Source: https://patents.justia.com/patent/9625360
Timestamp: 2019-08-21 19:53:39
Document Index: 549977933

Matched Legal Cases: ['Application No. 62', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61', 'Application No. 61']

US Patent for Apparatus, system, and method for collecting a target material Patent (Patent # 9,625,360 issued April 18, 2017) - Justia Patents Search
Justia Patents FloatUS Patent for Apparatus, system, and method for collecting a target material Patent (Patent # 9,625,360)
Oct 14, 2015 - RareCyte, Inc.
This disclosure is directed to an apparatus, system and method for retrieving a target material from a sample. A fraction-density-altering solution may be added to a vessel that contains the sample to change the density of a first fraction of the sample without changing the density of the target material or the density of any other sample fraction. A collector may be inserted into the vessel to funnel the target material from the sample into the collector or into a processing receptacle included in the collector. In one implementation, the collector may include a cannula which extends from a funnel into a chamber to hold the processing receptacle. In another implementation, the processing receptacle may be inserted into a bore within the collector.
This application claims the benefit of Provisional Application No. 62/068,480, filed Oct. 24, 2014; and this application is a continuation-in-part of application Ser. No. 14/665,368, filed Mar. 23, 2015, (now U.S. Pat. No. 9,217,687) which is a continuation-in-part of application Ser. No. 14/610,522, filed Jan. 30, 2015, which claims the benefit of Provisional Application No. 61/935,457, filed Feb. 4, 2014, and which is also a continuation-in-part of application Ser. No. 14/495,449, filed Sep. 24, 2014, (now U.S. Pat. No. 9,039,999) which is a continuation-in-part of application Ser. No. 14/090,337, filed Nov. 26, 2013, (now abandoned) which claims the benefit of Provisional Application No. 61/732,029, filed Nov. 30, 2012; Provisional Application No. 61/745,094, filed Dec. 21, 2012; Provisional Application No. 61/791,883, filed Mar. 15, 2013; Provisional Application No. 61/818,301, filed May 1, 2013; and Provisional Application No. 61/869,866, filed Aug. 26, 2013; and is also a continuation-in-part of application Ser. No. 14/266,939, filed May 1, 2014, (now abandoned) which claims the benefit of Provisional Application No. Provisional Application No. 61/818,301, filed May 1, 2013, Provisional Application No. 61/869,866, filed Aug. 26, 2013, and Provisional Application No. 61/935,457, filed Feb. 4, 2014.
FIG. 7 shows an example fluid layering device.
FIG. 8 shows a flow diagram of an example method for retrieving a target material.
FIGS. 9A-9D show example float and tube systems.
FIG. 10 shows a sample having undergone density-based separation.
FIGS. 11A-11B show a seal being formed by a clamp.
FIGS. 12A-12G show an example system retrieving a target material.
FIG. 13 shows a flow diagram of an example method for retrieving a target material.
This disclosure is directed to an apparatus, system and method for retrieving a target material from a suspension. A system includes a processing vessel, such as an Eppendorf tube, a syringe or a test tube, and a collector. The collector is sized and shaped to fit into a primary vessel, such as a test tube. The collector funnels the target material from the suspension through a cannula and into the processing vessel. The cannula extends into a cavity at a first end of the collector that holds the processing vessel. The collector includes a funnel at a second end in fluid communication with the cannula. In one implementation, the processing vessel includes at least one collection fluid to be expelled, such that the at least one collection fluid pushes the target material into the collector.
The main body can be composed of a variety of different materials including, but not limited to, a ceramic; metals; organic or inorganic materials; 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, polyamides, 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; and combinations thereof.
The collection fluid 412 has a greater density than the density of the target material of the suspension (the density may be greater than the density of a subset of suspension fractions or all of the suspension fractions) and is inert with respect to the suspension materials. The collection fluid 412 may be miscible or immiscible in the suspension fluid. Examples of suitable collection fluids include, but are not limited to, solution of colloidal silica particles coated with polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, an oil, a gas, and combinations thereof; olive oil, mineral oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil, fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide, and combinations thereof; organic solvents such as 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate, tetramethylene sulfones, and ionic liquids; polymer-based solutions; surfactants; perfluoroketones, such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones, hydrofluoroethers, hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon and silicon-based liquids, such as phenylmethyl siloxane; and combinations thereof.
FIG. 7 shows a fluid layering device 700. The fluid layering device 700 may include a motor 704 connected to a rod 708 which is also connected to a piston 710. The fluid layering device 700 may also include a switch 706 to activate or de-activate the motor 704. When the motor 704 is activated, the rod 708, which may be threaded, may rotate, thereby causing the piston 710 to move up and down, thereby creating a pressure gradient to expel a layering fluid 716 from the fluid layering device 700 a plug 714 when the fluid layering device 700 is inserted into the collector 200, such that the cannula 216 extends through the plug 714. Alternatively, the rod 708 may move up and down, thereby causing the piston 710 to move up and down, hence creating the pressure gradient. Alternatively, a cam mechanism, servomotor, diaphragm, or rack and pinion system may be used to either move the piston 710 up and down to create the pressure gradient or to create the pressure gradient without the piston 710.
The fluid layering device 700 may have a main body 702 with a tapered geometry that widens or narrows toward the plug 714; the fluid layering device 700 may have a generally cylindrical geometry; or, the fluid layering device 700 may have a generally cylindrical geometry in a first segment and a cone-shaped geometry in a second segment, where the first and second segments are connected and continuous with each other. Although at least one segment of the fluid layering device 700 has a circular cross-section, in other embodiments, the at least one segment can have elliptical, square, triangular, rectangular, octagonal, or any other suitable cross-sectional shape. The fluid layering device 700 can be composed of a transparent, semitransparent, opaque, or translucent material, such as plastic or another suitable material. The processing vessel includes a central axis, which when inserted into the cavity 212 of the collector 200 is coaxial with the central axis 202 of the collector 200. The plug 714 permits the introduction of the layering fluid 716 into the primary vessel via the cannula 216 of the collector 200.
The plug 714 may be composed of re-sealable rubber or other suitable re-sealable material that can be repeatedly punctured with a needle or other sharp implement to access the contents of the fluid layering device 700 interior or permit introduction of contents into the fluid layering device 700 and re-seals when the needle or implement is removed. The plug 714 may be inserted into the fluid layering device 700 such that a seal is maintained between the plug 714 and the fluid layering device 700, such as by an interference fit. Alternatively, the plug 714 can be formed in the closed end 404 of the fluid layering device 700 using heated liquid rubber that can be shaped while warm or hot and hardens as the rubber cools. An adhesive may be used to attach a plug 714 to the inner wall of the processing vessel can be a polymer-based adhesive, an epoxy, a contact adhesive or any other suitable material for bonding or creating a thermal bond. Alternatively, the plug 714 may be injected into the fluid layering device 700. Alternatively, the plug 714 may be thermally bonded to the fluid layering device 700.
The fluid layering device 700 may also include a controller (not shown) to control the output of the motor 704, such as the frequency and/or amplitude of the various strokes. The fluid layering device 700 may also include a battery (not shown) which may be chargeable or replaceable.
The layering fluid 716 may be miscible or immiscible with the suspension fluid and inert with respect to the suspension materials. Examples of suitable layering fluids include, but are not limited to, solution of colloidal silica particles coated with polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, an oil, a gas, and combinations thereof; olive oil, mineral oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil, fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide, and combinations thereof; organic solvents such as 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate, tetramethylene sulfones, and ionic liquids; polymer-based solutions; surfactants; perfluoroketones, such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones, hydrofluoroethers, hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon and silicon-based liquids, such as phenylmethyl siloxane; and combinations thereof.
FIG. 8 shows a flow diagram for an example method for retrieving a target material. In block 802, a sample, such as anticoagulated whole blood, is obtained, and is added to a primary vessel, such as a test tube. A float may also be added to the primary vessel. For the sake of convenience, the methods are described with reference to the float, but the methods described below are not intended to be so limited in their application and may be performed without the float.
A depletion reagent may be added to the sample to remove non-target material, such as by lysing the non-target material or changing the density of the non-target material. Examples of suitable depletion reagents include, but are not limited to, solution of colloidal silica particles coated with polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g. OptiPrep), a complex, branch glucan (e.g. Dextran), cesium chloride, sucrose, sugar-based solutions, polymer solutions, multi-phase polymer solutions, tetrameric antibody complexes (e.g. RosetteSep) or the like.
FIG. 9A shows an isometric view of an example primary vessel and float system 900. The system 900 includes a primary vessel 902 and a float 904 suspended within whole blood 906. In the example of FIG. 9A, the primary vessel 902 has a circular cross-section, a first open end 910, and a second closed end 908. The open end 910 is sized to receive a cap 912. The primary vessel may also have two open ends that are sized to receive caps, such as the example tube and separable float system 920 shown FIG. 9B. The system 920 is similar to the system 900 except the primary vessel 902 is replaced by a primary vessel 922 that includes two open ends 924 and 926 configured to receive the cap 912 and a cap 928, respectively. The primary vessels 902 and 922 have a generally cylindrical geometry, but may also have a tapered geometry that widens, narrows, or a combination thereof toward the open ends 910 and 924, respectively. Although the primary vessels 902 and 922 have a circular cross-section, in other embodiments, the primary vessels 902 and 922 can have elliptical, square, triangular, rectangular, octagonal, or any other suitable cross-sectional shape that substantially extends the length of the tube. The primary vessels 902 and 922 can be composed of a transparent, semitransparent, opaque, or translucent material, such as plastic or another suitable material. The primary vessels 902 and 922 each include a central axis 918 and 930, respectively. The primary vessel 902 may also include a septum 914, as seen in magnified view 916, at the closed end 908 to permit the removal of a fluid, the suspension, or a suspension fraction, whether with a syringe, a pump, by draining, or the like. The primary vessel 902 may have an inner wall and a first diameter.
The septum 914 may be composed of re-sealable rubber or other suitable re-sealable material that can be repeatedly punctured with a needle or other sharp implement to access the contents of the primary vessel 902 interior and re-seals when the needle or implement is removed. The septum 914 may be inserted into the primary vessel 902 such that a seal is maintained between the septum 914 and the primary vessel 902, such as by an interference fit. Alternatively, the septum 914 can be formed in the openings and/or the bottom interior of the tube using heated liquid rubber that can be shaped while warm or hot and hardens as the rubber cools. An adhesive may be used to attach the septum 914 to the wall of the opening and tube interior and can be a polymer-based adhesive, an epoxy, a contact adhesive or any other suitable material for bonding rubber to plastic or creating a thermal bond. Alternatively, the septum 914 may be thermally bonded to the primary vessel 902.
The float 904 includes a main body, two teardrop-shaped end caps, and support members radially spaced and axially oriented on the main body. Alternatively, the float 904 may not include any support members. Alternatively, the float 904 may include support members which do not engage the inner wall of the primary vessel 902.
In alternative embodiments, the number of support members, support member spacing, and support member thickness can each be independently varied. The support members can also be broken or segmented. The main body is sized to have an outer diameter that is less than the inner diameter of the primary vessel 902, thereby defining fluid retention channels between the outer surface of the main body and the inner wall of the primary vessel 902. The surfaces of the main body between the support members can be flat, curved or have another suitable geometry. The support members and the main body may be a singular structure or may be separate structures.
Embodiments include other types of geometric shapes for float end caps. The top end cap may be teardrop-shaped, dome-shaped, cone-shaped, or any other appropriate shape. The bottom end cap may be teardrop-shaped, dome-shaped, cone-shaped, or any other appropriate shape. In other embodiments, the main body of the float 904 can include a variety of different support structures for separating samples, supporting the tube wall, or directing the suspension fluid around the float during centrifugation. Embodiments are not intended to be limited to these examples. The main body may include a number of protrusions that provide support for the tube. In alternative embodiments, the number and pattern of protrusions can be varied. The main body may include a single continuous helical structure or shoulder that spirals around the main body creating a helical channel. In other embodiments, the helical shoulder can be rounded or broken or segmented to allow fluid to flow between adjacent turns of the helical shoulder. In various embodiments, the helical shoulder spacing and rib thickness can be independently varied. In another embodiment, the main body may include a support member extending radially from and circumferentially around the main body. In another embodiment, the support members may be tapered.
The float 904 can be composed of a variety of different materials including, but not limited to, metals; organic or inorganic materials; ferrous plastics; sintered metal; machined metal; plastic materials and combinations thereof. The primary vessel 902 may have an inner wall and a first diameter. The float 904 can be captured within the primary vessel 902 by an interference fit, such that under centrifugation, an inner wall of the tube expands to permit axial movement of the float 904. When centrifugation stops, the inner wall reduces back to the first diameter to induce the interference fit. Alternatively, the inner wall may not expand and the interference fit may not occur between the float 904 and the primary vessel 902, such that the float moves freely within the tube before, during, or after centrifugation. The end caps of the float may be manufactured as a portion of the main body, thereby being one singular structure, by machining, injection molding, additive techniques, or the like; or, the end caps may be connected to the main body by a press fit, an adhesive, a screw, any other appropriate method by which to hold at least two pieces together, or combinations thereof.
The cap 912 may be composed of a variety of different materials including, but not limited to, organic or inorganic materials; plastic materials; and combination thereof.
In block 804, the sample, the float, and the primary vessel undergo centrifugation. FIG. 10 shows an isometric view of the primary vessel and float system 900 having undergone density-based separation, such as by centrifugation. Suppose, for example, the centrifuged whole blood includes three fractions. For convenience sake, the three fractions include plasma, buffy coat, and red blood cells. However, when another suspension undergoes centrifugation, there may be more than, less than, or the same number of fractions, each fraction having a different density. The suspension undergoes axial separation into three fractions along the length the tube based on density, with red blood cells 1003 located on the bottom, plasma 1001 located on top, and buffy coat 1002 located in between, as shown in FIG. 10. The float 904 may have any appropriate density to settle within one of the fractions. The density of the float 904 can be selected so that the float 904 expands the buffy coat 1002 between the main body of the float and the inner wall of the primary vessel. The buffy coat 1002 can be trapped within an area between the float 904 and the primary vessel 902.
FIGS. 11A and 11B show a first seal being formed to prevent fluids from moving up or down within the primary vessel. For convenience, FIG. 11A shall be used to describe the method, though the method applies equally to FIG. 11B. The first seal also inhibits float movement. The first sealing ring 600 may be placed at approximately a lower end of the main body of the float 904. The first sealing ring 600 exerts circumferential or radial forces on the primary vessel 902, thereby causing the primary vessel 902 to collapse inwardly against the float 904. Magnified view 1102 shows the first sealing ring 600 tightened around the float and primary vessel system 900. The first sealing ring 600, having been placed at an interface of the buffy coat 1002 and the red blood cells 1003, causes the primary vessel 902 to collapse inwardly until a seal is formed between the primary vessel 902 and the float 904. An outer wall of the first sealing ring 600 may sit flush with an outer wall of the primary vessel 902; the outer wall of the first sealing ring 600 may extend past the outer wall of the primary vessel 902; or, the outer wall of the primary vessel 902 may extend past the outer wall of the first sealing ring 600. The first sealing ring 600 remains tightened to maintain the seal, which prevents fluids from moving past the seal in any direction. The first sealing ring 600 may also remain in tension. Alternatively, the first sealing ring 600 may be overtightened and then the force applied to the first sealing ring 600 is removed. The first sealing ring 600 may expand slightly, though still remains constricted.
To apply the first sealing ring 600 and thereby form the seal, a clamp may be used to circumferentially apply a force directed toward the central axis of the primary vessel 902 to the first sealing ring 600 and the float and primary vessel system 900. The first sealing ring 600 is placed around the float and primary vessel system 900 after the float and primary vessel system 900 have undergone density-based separation, such as by centrifugation. The first sealing ring 600 and float and primary vessel system 900 are then placed into the clamp. The clamp may include a shelf to support the first sealing ring 600 against the primary vessel 902. Operation of the clamp may be automated or may be performed manually. Alternatively, the clamp may form a seal between the float 904 and primary vessel 902 without the inclusion of the first sealing ring 600. Alternatively, a seal may be formed between the float 904 and the primary vessel 902 such as by ultrasonic welding; or by applying heat or a temperature gradient to deform and/or melt the primary vessel 902 to the float 904. For the sake of convenience, the methods are described with reference to the seal between the float and the primary vessel, but the methods described below are not intended to be so limited in their application and may be performed without the seal.
The plasma 1001 may be removed from the primary vessel 902, as shown in FIG. 12A, such as by pipetting, suctioning, pouring, or the like. Returning to FIG. 8, in block 806, a clearing fluid 1202 may be added to the primary vessel 902 and the system undergoes centrifugation again, as shown in FIGS. 12B-12C. The clearing fluid 1202 has a density greater than the buffy coat 1002, but may be layered on top of the buffy coat 1002. It may be desirous to gently layer the clearing fluid 1202 on top of the buffy coat 1002 to inhibit mixing of the clearing fluid 1202 with the buffy coat 1002. During centrifugation, the clearing fluid 1202 moves underneath the buffy coat 1002 but remains above the clamp 600. After centrifugation, the buffy coat 1002, having a density less than the clearing fluid 1202, rests on top of the clearing fluid 1202. An appropriate amount of the clearing fluid 1202, such as up to 500 milliliters, may be added to the primary vessel 902, such that the buffy coat 1002 is not between the main body of the float 904 and the inner wall of the primary vessel 902. The clearing fluid 1202 may be miscible or immiscible with the suspension fluid and inert with respect to the suspension materials. Examples of suitable clearing fluids include, but are not limited to, solution of colloidal silica particles coated with polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, an oil, a gas, and combinations thereof; olive oil, mineral oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil, fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide, and combinations thereof; organic solvents such as 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate, tetramethylene sulfones, and ionic liquids; polymer-based solutions; surfactants; perfluoroketones, such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones, hydrofluoroethers, hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon and silicon-based liquids, such as phenylmethyl siloxane; and combinations thereof.
A second sealing ring 600 may be placed at approximately an upper end of the main body of the float 904, as shown in FIG. 12D. Returning to FIG. 8, in block 808, a density-altering agent may be added to the primary vessel 902 to change the density of non-target material relative to the density the of target material. For example, the target material may be circulating tumor cells or fetal cells. The density-altering agent may be added to change the density of the fluid within which these cells float without changing the density of the cells. The density of the fluid and any additional non-target materials may be made greater than the density of the target material, such as the cells. Examples of suitable fraction-changing-density solutions include, but are not limited to, solution of colloidal silica particles coated with polyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g. OptiPrep), a complex, branch glucan (e.g. Dextran), cesium chloride, sucrose, sugar-based solutions, polymer solutions, multi-phase polymer solutions, tetrameric antibody complexes (e.g. RosetteSep) or the like.
To be imaged, a solution containing a fluorescent probe may be used to label the target material, thereby providing a fluorescent signal for identification and characterization, such as through imaging. The fluorescent probe may be added to the primary vessel after the second sealing ring has been applied or after at least one non-target material, such as the plasma, has been removed. The solution containing the fluorescent probe may be added to the suspension before the suspension is added to the vessel, after the suspension is added to the vessel but before centrifugation, or after the suspension has undergone centrifugation. The fluorescent probe includes a fluorescent molecule bound to a ligand. The target material may have a number of different types of surface markers. Each type of surface marker is a molecule, such an antigen, capable of attaching a particular ligand, such as an antibody. As a result, ligands may be used to classify the target material and determine the specific type of target materials present in the suspension by conjugating ligands that attach to particular surface markers with a particular fluorescent molecule. Examples of suitable fluorescent molecules include, but are not limited to, quantum dots; commercially available dyes, such as fluorescein, Hoechst, FITC (“fluorescein isothiocyanate”), R-phycoerythrin (“PE”), Texas Red, allophycocyanin, Cy5, Cy7, cascade blue, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC (“tetramethylrhodamine isothiocyanate”); combinations of dyes, such as CY5PE, CY7APC, and CY7PE; and synthesized molecules, such as self-assembling nucleic acid structures. Many solutions may be used, such that each solution includes a different type of fluorescent molecule bound to a different ligand.
In block 810, a collector, such as the collector 200, may be inserted into the primary vessel 902. In block 812, a fluid layering device 700 may be inserted into the collector 200. FIG. 12D shows the collector 200 inserted into the primary vessel 902 and also forming a seal 1206 between the second end 208 of the collector 200 and the inner wall of the primary vessel 902. The layering fluid 716 may be added to the primary vessel 902 by the fluid layering device 700 via the cannula 216 of the collector 200, as shown in FIG. 12E.
Returning to FIG. 8, in block 814, the fluid layering device 700 is removed from the collector 200 and a processing vessel 402 including the collection fluid 412 is inserted into the collector 200, as also shown in FIG. 12E. The layering fluid 716 added by the fluid layering device 700 fills any air gap that exists extending from the cannula 216 of the collector to the uppermost layer of fluid or sample material within the primary vessel 902. It should be noted that the collection fluid 412 and the layering fluid 716 may be the same type of fluid or may be a different type of fluid. The collection fluid 412 and the layering fluid 716 are referenced differently herein, even when the collection fluid 412 and the layering fluid 716 are the same, to depict that the collection fluid 412 and the layering fluid 716 are introduced to the system and method by different mechanisms, vessels, or devices.
Returning to FIG. 8, in block 816, the vessel, the collector, and the processing receptacle are centrifuged. During centrifugation, the additional collection fluid 412 moves from the processing vessel 402 into the primary vessel 902 via the cannula 216 of the collector 200 and displaces the buffy coat 1002 from the primary vessel 902 into the processing vessel 402 via cannula 216 the collector 200. The processing vessel 402, now including the buffy coat 1002, may be removed from the collector 200.
The processing receptacle 302 may also include a processing solution to effect a transformation on the target material when the target material enters the processing receptacle 302. The processing solution may be a preservative, a fixative, a cell adhesion solution, a dye, a freezing stabilization media, or the like. Unlike the collection fluid, most, if not all, of the processing solution remains within the processing receptacle 302 upon centrifugation, thereby effecting the transformation on the target material in one manner or another (i.e. preserving, fixing, increasing adhesion properties, or the like) in the processing receptacle 302. The processing solution may be introduced as a liquid or as a liquid container in a casing. The casing may be dissolvable in an aqueous solution but not in the collection fluid (such as a gel cap); or, the casing may be breakable, such that the casing breaks when the processing receptacle 302 is shaken in a vortex mixer. Additionally, more than one processing solution may be used.
Furthermore, when the vessel includes a septum in the closed end, the plasma, for example, may be removed through the septum with a needle, syringe, by draining, or the like. The plasma may then be further processed and analyzed.
FIG. 13 shows an example method 1300 for retrieving a target material using sequential density fractionation. The example method 1300 is similar to the example method 800, except the example method 1300 collects the target material by sequential density fractionation. After the steps of block A have been performed, as shown in FIG. 8, sequential density fractionation is performed, as seen in block 1302. Block 1302 is also a snapshot of the sequential density fractionation steps. In block 1304, an nth processing receptacle including an nth collection fluid is inserted into the collector, such that nth is greater than or equal to first (i.e. second, third, fourth, and so on). In block 1306, the system is centrifuged to collect a fraction or sub-fraction and the nth processing receptacle is removed. In block 1308, the operator determines whether or not the desired fraction or sub-fraction is obtained. When the desired fraction or sub-fraction is obtained, the process may stop as shown in block 1310, though the process may continue until all fractions or sub-fractions are obtained. When the desired fraction or sub-fractions is not yet obtained, the process restarts at block 1304. The processing receptacles may also include a processing solution to effect a change on the respective sub-fractions. Two or more processing receptacles and respective collection fluids may be used depending on the number of fractions or sub-fractions desired for separation and collection. Each successive collection fluid is denser than the preceding collection fluid. Similarly, each successive fraction or sub-fraction is denser than the preceding fraction or sub-fraction. Once collected, the consecutive sub-fractions may be analyzed, such as for diagnostic, prognostic, research purposes, to determine components characteristics (i.e. a complete blood count), how those characteristics change over time, or the like.
The target material may be analyzed using any appropriate analysis method or technique, though more specifically extracellular and intracellular analysis including intracellular protein labeling; chromogenic staining; molecular analysis; genomic analysis or nucleic acid analysis, including, but not limited to, genomic sequencing, 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); polymerase 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 may require fixation, permeabilization, and isolation of the target material 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, Twist1, 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.
After collection, the target material may also be imaged or may undergo flow cytometry.
The following is an example method for retrieving a target material when the density of the plasma is changed to be greater than the density of the buffy coat but less than the density of the red blood cells:
1. Add sample to a tube with a float.
2. (Optional) Add depletion reagent to the sample.
3. Centrifuge to effect a density-based separation of the sample and to align the float with a target material.
4. Apply a first sealing ring to the tube at a location near the lower end of a main body of the float to form a first seal between the tube and the float.
5. Remove at least a first fraction of the sample from the tube
6. Add clearing fluid to the tube.
7. Centrifuge to push target material at least above the main body of the float.
8. (Optional) Apply a second sealing ring to the tube at a location near the upper end of the main body of the float to form a second seal between the tube and the float.
9. (Optional) Add fixative and/or permeabilizing agent to the tube to fix and/or permeabilize the target material. Incubation may be performed, if it is desirous to do so.
10. Add at least one stain to the target material (alternatively, the at least one stain may be added with the sample in Step 1).
11. Add density-altering agent to the tube.
12. Insert collector into tube.
13. Insert fluid layering device into collector.
14. Fill any air gap from cannula to uppermost layer of fluid or sample material within the tube with layering fluid via the fluid layering device.
15. Remove fluid layering device and insert processing receptacle including collection fluid into collector.
16. Centrifuge to collect target material.
providing a primary vessel comprising an open end, and a sample comprising a target material and at least one other sample fraction;
adding at least one fluorescent probe to the primary vessel;
adding a density-altering agent to the vessel to change the density of the at least one other sample fraction such that the density of the target material is less than a changed density of the at least one other sample fraction;
inserting a collector into the open end of the primary vessel;
adding a layering fluid to the primary vessel, the layering fluid having a density greater than the target material;
inserting a first processing vessel into the collector, wherein the first processing vessel includes a first collection fluid having a density greater than at least a first sub-fraction of the target material; and
centrifuging the primary vessel, the collector, and the first processing vessel, such that during centrifugation the first collection fluid flows into the primary vessel via a cannula of the collector and at least the first sub-fraction of the target material flows into the first processing vessel via the cannula of the collector,
wherein centrifuging the primary vessel, the collector, and the first processing vessel is performed after inserting the collector into the primary vessel and after inserting the first processing vessel into the collector.
2. The method of claim 1, wherein the density-altering agent is selected from the group consisting of a solution of colloidal silica particles coated with polyvinylpyrrolidone, a polysaccharide solution, iodixanol, a complex branched glucan, cesium chloride, sucrose, a sugar-based solution, a polymer solution, and a multi-phase polymer solution.
removing the first processing vessel comprising at least the first sub-fraction from the collector;
inserting a second processing vessel comprising a second collection fluid into the collector; and
re-centrifuging the primary vessel with the collector and the second processing vessel,
wherein the second collection fluid flows into the primary vessel via the cannula of the collector and a second sub-fraction of the target material flows into the second processing receptacle via the cannula of the collector,
wherein the second collection fluid has a density greater than the second sub-fraction and the first collection fluid, and wherein the second sub-fraction has a density greater than the density of the first sub-fraction.
4. The method of claim 3, wherein the removing, inserting, and re-centrifuging steps are repeated with an nth processing vessel including an nth collection fluid, wherein nth is equal to or greater than 3rd, and wherein the removing, inserting, and re-centrifuging steps are repeated until all desired sub-fractions of the target material are obtained from the sample, wherein each successive collection fluid has a density greater than each preceding collection fluid.
5. The method of claim 1, wherein the first processing vessel includes a plug in a closed end.
6. The method of claim 5, wherein the first processing vessel includes cap to seal an open end.
7. The method of claim 5, wherein the cannula of the collector extends at least partially through the plug.
8. The method of claim 1, wherein adding the layering fluid is performed before inserting the collector into the primary vessel.
9. The method of claim 1, wherein adding the layering fluid is performed after inserting the collector into the primary vessel by inserting a fluid laying device into the collector and forcing the layering fluid through the cannula of the collector and into the primary vessel.
10. The method of claim 1, further comprising the step of adding a depletion reagent to the primary vessel.
11. The method of claim 10, wherein the depletion reagent lyses at least one non-target material.
12. The method of claim 10, wherein the depletion reagent changes the density of at least one non-target material.
13. The method of claim 1, wherein the first collection fluid and the layering fluid are selected from the group consisting of: a solution of colloidal silica particles coated with polyvinylpyrrolidone, a polysaccharide solution, iodixanol, an organic solvent, a liquid wax, an oil, a gas, olive oil, silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil, fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene, perfluorooctylbromide, 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane, propylene carbonate, tetramethylene sulfones, ionic liquids, a polymer-based solution, a surfactant, a perfluoroketone, perfluorocyclopentanone, perfluorocyclohexanone, a fluorinated ketone, a hydrofluoroether, a hydrofluorocarbon, a perfluorocarbon, a perfluoropolyether, silicon, a silicon-based liquid, phenylmethyl siloxane, and combinations thereof.
14. The method of claim 1, the primary vessel further comprising a float comprising a main body.
15. The method of claim 14, further comprising the step of centrifuging the primary vessel comprising the sample and the float thereby effecting a density-based separation of the sample into the target material and the at least one other sample fraction and trapping the target material between the main body of the float and an inner wall of the primary vessel,
wherein the centrifuging to effect the density-based separation is performed after providing the primary vessel comprising the sample and the float, and before adding the at least one fluorescent probe.
16. The method of claim 15, further comprising the step of adding a clearing fluid having a density greater than the target material to the primary vessel, wherein the clearing fluid is added to the primary vessel after centrifuging to effect the density-based separation.
17. The method of claim 16, further comprising the step of forming a first seal between a lower end of the main body of the float and the inner wall of the primary vessel, wherein forming the first seal is performed after centrifuging to effect the density-based separation and before adding clearing fluid.
18. The method of claim 17, further comprising the step of moving the target material at least above the main body of the float by re-centrifuging the primary vessel, wherein the moving step is performed after the adding the clearing fluid, after centrifuging to effect the density-based separation, and before adding the at least one fluorescent probe.
19. The method of claim 18, further comprising the step of forming a second seal between an upper end of the main body of the float and the inner wall of the primary vessel, wherein forming the second seal is performed after the moving step and before adding the at least one fluorescent probe to the primary vessel.
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Axis-Shield, OptiPrep Leaflet.
Patent Publication Number: 20160041077
Inventors: Lance U'Ren (Seattle, WA), Elizabeth Chang (Mercer Island, WA), Jennifer Chow (Seattle, WA)
Application Number: 14/883,071
International Classification: G01N 1/40 (20060101); B01L 3/00 (20060101); C12Q 1/68 (20060101); B01D 21/26 (20060101); G01N 1/34 (20060101); B01L 9/00 (20060101);