Apparatus, system, and method for collecting a target material

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.

TECHNICAL FIELD

This disclosure relates generally to density-based fluid separation and, in particular, to retrieving a target material from a suspension.

BACKGROUND

Suspensions often include materials of interests 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 ova, fetal cells, endothelial cells, parasites, bacteria, and inflammatory cells, and viruses, including HIV, cytomegalovirus, hepatitis C virus, and Epstein-Barr virus. 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 that occur in a suspension with very low concentrations 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. However, CTCs occur in very low numbers in peripheral whole blood samples. For instance, a 7.5 ml sample of peripheral whole blood sample that contains as few as 5 CTCs is considered clinically relevant for the diagnosis and treatment of a cancer patient. In other words, detecting 5 CTCs in a 7.5 ml blood sample is equivalent to detecting 1 CTC in a background of about 40-60 billion red and white blood cells, which is extremely time consuming, costly and difficult to accomplish using blood film analysis.

As a result, practitioners, researchers, and those working with suspensions continue to seek systems and methods for accurate analysis of suspensions for the presence or absence rare materials of interest.

DETAILED DESCRIPTION

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.

Collector

FIG. 1Ashows an isometric view of a collector100.FIG. 1Bshows a cross-sectional view of the collector100taken along the line I-I shown inFIG. 1A. Dot-dashed line102represents the central or highest-symmetry axis of the collector100. The collector100may be sized and shaped to fit within a primary vessel containing or capable of holding a suspension, the suspension suspected of including a target material. The collector100funnels the target material from the suspension through a cannula106and into a processing vessel (not shown) to be located within a cavity108. The collector100includes the main body104which includes a first end110and a second end112. A seal may be formed between the second end112and an inner wall of the primary vessel to maintain a fluid-tight sealing engagement before, during, and after centrifugation and to inhibit any portion of the suspension from being located or flowing between an inner wall of the primary vessel and a main body104of the collector100. The seal may be formed by an interference fit, a grease (such as vacuum grease), an adhesive, an epoxy, by bonding (such as by thermal bonding), by welding (such as by ultrasonic welding), by clamping (such as with a ring or clamp), an insert (such as an O-ring or a collar) that fits between the second end112and the inner wall of the primary vessel, or the like. The main body104may be any appropriate shape, including, but not limited to, cylindrical, triangular, square, rectangular, or the like. The collector100also includes an internal funnel114which is a concave opening. The funnel114may taper toward the cannula106from the second end112. The funnel114channels a target material from below the second end112into the cannula106which is connected to, and in fluid communication with, an apex of the funnel114. The apex of the funnel114has a smaller diameter than the mouth of the funnel114. The funnel114is formed by a tapered wall that may be straight, curvilinear, arcuate, or the like. The funnel114may be any appropriate shape, including, but not limited to, tubular, spherical, domed, conical, rectangular, pyramidal, or the like. Furthermore, the outermost diameter or edge of the funnel114may be in continuous communication or constant contact (i.e. sit flush) with the inner wall of the primary vessel such that no dead space is present between the second end112of the collector100and the inner wall of the primary vessel.

The cannula106, such as a tube or a needle, including, but not limited to a non-coring needle, extends from the apex of the funnel114and into the cavity108. In the example ofFIG. 1, the cavity108is a concave opening extending from the first end110into the main body104and may accept and support the processing vessel (not shown). The cavity108may be any appropriate depth to accept and support the processing vessel (not shown). The cannula106may extend any appropriate distance into the cavity108in order to puncture the base of, or be inserted into, the processing vessel (not shown). The cannula106may include a flat tip, a beveled tip, a sharpened tip, or a tapered tip. Furthermore, the cavity108may be any appropriate shape, including, but not limited to, tubular, spherical, domed, conical, rectangular, pyramidal, or the like. The cavity108may be threaded to engage a threaded portion of the processing vessel (not shown).

The collector100may also include a retainer (not shown) to prevent the collector100from sliding relative to the primary vessel, thereby keeping the collector100at a pre-determined height within the primary vessel. The retainer (not shown) may be a shoulder extending radially from the first end110, a clip, a circular protrusion that extends beyond the circumference of the cylindrical main body104, a detent, or the like.

FIG. 2Ashows an isometric view of a collector200.FIG. 2Bshows a cross-section view of the collector200taken along the line II-II shown inFIG. 2A. Dot-dashed line202represents the central or highest-symmetry axis of the collector200. The collector200is similar to the collector100, except that the collector200includes a main body204that is more elongated than the main body104of the collector100in order to accommodate a greater portion of the processing vessel (not shown). The main body204includes a first end206and a second end208. A seal may be formed between the second end208and an inner wall of the primary vessel to maintain a fluid-tight sealing engagement before, during, and after centrifugation and to inhibit any portion of the suspension flowing between an inner wall of the primary vessel and the main body204of the collector200. The seal may be formed by an interference fit, a grease (such as vacuum grease), an adhesive, an epoxy, by bonding (such as thermal bonding), by welding (such as ultrasonic welding), clamping (such as with a ring or clamp), an insert (such as an O-ring or a collar) that fits between the second end208and the inner wall of the primary vessel, or the like.

The first end206includes a cavity212dimensioned to accept and hold at least a portion of the processing vessel (not shown). The cavity212may have a tapered or stepped bottom end220on which the processing vessel (not shown) may rest. The first end206may also include at least one cut-out210to permit proper grip of the processing vessel (not shown) for insertion and removal. The collector200funnels the target material from the suspension into an internal funnel222at the second end208, through a cannula214, and into a processing vessel (not shown) located within the cavity212. The cannula214may rest on a shelf224so that an inner bore of the cannula214sits flush with an inner wall of the funnel222, as shown inFIG. 2B.

The collector200may include a shoulder216, which extends circumferentially around the main body204. The shoulder216may be larger than the inner diameter of the primary vessel so as to rest on the open end of the primary vessel and, upon applying a lock ring (not shown) to the outside of the primary vessel and the shoulder216, to inhibit movement of the collector200relative to the primary vessel. The lock ring (not shown) applies pressure to the primary vessel along the shoulder216. The lock ring may be a two-piece ring, a one piece ring wrapping around the full circumference of the primary vessel, or a one piece ring wrapping around less than the full circumference of the primary vessel, such as one-half (½), five-eighths (⅝), two-thirds (⅔), three-quarters (¾), seven-eighths (⅞), or the like. Alternatively, the shoulder216may fit within the primary vessel. Alternatively, the shoulder216may be a clip, such that the shoulder216may include a catch into which the primary vessel may be inserted to inhibit movement of the collector200relative to the primary vessel. Alternatively, the shoulder216may form an interference fit with the inner wall of the primary vessel around which a seal ring may be placed.

As shown inFIG. 2A, the collector200may include at least one window218to access the cavity212through an inner wall of the main body204. The at least one window218permits an operator to confirm proper placement of the processing vessel (not shown) within the cavity212. The at least one window218also allows fluid discharged from the cannula214to flow out of the collector200and into a space formed between the collector200and the primary vessel (not shown) and above the seal between the second end208and the inner wall of the primary vessel.

FIG. 2Cshows an isometric view of a collector230.FIG. 2Dshows a cross-section view of the collector230taken along the line III-III shown inFIG. 2C. The collector230is similar to the collector200, except that the collector230includes a main body238including an extension234extending away from a first end232and a lid236to at least temporarily seal an opening240within the extension234. The opening240may be in fluid communication with the cavity212at the first end232. The lid236may removable, puncturable and resealable (e.g. a flap lid), or puncturable and non-resealable (e.g. a foil lid). The extension234may be sized to accept the lid236when punctured such that a portion of the lid236does not extend into the cavity212at the first end232. Note that the collector230does not include the at least one cut-out210.

FIG. 3Ashows an isometric view of a collector300. Dot-dashed line302represents the central or highest-symmetry axis of the collector300. The collector300includes a first side304, a second side306, and a bore308. An inner wall312to form the bore308,324, and334may be tapered (i.e. becoming narrower from the first side304to the second side306; or becoming wider from the first side304to the second306), as shown inFIG. 3A.

The first and second sides304and306may be connected to the inner wall312via straight walls (i.e. first and second sides304and306are planar), tapered walls, or at least partially arcuate walls.

The collector300may be sized and shaped to fit within a vessel containing or capable of holding a suspension. The collector300fits against an inner wall of the vessel, such that no portion of the suspension may be located between the inner wall of the vessel and the main body310of the collector300. The collector300gathers a sample within the bore308. The bore308may be expandable, such that the diameter of the bore308may increase during centrifugation and then return to a resting diameter when not under centrifugation. Expanding the diameter may allow for less constricted flow of fluid and suspension components during centrifugation. The collector300may be composed of a ceramic, metal, polymer, flexible polymer, glass, organic or inorganic materials, or the like.

FIG. 3Bshows an isometric view of a collector320. The collector320is similar to the collector300, except that an inner wall322of the collector320may be straight (i.e. having a uniform diameter from the first side304to the second side306).FIG. 3Cshows an isometric view of a collector330. The collector330is similar to the collector300, except that an inner wall332of the collector330may be at least partially arcuate (i.e. concave, convex, or curvilinear).

The collector may also include a filter (not shown). The filter (not shown) may be located at the second side or in the bore. The filter (not shown) is configured to provide a more pure sample by permitting a target material to pass through, while inhibiting non-target material from passing through.

The cannula 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 a polypropylene, acrylic, polycarbonate, or the like; and combinations thereof. The cannula may have a tip along a longitudinal axis of the cannula.

Collector-Processing Vessel System

FIG. 4Ashows an exploded view of the example collector200and processing vessel402.FIG. 4Bshows a cross-sectional view of the processing vessel402inserted into the cavity212at the first end206of the collector200taken along the line IV-IV shown inFIG. 4A. The collector200and processing vessel402form a collector-processing vessel system400. The processing vessel402may be an Eppendorf tube, a syringe, or a test tube and has a closed end404and an open end406. The open end406is sized to receive a cap408. The cap408may 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 stored in the processing vessel402interior and re-seals when the needle or implement is removed. Alternatively, the processing vessel402may also have two open ends that are sized to receive caps. The processing vessel402may have a tapered geometry that widens or narrows toward the open end406; the processing vessel402may have a generally cylindrical geometry; or, the processing vessel402may 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 processing vessel402has 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 processing vessel402can be composed of a transparent, semitransparent, opaque, or translucent material, such as plastic or another suitable material. The processing vessel includes a central axis414, which when inserted into the cavity212is coaxial with the central axis202of the collector200. The processing vessel402may also include a plug410at the closed end404to permit the introduction of the target material or to exchange or replace the target material with a collection fluid412. The closed end404may be threaded to provide for a threaded connection with a threaded cavity212of the collector200. The processing vessel402may be composed of glass, plastic, or other suitable material.

The plug410may 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 processing vessel402interior or permit introduction of contents into the processing vessel402and re-seals when the needle or implement is removed. The plug410may be inserted into the processing vessel402such that a seal is maintained between the plug410and the processing vessel402, such as by an interference fit. Alternatively, the plug410can be formed in the closed end404of the processing vessel402using 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 plug410to 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 plug410may be injected into the processing vessel402. Alternatively, the plug410may be thermally bonded to the processing vessel402.

In the example ofFIG. 4B, the cannula214has a tapered tip that punctures the plug410and extends into an inner cavity of the processing vessel402with the shaft of the cannula214not extending into the inner cavity of the processing vessel402. As explained in greater detail below, the inner cavity of the processing vessel402holds the target material. The cannula214may be covered by a resealable sleeve (not shown) to prevent the target material from flowing out unless the processing vessel402is in the cavity212to a depth that allows the cannula214to just penetrate the processing vessel402. The resealable sleeve (not shown) covers the cannula214, is spring-resilient, can be penetrated by the cannula214, and is made of an elastomeric material capable of withstanding repeated punctures while still maintaining a seal.

As shown inFIGS. 4A-4B, the processing vessel402may be loaded with a collection fluid412prior to insertion into the collector200. The collection fluid412displaces the target material, such that when the collector200and processing vessel402are inserted into the primary vessel (not shown) including the target material, and the collector, processing vessel, and primary vessel undergo centrifugation, the collection fluid412flows out of the processing vessel402and into the primary vessel, and, through displacement, such as through buoyant displacement (i.e. lifting a material upwards), pushes the target material through the cannula214and into the processing vessel402.

The collection fluid412has 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 fluid412may 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.

The processing vessel402may also include a processing solution (not shown) to effect a transformation on the target material when the target material enters the processing vessel402. The processing solution (not shown) may be a preservative, a cell adhesion solution, a dye, or the like. Unlike the collection fluid412, most, if not all, of the processing solution (not shown) remains within the processing vessel402upon centrifugation, thereby effecting the transformation on the target material in one manner or another (i.e. preserving, increasing adhesion properties, or the like). The processing solution (not shown) may be introduced as a liquid or as a liquid contained in a casing. The casing may be dissolvable in an aqueous solution but not in the collection fluid412(such as gel cap); or, the casing may be breakable, such that the casing breaks when the processing vessel402is shaken in a vortex mixer. Additionally, more than one processing solution may be used.

The processing vessel402may include a flexible cap that can be pushed to dispense a pre-determined volume therefrom and onto a substrate, such as a slide or a well plate. The cap408may be flexible or the cap408may be removed and the flexible cap inserted into the open end406. Alternatively, the processing vessel402may be attached to (i.e. after accumulating the target material) or may include a dispenser, which is capable of dispensing a pre-determined volume of target material from the processing vessel402onto another substrate, such as a microscope slide. The dispenser may repeatedly puncture the re-sealable cap408or compress the material within the processing vessel402to withdraw and dispense the pre-determined volume of target material onto the substrate. Alternatively, the cap408may be removed and the dispenser (not shown) may be inserted directly into the processing vessel402to dispense the buffy coat-processing solution mixture.

FIG. 4Cshows an example collection system420including the collector300and a processing vessel422. The processing vessel422is similar to the processing vessel402. The processing vessel422may be inserted into the collector300. The processing vessel422may receive the target material or the portion of the target material. Furthermore, the processing vessel422may be removed from the collector300and then placed into another vessel, such as a tube, an Eppendorf tube or a slide, to transfer the target material or the portion of the target material to the other vessel, such as by centrifugation, for further processing. Alternatively, an adapter, such as a ferrule, may be included to connect the processing vessel422to the collector300. The adapter may be metal, plastic, glass, or the like.

FIG. 5Ashows an exploded view of the example collector200and canopy502.FIG. 5Bshows a cross-sectional view of the processing vessel502inserted into the cavity212of the collector200taken along the line V-V shown inFIG. 4A. The collector200and canopy502form a collector-canopy system500. The canopy502is similar to the processing vessel402, except that the canopy has a second open end504. When the collector-canopy system500is inserted into the primary vessel, some fluid within the primary vessel, such as a portion of the suspension, a portion of a suspension fraction, a portion of a clearing fluid, or the like, may be discharged through the cannula214. The canopy502inhibits a portion of the fluid in the primary vessel that may be discharged through the cannula214from escaping from the opening of the first end206of the collector200. The discharged fluid, having been blocked by the canopy502, flows out of the second open end504, and out of the window218. Dashed lines506show fluid flow as the fluid is discharged through the cannula214and retained by the canopy502.

Alternatively, when the collector230is used, the lid236of the collector230inhibits a portion of the fluid in the primary vessel that may be discharged through the cannula214from escaping from the opening of the first end206of the collector200in a manner similar to that of the canopy502.

Sealing Ring

FIG. 6Ashows an isometric view of a sealing ring600.FIG. 6Bshows a top down view of the sealing ring600. Dot-dashed line602represents the central or highest-symmetry axis of the sealing ring600. The sealing ring600includes an inner wall604, an outer wall606, and a cavity608. InFIG. 6B, RIWrepresents the radial distance from the center of the sealing ring600to the inner wall604, and ROWrepresents the radial distance from the center of the sealing ring600to the outer wall606. The sealing ring600is configured to fit around a primary vessel, such as a tube. The cavity608is sized and shaped to receive the primary vessel. The sealing ring600may be tightened, such that the size of the cavity608and the radii of the inner and outer walls604and606are reduced by circumferentially applying an approximately uniform, radial force, such as the radial force created by a clamp, around the outer wall606directed to the central axis602of the sealing ring600. When the sealing ring600is tightened around the primary vessel, the uniform force applied to the sealing ring600is applied to the primary vessel, thereby causing the primary vessel to constrict. When the radial force is removed from the sealing ring600, the sealing ring600remains tightened and in tension around the primary vessel.

The sealing ring may be any shape, including, but not limited to, circular, triangular, or polyhedral.FIG. 6Cshows an isometric view of a sealing ring610.FIG. 6Dshows a top down view of the sealing ring610. Sealing ring610is similar to sealing ring600, except sealing ring610is polyhedral. Dot-dashed line612represents the central or highest-symmetry axis of the sealing ring610. The sealing ring610includes an inner wall614, an outer wall616, and a cavity618. The sealing ring may be composed of a metal, such as brass, a polymer, or combinations thereof.

Alternatively, as shown inFIG. 6E, a sealing ring620may be composed of a piezoelectric material.FIG. 6Fshows a top down view of the sealing ring620. Dot-dashed line622represents the central or highest-symmetry axis of the sealing ring620. The sealing ring620may be connected to an electric potential source628, such as a battery, via a first lead624and a second lead626. The electric potential source628creates a mechanical strain that causes the sealing ring620to tighten (i.e. sealing ring620radii decrease). The sealing ring620includes an inner wall630, an outer wall632, and a cavity634. InFIG. 6F, RIWrepresents the radial distance from the center of the sealing ring620to the inner wall630, and ROWrepresents the radial distance from the center of the sealing ring620to the outer wall632. Alternatively, the sealing ring620may be in a naturally tightened stated. When applying the electric potential the sealing ring620expands. Alternatively, a portion of the sealing ring may be composed of the piezoelectric material, such that the piezoelectric portion acts as an actuator to cause the other portion of the sealing ring to tighten and apply the substantially uniform circumferential pressure on the primary vessel, thereby constricting the primary vessel to form the seal.

FIG. 6Gshows an isometric view of a sealing ring640. The sealing ring includes an adjustment mechanism648to adjust the inner diameter RID. The collapsible ring includes a processing vessel adapter642and a primary vessel adapter646, the first and primary vessel adapters642and646being joined by a band portion644. The first and primary vessel adapters642and646include complementary portions of the adjustment mechanism648. The adjustment mechanism648includes, but is not limited to, a ratchet, tongue and groove, detents, or the like.

The sealing ring may also include a thermal element, such as a heated wire. The thermal element may soften the primary vessel for constriction. Alternatively, the thermal element may melt the primary vessel to provide a more adherent seal. Alternatively, the thermal element may cause the sealing ring to compress, thereby forming a seal between the primary vessel and float.

Sealing Ring

FIG. 7shows a fluid layering device700. The fluid layering device700may include a motor704connected to a rod708which is also connected to a piston710. The fluid layering device700may also include a switch706to activate or de-activate the motor704. When the motor704is activated, the rod708, which may be threaded, may rotate, thereby causing the piston710to move up and down, thereby creating a pressure gradient to expel a layering fluid716from the fluid layering device700a plug714when the fluid layering device700is inserted into the collector200, such that the cannula216extends through the plug714. Alternatively, the rod708may move up and down, thereby causing the piston710to 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 piston710up and down to create the pressure gradient or to create the pressure gradient without the piston710.

The fluid layering device700may have a main body702with a tapered geometry that widens or narrows toward the plug714; the fluid layering device700may have a generally cylindrical geometry; or, the fluid layering device700may 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 device700has 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 device700can 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 cavity212of the collector200is coaxial with the central axis202of the collector200. The plug714permits the introduction of the layering fluid716into the primary vessel via the cannula216of the collector200.

The plug714may 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 device700interior or permit introduction of contents into the fluid layering device700and re-seals when the needle or implement is removed. The plug714may be inserted into the fluid layering device700such that a seal is maintained between the plug714and the fluid layering device700, such as by an interference fit. Alternatively, the plug714can be formed in the closed end404of the fluid layering device700using 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 plug714to 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 plug714may be injected into the fluid layering device700. Alternatively, the plug714may be thermally bonded to the fluid layering device700.

The fluid layering device700may also include a controller (not shown) to control the output of the motor704, such as the frequency and/or amplitude of the various strokes. The fluid layering device700may also include a battery (not shown) which may be chargeable or replaceable.

Methods

For the sake of convenience, the methods are described with reference to an example suspension of anticoagulated whole blood. But the methods described below are not intended to be so limited in their scope of application. The methods, in practice, may be used with any kind of sample, such as a suspension or other biological fluid. For example, a sample may be urine, blood, bone marrow, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, synovial fluid, nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucus membrane secretions, aqueous humor, vitreous humor, vomit, a suspension derived from a tissue sample or a culture sample, and any other physiological fluid or semi-solid. It should also be understood that a target material may be a fraction of a sample or a sub-fraction of a fraction, such as a portion of buffy coat. The target material may include an analyte, such as a cell, such as ova, a nucleated red blood cell, or a circulating tumor cell (“CTC”), a circulating endothelial cell, a fetal 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, a parasite (e.g. spirochetes, such asBorrelia burgdorferi), a microorganism, a virus, or an inflammatory cell; or, the target material may be the analytes.

FIG. 8shows a flow diagram for an example method for retrieving a target material. In block802, 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. 9Ashows an isometric view of an example primary vessel and float system900. The system900includes a primary vessel902and a float904suspended within whole blood906. In the example ofFIG. 9A, the primary vessel902has a circular cross-section, a first open end910, and a second closed end908. The open end910is sized to receive a cap912. The primary vessel may also have two open ends that are sized to receive caps, such as the example tube and separable float system920shownFIG. 9B. The system920is similar to the system900except the primary vessel902is replaced by a primary vessel922that includes two open ends924and926configured to receive the cap912and a cap928, respectively. The primary vessels902and922have a generally cylindrical geometry, but may also have a tapered geometry that widens, narrows, or a combination thereof toward the open ends910and924, respectively. Although the primary vessels902and922have a circular cross-section, in other embodiments, the primary vessels902and922can have elliptical, square, triangular, rectangular, octagonal, or any other suitable cross-sectional shape that substantially extends the length of the tube. The primary vessels902and922can be composed of a transparent, semitransparent, opaque, or translucent material, such as plastic or another suitable material. The primary vessels902and922each include a central axis918and930, respectively. The primary vessel902may also include a septum914, as seen in magnified view916, at the closed end908to 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 vessel902may have an inner wall and a first diameter.

The septum914may 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 vessel902interior and re-seals when the needle or implement is removed. The septum914may be inserted into the primary vessel902such that a seal is maintained between the septum914and the primary vessel902, such as by an interference fit. Alternatively, the septum914can 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 septum914to 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 septum914may be thermally bonded to the primary vessel902.

The float904includes a main body, two teardrop-shaped end caps, and support members radially spaced and axially oriented on the main body. Alternatively, the float904may not include any support members. Alternatively, the float904may include support members which do not engage the inner wall of the primary vessel902.

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 vessel902, thereby defining fluid retention channels between the outer surface of the main body and the inner wall of the primary vessel902. 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 float904can 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 float904can 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 vessel902may have an inner wall and a first diameter. The float904can be captured within the primary vessel902by an interference fit, such that under centrifugation, an inner wall of the tube expands to permit axial movement of the float904. 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 float904and the primary vessel902, 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 cap912may be composed of a variety of different materials including, but not limited to, organic or inorganic materials; plastic materials; and combination thereof.

In block804, the sample, the float, and the primary vessel undergo centrifugation.FIG. 10shows an isometric view of the primary vessel and float system900having 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 cells1003located on the bottom, plasma1001located on top, and buffy coat1002located in between, as shown inFIG. 10. The float904may have any appropriate density to settle within one of the fractions. The density of the float904can be selected so that the float904expands the buffy coat1002between the main body of the float and the inner wall of the primary vessel. The buffy coat1002can be trapped within an area between the float904and the primary vessel902.

FIGS. 11A and 11Bshow a first seal being formed to prevent fluids from moving up or down within the primary vessel. For convenience,FIG. 11Ashall be used to describe the method, though the method applies equally toFIG. 11B. The first seal also inhibits float movement. The first sealing ring600may be placed at approximately a lower end of the main body of the float904. The first sealing ring600exerts circumferential or radial forces on the primary vessel902, thereby causing the primary vessel902to collapse inwardly against the float904. Magnified view1102shows the first sealing ring600tightened around the float and primary vessel system900. The first sealing ring600, having been placed at an interface of the buffy coat1002and the red blood cells1003, causes the primary vessel902to collapse inwardly until a seal is formed between the primary vessel902and the float904. An outer wall of the first sealing ring600may sit flush with an outer wall of the primary vessel902; the outer wall of the first sealing ring600may extend past the outer wall of the primary vessel902; or, the outer wall of the primary vessel902may extend past the outer wall of the first sealing ring600. The first sealing ring600remains tightened to maintain the seal, which prevents fluids from moving past the seal in any direction. The first sealing ring600may also remain in tension. Alternatively, the first sealing ring600may be overtightened and then the force applied to the first sealing ring600is removed. The first sealing ring600may expand slightly, though still remains constricted.

To apply the first sealing ring600and thereby form the seal, a clamp may be used to circumferentially apply a force directed toward the central axis of the primary vessel902to the first sealing ring600and the float and primary vessel system900. The first sealing ring600is placed around the float and primary vessel system900after the float and primary vessel system900have undergone density-based separation, such as by centrifugation. The first sealing ring600and float and primary vessel system900are then placed into the clamp. The clamp may include a shelf to support the first sealing ring600against the primary vessel902. Operation of the clamp may be automated or may be performed manually. Alternatively, the clamp may form a seal between the float904and primary vessel902without the inclusion of the first sealing ring600. Alternatively, a seal may be formed between the float904and the primary vessel902such as by ultrasonic welding; or by applying heat or a temperature gradient to deform and/or melt the primary vessel902to the float904. 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.

When operation of the clamp is automated, a motor causes translation of either a collet, including collet fingers, or a pressure member to cause compression of the collet fingers. The motor may be connected to the collet or the pressure member by a shaft, such as a cam shaft, and one or more gears. A base engages and holds the object. When the collet is driven by the motor, the pressure member remains stationary. When the pressure member is driven by the motor, the collet remains stationary. The clamp may include a release, so as to cause the pressure member to slide off of the collet fingers904, thereby removing the clamping force.

Alternatively, the clamp may be, but is not limited to, a collet clamp, an O-ring, a pipe clamp, a hose clamp, a spring clamp, a strap clamp, or a tie, such as a zip tie. The clamp may be used without a first sealing ring to provide a seal between a float and a tube.

The plasma1001may be removed from the primary vessel902, as shown inFIG. 12A, such as by pipetting, suctioning, pouring, or the like. Returning toFIG. 8, in block806, a clearing fluid1202may be added to the primary vessel902and the system undergoes centrifugation again, as shown inFIGS. 12B-12C. The clearing fluid1202has a density greater than the buffy coat1002, but may be layered on top of the buffy coat1002. It may be desirous to gently layer the clearing fluid1202on top of the buffy coat1002to inhibit mixing of the clearing fluid1202with the buffy coat1002. During centrifugation, the clearing fluid1202moves underneath the buffy coat1002but remains above the clamp600. After centrifugation, the buffy coat1002, having a density less than the clearing fluid1202, rests on top of the clearing fluid1202. An appropriate amount of the clearing fluid1202, such as up to 500 milliliters, may be added to the primary vessel902, such that the buffy coat1002is not between the main body of the float904and the inner wall of the primary vessel902. The clearing fluid1202may 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 ring600may be placed at approximately an upper end of the main body of the float904, as shown inFIG. 12D. Returning toFIG. 8, in block808, a density-altering agent may be added to the primary vessel902to 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 block810, a collector, such as the collector200, may be inserted into the primary vessel902. In block812, a fluid layering device700may be inserted into the collector200.FIG. 12Dshows the collector200inserted into the primary vessel902and also forming a seal1206between the second end208of the collector200and the inner wall of the primary vessel902. The layering fluid716may be added to the primary vessel902by the fluid layering device700via the cannula216of the collector200, as shown inFIG. 12E.

Returning toFIG. 8, in block814, the fluid layering device700is removed from the collector200and a processing vessel402including the collection fluid412is inserted into the collector200, as also shown inFIG. 12E. The layering fluid716added by the fluid layering device700fills any air gap that exists extending from the cannula216of the collector to the uppermost layer of fluid or sample material within the primary vessel902. It should be noted that the collection fluid412and the layering fluid716may be the same type of fluid or may be a different type of fluid. The collection fluid412and the layering fluid716are referenced differently herein, even when the collection fluid412and the layering fluid716are the same, to depict that the collection fluid412and the layering fluid716are introduced to the system and method by different mechanisms, vessels, or devices.

Returning toFIG. 8, in block816, the vessel, the collector, and the processing receptacle are centrifuged. During centrifugation, the additional collection fluid412moves from the processing vessel402into the primary vessel902via the cannula216of the collector200and displaces the buffy coat1002from the primary vessel902into the processing vessel402via cannula216the collector200. The processing vessel402, now including the buffy coat1002, may be removed from the collector200.

The collection fluid may be miscible or immiscible with the suspension fluid and inert with respect to the suspension materials. The collection fluid412has a greater density than the density of the target material of the suspension (the density may be less than the density of at least one other suspension fraction or the density may be greater than all of the suspension fractions) and is inert with respect to the suspension materials. 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), a complex, branch glucan (e.g. Dextran), cesium chloride, sucrose, sugar-based solutions, polymer-based solutions, surfactants, an organic solvent, a liquid wax, an oil, olive oil, mineral oil, silicone oil, and ionic liquids; perfluoroketones, such as perfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones, hydrofluoroethers, hydrofluorocarbons, perfluorocarbons, perfluoropolyethers, silicon and silicon-based liquids, such as phenylmethyl siloxane.

The processing receptacle302may also include a processing solution to effect a transformation on the target material when the target material enters the processing receptacle302. 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 receptacle302upon 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 receptacle302. 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 receptacle302is 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.

Sequential density fractionation is the division of a sample into fractions or of a fraction of a sample into sub-fractions by a step-wise or sequential process, such that each step or sequence results in the collection or separation of a different fraction or sub-fraction from the preceding and successive steps or sequences. In other words, sequential density fractionation provides individual sub-populations of a population or individual sub-sub-populations of a sub-population of a population through a series of steps. For example, buffy coat is a fraction of a whole blood sample. The buffy coat fraction may be further broken down into sub-fractions including, but not limited to, reticulocytes, granulocytes, lymphocytes/monocytes, and platelets. These sub-fractions may be obtained individually by performing sequential density fractionation.

FIG. 13shows an example method1300for retrieving a target material using sequential density fractionation. The example method1300is similar to the example method800, except the example method1300collects the target material by sequential density fractionation. After the steps of block A have been performed, as shown inFIG. 8, sequential density fractionation is performed, as seen in block1302. Block1302is also a snapshot of the sequential density fractionation steps. In block1304, an nthprocessing receptacle including an nthcollection fluid is inserted into the collector, such that nthis greater than or equal to first (i.e. second, third, fourth, and so on). In block1306, the system is centrifuged to collect a fraction or sub-fraction and the nth processing receptacle is removed. In block1308, 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 block1310, 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 block1304. 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.

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 tube6. 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.

The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the disclosure. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the systems and methods described herein. The foregoing descriptions of specific embodiments are presented by way of examples for purposes of illustration and description. They are not intended to be exhaustive of or to limit this disclosure to the precise forms described. 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 this disclosure and practical applications, to thereby enable others skilled in the art to best utilize this disclosure and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of this disclosure be defined by the following claims and their equivalents: