Patent Application
20140349828
This disclosure relates generally to density-based fluid separation and, in particular, to retrieving a target material from a suspension.
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.
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 into a chamber at a first end of the collector and a funnel at a second end that that is in fluid communication with the cannula. The chamber is sized and shaped to hold the processing receptacle. In another implementation, the processing receptacle may be inserted into a bore within the collector.
The collector 100 includes a primary body 102. The primary body 102 includes a first end 104 and a second end 106. The primary body 102 may be any appropriate shape, including, but not limited to, cylindrical, triangular, square, rectangular, or the like. The collector 100 also includes a funnel 108 which is a concave cavity extending from the second end 106 into the primary body 102. The funnel 108 channels a target material from below the second end 106 into a cannula 110 which is connected to and in fluid communication with a apex of the funnel 108. The apex of the funnel 108 has a smaller diameter than the mouth of the funnel 108. The funnel 108 is formed by a tapered wall that may be straight, curvilinear, arcuate, or the like.
The cannula 110, such as a tube or a needle, including, but not limited to a non-coring needle, extends from the apex of the funnel 108 and into the chamber 112. The chamber 112 is a cavity to accept and support the processing receptacle (not shown). The chamber 112 may be threaded to engage a threaded portion of the processing receptacle (not shown). The cannula 110 may extend any appropriate distance into the chamber 112 in order to puncture or be inserted into the processing receptacle (not shown). The cannula 110 may include a flat tip or a tapered tip. The chamber 112 is a concave cavity extending from the first end 104 into the primary body 102. The chamber 112 may be any appropriate depth to accept and support the processing receptacle (not shown). Furthermore, the chamber 112 may be any appropriate shape, including, but not limited to, semi-spherical, conical, pyramidal, or the like.
The collector 100 may also include a retainer (not shown) to prevent the collector 100 from sliding relative to the vessel, thereby keeping the collector 100 at a pre-determined height within the vessel. The retainer (not shown) may be a shoulder extending radially from the first end 104, a clip, a circular protrusion that extends beyond the circumference of the cylindrical primary body 102, a detent, or the like.
The primary body may 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, polyimides, polymethacrylate, polyolefins (e.g., polyethylene, polypropylene), polyallomers, polyoxadiazole, polyparaxylene, polyphenylene oxides (PPO), modified PPOs, polystyrene, polysulfone, fluorine containing polymer such as polytetrafluoroethylene, polyurethane, polyvinyl acetate, polyvinyl alcohol, polyvinyl halides such as polyvinyl chloride, polyvinyl chloride-vinyl acetate copolymer, polyvinyl pyrrolidone, polyvinylidene chloride, specialty polymers, polystyrene, polycarbonate, polypropylene, acrylonitrite butadiene-styrene copolymer, butyl rubber, ethylene propylene diene monomer; and combinations thereof.
The cannula may 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.
The first and second sides 204 and 206 may be connected to the inner wall 212 via straight walls (i.e. first and second sides 204 and 206 are planar), tapered walls, or at least partially arcuate walls.
The collector 200 may be sized and shaped to fit within a vessel containing or capable of holding a suspension. The collector 200 fits 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 outer wall 210 of the collector 200. The collector 200 gathers a sample within the bore 208. The bore 208 may be expandable, such that the diameter of the bore 208 may 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 collector 200 may be composed of a ceramic, metal, polymer, flexible polymer, glass, organic or inorganic materials, or the like.
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 plug 306 may be composed of re-sealable rubber or other suitable re-sealable material that may be repeatedly punctured with a needle or other sharp implement to access the contents of the processing receptacle 302 interior or permit introduction of contents into the processing receptacle 302 and re-seals when the needle or implement is removed. The plug 306 may be formed in the closed end 310 of the processing receptacle 302 using heated liquid rubber that may be shaped and hardens as the rubber cools. The adhesive used to attach a plug to the wall may 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 306 may be injected into the processing receptacle 302. Alternatively, the plug 306 may be pre-molded and then inserted into the processing receptacle 302.
When the cannula 130 includes a tapered tip, a portion of the tapered tip may extend into an inner cavity of the processing receptacle 302, whereas another portion of the tapered tip may not enter the inner cavity of the processing receptacle 302. The inner cavity of the processing receptacle 302 is the portion of the processing receptacle 302 to hold the suspension. The cannula 130 may be covered by a resealable sleeve (not shown) to prevent the target material from flowing out unless the processing receptacle 302 is in the chamber 138 and is inserted to a depth appropriate enough for the cannula 130 to penetrate the processing receptacle 302. The resealable sleeve (not shown) covers the cannula 130, is spring-resilient, may be penetrated by the cannula 130, and is made of an elastomeric material capable of withstanding repeated punctures while still maintaining a seal.
The processing receptacle 302 may include a flexible cap that may be pushed to dispense a pre-determined volume therefrom and onto the substrate. The cap 312 may be flexible or the cap 312 may be removed and the flexible cap inserted into the open end 308. Alternatively, the processing receptacle 302 may 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 receptacle 302 onto another substrate, such as a microscope slide. The dispenser may repeatedly puncture the re-sealable cap 312 or compress the material within the processing receptacle 302 to withdraw and dispense the pre-determined volume of target material onto the substrate. Alternatively, the cap 312 may be removed and the dispenser (not shown) may be inserted directly into the processing receptacle 302 to dispense the sample-processing solution mixture.
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 as Borrelia burgdorferi), a microorganism, a virus, or an inflammatory cell; or, the target material may be the analytes.
In one instance, the density of the plasma may be changed to be greater than the density of the buffy coat but less than the density of the red blood cells. 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, the hematocrit of the blood may be obtained to calculate the amount of plasma-density-changing solution to be added to the blood sample based on the amount of plasma within the blood sample. In another instance, the density of the plasma may be changed to be greater than the densities of both the buffy coat and the red blood cells. Alternatively, more than one fraction-density-changing solution may be added to change the density of another non-target material, such as the red blood cells, to effect a greater demarcation between the target material and the non-target materials. The fraction-density-changing solution may be isotonic or non-isotonic, so as to alter the density of a target material or a non-target material.
The fraction-density-altering solution may be miscible with the suspension fluid and inert with respect to the suspension materials. 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, and multi-phase polymer solutions.
In block 506, a delineation fluid may be added to the vessel. The delineation fluid may be layered on top of the blood sample. It may be desirous to gently layer the delineation fluid on top of the blood sample to inhibit mixing of the delineation fluid with the blood sample. The delineation fluid may be used to cause further separation between the target material and any non-target material below the target material after centrifugation, for example, to further separate the buffy coat and the plasma when the density of the plasma is between the densities of the buffy coat and the red blood cells, or to further separate the buffy coat from the red blood cells when the density of the red blood cells is between the densities of the buffy coat and the plasma. The delineation fluid may lyse or congeal a non-target material, such as red blood cells of a whole blood sample. The delineation fluid may be isotonic or non-isotonic, so as to alter the density of a target material or a non-target material.
The delineation fluid may be miscible or immiscible with the suspension fluid and inert with respect to the suspension materials. Examples of delineation 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, an oil, olive oil, mineral oil, silicone oil, silicon and silicon-based liquids, such as phenylmethyl siloxane.
Returning to
Returning to
The displacement fluid may be miscible or immiscible with the suspension fluid and inert with respect to the suspension materials. The displacement fluid 208 has 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 displacement 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 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 displacement 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 displacement 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.
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.
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. 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 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.
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:
The following is an example method for retrieving a target material when the density of the plasma is changed to be greater than the densities of both the buffy coat and the red blood cells:
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:
This application 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.
| Number | Date | Country | |
|---|---|---|---|
| 61818301 | May 2013 | US | |
| 61869866 | Aug 2013 | US | |
| 61935457 | Feb 2014 | US |
