Patent Application
20130344507
This disclosure relates generally to immunomagnetic separation and, in particular, to systems and methods for separation of a target analyte of a suspension from the other component materials of the suspension with a magnetic field or magnetic gradient.
Suspensions often include materials of interest that are difficult to detect, extract and isolate for analysis. For instance, whole blood is a suspension of materials in a fluid. The materials include billions of red and white blood cells and platelets in a proteinaceous fluid called plasma. Whole blood is routinely examined for the presence of abnormal organisms or cells, such as fetal cells, endothelial cells, epithelial cells, parasites, bacteria, and inflammatory cells, and viruses, including HIV, cytomegalovirus, hepatitis C virus, and Epstein-Barr virus and nucleic acids. Currently, practitioners, researchers, and those working with blood samples try to separate, isolate, and extract certain components of a peripheral blood sample for examination. Typical techniques used to analyze a blood sample include the steps of smearing a film of blood on a slide and staining the film in a way that enables certain components to be examined by bright field microscopy.
On the other hand, materials of interest composed of particles that occur in very low numbers are especially difficult if not impossible to detect and analyze using many existing techniques. Consider, for instance, circulating tumor cells (“CTCs”), which are cancer cells that have detached from a tumor, circulate in the bloodstream, and may be regarded as seeds for subsequent growth of additional tumors (i.e., metastasis) in different tissues. The ability to accurately detect and analyze CTCs is of particular interest to oncologists and cancer researchers, but CTCs occur in very low numbers in peripheral whole blood samples. For instance, a 7.5 ml sample of peripheral whole blood that contains as few as 3 CTCs is considered clinically relevant in the diagnosis and treatment of a cancer patient. However, detecting even 1 CTC in a 7.5 ml blood sample may be clinically relevant and is equivalent to detecting 1 CTC in a background of about 40-50 billion red and white blood cells. Using existing techniques to find, isolate and extract as few as 3 CTCs of a whole blood sample is extremely time consuming, costly and is extremely difficult to accomplish.
As a result, practitioners, researchers, and those working with suspensions continue to seek systems and methods to more efficiently and accurately detect, isolate and extract target materials of a suspension.
This disclosure is directed to systems and methods for immunomagnetic separation of a target analyte of a suspension from the other component materials of the suspension. The system may be composed of a tube, a magnetizable float, and a magnet. The magnetizable float is configured to propagate or introduce a magnetic field. In one aspect, the magnet is included in the magnetizable float. In another aspect, the magnet is external to the magnetizable float. The system may also include a solution containing a particle to conjugate with the target analyte to form a target analyte-particle complex. The particle is capable of being attracted by the magnetic field or magnetic gradient introduced by the magnet. The target analyte-particle complex may be attracted to the magnetizable float. The systems and methods may allow for positive selection (separating desired material from the other component materials of the suspension) or negative selection (separating the non-desired material from the other component materials of the suspension).
Immunomagnetic separation provides a high level of sensitivity and specificity for rare cells in a biological sample, such as circulating tumor cells (“CTCs”) in blood.
Current immunomagnetic separation technology introduces ferromagnetic particles, which may be attracted by an applied magnetic field, into a blood sample, where antibodies directed to a surface antigen of a cell are coated onto or attached to the magnetic particles. Magnetic separation then occurs, whereby a magnet, having a magnetic field, separates the cells that have attached to or interacted with the ferromagnetic particles from the remaining sample. These systems and methods, however, may attract unwanted cells or analytes that have non-specifically attached to the magnetic particles or interacted with the magnetic particles through processes such as phagocytosis. In doing so, the target analytes of the original sample will still be intermixed with unwanted analytes. Further, not all magnetic bound cells may be removed, as only those magnetically bound cells that are in proximate distance to the magnet will be captured.
Furthermore, some current immunomagnetic separation technologies require that the magnetically separated cells be present in a monolayer for imaging. The cells that are not located in the monolayer are not detected.
A blood sample also includes red blood cells (“RBCs”), which are viscous and may cause interference when removing the magnetically bound cells. The RBCs provide another layer or component which makes imaging the target analytes more difficult.
Separating by density, such as by centrifugation, then introducing a magnetic field to a magnetizable float reduces the prevalence of non-specifically attached magnetic particles and non-target analytes that will be attached to a magnetizable float once magnetized. The unbound magnetic particles will be too far away (due to their respective densities and the prior separation through centrifugation) from the applied or transmitted magnetic field of the magnetizable float. The non-target analytes may have attached to magnetic particles or interacted with the magnetic particles through phagocytosis or other processes. The non-target analytes and non-specifically bound magnetic particles which are attracted to the magnetizable float once it is magnetized may be identified and eliminated from analysis through subsequent imaging. The systems and methods permit more specific detection and characterization through imaging, thereby permitting specific target analytes to be removed from the magnetizable float. Imaging allows for the determination of various characteristics of the analyte that is magnetically attracted and bound to the magnetizable float. Additionally, density separation by centrifugation separates the RBCs from the sample, which makes imaging and attraction more efficient, as there is no concern for imaging through an additional component.
Furthermore, imaging techniques, such as multiple z-stacks or optical axis integration, of this method permit the target analytes to be imaged in a multitude of focal planes. The target analyte may exist at any depth between the magnetizable float and tube for imaging.
Additionally, attracting a target analyte to a magnetizable float with the introduction of a magnetic field may permit using fluidics to wash, fix, permeabilize, and label the target analyte. The force of the magnetic field, being greater than the forces produced by the flow-thru, may hold the target analyte while the flow-thru force will remove the non-target analytes.
It should be understood that “propagate” refers to a characteristic of material, element, structure, component or the like to carry, transmit, or maintain a magnetic field or a magnetic gradient.
It should be understood that “magnetizable” refers to the capability of a material, element, structure, component, or the like to propagate or introduce a magnetic field. “Magnetizable” further includes a material, element, structure, component, or the like which has its own magnetic field or magnetic gradient.
Methods and systems for separating component materials of a suspension are disclosed. The detailed description is organized into two subsections: (1) A general description of various tube and float systems is provided in a first subsection. (2) Examples of methods and systems for separating component materials of suspensions using tube and float systems in which the tube is magnetizable are provided in a second subsection.
The coil 138 may be comprised of any conductive material, including but not limited to metal, including, but not limited to, aluminum, brass, gold, silver, tin, copper, bronze, chromium, cobalt, nickel, lead, iron, steel, manganese, zinc, and combinations thereof; conductive fabric; conductive thread; conductive foam; conductive wool; conductive tape; and combinations thereof.
Embodiments include other types of geometric shapes for end caps.
In other embodiments, the main body of the magnetizable float 104 can include a variety of different support structures for separating target materials, supporting the tube wall, or directing the suspension fluid around the magnetizable float during centrifugation.
The magnetizable float may include an internal magnet, such as a permanent magnet, an electromagnet, or a switchable magnet. Alternatively, a magnet, such as a permanent magnet, an electromagnet, or a switchable magnet, may be external to the magnetizable float, such that the magnetizable float propagates, transmits, and/or carries the magnet field or magnetic gradient introduced by the magnet.
When the magnetizable core is not a magnet, the magnetizable core may be composed of a metal, including, but not limited to, aluminum, brass, gold, silver, tin, copper, bronze, chromium, cobalt, nickel, lead, iron, steel, manganese, zinc, and combinations thereof.
The magnetizable float can be composed of a variety of different materials. The magnetizable float can be composed of a metal, including, but not limited to, aluminum, brass, gold, silver, tin, copper, bronze, chromium, cobalt, nickel, lead, iron, steel, manganese, zinc, neodymium, and combinations thereof The magnetizable float can be composed of a organic or inorganic materials; ferrous plastics; sintered metal; machined metal; and plastic materials, such as polyoxymethylene (“Delrin®”), polystyrene, acrylonitrile butadiene styrene (“ABS”) copolymers, aromatic polycarbonates, aromatic polyesters, carboxymethylcellulose, ethyl cellulose, ethylene vinyl acetate copolymers, nylon, polyacetals, polyacetates, polyacrylonitrile and other nitrile resins, polyacrylonitrile-vinyl chloride copolymer, polyamides, aromatic polyamides (“aramids”), polyamide-imide, polyarylates, polyarylene oxides, polyarylene sulfides, polyarylsulfones, polybenzimidazole, polybutylene terephthalate, polycarbonates, polyester, polyester imides, polyether sulfones, polyetherimides, polyetherketones, polyetheretherketones, polyethylene terephthalate, polyimides, polymethacrylate, polyolefins (e.g., polyethylene, polypropylene), polyallomers, polyoxadiazole, polyparaxylene, polyphenylene oxides (PPO), modified PPOs, polystyrene, polysulfone, fluorine containing polymer such as polytetrafluoroethylene, polyurethane, polyvinyl acetate, polyvinyl alcohol, polyvinyl halides such as polyvinyl chloride, polyvinyl chloride-vinyl acetate copolymer, polyvinyl pyrrolidone, polyvinylidene chloride, specialty polymers, polystyrene, polycarbonate, polypropylene, acrylonitrite butadiene-styrene copolymer, butyl rubber, ethylene propylene diene monomer, others, and combinations thereof.
When the magnetizable float includes the magnetizable coating, the magnetizable coating may be composed of a metal, including, but not limited to, aluminum, brass, gold, silver, tin, copper, bronze, chromium, cobalt, nickel, lead, iron, steel, manganese, zinc, neodymium, and combinations thereof.
The support members may be included during the molding, forming or machining processes or added after the molding, forming and machining processes are complete.
The tube may have a sidewall and a first diameter, the sidewall being elastically radially expandable to a second diameter in response to an axial load, pressure due to centrifugation, or internally-introduced pressure, the second diameter being sufficiently large to permit axial movement of the magnetizable float in the tube during centrifugation.
In block 806, a magnetizable float is added to the vessel and a cap seals the vessel. For the sake of convenience, the suspension discussed herein is blood, though the suspension can be urine, blood, bone marrow, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucus membrane secretions, aqueous humor, vitreous humor, vomit, and any other physiological fluid or semi-solid. Furthermore, for the sake of convenience, the target analyte discussed herein is a circulating tumor cell (“CTC”), though the target analyte can be a cell, such as ova, a circulating endothelial cell, a vesicle, a liposome, a protein, a nucleic acid, a biological molecule, a naturally occurring or artificially prepared microscopic unit having an enclosed membrane, parasites, microorganisms, viruses, or inflammatory cells.
In block 808, the magnetizable float, the tube, and the suspension undergo density-based separation, such as by centrifugation, thereby permitting separation of the suspension into density-based fractions along an axial position in the tube based on density. In block 810, the target analyte-particle complex is attracted to the magnetizable float after centrifugation. Alternatively, the target analyte-particle complex may be attracted to the magnetizable float during centrifugation.
A doubly-conjugated ligand is a ligand which has been conjugated with two distinct molecules, such that the two molecules are not of the same composition or structure. The doubly-conjugated ligand provides a wide range of applications, such as a fluorescent linker between two articles, such that it is conjugated with a fluorescent marker and a distinct molecule so that the fluorescent marker emits a light signal upon excitation. The doubly-conjugated ligand provides a more functional way of linking two or more articles. The doubly-conjugated ligand may also amplify the detectable signal of the labeled article. To doubly conjugate the ligand, an unlabeled ligand may first be added to a reactive solution containing both a reactive first molecule and a reactive second molecule, such that the reactive first and second molecules are not of the same composition or structure. Next, the solution is incubated. The reactive second and third molecules compete for attachment to the unfilled conjugation sites of the ligand, thereby conjugating the ligand with some of the reactive second molecule and some of the reactive third molecule. In order to do this, however, a portion of the molar concentrations of each of the second and third molecules will be used, such that the total molar amount will be the same as when a single labeling reaction were done.
Alternatively, to doubly conjugate a ligand, the ligand may be pre-labeled with the third molecule and then added to a solution comprising the reactive second molecule, such that the third molecule and the reactive second molecule are not of the same composition or structure. Next, the solution is incubated. The reactive second molecule will attach to at least one of the unfilled conjugation sites on the ligand pre-labeled with the third molecule.
Regarding
The particle may come in any form, including, but not limited to, a bead, a nanoparticle (such as a quantum dot), a shaving, a filing, or the like, such that the particle is capable of being attracted by a magnetic field or magnetic gradient introduced by a magnet. The particle may itself be magnetic, diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic. The particle 1014 may be composed of a variety of different materials including, but not limited to, metals, including, but not limited to, aluminum, brass, gold, silver, tin, copper, bronze, chromium, cobalt, nickel, lead, iron, steel, manganese, zinc, neodymium, and combinations thereof; at least one organic material, at least one inorganic material, at least one mineral, at least one ferrofluid, at least one plastic, at least one polymer and combinations thereof.
Returning to
The excitation light is focused by an objective 1204 onto the analysis area, which is a space between the magnetizable float and tube in which a target analyte may be retained or trapped. The different wavelengths excite different fluorescent markers, causing the fluorescent markers to emit light at lower energy wavelengths. A portion of the light emitted by the fluorescent markers is captured by the objective 1204 and transmitted to a detector 1206 that generates images that are processed and analyzed by a computer or associated software or programs. The images formed from each of the channels can be overlaid when a plurality of fluorescent markers, having bound themselves to the target analyte, are excited and emit light. The light source 1202 and the objective 1204 may be separate pieces or may be one piece. The light source 1202 and the objective 1204 may be coaxial or may be located on different planes. The target analyte 904 can then be characterized, and its location identified, based on the light emission(s) from the fluorescent marker(s) 912 attached to the target analyte 904.
Returning to
Alternatively, when the magnetizable float is removed from the tube, a second force may be introduced by an extraction device—such as by a magnet, a pipette, or the like—to remove the target analyte-particle complex from the magnetizable float. The second force may be stronger than the force of the magnetic field or the magnetic gradient, so as to overcome the magnetic force, thereby attracting or pulling the target analyte off of the magnetizable float. The second force may be magnetic, electrical, pressure (such as by suction or a vacuum), or the like.
Alternatively, a dissipating magnetic field or a fluctuating magnetic field with decreasing amplitude may also permit isolation of a target analyte-particle complex, such as one that may be implemented to reduce or eliminate hysteresis. When the magnetic field is temporarily applied (i.e. through the use of a switchable magnet, an electromagnet, or a magnetizable core/coating with a magnetic field being introduced), once the magnetic field is turned off or removed in some manner, the magnetizable float is no longer magnetic and may no longer magnetically attract the particle. After the magnetic field is eliminated or reduced, the magnetizable float may have a residual magnetism, or hysteresis, which will still permit the magnetizable float to magnetically attract certain particles. Hysteresis, however, may be reduced or eliminated by introducing or applying a magnetic field in the opposite direction that of the residual magnetic field. The magnetizable float may be permanently magnetized by adding a magnet to an inner cavity of the magnetizable float, making the magnetizable float out of a magnetic material, or having the outer surface of the magnetizable float comprising at least one magnet. Referring back to the magnetizable float having at least one coil within or around the magnetizable float, the at least one coil may comprise a plurality of smaller coils, each coil thereby producing a smaller magnetic field which may aggregate to form a larger, stronger magnetic field. As the intensity of the magnetic field decreases, more particles will be required to hold the attracted analyte to the magnetizable float. The magnetic field may be denoted as B. The magnetic field has a maximum amplitude, BMAX, and a minimum amplitude, B0, where B0 is the point at which the magnetic field is lost, has completely dissipated, or does not exist.
As an example of the number of particles required for attraction versus the amplitude of the magnetic field, an analyte that only has been conjugated with one particle may fall off of the magnetizable float when B at a first time (BT1) is 75% of BMAX, or 0.75BMAX. An analyte that has been conjugated with two particles may fall off when at a second time (BT2) is 60% of BMAX, or 0.60BMAX. An analyte that has been conjugated with three particles may fall off when at a third time (BT3) is 50% of BMAX, or 0.50BMAX. An analyte that has been conjugated with four particles may fall off when at a fourth time (BT4) is 33% of BMAX, or 0.33BMAX. When a specific target analyte has been conjugated with a known number of particles, it may be desirous to wait until the magnetic field is at a level which no longer attracts the conjugate. When the specific target analyte has been conjugated with an unknown number of particles, further analysis may be performed on the conjugate to determine its classification. It should be noted that the numbers and values discussed herein are used to show the principles and may be different in practice or with each conjugation.
To retrieve the target analyte-particle complex from the tube, a hole may be bored through the sidewall of the tube. The hole may be introduced by drilling, cutting, or puncturing the sidewall of the tube at a location where the target analyte is located, as may be determined by imaging. When the hole is formed, the extraction device may be introduced to retrieve the target analyte-particle complex by introducing the second force to overcome the magnetic force of the magnetizable float.
Referring now to
Referring now to
The target analyte may be analyzed using any appropriate analysis method or technique, though more specifically extracellular and intracellular analysis including intracellular protein labeling; nucleic acid analysis, including, but not limited to, DNA arrays, expression arrays, protein arrays, and DNA hybridization arrays; in situ hybridization (“ISH”—a tool for analyzing DNA and/or RNA, such as gene copy number changes); polymer chain reaction (“PCR”); reverse transcription PCR; or branched DNA (“bDNA”—a tool for analyzing DNA and/or RNA, such as mRNA expression levels) analysis. These techniques require fixation, permeabilization, and isolation of the target analyte prior to analysis. Some of the intracellular proteins which may be labeled include, but are not limited to, cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin, FtsZ, myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc, caspases, survivin, p27kip, FOXC2, BRAF, Phospho-Akt1 and 2, Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twist', Snail1, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3, phosphorylated receptor kinases, modified histones, chromatin-associated proteins, and MAGE. To fix, permeabilize, or label, fixing agents (such as formaldehyde, formalin, methanol, acetone, paraformaldehyde, or glutaraldehyde), detergents (such as saponin, polyoxyethylene, digitonin, octyl β-glucoside, octyl β-thioglucoside, 1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO, (1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol or octylphenol ethylene oxide), or labeling agents (such as fluorescently-labeled antibodies, enzyme-conjugated antibodies, Pap stain, Giemsa stain, or hematoxylin and eosin stain) may be used.
In direct conjugation, the first molecule of the particle bonds directly with the CTC. In first-degree indirect conjugation, the particle having a first molecule is conjugated to the CTC via a doubly-conjugated ligand. The doubly-conjugated ligand is bound to a second molecule and a third molecule. The second molecule is a complementary molecular conjugate to that of the first molecule such that the first molecule and the second molecule bond to each other. In second-degree indirect conjugation, the CTC is conjugated to the particle via the first intermediary particle bound to a second molecule and the fluorescent marker-conjugated second intermediary particle while in the tube or prior to introduction into the tube.
The first molecule of the particle, whether introduced to the particle through binding, coating, or attaching, may include, but is not limited to, an avidin, such as streptavidin or neutravidin; Protein A, Protein G, Protein L; biotin; an aptamer; a primary antibody that binds to biomarkers, including but not limited to, EpCAM, AMACR, Androgen receptor, CD 146, CD227, CD235, CD24, CD30, CD44, CD45, CD56, CD71, CD105, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2, Mammaglobin, or PSMA; a ligand, such as EGF, HGF, TGFα, TGFβ superfamily of ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands, amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family of ligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands; IgG, IgM; scFv, Fab, sdAb; an antibody-like molecule that binds to a biomarker; or a second antibody.
The first and second molecules are complementary molecular conjugates that will indirectly bind and attach any particle to which they are already attached by binding and attaching to each other. The second molecule may include, but is not limited to, an avidin, such as streptavidin or neutravidin; Protein A, Protein G, Protein L; biotin; an aptamer; a primary antibody that binds to biomarkers, including but not limited to, EpCAM, AMACR, Androgen receptor, CD146, CD227, CD235, CD24, CD30, CD44, CD45, CD56, CD71, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2, Mammaglobin, or PSMA; a ligand, such as EGF, HGF, TGFα, TGFβ superfamily of ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands, amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family of ligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands; IgG, IgM; scFv, Fab, sdAb; an antibody-like molecule that binds to a biomarker; or a second antibody.
On a doubly-conjugated ligand, the third molecule may include, but is not limited to, a fluorescent marker; alkaline phosphatase; an avidin, such as streptavidin or neutravidin; Protein A, Protein G, Protein L; biotin; an aptamer; a primary antibody that binds to biomarkers, including but not limited to, EpCAM, AMACR, Androgen receptor, CD 146, CD227, CD235, CD24, CD30, CD44, CD45, CD56, CD71, CD105, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2, Mammaglobin, or PSMA; a ligand, such as EGF, HGF, TGFα, TGFβ superfamily of ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands, amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family of ligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands; IgG, IgM; scFv, Fab, sdAb; an antibody-like molecule that binds to a biomarker; or a second antibody.
The first and second intermediaries may include, but are not limited to an avidin, such streptavidin or neutravidin; biotin; a protein; an antibody, including but not limited to, EpCAM, AMACR, Androgen receptor, CD146, CD227, CD235, CD24, CD30, CD44, CD45, CD56, CD71, CD105, CD324, CD325, MUC1, CEA, cMET, EGFR, Folate receptor, HER2, Mammaglobin, or PSMA; a ligand, such as EGF, HOF, TGFα, TGFβ superfamily of ligands, IGF1, IGF2, Wnt signaling proteins, FGF signaling ligands, amphiregulin, HB-EGF, neuregulin signaling ligands, MSP, VEGF family of ligands, betacellulin, epiregulin, epigen, hedgehog signaling ligands; or an aptamer.
The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the invention. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The foregoing descriptions of specific embodiments of the present invention are presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalents:
This application claims the benefit of Provisional Application No. 61/618,143, filed Mar. 30, 2012, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61618143 | Mar 2012 | US |
