comprises two or more sizing modules that are fluidly connected in parallel with each other.   2. The system of claim 1 adapted for high throughput analysis of at least 10 mL / hour of the liquid sample.   The system of claim 1, further comprising one or more capture modules fluidly connected to the sizing sorting module, wherein the capture module selectively selects the first analyte or the second analyte from the sample. A system characterized by capturing.   15. The system of claim 14, wherein each of the capture modules comprises a two dimensional obstacle array.   15. The system of claim 14, wherein each of the capture modules comprises a two-dimensional obstacle array that is coupled to one or more antibodies.   17. The system of claim 16, wherein the antibody selectively binds red blood cells, white blood cells, fetal blood cells, fetal nucleated blood cells, cancer cells, epithelial cells, stem cells, progenitor cells, foam cells, or platelets. Feature system.   17. The system of claim 16, wherein the antibody is selected from the group consisting of anti-CD71, anti-CD36, anti-CD451, anti-GPA, anti-selectin, anti-carbohydrate, anti-antigen I, and anti-EpCam.   2. The system of claim 1, wherein the liquid sample is a blood sample and the first analyte is selected from the group consisting of epithelial cells, endothelial cells, progenitor cells, stem cells, foam cells, or cancer cells. System.   2. The system of claim 1, wherein the liquid sample is a blood sample and is 5 mL or less.   2. The system of claim 1 wherein the liquid sample is a blood sample of a woman gestating 12 weeks or less.   The system of claim 1, wherein a gap between obstacles in the sorting area is 1000 microns or less.   2. The system of claim 1 further comprising a reservoir liquid-coupled to the sorting area or capture area and containing magnetic particles.   The system of claim 1, further comprising a magnetic bead configured to selectively bind the first analyte or the second analyte.   25. The system of claim 24, wherein the magnetic bead is specifically bound to an antibody that binds the first analyte.   The system of claim 1, further comprising a reservoir in liquid connection with the sizing sorting module, the reservoir comprising magnetic particles configured to preferentially bind the first analyte. System.   The system of claim 2, wherein the analyzer comprises a microarray configured to detect one or more SNPs in the first sample.   The system of claim 2, wherein the analyzer comprises a microarray configured to detect mRNA levels in the first sample.   10. The system of claim 9, wherein the obstacles are separated by a gap that guides the sample flow non-uniformly to the next gap.   A system comprising a sorting module adapted to remove more than 99.5% of enucleated cells from a blood sample and retain more than 99% of nucleated cells from the blood sample.   32. The system of claim 31, further comprising an analyzer fluidly connected to the sorting module and configured to analyze one or more nucleated cells, and a database storing analysis data. system.   A plurality of two-dimensional obstacles that are one or more first concentration regions that define a first fluid flow path for a first specimen and a second fluid flow path for a second specimen; In addition, the first specimen and the second specimen are liquid-connected to the concentrated area where the hydrodynamic diameters of the first specimen and the second specimen are different, and data of the first specimen or the second specimen is acquired. And a database for storing the data.   33. The system of claim 32, wherein the first specimen comprises red blood cells (RBC), nucleated red blood cells (NRBC), fetal RBC, fetal nucleated RBC (fNRBC), trophoblast, fetal fibroblasts, white blood cells (WBC). ), An infected WBC, stem cells, epithelial cells, endothelial cells, stem cells, cancer cells, viral cells, bacterial cells, and protozoa. 33. The system of claim 32, wherein the cell type is found in vivo at a concentration of 1 × 10 ⁻³ cells / μL or less.   33. The system of claim 32, wherein the gap between obstacles in the first enrichment region is a maximum of 1000 microns.   34. The system of claim 32, further comprising one or more second enrichment regions, wherein the second enrichment region captures the first analyte or the second analyte, and the second enrichment region comprises A system in liquid connection to the first enrichment region.   35. The system of claim 32, wherein the one or more first enrichment regions are configured to hold 99% or more of the first analyte.   34. The system of claim 32, wherein the one or more first enrichment regions are configured to increase the concentration of the first analyte by a factor of at least 100,000. A method for identifying characteristics associated with a patient condition comprising:
Obtaining a plurality of control samples;
Obtaining a plurality of case samples;
Applying each of the samples to a device comprising a plurality of obstacles for deflecting the first specimen from the sample in a direction different from the second specimen from the blood sample, A step in which the diameter of the second specimen is different;
Analyzing the first analyte from the sample to identify characteristics of the first analyte;
Performing a related study based on said characteristics.   40. The method of claim 39, wherein the characteristic is the presence or absence of the first analyte.   40. The method of claim 39, wherein the characteristic is the number of the first analyte.   40. The method of claim 39, wherein the characteristic is a morphology of the first specimen or a phenotype of the first specimen.   40. The method of claim 39, wherein the characteristic is a genotype of the first specimen.   40. The method of claim 39, wherein the characteristic is protein metabolism of the first analyte.   40. The method of claim 39, wherein the characteristic is an RNA structure of the first analyte.   40. The method of claim 39, wherein the characteristic is a gene expression level of the first specimen.   40. The method of claim 39, wherein the plurality of control samples comprises at least 100 control samples.   40. The method of claim 39, wherein the plurality of case samples includes at least 100 case samples.   40. The method of claim 39, wherein the control sample and case sample are blood samples.   50. The method of claim 49, wherein the blood of each blood sample is 100 mL or less.   40. The method of claim 39, wherein the specimen is a cell type.   52. The method of claim 51, wherein the specimen is an epithelial cell, a cancer cell, or a fetal cell.   33. The method of claim 32, wherein the obstacle deterministically directs the liquid flow of the first and second analytes.   54. The method of claim 53, wherein the obstacle array non-uniformly defines a gap leading the sample flow to a next gap. A method for detecting a biohazard specimen in a sample, wherein the biohazard specimen is found in a sample at a concentration of 1 × 10 ⁻³ specimen / μL or less, and the concentration of the specimen is increased by at least 10,000 times Applying to a flow-through concentrator configured to cause and analyzing the concentrated biohazard analyte.   56. The method of claim 55, wherein the concentrator is configured to retain 99% or more of the biohazard specimen.   56. The method of claim 55, wherein the concentrator is configured to concentrate the biohazard specimen by a factor of at least 100,000.   56. The method of claim 55, wherein the concentrator has a specificity of 98% or higher and a sensitivity of 98% or higher.   56. The method of claim 55, wherein the biohazard specimen is a pathogen selected from the group comprising bacteria, protozoa, and viruses.   56. The method of claim 55, wherein the biohazard specimen comprises: plague, anthrax, botulinum, wild gonorrhoeae, coxiella, brucella, burkholderia bone, burkholderia pseudobone, streptococci, ebola virus, Pathogen selected from the group consisting of Lassa virus, SARS, acne, alphavirus, rash typhoid rickettsia, parrot disease Chlamydia, Salmonella species, Escherichia coli O157: H7, Vibrio cholerae, Cryptosporidium parvum, Nipah virus, Hantavirus A method characterized in that   56. The method of claim 55, wherein the sample is a water sample, an air sample, a plant or animal sample, or a soil sample.   56. The method of claim 55, wherein the sample is a fluid sample.   56. The method of claim 55, further comprising melting the sample into a fluid sample.   56. The method of claim 55, wherein the concentrator removes 99% or more of the second analyte from the sample.   56. The method of claim 55, wherein the biohazard specimen is a cell type.   56. The method of claim 55, wherein the biohazard specimen is a toxin.   56. The method of claim 55, wherein the series of analyzes is performed by computer-executable logic.   56. The method of claim 55, wherein the concentrating device guides the biohazard specimen deterministically in a first direction and guides a second specimen in the sample in a second direction. A method comprising a dimensional array.   A method for identifying fetal abnormalities, wherein a maternal blood sample from a pregnant woman is passed through a flow-through module that deterministically separates fetal cells in the sample, wherein the separated fetal cells are extracted from the blood sample. Providing to an analyzer configured to take an image of the enriched one or more fetal cells; analyzing a signal from one or more nucleic acid probes that bind fetal nucleic acid; A method comprising: analyzing a signal; and generating a diagnostic output based on the analyzing step.   70. The method of claim 69, wherein the probe is for a chromosome.   71. The method of claim 70, wherein the chromosome is selected from the group consisting of an X chromosome, a Y chromosome, a chromosome 21, a chromosome 13, and a chromosome 18.   72. The method of claim 70, wherein the analysis identifies the number of the probe signals, the size of the probe signals, the shape of the probe signals, and the aspect ratio of the probe signals. Or identifying a distribution of the probe signal. In computer program products that detect fetal conditions,
A computer code for detecting fetal nucleated red blood cells (fnRBC) in the sample;
Computer code for receiving a probe signal from one or more nucleic acid probes that bind a nucleic acid of interest;
Computer code for analyzing the probe signal to detect one or more fetal cells;
Computer code for analyzing the probe signal to detect the state of the one or more fetal cells;
A computer program product comprising a computer readable medium for storing the computer code.   74. The computer program product of claim 73, wherein the computer readable medium is a memory, hard drive, floppy disk, CD-ROM, flash memory, or tape.   75. The computer program product of claim 73, wherein the probe is dedicated to chromosomes.   76. The computer program product of claim 75, wherein the chromosome is selected from the group consisting of an X chromosome, a Y chromosome, a chromosome 21, a chromosome 13, and a chromosome 18.   74. The computer program product of claim 73, wherein the probe is a colorimetric probe.   74. The computer program product of claim 73, wherein the probe is a fluorescent probe.   70. The method of claim 69, wherein the pregnancy period of the pregnant woman is 12 weeks or less.   70. The method of claim 69, wherein the flow-through module comprises one or more two-dimensional obstacle arrays, the obstacles defining gaps that non-uniformly direct the sample flow to the next gap. A method characterized by that. A pre-natal test kit,
A sizing module configured to separate one or more first type cells from a maternal blood sample, wherein the first type cells are less than 1% of all blood cells in a pregnant woman's body A kit comprising a series of instructions for analyzing the one or more enriched cells for prenatal diagnosis of the fetus. A pre-natal test kit,
A sizing module configured to separate one or more first type cells from a neonatal blood sample, wherein the first type cells are less than 1% of all blood cells in vivo. A kit comprising a set of instructions for analyzing a prenatal diagnosis of a fetus by analyzing the one or more enriched cells as seen at a concentration.   84. The kit according to claim 81 or 82, further comprising one or more reagents selected from the group consisting of PCR reagents, lysis reagents, nucleic acid probes, and labeling reagents.   83. The kit according to claim 81 or 82, wherein the labeling reagent is a FISH reagent.   83. The kit according to claim 81 or 82, wherein the FISH reagent specifically binds to a chromosome selected from the group consisting of X chromosome, Y chromosome, chromosome 13, chromosome 18, and chromosome 21.   83. The kit according to claim 81 or 82, further comprising a microarray.   83. A kit according to claim 81 or 82, wherein the sizing sorting module deterministically guides the one or more first types of cells in a first direction, and the one or more first types. A kit comprising a two-dimensional obstacle array for guiding two types of cells in a second direction.   88. The kit of claim 87, wherein the first type of cell is a fetal cell.   88. The kit of claim 87, wherein the second type of cells is enucleated erythrocytes.   90. The kit of claim 89, wherein the size selection module retains 99% or more of the first type of cells and removes 99% of the enucleated erythrocytes.   83. The kit of claim 81 or 82, further comprising an obstacle array, wherein the obstacle is selected from the group consisting of anti-CD71, anti-CD36, anti-selectin, anti-GPA, anti-CD45, and anti-antibody i. A kit characterized by being bound to an antibody.   83. The kit according to claim 81 or 82, wherein the prenatal diagnosis includes determining the sex of the fetus.   84. The kit of claim 81 or 82, wherein the prenatal diagnosis comprises chromosome 13, chromosome 18, chromosome 21 (Down's syndrome), Turner syndrome (X chromosome damage), Klinefelter syndrome (XXY), or other sex chromosomes. Alternatively, a kit comprising determination of the presence of an autosomal number abnormality.   82. The kit of claim 81, wherein the prenatal diagnosis comprises Wolf-Hirschhorn syndrome (4p-), cat cry syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13). ), Angelman Syndrome (15q11.2-q13), Miller-Dieker Syndrome (17q13.3), Smith-Magenis Syndrome (17p11.2), DiGeorge Syndrome and Membrane-Heart-Facial Syndrome (22q11.2), Kalman Characterized by comprising a determination of a condition selected from the group consisting of syndrome (Xp22.3), steroid sulfatase deficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22.3), and retinoblastoma Kit to do.   83. The kit according to claim 81 or 82, wherein the sorting module guides the first specimen deterministically in a first direction and the second specimen in a second direction. kit.   83. The kit of claim 81 or 82, wherein the sorting module comprises one or more two-dimensional obstacle arrays that define a plurality of gaps that guide flow non-uniformly to the subsequent gaps. Kit to do.   82. The kit according to claim 81, wherein the prenatal diagnosis includes determination of the sex of the fetus.   82. The kit according to claim 81, wherein the prenatal diagnosis includes determination of the presence of chromosome 13.   83. The kit of claim 82, wherein the postpartum diagnosis includes determining the presence or number of circulating nucleated red blood cells. A business method of testing prenatal diagnosis,
Obtaining a blood sample from a pregnant or / or pregnant mammal;
Concentrating one or more fetal cells from the blood sample;
Analyzing the fetal cells to determine the status of the fetus;
Providing the status report in exchange for a service fee.   101. The business method of claim 100, wherein the business entrusts execution of the analysis step to a CLIA laboratory.   101. The business method of claim 100, wherein the enrichment step is performed in a fluidic system configured to separate the fetal cells from maternal cells by guiding the cells in different directions depending on size. Business way.   101. The business method of claim 100, wherein the business provides the service.   101. The business method of claim 100, wherein the condition is trisomy 13, trisomy 18, trisomy 21 (Down syndrome), Turner syndrome (X chromosome damage), Kleinfelter syndrome (XXY), and the number of sex chromosomes or autosomes. A business method characterized by being selected from the group consisting of abnormalities.   101. The business method of claim 100, wherein the condition is Wolf-Hirschhorn syndrome (4p-), cat cry syndrome (5p-), Williams syndrome (7q11.23), Prader-Willi syndrome (15q11.2-q13). Angelman syndrome (15q11.2-q13), Miller-Dieker syndrome (17q13.3), Smith-Magenis syndrome (17p11.2), DiGeorge syndrome and membrane-cardio-facial syndrome (22q11.2), Kalman syndrome (Xp22.3), steroid sulfatase deficiency (STS) (Xp22.3), X-linked ichthyosis (Xp22.3), and retinoblastoma (13q14) .   101. The business method of claim 100, wherein the report is created for a health care provider or a health insurance company.   101. The business method of claim 100, wherein the business entrusts execution of the enrichment step to a CLIA laboratory.   A business method comprising commercializing a diagnostic product for performing a prenatal test for a genetic defect in a fetus, said diagnostic product enriching fetal cells from a maternal blood sample.   109. The business method of claim 108, wherein the business manufactures the diagnostic product.   109. The business method of claim 108, wherein the diagnostic product is manufactured from a polymer material.   109. The business method of claim 108, wherein the diagnostic product is disposable.   109. The business method of claim 108, wherein the diagnostic product selectively separates fetal nucleated red blood cells (fnRBC) from enucleated red blood cells.   109. The business method according to claim 108, wherein the diagnostic product detects a genetic abnormality from the fetal cells.   114. The business method according to claim 113, wherein the genetic abnormality is detected using a label that binds a nucleic acid.   115. The business method of claim 114, wherein the label is a fluorescent label.   116. The business method of claim 115, wherein the label is a colorimetric label. A business method for separating fetal cells from maternal blood,
Obtaining a blood sample from a mammal that is pregnant or carrying a fetus;
Concentrating one or more fetal cells from the blood sample at cost and expense.

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